EP4179108A1

EP4179108A1 - Methods analysing 5'-monophosphorylated mrna fragments in prokaryotic cells

Info

Publication number: EP4179108A1
Application number: EP21742068.6A
Authority: EP
Inventors: Vicente Pelechano GARCIA
Original assignee: 3n Bio AB
Current assignee: 3n Bio AB
Priority date: 2020-07-07
Filing date: 2021-07-01
Publication date: 2023-05-17
Also published as: GB202010429D0; WO2022008341A1; GB2596822A; CN116096918A

Abstract

The present invention generally relates to methods of determining the identity of one or more 5'-monophosphorylated messenger ribonucleic acid (mRNA) sequence being translated in a prokaryotic cell in which 5'-3' co-translational degradation of mRNA occurs. The invention also relates to corresponding uses, kits of parts and libraries of sequences.

Description

METHODS ANALYSING 5'-MONOPHOSPHORYLATED MRNA FRAGMENTS IN PROKARYOTIC CELLS

The present invention generally relates to methods of determining the identity of one or more messenger ribonucleic acid (rrsRNA) sequence being translated in a prokaryotic cell. The invention also relates to corresponding uses, kits of parts and libraries of sequences.

Prokaryotes, and bacteria in particular, are organisms that place a large burden on society, such as by causing disease and spoilage of food stuff. Important in managing the impact of prokaryotes on society is being able to quickly identify problematic prokaryotes and/or identify characteristics of prokaryotes. Being able to conduct such tests can allow for timely action to be taken in response to the challenges presented by the prokaryotes, such as the fast and accurate diagnosis of a disease and/or testing for antibiotic resistance.

Although there are genetic tests for identifying prokaryotes and/or their characteristics, these often focus on the prokaryotic genome or the presence of mRNA. However, such methods do not identify if the genome or mRNA results in the production of functional proteins, and it is often the existence and/or identity of the functional proteins that is important for identifying prokaryotes and/or their characteristics, and whether they are actually viable and/or replicating. Additionally, known methods are not suitable for identifying what specific prokaryotes are viable and/or replicating in a complex sample (such as a soil sample or a sample from a patient) and are often slow, as they require numerous rounds of culturing the prokaryotes in order to conduct the testing.

Some known methods involve measuring ribosome dynamics using ribosome profiling (Brar and Weissman 2015, Nat. Rev. Mol. Cell Biol., 16: 651-664). However, ribosome profiling only allows identification of very small fragments of RNA which makes it difficult to identify the sequences the fragments are from. Additionally, the process of ribosome profiling is complex and requires fresh cells, so is expensive, time consuming and not useable for complex samples.

Accordingly, there is a need for new methods for identifying prokaryotes and their characteristics.

Against this background, the inventor has surprisingly discovered that some prokaryotic cells undertake 5’-3’ co-translational degradation, and that in such prokaryotic cells it is possible to determine one or more mRNA sequences which are actually being translated, by determining the sequence of one or more 5’-monophosphorylated fragments of mRNA. Furthermore, the inventor has surprisingly discovered that such methods of identifying translated mRNA are particularly useful in determining the identity of a prokaryotic cell in a sample, providing a diagnosis and/or prognosis in a patient of a condition caused by one or more prokaryotic organism, identifying the effect of an agent on the mRNA sequences being translated in a prokaryotic cell, identifying the physiological status of one or more prokaryotic cell, and in determining the effect of an environmental condition on one or more prokaryotic cell.

In a first aspect, the invention provides a method for determining the identity of one or more mRNA sequence being translated in a prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(ii) determining, from the one or more prokaryotic cell, the sequence of one or more 5’- monophosphorylated fragment of mRNA;

(iii) determining, from the sequence information in step (ii), the identity of one or more mRNA sequence being translated in the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5 3 co-translational degradation of mRNA occurs.

By “identity of one or more mRNA sequence being translated”, we include that the sequence of RNA nucleotides is translated from the mRNA to a protein in the prokaryotic cell. As would be known to one skilled in molecular biology, not all mRNA sequences transcribed in a prokaryotic cell are actually translated into protein; this method identifies the mRNA sequences that are translated into protein. We also include that the “identity of the one or more mRNA sequence being translated” (which includes the sequence or partial sequence of RNA nucleotides) is sufficient to identify the gene from which the mRNA was transcribed and/or the protein to which the mRNA will be translated.

One skilled in molecular biology would understand the cellular processes of transcription and translation, which both fall within the overall term “gene expression”. Transcription includes the production of an RNA molecule based on a particular deoxyribonucleic acid (DNA) sequence, mediated by RNA polymerase enzymes. In the context of the invention, the DNA sequence will be a gene from which an mRNA molecule, an RNA copy of the DNA sequence, is produced. RNA is a polymeric nucleic acid molecule made from monomers called nucleotides - the order of the particular nucleotides in an RNA molecule is the RNA sequence. RNA nucleotides include guanine (G), uracil (U), adenine (A) and cytosine (C). An RNA sequence is often a linear structure, with the first nucleotide in the sequence being termed as the 5’ nucleotide and the last nucleotide in the sequence being termed as the 3’ nucleotide. The terms 5’ and 3’ are derived from the names of the 5’ and 3’ carbons on the RNA ribose sugar which are normally attached to adjacent nucleotides in an RNA sequence, but in the first nucleotide of a sequence the 5’ carbon is not connected (hence the 5’ nucleotide) and in the last nucleotide of a sequence the 3’ carbon is not connected (hence the 3’ nucleotide).

The RNA sequence defines what protein will be produced from it, via translation. Accordingly, translation includes the production of a protein from the mRNA molecule, which is mediated by a ribosome. The ribosome reads the RNA nucleotide sequence three nucleotides at a time from the 5’ end to the 3’ end (the three nucleotide sequences are termed “codons”). The ribosome interprets different codons as different amino acids, and forms a protein sequence of different amino acids in the order of the corresponding codons in the mRNA sequence. The 5’ nucleotide of an mRNA sequence encoding a protein is often termed the start codon, and in prokaryotes it generally encodes an amino acid that is a derivative of methionine (such as N- Formylmethionine (fMet)). The 3’ nucleotide of a mRNA sequence is often termed the stop codon, which includes the codon sequences of UAG, UAA and UGA. The stop codon does not encode an amino acid, but leads to the ribosome dissociating from the mRNA molecule.

Accordingly, by “partial sequence of RNA nucleotides” we include that a sequence of RNA nucleotides that does not have a start codon and/or does not have a stop codon. Alternatively, a partial sequence of RNA nucleotides might have both a start codon and a stop codon but be lacking a sequence of RNA nucleotides between the start codon and the stop codon. The method of the invention includes determining the sequence of one or more 5’- monophosphorylated fragment of mRNA, but those fragments comprise the whole or nearly whole RNA sequence. As outlined above, the sequence of RNA nucleotides needs to be sufficient to identify the gene from which the mRNA was transcribed and/or the protein to which the mRNA will be translated. As discussed herein, it would be known to the person skilled in molecular biology how to conduct such an identification, such as from a partial sequence of RNA nucleotides.

By “whole sequence of RNA nucleotides”, we include that a sequence of RNA nucleotides that has a start codon and a stop codon and does not lack a sequence of RNA nucleotides between the start codon and the stop codon. It would be known to the skilled person in molecular biology how to identify a particular gene or a particular protein based on an RNA sequence or a partial RNA sequence. For example, various freely-available databases of bacterial genomes are available, such as those published on the National Center for Biotechnology Information Assembly database (https://www.ncbi.nlm.nih.gov/assemblv/^') as discussed further in the Example. It would also be known by one skilled in molecular biology and/or bioinformatics how to align the identified sequences with the sequences in available databases, such as how is described in the Example.

By “5’-monophosphorylated fragment of mRNA”, we include a part of an mRNA molecule, such as a degradation product, on which the 5’ carbon of the 5’ nucleotide is attached to one phosphate group. The 5’-monophosphorylated fragment of mRNA arises through the process of 5’-3’ co-translational degradation, as described herein, in which the degradation of the mRNA molecule leads to a shorter mRNA fragment on which the 5’ carbon is phosphorylated. In one embodiment, the 5’-monophosphorylated fragment of mRNA comprises part of the 5’ untranslated region (UTR) and/or the start codon. In an alternative embodiment, the 5’- monophosphorylated fragment of mRNA does not comprise the start codon; in this embodiment, the 5’-monophosphorylated fragment of mRNA may have been generated by 5’- 3’ co-translational degradation from a break in the middle of the mRNA (such as an endonucleolytic cleavage site), as described herein. In one preferred embodiment of the invention, the one or more monophosphorylated fragments of mRNA are from different parts of the mRNA molecule so have different sequences. In a further preferred embodiment, the one or more monophosphorylated fragments of mRNA are from different mRNA sequences from the one or more prokaryotic cells. By “5’-monophosphorylated fragment of mRNA”, we include that the mRNA is a fragment that is at least about 1 % shorter than the mRNA molecule; for example, at least about 2%; at least about 3%; at least about 4%; at least about 5%; at least about 6%; at least about 7%; at least about 8%; at least about 9%; at least about 10%; at least about 15%; at least about 20%; at least about 25%; at least about 30%; at least about 35%; at least about 40%; at least about 45%; at least about 50%; at least about 55%; at least about 60%; at least about 65%; at least about 70%; at least about 75%; at least about 80%; at least about 85%; at least about 95%; or about 99% shorter than the mRNA molecule.

The term “5’-3’ co-translational degradation” would be understood by a person skilled in molecular biology. By “5’-3’ co-translational degradation”, we include that the mRNA molecule is degraded as it is being translated, and that the degradation starts at least at the 5’ nucleotide (as described herein, the 5’ nucleotide can be at an original termini of the mRNA molecule or at a break in the middle of the mRNA molecule, with that break becoming a new 5’ termini); for example, we include that the mRNA molecule is degraded while it is attached to a ribosome or is interacting with a ribosome, and that the degradation starts at least at the 5’ nucleotide. Accordingly, by “prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs” we include that the prokaryotic cell is capable of 5’-3’ co-translational degradation; for example, the prokaryotic cell comprises cellular machinery which can mediate 5’-3’ co- translational degradation.

The process of 5’-3’ co-translational degradation has been known to occur in eukaryotic cells, in which it is mediated by the exonuclease enzyme XRN1 (Pelechano et a!., 2015, Cell, 161 :1400-1412 and Pelechano et al., 2016, Nat. Protoc., 11 :359-376). However, it was previously not thought that the process of co-translational degradation occurred in prokaryotic cells. Through the inventor’s surprising discovery that 5’-3’ co-translational degradation does occur in prokaryotic cells, it has been possible to develop the methods of the invention. As discussed herein, 5’-3’ co-translational degradation may be characterised by a 3-nucleotide periodicity in the mRNA sequences identified.

In a second aspect, the invention provides a method for determining the presence and/or identity of one or more prokaryotic cell in a sample, comprising the steps of:

(i) providing a sample;

(ii) determining, from the sample, the sequence of one or more 5’-monophosphorylated fragment of mRNA;

(iii) determining, from the sequence information in step (ii), the presence and/or identity of one or more prokaryotic cell in the sample; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

By “determining the presence of one or more prokaryotic cell in a sample”, we include determining whether there is one or more prokaryotic cells in the sample. In a preferred embodiment, we include that “determining the presence of one or more prokaryotic cell in a sample” comprises determining if the one or more prokaryotic cell is viable and/or replicating. In one embodiment (for example, of the second aspect of the invention), the identity of the one or more prokaryotic cells, the presence of which is to be determined, in the sample is known before the method is undertaken. In an alternative embodiment (for example, of the second aspect of the invention), the identity of the one or more prokaryotic cells, the presence of which is to be determined, in the sample is not known before the method is undertaken.

By “identity of the one or more prokaryotic cells”, we include that that identity is defined by the phylum, class, order, family, genus, species and/or strain of the one or more prokaryotic cells. Accordingly, by “determining the identity of one or more prokaryotic cell in a sample” we include determining the phylum, class, order, family, genus, species and/or strain of the one or more prokaryotic cells in the sample.

In one embodiment (for example, of the second aspect of the invention), the one or more prokaryotic cell comprises about two or more prokaryotic cells; for example, about three or more; about four or more; about five or more; about six or more; about seven or more; about eight or more; about nine or more; about 10 or more; about 15 or more; about 20 or more; about 25 or more; about 30 or more; about 35 or more; about 40 or more; about 50 or more; about 60 or more; about 70 or more; about 80 or more; about 90 or more; about 100 or more; about 150 or more; about 200 or more; about 250 or more; about 300 or more; about 350 or more; about 400 or more; about 500 or more; about 550 or more; about 600 or more; about 750 or more; about 800 or more; about 850 or more; about 900 or more; about 950 or more; about 1 ,000 or more; about 2,000 or more; about 3,000 or more; about 4,000 or more; about 5,000 or more; about 6,000 or more; about 7,000 or more; about 8,000 or more; about 9,000 or more; about 10,000 or more; about 20,000 or more; about 30,000 or more; about 40,000 or more; about 50,000 or more; about 60,000 or more; about 70,000 or more; about 80,000 or more; about 90,000 or more; about 100,000 or more; about 200,000 or more; about 300,000 or more; about 400,000 or more; about 500,000 or more; about 600,000 or more; about 700,000 or more; about 800,000 or more; about 900,000 or more; about 1 ,000,000 or more; about 2,000,000 or more; about 3,000,000 or more; about 4,000,000 or more; about 5,000,000 or more; about 6,000,000 or more; about 7,000,000 or more; about 8,000,000 or more; about 9,000,000 or more; about 10,000,000 or more; about 100,000,000 or more; about 1 ,000,000,000 or more; about 10,000,000,000 or more; or about 100,000,000,000 or more prokaryotic cells.

In one embodiment, the one or more mRNA sequence comprises about two or more mRNA sequences; for example, about three or more; about four or more; about five or more; about six or more; about seven or more; about eight or more; about nine or more; about 10 or more; about 15 or more; about 20 or more; about 25 or more; about 30 or more; about 35 or more; about 40 or more; about 50 or more; about 60 or more; about 70 or more; about 80 or more; about 90 or more; about 100 or more; about 150 or more; about 200 or more; about 250 or more; about 300 or more; about 350 or more; about 400 or more; about 500 or more; about 550 or more; about 600 or more; about 750 or more; about 800 or more; about 850 or more; about 900 or more; about 950 or more; about 1 ,000 or more; about 2,000 or more; about 3,000 or more; about 4,000 or more; about 5,000 or more; about 6,000 or more; about 7,000 or more; about 8,000 or more; about 9,000 or more; about 10,000 or more; about 20,000 or more; about 30,000 or more; about 40,000 or more; about 50,000 or more; about 60,000 or more; about 70,000 or more; about 80,000 or more; about 90,000 or more; about 100,000 or more; about 200,000 or more; about 300,000 or more; about 400,000 or more; about 500,000 or more; about 600,000 or more; about 700,000 or more; about 800,000 or more; about 900,000 or more; about 1 ,000,000 or more; about 2,000,000 or more; about 3,000,000 or more; about 4,000,000 or more; about 5,000,000 or more; about 6,000,000 or more; about 7,000,000 or more; about 8,000,000 or more; about 9,000,000 or more; or about 10,000,000 or more mRNA sequences.

In one embodiment, the one or more 5’-monophosphorylated fragment comprises about two or more 5’-monophosphorylated fragments; for example, about three or more; about four or more; about five or more; about six or more; about seven or more; about eight or more; about nine or more; about 10 or more; about 15 or more; about 20 or more; about 25 or more; about 30 or more; about 35 or more; about 40 or more; about 50 or more; about 60 or more; about 70 or more; about 80 or more; about 90 or more; about 100 or more; about 150 or more; about 200 or more; about 250 or more; about 300 or more; about 350 or more; about 400 or more; about 500 or more; about 550 or more; about 600 or more; about 750 or more; about 800 or more; about 850 or more; about 900 or more; about 950 or more; about 1 ,000 or more; about 2,000 or more; about 3,000 or more; about 4,000 or more; about 5,000 or more; about 6,000 or more; about 7,000 or more; about 8,000 or more; about 9,000 or more; about 10,000 or more; about 20,000 or more; about 30,000 or more; about 40,000 or more; about 50,000 or more; about 60,000 or more; about 70,000 or more; about 80,000 or more; about 90,000 or more; about 100,000 or more; about 200,000 or more; about 300,000 or more; about 400,000 or more; about 500,000 or more; about 600,000 or more; about 700,000 or more; about 800,000 or more; about 900,000 or more; about 1 ,000,000 or more; about 2,000,000 or more; about 3,000,000 or more; about 4,000,000 or more; about 5,000,000 or more; about 6,000,000 or more; about 7,000,000 or more; about 8,000,000 or more; about 9,000,000 or more; or about 10,000,000 or more 5’-monophosphorylated fragments. In a particular embodiment, it might not be specifically known how many more than one 5’-monophosphorylated fragment is present prior to undertaking the method of any aspect of the invention, so we include that the two or more 5’-monophosphorylated fragments can be a plurality of 5’-monophosphorylated fragments. Preferably, the one or more 5’-monophosphorylated fragment comprises about 50 or more 5’-monophosphorylated fragments.

In a further embodiment, the one or more 5’-monophosphorylated fragment comprises about one to about 10,000,000,000 5’-monophosphorylated fragments; for example, about one to about 10,000,000; about 50 to about 10,000,000,000; or about 50 to about 10,000,000 5’- monophosphorylated fragments

In one embodiment (for example, of the second aspect of the invention), the method comprises determining the presence and/or identity of two or more prokaryotic cells in the sample, wherein the method further comprises the step of:

(iv) identifying, from the information in step (iii), whether the two or more prokaryotic cells belong to the same genus and/or species and/or strain or two or more different genus and/or species and/or strains.

In a further embodiment (for example, of the second aspect of the invention), the method comprises determining the presence and/or identity of two or more prokaryotic cells in the sample, wherein the method further comprises the step of:

(iv) identifying, from the information in step (iii), whether the two or more prokaryotic cells belong to the same phylum, class, order, family, genus, species and/or strain or two or more different phylum, classes, orders, families, genus, species and/or strains.

In a third aspect, the invention provides a method for the diagnosis and/or prognosis of a condition in a patient, comprising the steps of:

(i) providing a sample from the patient;

(ii) determining, from the sample, the sequence of one or more 5’-monophosphorylated fragments of mRNA;

(iii) determining, from the sequence information in step (ii), the presence and/or identity of one or more prokaryotic organism in the sample;

(iv) providing a diagnosis and/or prognosis in the patient on the basis of the one or more prokaryotic organism identified in step (iii); wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

By “diagnosis of a condition in a patient”, we include identifying the condition and/or the cause of the condition (for example, the identity of the one or more prokaryotic organism).

By “prognosis of a condition in a patient”, we include predicting the course of the condition; for example, identifying if the condition is likely to get more severe or less severe and/or identifying the likely duration of the condition.

In one embodiment (for example, of the third aspect of the invention), the condition is caused by the one or more prokaryotic organism.

In a particular embodiment (for example, of the third aspect of the invention), the condition is the physiological response of the patient. For example, the method of the third aspect of the invention can be used to assess how the patient has reacted to a drug or agent (as described herein), wherein the drug or agent might have had an indirect effect on the patient’s microbiome (including the one or more prokaryotic cell) (for example, as discussed in Maier, L, Pruteanu, M., Kuhn, M., Zeller, G., Telzerow, A., Anderson, E. E., et al. (2018). Extensive impact of non-antibiotic drugs on human gut bacteria. Nature, 555(7698), 623-628).

By “the condition is caused by the one or more prokaryotic organism”, we include that the presence of the one or more prokaryotic organism has led to condition and/or an infection of the one or more prokaryotic organism has led to condition and/or that the prokaryotic organism has caused one or more symptoms associated with the condition.

In one embodiment (for example, of the third aspect of the invention), the one or more prokaryotic organism is one or more pathogenic prokaryotic organism.

In one embodiment, the pathogenic prokaryotic organism is selected from the list consisting of: Clostridioides difficile; Campylobacter; Enterococcus; Staphylococcus aureus; Enterobacteriaceae; Neisseria; Acinetobacter; Pseudomonas aeruginosa; Tuberculosis; Streptococcus; Salmonella; Shigella; and Streptococcus. Most preferably, the pathogenic prokaryotic organism is selected from the list consisting of: Clostridioides difficile; Campylobacter; Enterococcus; Staphylococcus aureus; Enterobacteriaceae; Acinetobacter; Pseudomonas aeruginosa; Tuberculosis; Streptococcus; and Streptococcus. In a particular embodiment, the pathogenic prokaryotic organism is not selected from the list consisting of: Neisseria; Salmonella; and Shigella.

In one embodiment (for example, of the third aspect of the invention), the condition caused by the one or more prokaryotic organism is one or more condition selected from the list consisting of: acne; rhinosinusitis; typhus; bacterial meningitis; bacterial vaginosis; tuberculosis; tetanus; syphilis; bacterial pneumonia; gastroenteritis; food poisoning; and gonorrhoea.

In an alternative embodiment (for example, of the third aspect of the invention), the condition is not caused by the one or more prokaryotic organism. Although prokaryotic organisms can be pathogenic, commensal non-pathogenic prokaryotic organisms exist in patients. If the patient has a condition it might also affect such prokaryotic organisms, which is likely to change the behaviour and/or gene expression of that prokaryotic organism. Accordingly, by analysing the prokaryotic organisms in a patient it can be possible to provide a diagnosis and/or prognosis of a condition in the patient, even if the condition is not caused by the one or more prokaryotic organism.

In one embodiment (for example, of the third aspect of the invention), the condition not caused by the one or more prokaryotic organism is one or more condition selected from the list consisting of: a non-prokaryotic infectious condition; a condition caused by a prion; a condition caused by a fungi; a condition caused by a parasite; a condition caused by a virus; an autoimmune disease; diabetes (such as Type 1 diabetes and/or Type 2 diabetes); cancer; an allergy; Celiac disease; Crohn’s disease; colitis; and irritable bowel syndrome.

In a particular embodiment (for example, of the third aspect of the invention), the one or more prokaryotic organism is one or more prokaryotic cell.

In one embodiment (for example, of the third aspect of the invention), the method further comprises the step of:

(v) from the diagnosis and/or prognosis in step (iv), treating the condition.

In one embodiment (for example, of the third aspect of the invention), treating the condition comprises administering to the patient an agent for treating the condition.

Preferably, the agent for treating the condition is an anti-prokaryotic agent, such as an antibacterial agent. Preferably, the anti-bacterial agent is an antibiotic. More preferably, the anti- bacterial agent is one or more agent selected from the list consisting of: a b-Lactam; a penicillin; a cephalosporin; chloramphenicol; mupirocin; an aminopenicillins; am aminoglycoside (for example, streptomycin, gentamicin, sisiomicin, netilmicin, kanamycin, amikacin, neomycin, tobramycin, toframycin, spectinolycin, or paromonucin); a macrolide (for example, erythromycin or roxithromycin); a polyketide; a quinolone (for example ciprofloxacin, levofloxacin, or trovafloxacin); a flouroquinolone; a streptogramin antibiotic; a sulphonamide; a Tetracycline; and a nitroimidazole. Preferably, the agent is mupirocin and/or chloramphenicol.

Preferably, the agent for treating the condition is one or more agent selected from the list consisting of: a cell wall synthesis inhibitor; an agent that modifies ribosome function; an agent that effects prokaryotic cell physiology; an agent that affects prokaryotic cell physiology but does not modify ribosome function; an inhibitor of membrane function; a protein synthesis inhibitor; an inhibitors of nucleic acid synthesis (such as an inhibitor of DNA synthesis and/or an inhibitor of RNA synthesis); a human drug which affects prokaryotes; a veterinary which affects prokaryotes; a plant drug which affects prokaryotes; an agent that competes with and/or destroys prokaryotes (for example, bacteriophages); antibacterial nanomaterials; and engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) agents targeting prokaryotes.

In a fourth aspect, the invention provides a method for determining the effect of an agent on the mRNA sequences being translated in a prokaryotic cell, comprising the steps of:

(i) contacting the agent with one or more prokaryotic cell;

(ii) determining, from the one or more prokaryotic cell, the sequence of one or more 5’- monophosphorylated fragments of mRNA;

(iii) determining, from the sequence information in step (ii), the effect of the agent on the mRNA sequences being translated in the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5 3 co-translational degradation of mRNA occurs.

By “determining the effect of an agent on the mRNA sequences being translated in a prokaryotic cell”, we include that the effect of the agent on the mRNA sequences being translated indicates that: • the agent has caused a change in the physiology of the prokaryotic cell and/or

• the pathogenicity of the prokaryotic cell (for example, the agent has reduced how pathogenic the prokaryotic cell is and/or has stopped the prokaryotic cell from being pathogenic and/or has reduced how infective the prokaryotic cell is and/or has stopped the prokaryotic cell from being infectious) and/or

• the viability of the prokaryotic cell (for example, the agent has made the prokaryotic cell less viable and/or the agent has made the prokaryotic cell more viable and/or the agent has stopped the prokaryotic cell dividing and/or the agent has reduced the rate at which the prokaryotic cell divides and/or the agent has increased the rate at which the prokaryotic cell divides and/or indicates that the agent will kill the prokaryotic cell) and/or

• metabolic state of the prokaryotic cell and/or

• has increased the gene expression (for example, transcription and/or translation) of the prokaryotic cell and/or has decreased the gene expression (for example, transcription and/or translation) of the prokaryotic cell and/or

• has changed the genes expressed in the prokaryotic cell.

In an embodiment of any of the aspects of the invention, the one or more prokaryotic cell is one or more pathogenic prokaryotic cell.

In one embodiment (for example, of the fourth aspect of the invention), the agent is one or more agent for treating the condition as described for the third aspect of the invention.

In one embodiment (for example, of the fourth aspect of the invention), the agent is a disinfectant. Preferably, the disinfectant is one or more disinfectant selected from the list consisting of: an alcohol; an aldehyde; an oxidizing agent; a phenolic agent; a quaternary ammonium compound; a chlorine; an iodine; an agent that changes pH (such as an acid and/or a base); a soap; and a detergent.

In one embodiment (for example, of the fourth aspect of the invention), the agent is a food additive. Preferably, the food additive is one or more food additive selected from the list consisting of: an acidity regulator; an antioxidant; a colourant; a preservative; a stabiliser; a sweetener; a flavour enhancer; a vitamin; a mineral; or an agent for controlling food texture (for example, an emulsifier, a foaming agent, an antifoaming agent, a humectant, a gelling agent, a glazing agent, an anticaking agent, a bulking agent, a carbonating agent, a firming agent, or a thickener). In one embodiment (for example, of the fourth aspect of the invention), the agent is an agricultural product. Preferably, the agricultural product is one or more agricultural product selected from the list consisting of: an insecticide; a herbicide; a fungicide; a fertiliser; or a compost.

In one embodiment (for example, of the fourth aspect of the invention), the agent is a veterinary agent. Preferably, the veterinary agent is one or more agent for treating the condition as described for the third aspect of the invention.

In one embodiment, the agent is one or more heavy metal. Preferably, the heavy metal is one or more heavy metal selected from the list consisting of: antimony; cerium; dysprosium; erbium; europium; gadolinium; gallium; germanium; holmium; indium; lanthanum; lutetium; neodymium; niobium; praseodymium; samarium; tantalum; terbium; thulium; tungsten; uranium; ytterbium; iridium; osmium; palladium; platinum; rhodium; ruthenium; gold; silver; chromium; cobalt; copper; iron; lead; molybdenum; nickel; tin; zinc; arsenic; bismuth; cadmium; hafnium; manganese; mercury; protactinium; rhenium; selenium; tellurium; thallium; thorium; vanadium; and zirconium.

By “contacting the agent with one or more prokaryotic cell”, we include exposing the one or more prokaryotic cell to the agent (in particular, so that the agent can have an effect on the one or more prokaryotic cell) and/or placing the agent in the proximity of the one or more prokaryotic agent (in particular, so that the agent can have an effect on the one or more prokaryotic cell); for example, by adding an agent to a container or a liquid comprising the one or more prokaryotic cell or applying the agent to a surface comprising the one or more prokaryotic cell.

By “determining, from the sequence information in step (ii), the effect of the agent on the mRNA sequences being translated in the one or more prokaryotic cell”, we include that particular sequence information might indicate the effect of the agent on the one or more prokaryotic cell. For example, if an agent leads to specific sequence information for a particular mRNA sequence in the one or more prokaryotic cell then that agent is likely to have had an effect on the one or more prokaryotic cell; however, if there is no change in the sequence information of a particular mRNA sequence then the agent is unlikely to have had an effect on the one or more prokaryotic cell. As will be appreciated, how the effect is to be interpreted will depend on the particular mRNA sequence (for example, based on the function of the protein translated from that mRNA sequence) and/or the known or suspected function of the agent - it would be known to one skilled in molecular biology how to interpret such a result.

In one embodiment (for example, of the fourth aspect of the invention), step (i) further comprises providing one or more prokaryotic cell which has not been contacted with the agent; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not contacted with the agent, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) comprises determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) for the one or more prokaryotic cell contacted with the agent with the sequence information in step (ii) for the one or more prokaryotic cell not contact with the agent.

By “one or more prokaryotic cell which has not been contacted with the agent”, we include not exposing the one or more prokaryotic cell to the agent (in particular, so that the agent does not have an effect on the one or more prokaryotic cell) and/or not placing the agent in the proximity of the one or more prokaryotic agent (in particular, so that the agent does not have an effect on the one or more prokaryotic cell). We further include that the “one or more prokaryotic cell which has not been contacted with the agent” is a negative control, which is concept that would be known to one skilled in molecular biology.

By “determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) for the one or more prokaryotic cell contacted with the agent with the sequence information in step (ii) for the one or more prokaryotic cell not contact with the agent”, we include that the comparison between the sequence information of the one or more prokaryotic cell contacted with the agent and the sequence information of one or more prokaryotic cell not contacted with agent indicates the effect of the agent on the one or more prokaryotic cell. For example, if an agent changes the sequence information for a particular mRNA sequence in the one or more prokaryotic cell contacted with the agent when compared to the sequence information for that particular mRNA sequence in the one or more prokaryotic cell not contacted with the agent then that agent is likely to have had an effect on the one or more prokaryotic cell; however, if there is no change in the sequence information of a particular mRNA sequence then the agent is unlikely to have had an effect on the one or more prokaryotic cell. As will be appreciated, how the effect is to be interpreted will depend on the particular mRNA sequence (for example, based on the function of the protein translated from that mRNA sequence) and/or the known or suspected function of the agent - it would be known to one skilled in molecular biology how to interpret such a result.

In one embodiment (for example, of the fourth aspect of the invention), the one or more prokaryotic cell which has been contacted with the agent and one or more prokaryotic cell which has not been contacted with the agent are from the same phylum, class, order, family, genus, species and/or strain. In an alternative embodiment (for example, of the fourth aspect of the invention), the one or more prokaryotic cell which has been contacted with the agent and one or more prokaryotic cell which has not been contacted with the agent are from a different phylum, class, order, family, genus, species and/or strain.

In one embodiment (for example, of the fourth aspect of the invention), step (iii) comprises determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) with a control for the effect of the agent on the one or more prokaryotic cell.

Preferably, the control for the effect of the agent on the one or more prokaryotic cell is selected from the list consisting of: sequence information from a database; sequence information of the effect of the agent on one or more prokaryotic cell from a database; sequence information of one or more prokaryotic cell which has not been contacted with the agent; sequence information of one or more prokaryotic cell which has been contacted with the agent; sequence information of one or more prokaryotic cell which has not been contacted with the agent from a database; or sequence information of one or more prokaryotic cell which has been contacted with the agent from a database.

By “determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) with a control for the effect of the agent on the one or more prokaryotic cell”, we include that the comparison between the sequence information of the one or more prokaryotic cell contacted with the agent and the control for the effect of the agent on the one or more prokaryotic cell indicates the effect of the agent on the one or more prokaryotic cell.

In a fifth aspect, the invention provides a method for determining the physiological status of one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell; (ii) determining, from the one or more prokaryotic cell, the sequence of one or more 5’- monophosphorylated fragments of rrsRNA;

(iii) determining, from the sequence information in step (ii), the physiological status of the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

By “determining the physiological status of one or more prokaryotic cell”, we include:

• identifying the growth rate of the one or more prokaryotic cell; and/or

• identifying growth status of the one or more prokaryotic cell; and/or

• identifying metabolic rate of the one or more prokaryotic cell; and/or

• identifying metabolic status of the one or more prokaryotic cell; and/or

• identifying pathogenicity of the one or more prokaryotic cell; and/or

• identifying the respiratory status of the one or more prokaryotic cell; and/or

• identifying the ability of the one or more prokaryotic cell to consume amino acids and/or nutrients; and/or

• identifying the ability of the one or more prokaryotic cell to adapt to an environmental stress; and/or

• identifying the ability of the one or more prokaryotic cell to sporulate and/or aggregate and/or form biofilms and/or enter in quiescent state; and/or

• identifying the ability of the one or more prokaryotic cell to respond to agents (such as those disclosed herein, including drugs, pharmaceutical components, and/or additives (such as food additives); and/or

• identifying the ability of the one or more prokaryotic cell to respond to the presence of other microorganisms (such as other prokaryotes, fungi, and/or protozoa); and/or

• identifying the ability of the one or more prokaryotic cell to react to the immune system response of an animal (such as the human patients and/or non-human patients described herein) or plants; and/or

• identifying the translational activation and/or translation repression and/or RNA decay of genes (and/or particular groups of genes) in the one or more prokaryotic cell. By “determining, from the sequence information in step (ii), the physiological status of the one or more prokaryotic cell”, we include that particular sequence information might indicate physiological status of one or more prokaryotic cell. As will be appreciated, how the effect is to be interpreted will depend on the particular sequence information - it would be known to one skilled in molecular biology how to interpret such a result.

In one embodiment (for example, of the fifth aspect of the invention), step (iii) comprises determining the physiological status by comparing the sequence information in step (ii) with a control for the physiological status of the one or more prokaryotic cell.

Preferably, the control for the physiological status of the one or more prokaryotic cell is selected from the list consisting of: sequence information from a database; and sequence information of the physiological status of one or more prokaryotic cell from a database.

By “determining the physiological status by comparing the sequence information in step (ii) with a control for the physiological status of the one or more prokaryotic cell”, we include that the comparison between the sequence information of the one or more prokaryotic cell and the control for the physiological status of the one or more prokaryotic cell indicates the physiological status of the one or more prokaryotic cell.

In a sixth aspect, the invention provides a method for determining the effect of an environmental condition on one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the effect of an environmental condition on the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5 3 co-translational degradation of mRNA occurs.

By “for determining the effect of an environmental condition on one or more prokaryotic cell”, we include that the method indicates that: • the environmental condition has caused a change in the physiology of the prokaryotic cell and/or the pathogenicity of the prokaryotic cell (for example, the environmental condition has reduced how pathogenic the prokaryotic cell is and/or has stopped the prokaryotic cell from being pathogenic and/or has reduced how infective the prokaryotic cell is and/or has stopped the prokaryotic cell from being infectious); and/or

• the viability of the prokaryotic cell (for example, the environmental condition has made the prokaryotic cell less viable; and/or

• the environmental condition has made the prokaryotic cell more viable; and/or

• the environmental condition has stopped the prokaryotic cell dividing; and/or

• the environmental condition has reduced the rate at which the prokaryotic cell divides (for example, the prokaryotic cell sporulate and/or the prokaryotic cell is dormant and/or the prokaryotic cell is quiescent); and/or

• the environmental condition has increased the rate at which the prokaryotic cell divides and/or indicates that the environmental condition will kill the prokaryotic cell); and/or

• metabolic state of the prokaryotic cell; and/or

• has increased the gene expression (for example, transcription and/or translation) of the prokaryotic cell; and/or

• has decreased the gene expression (for example, transcription and/or translation) of the prokaryotic cell and/or has changed the genes expressed in the prokaryotic cell.

By “determining, from the sequence information in step (ii), the effect of an environmental condition on the one or more prokaryotic cell”, we include that particular sequence information might indicate the effect of the environmental condition on the one or more prokaryotic cell. As will be appreciated, how the effect is to be interpreted will depend on the particular sequence information, and one skilled in molecular biology would appreciate how to interpret such a result in light of the teaching herein.

In one embodiment of the sixth embodiment of the invention, step (i) further comprises providing the one or more prokaryotic cell in the environmental condition or isolated from the environmental condition.

By “the one or more prokaryotic cell in the environmental condition”, we include that the one or more prokaryotic cell is in the environmental condition as the method is undertaken; for example, the one or more prokaryotic cell is physically in the environmental condition and/or the one or more prokaryotic cell is contacted with the environmental condition and/or the one or more prokaryotic cell is exposed to the environmental condition. By “the one or more prokaryotic cell is isolated from the environmental condition”, we include that the one or more prokaryotic cell was in the environmental condition, but has been removed from the environmental condition prior to the method being undertaken; for example, the one or more prokaryotic cell was physically in the environmental condition, but has been removed from the environmental condition prior to the method being undertaken and/or the one or more prokaryotic cell was contacted with the environmental condition, but is no longer contacted with the environmental condition when the method is undertaken and/or the one or more prokaryotic cell was exposed to the environmental condition, but is no longer exposed to the environmental condition when the method is undertaken.

In one embodiment of the sixth embodiment of the invention the environmental condition is one or more selected from the list consisting of: a mixture comprising about two or more prokaryotic species, including the one or more prokaryotic cell; a mixture comprising the one or more prokaryotic cell and one or more eukaryotic cells; a mixture comprising the one or more prokaryotic cell and one or more virus; a mixture comprising the one or more prokaryotic cell and one or more prions; a sample; heat; cold; a temperature range; a pH; a pH range; a radiation; a level of radiation; a level of a contaminant; a change in nutrition; stationary phase; sporulation; and a level of one or more heavy metal. Preferably, the mixture is a liquid comprising the mixture.

As would be known to one skilled in microbiology, the stationary phase is when a prokaryotic (in particular, a bacterial) cell is not in the process of growing, but is metabolically active.

As an alternative embodiment, the environmental condition is one or more selected from the list consisting of: a mixture of about two or more prokaryotic phylum, including the one or more prokaryotic cell; a mixture of about two or more prokaryotic order, including the one or more prokaryotic cell; a mixture of about two or more prokaryotic family, including the one or more prokaryotic cell; a mixture of about two or more prokaryotic genus, including the one or more prokaryotic cell; and/or a mixture of about two or more prokaryotic strain, including the one or more prokaryotic cell. Preferably, the environmental condition is a mixture of about two or more prokaryotic strain, including the one or more prokaryotic cell; and/or the environmental condition is a mixture of about two or more prokaryotic species, including the one or more prokaryotic cell.

In one embodiment, the environmental condition is an environmental condition that modifies ribosome function. In a particular embodiment, the about two or more prokaryotic phylum, order, family, genus, species and/or strain, including the one or more prokaryotic cell is about three or more prokaryotic phylum, order, family, genus, species and/or strain, including the one or more prokaryotic cell; for example: about four or more; about five or more; about six or more; about seven or more; about eight or more; about nine or more; about 10 or more; about 11 or more; about 12 or more; about 13 or more; about 14 or more; about 15 or more; about 16 or more; about 17 or more; about 18 or more; about 19 or more; about 20 or more; about 25 or more; about 30 or more; about 35 or more; about 40 or more; about 45 or more; about 50 or more; about 60 or more; about 70 or more; about 80 or more; about 90 or more; about 100 or more; about 150 or more; about 200 or more; about 250 or more; about 300 or more; about 350 or more; about 400 or more; about 450 or more; about 500 or more; about 550 or more; about 600 or more; about 650 or more; about 700 or more; about 750 or more; about 800 or more; about 850 or more; about 900 or more; about 950 or more; about 1 ,000 or more; about 1 ,500 or more; about 2,000 or more; about 2,500 or more; about 3,000 or more; about 3,500 or more; about 4,000 or more; about 4,500 or more; about 5,000 prokaryotic phylum, order, family, genus, species and/or strain, including the one or more prokaryotic cell. Preferably, the about two or more prokaryotic phylum, order, family, genus, species and/or strain, including the one or more prokaryotic cell is about 10 or more; or about 100 or more; or about 1 ,000 or more prokaryotic phylum, order, family, genus, species and/or strain, including the one or more prokaryotic cell.

Preferably, “heat” is a temperature of about 20 ^°C or more; for example: about 30 ^°C or more; about 37 ^°C or more; about 40 ^°C or more; about 50 ^°C or more; about 60 ^°C or more; about 70 ^°C or more; about 80 ^°C or more; about 90 ^°C or more; about 100 ^°C or more; about 110 ^°C or more; about 120^°C or more; about 130^°C or more; about 140 ^°C or more; about 150^°C or more; about 160^°C or more; about 170^°C or more; about 180^°C or more; about 190^°C or more; or about 200 ^°C or more. Preferably, “heat” is a temperature of about 20 ^°C or more (for example, for prokaryotic cells normally found in soil or water) or about 37 ^°C or more (for example, for prokaryotic cells normally found in a human patient, such as in the human patient’s digestive system).

Preferably, “cold” is a temperature of about 30 ^°C or less; for example: about 20 ^°C or less; about 10 ^°C or less; about 0^°C or less; about -10^°C or less; about -20 ^°C or less; about -30 ^°C or less; about -40 ^°C or less; about -50 ^°C or less; about -60 ^°C or less; about -70 ^°C or less; about -80 ^°C or less; about -90 ^°C or less; about -100 ^°C or less; about -110 ^°C or less; about -120 ^°C or less; about -130 ^°C or less; about -140 ^°C or less; about -150 ^°C or less; about -160 ^°C or less; about - 170 ^°C or less; about -180 ^°C or less; about -190 ^°C or less; or about -200 ^°C or less.

Preferably, the temperature range is: about -200 ^°C to about 0^°C; about -200 ^°C to about -150 ^°C; about -200 ^°C to about -100^°C; about -200 ^°C to about -50 ^°C; about -150^°C to about 0^°C; about - 150 ^°C to about -100 ^°C; about - 150 ^°C to about -50 ^°C; about -100 ^°C to about 0 ^°C; about - 100 ^°C to about -50 ^°C; about -50 ^°C to about 0^°C; about 0 ^°C to about 50 ^°C; about 0^°C to about 100^°C; about 0 ^°C to about 200 ^°C; about 50 ^°C to about 100 ^°C; about 50 ^°C to about 200 ^°C; about 100 ^°C to about 200 ^°C; about 100^°C to about 1000 ^°C; about 200 ^°C to about 1000^°C; about 300 ^°C to about 1000^°C; about 400 ^°C to about 1000^°C; about 500 ^°C to about 1000 ^°C; about 600 ^°C to about 1000^°C; about 700 ^°C to about 1000^°C; about 800 ^°C to about 100013; or about 900 ^°C to about 1000^°C.

As would be appreciated by one skilled in microbiology, how ‘heat’ and ‘cold’ might be relevant to a particular prokaryote could depend on in which habitat that prokaryote is normally found; for example, for a prokaryotic cell normally found in the digestive system (such as the gut) of a human patient, ‘heat’ could be a temperature of about 37 ^°C or more and ‘cold’ could be a temperature of about 30 ^°C or less.

Preferably, the pH is an acidic pH or an alkaline pH. Preferably, the pH is a pH of about 1 or more; for example: about 2 or more; about 3 or more; about 4 or more; about 5 or more; about 6 or more; about 7 or more; about 8 or more; or about 9 or more. In an alternative preferred embodiment, the pH is a pH of about 9 or less; for example: about 8 or less; about 7 or less; about 6 or less; about 5 or less; about 4 or less; about 3 or less; about 2 or less; or about 1 or less.

Preferably, the pH range is a pH of about 1 to a pH of about 9; for example: a pH of about 1 to a pH of about 6; a pH of about 1 to a pH of about 3; a pH of about 1 to a pH of about 2; a pH of about 7 to a pH of about 9; or a pH of about 8 to a pH of about 9.

Preferably, the radiation is one or more selected from the list consisting of: visible light; electromagnetic radiation; alpha radiation; beta radiation; gamma radiation; x radiation; microwaves; infrared radiation; ultraviolet light; and ionizing radiation, most preferably ultraviolet light and/or ionizing radiation.

Preferably, the level of radiation is about one rad (as a unit of absorbed radiation dose) or more; for example: about 10 rads or more; about 20 or more; about 30 or more; about 40 or more; about 50 or more; about 60 or more; about 70 or more; about 80 or more; about 90 or more; about 100 or more; about 150 or more; about 200 or more; about 300 or more; about 400 or more; about 500 or more; about 600 or more; about 700 or more; about 800 or more; about 900 or more; about 1000 or more; about 2000 or more; about 3000 or more; about 4000 or more; about 5000 or more; about 6000 or more; about 7000 or more; about 8000 or more; about 9000 or more; or about 10000 or more.

Preferably, the change nutrition is selected from the list consisting of: a reduction in nutrition; an increase in nutrition; an excess of nutrition; and an absence of nutrition.

Preferably, the level of one or more heavy metal is a toxic level (or a toxic amount) of one or more heavy metal or a sub-toxic level (or sub-toxic amount) of one or more heavy metal. As will be appreciated, the toxic or sub-toxic level of a heavy metal will depend on the type of prokaryotic cell and/or the type of heavy metal. This will be known to one skilled in molecular biology, who would be able to identify whether a certain level or amount of the heavy metal will lead to toxicity.

In one embodiment of the methods of the invention, in step (i) the one or more prokaryotic cell is in a sample.

In one embodiment of the methods of the invention, the sample is one or more selected from the list consisting of: a sample from a patient; an environmental sample; and a laboratory sample.

Samples such as a sample from a patient and an environmental sample are examples of complex samples, in that they often contain many different components and often a comparatively small amount of the one or more prokaryotic cell. Accordingly, for conventional methods it is often very difficult to conduct tests for, and on, prokaryotic cells directly on a complex sample and it often necessary to purify the prokaryotic cells and/or grow up the prokaryotic cells from the sample prior to testing. The need to purify and/or grow the prokaryotic cells of interest prior to testing often leads to increases in cost, complexity, and testing time. As discussed herein, the current invention allows testing (such as by the methods of the invention) directly on a complex sample, which makes it particularly advantageous when compared to the conventional methods already known.

Preferably, the sample from the patient is one or more selected from the list consisting of: a biopsy; skin; a skin swab; mucus; vomit; faeces; blood; tissue; urine; sweat; bodily fluid; semen; vaginal discharge; a vaginal swab; a mouth swab; buccal cells; a buccal swab; an eye swab; an ear swab; a nose swab; a sample from under one or more nail; a pulmonary lavage; a gastric washing; a sample from the gastrointestinal tract; oral fluid; a throat swab; a wound swab; a cough swab; a sinus washout; and saliva. More preferably, the sample from the patient is faeces, urine, a mucosal swab and/or a skin swab. It would be known to the person skilled in medicine how to obtain any of the above patient samples, in a form suitable to be used as part of the present invention.

In one embodiment, the patient (for example, the patient of the third aspect of the invention or the patient from which the patient sample is taken for the sixth aspect of the invention) is a human patient or a non-human patient. Most preferably, the patient is a human patient. Preferably, the non-human patient is a veterinary patient. More preferably, the non-human patient is one or more selected from the list consisting of: a mammal; a bird; a fish; a non-fish aquatic animal; and an insect. Preferably, the mammal is one or more selected from the list consisting of: cattle; a pig; a horse; a donkey; a yak; a water buffalo; a gayal; a sheep; a goat; a reindeer; a moose; a camel; a dromedary; a llama; an alpaca; a rabbit; a kangaroo; a dog; a cat; a ferret; a mouse; a rat; a guinea pig; a non-human primate; an ape (for example, a chimpanzee, a gorilla, a gibbon, or an orangutan); or a monkey (for example, a rhesus macaque). Preferably, the bird is one or more selected from the list consisting of: a chicken; a turkey; a duck; an ostrich; a pigeon; or a goose. Preferably, the fish is a fish for scientific research and/or a fish farmed for human consumption. More preferably, the fish is one or more selected from the list consisting of: a zebrafish; a grass carp; a silver carp; a common carp; a nile tilapia; a bighead carp; a catla (i.e. Indian carp); a crucian carp; an atlantic salmon; a Roho labeo; a milkfish; a rainbow trout; a wuchang bream; a black carp; a northern snakehead; or an amur catfish. Preferably, the non-fish aquatic animal is one or more selected from the list consisting of: a mollusc; a shellfish; a shrimp; a prawn; a crab; an oyster; a mussel; a sea cucumber; or a jellyfish. Preferably, the insect is one or more selected from the list consisting of: a honey bee; a bee; a silkworm; a grasshopper; or an ant.

In one embodiment (for example, an embodiment of the sixth aspect), the environmental sample is one or more selected from the list consisting of: water; fresh water; salt water; tap water; drinking water; a liquid; a plant; soil; compost; a sample from a bioreactor; a sample from a landfill site; fertiliser; a swab or sample from a non-patient surface; a non-patient swab; and a food stuff.

Preferably, the swab or sample from a non-patient surface is a swab or sample from a nonpatient surface that is exposed and/or interacts with the environment. For example, by the swab or sample from a non-patient surface we include one or more selected from the list consisting of: a solar panel; a vehicle (for example, a motor vehicle such as a car or a rocket); a surface in a room for food preparation; a surface in a room in a hospital (for example, a bed in a medical ward and/or a operating theatre); a surface in a residence (for example, a domestic residence or a hotel, such as a bedroom, a bathroom, a lounge, a dining room, and/or a kitchen); and a surface in a room for a non-human animal (i.e. a non-human patient described herein) (for example, a room on a farm, such as a chicken coop; a pig sty; a barn; and/or a shed).

In one embodiment, a sample from a bioreactor is a by-product in the production of a biofuel or the product in the production of a biofuel.

In one embodiment, the food stuff is a food for human consumption or a food for non-human (i.e. a non-human patient described herein) consumption. In a particular embodiment, the food stuff is one or more selected from the list consisting of: a dairy product (such as cheese or milk); wine; beer; fermented products; vegetables; fruit; meat; fish; and non-fish seafood. In a particular embodiment, the foodstuff is prepared for consumption (for example, it is cooked) or it is not yet prepared for consumption (for example, it is raw).

In one embodiment, the mRNA is being degraded by 5’-3’ co-translational degradation.

In one embodiment, the co-translational degradation of mRNA is exonucleolytic co- translational degradation.

The term “exonucleolytic” would be understood by one skilled in molecular biology. By “exonucleolytic co-translational degradation” we include that the co-translational degradation proceeds from the terminus of the RNA (such as mRNA) and not in the middle of the RNA. As particularly relevant to the present invention, by the “5’-3’ co-translational degradation” being exonucleolytic we include that the RNA molecule is degraded from the 5’ phosphate (5’P) in a 5’-3’ direction, trimming an upstream 5’P molecule towards a downstream 5’P position, most preferably until the degradation reaches a ribosome associated with the mRNA. As the degradation is co-translational it occurs while the mRNA molecule is still associated with at least one ribosome. Therefore, the 5’-3’ co-translational degradation produces a footprint of the ribosome position degrading the RNA until the last exposed 5’P that remains accessible. 5’-3’ exonucleolytic co-translational degradation can start at an original termini of the mRNA molecule or at a break in the middle of the mRNA molecule, with that break becoming a new 5’ termini (such as at a site of endonucleolytic cleavage or exonucleolytic cleavage). Regardless of the start site of the 5’-3’ co-translational degradation, it proceeds by degrading the most 5’ exposed RNA nucleotide of the 5’P RNA fragment.

In one embodiment, the co-translational degradation is not 3’-5’ co-translational degradation, preferably the co-translational degradation is not exonucleolytic 3’-5’ co-translational degradation.

In one embodiment, the co-translational degradation of mRNA is not mediated by 5’-3’ decay. It will be appreciated that 5’-3’ decay is different to 5’-3’ co-translational degradation, as it is not mediated by an exonuclease with 5’-3’ activity. 5’-3’ decay might occur in a prokaryotic cell which is not capable of 5’-3’ co-translational degradation (for example, a prokaryotic cell that does not have an exonuclease that has 5’ to 3’ exonuclease activity).

In one embodiment, the one or more prokaryotic cell comprises one or more exonuclease comprising 5’ to 3’ exonuclease activity.

In one embodiment, the co-translational degradation of mRNA is mediated by one or more exonuclease comprising 5’ to 3’ exonuclease activity.

By “exonuclease comprising 5’ to 3’ exonuclease activity”, we include a nuclease enzyme that degrades a nucleic acid (in particular, RNA) starting at least at the 5’ termini (or solely at the 5’ termini) of the nucleic acid molecule. Accordingly, we include that an exonuclease comprising 5’ to 3’ exonuclease activity will remove the 5’ terminal nucleotide of the nucleic acid which will leave the previously-3’ adjacent nucleotide as the new 5’ terminal nucleotide. The adjacent nucleotide can then be removed by the exonuclease comprising 5’ to 3’ exonuclease activity, and so on.

In a particular embodiment, the one or more exonuclease comprising 5’ to 3’ exonuclease activity is one or more exonuclease that consists of 5’ to 3’ exonuclease activity. By “exonuclease that consists of 5’ to 3’ exonuclease activity” we include that the nuclease is only able to mediate 5’ to 3’ exonuclease activity, so the exonuclease does not have 3’ to 5’ exonuclease activity or endonuclease activity.

In a particular embodiment, the one or more exonuclease comprising 5’ to 3’ exonuclease activity is one or more RNA (such as, mRNA) exonuclease comprising 5’ to 3’ exonuclease activity. In one embodiment, the exonuclease is an exonuclease stimulated by 5'-monophosphate.

By “an exonuclease stimulated by 5'-monophosphate”, we include that the exonuclease is able to recognise a 5'-monophosphate on a nucleic acid and/or will mediate of a degradation of a nucleic acid based on the presence of a 5'-monophosphate on the nucleic acid.

In one embodiment, the exonuclease is one or more selected from the list consisting of: an RNase J; an analogue of RNase J (also known as RNJ or Ribonuclease J),; a homologue of RNase J; a paralog of RNase J; an orthologue of RNase J; an analogue ofXRNI ; a homologue of XRN1 ; a paralog of XRN1 ; an orthologue of XRN1 ; an analogue of XRN2; a homologue of XRN2; a paralog of XRN2; and an orthologue of XRN2. Preferably, the exonuclease is one or more selected from the list consisting of: an RNase J; an analogue of RNase J; a homologue of RNase J; a paralog of RNase J; and an orthologue of RNase J. Most preferably, the exonuclease is an RNase J.

In a particular embodiment, the RNase J, analogue of RNase J, homologue of RNase J, paralog of RNase J and/or orthologue of RNase J is: a bacterial RNase J, a bacterial homologue of RNase J, a bacterial paralog of a RNase J and/or a bacterial orthologue of RNase J; or an archeal RNase J, an archeal analogue of RNase J, an archeal homologue of RNase J, an archeal paralog of RNase J and/or an archeal orthologue of RNase J.

RNase J is a bacterial and/or archeal exonuclease with 5’-3’ exonuclease activity.

XRN1 and XRN2 are eukaryotic exonucleases with 5’-3’ exonuclease activity, which might have bacterial and/or archeal analogues, homologues, paralogs, and/or orthologues which would function as part of the invention. XRN2 is referred to as Dhm1 in mice and Rati in budding yeast.

One skilled in molecular biology would understand what is meant by the terms “analogue”, “homologue”, “paralog” and “orthologue”.

In one embodiment, the RNase J is RNase J1 (also known as RNJA or Ribonuclease J1) and/or RNase J2 (also known as RNJB or Ribonuclease J2).

In an alternative embodiment, the one or more prokaryotic cells do not comprise RNase E and RNase J (also known as RNE or Ribonuclease E). The inventor has surprisingly discovered that co-translational degradation occurs in prokaryotic cells that lack RNase J, if those cells also lack RNase E,

One skilled in microbiology and/or molecular biology would be able to identify the necessary sequences (both amino acid sequences and nucleic acid sequences) of the nucleases described herein, for example by using the GenBank database https://www.ncbi.nlm.nih.gov/qenbank/ or the UniProt database - https://www.uniprot.org/. Exemplary sequences of the nucleases described herein can be found as follows: Ribonuclease J1 from Bacillus subtilis (strain 168) - amino acid sequence Q45493 on the UniProt database and nucleotide sequence NC 00964.3x1524785-1523118 on the GenBank database; Ribonuclease J2 from Bacillus subtilis (strain 168) - amino acid sequence 031760 on the UniProt database and nucleotide sequence NC_000964.3: 1749418-1751085 on the GenBank database; Ribonuclease J from Methanolobus psychrophilus - amino acid sequence Mpsy_0886 K12574 on the GenBank database and nucleotide sequence Mpsy_0886 K12574 on the GenBank database; Ribonuclease E from Escherichia coli (strain K12) amino acid sequence P21513 on the UniProt database and nucleotide sequence

NC_000913.3x1144367-1141182 on the GenBank database; XRN1 from Saccharomyces cerevisiae - amino acid sequence YGL173C SGDID:S000003141 on the UniProt database and on the Yeast Genome database (https://www.veastqenome.org/) and nucleotide sequence YGL173C SGDID:S000003141 on the Yeast Genome database; RAT1 (/.e. XRN2) from Saccharomyces cerevisiae - amino acid sequence YOR048C SGDID:S000005574 on the UniProt database and on the Yeast Genome database and nucleotide sequence YOR048C SGDID:S000005574 on the Yeast Genome database; XRN1 from Homo sapiens - amino acid sequence Q8IZH2, Q8IZH2-2 and Q8IZH2-3 on the UniProt database and nucleotide sequence NC_000003.12x142448062- 142306610 (chromosome 3 GRCh38.p13) on the GenBank database; and XRN2 from Homo sapiens - amino acid sequence Q9H0D6 and Q9H0D6-2 on the UniProt database and nucleotide sequence NCJD00020.11 :21303313- 21389825 (chromosome 20, GRCh38.p13) on the GenBank database.

In one embodiment, the one or more prokaryotic cell comprises at least two prokaryotic cells, and wherein the at least two prokaryotic cells belong to the same genus and/or species or two or more different genus and/or species.

In one embodiment, the method further comprises step (i)(a) performed after step (i), wherein step (i)(a) comprises subjecting the one or more prokaryotic cell to an agent and/or environmental condition that modifies ribosome function. In one particular embodiment, the agent is one or more agent for treating the condition as described for the third aspect of the invention. In one embodiment, environmental condition that modifies ribosome function is one or more environmental condition described herein.

By “subjecting the one or more prokaryotic cell to an agent and/or environmental condition that modifies ribosome function”, we include:

• exposing the one or more prokaryotic cell to the agent (in particular, so that the agent can have an effect on the one or more prokaryotic cell); and/or

• the environmental condition that modifies ribosome function (in particular, so that the environmental condition can have an effect on the one or more prokaryotic cell); and/or

• placing the agent and/or the environmental condition that modifies ribosome function in the proximity of the one or more prokaryotic agent (in particular, so that the agent and/or environmental condition that modifies ribosome function can have an effect on the one or more prokaryotic cell); for example, by adding an agent to a container or a liquid comprising the one or more prokaryotic cell or applying the agent to a surface comprising the one or more prokaryotic cell.

In one embodiment, the method comprises: step (i) further comprises providing one or more prokaryotic cell, wherein the one or more prokaryotic cell has not been subjected to the agent and/or environmental condition that modifies ribosome function; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not subjected to the agent and/or environmental condition that modifies ribosome function, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) further comprises providing the determination by comparing the sequence information in step (ii) for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function with the sequence information in step (ii) for the one or more prokaryotic cell not subjected to agent and/or environmental condition that modifies ribosome function.

By “one or more prokaryotic cell has not been subjected to the agent and/or environmental condition that modifies ribosome function”, we include: • not exposing the one or more prokaryotic cell to the agent (in particular, so that the agent does not have an effect on the one or more prokaryotic cell); and/or

• the environmental condition that modifies ribosome function (in particular, so that the environmental condition does not have an effect on the one or more prokaryotic cell); and/or

• not placing the agent and/or environmental condition that modifies ribosome function in the proximity of the one or more prokaryotic agent (in particular, so that the agent and/or environmental condition that modifies ribosome function does not have an effect on the one or more prokaryotic cell).

We further include that the “one or more prokaryotic cell has not been subjected to the agent and/or environmental condition that modifies ribosome function” is a negative control, which is concept that would be known to one skilled in molecular biology.

By “providing the determination by comparing the sequence information in step (ii) for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function with the sequence information in step (ii) for the one or more prokaryotic cell not subjected to agent and/or environmental condition that modifies ribosome function”, we include that the comparison between the sequence information of the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function and the sequence information of one or more prokaryotic cell not subjected to the agent and/or environmental condition that modifies ribosome function. For example, if an agent and/or environmental condition that modifies ribosome function changes the sequence information for a particular mRNA sequence in the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function when compared to the sequence information for that particular mRNA sequence in the one or more prokaryotic cell not subjected to the agent and/or environmental condition that modifies ribosome function then that agent and/or environmental condition that modifies ribosome function is likely to have had an effect on the one or more prokaryotic cell; however, if there is no change in the sequence information of a particular mRNA sequence then the agent and/or environmental condition that modifies ribosome function is unlikely to have had an effect on the one or more prokaryotic cell. As will be appreciated, how the effect is to be interpreted will depend on the particular mRNA sequence (for example, based on the function of the protein translated from that mRNA sequence) and/orthe known or suspected function of the agent and/or environmental condition that modifies ribosome function - it would be known to one skilled in molecular biology how to interpret such a result. In one embodiment, step (iii) further comprises providing the determination by comparing the sequence information in step (ii) with a known standard for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function.

Preferably, the known standard is selected from the list consisting of: sequence information from a database; and sequence information of one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function.

In one embodiment, the agent that modifies ribosome function is an antibiotic, as described herein. In a further embodiment, the agent that modifies ribosome function is an anti- prokaryotic agent, as described herein. Preferably, the antibiotic is one or more antibiotic selected from the list consisting of: mupirocin; and chloramphenicol.

In one embodiment, the method further comprises step (i)(b) performed after step (i), wherein step (i)(b) comprises isolating, from the one or more prokaryotic cell, one or more 5’- monophosphorylated fragment of mRNA.

In one embodiment, the isolating comprises purifying, from the one or more prokaryotic cell, one or more 5’-monophosphorylated fragment of mRNA.

As would be known to one skilled in molecular biology, isolating the one or more 5’- monophosphorylated fragment of mRNA from the one or more prokaryotic cell may comprise first extracting all, or substantially all, of the RNA from the one or more prokaryotic cell, which can be done using methods such as phenol/chloroform extraction and/or centrifugation, such as shown in the Example.

In one embodiment, the purifying comprises purifying the 5’-monophosphorylated fragment of mRNA from one or more of the following: proteins; lipids; small molecule metabolites; other cellular components; other components in the sample; and salts.

In one embodiment, the method further comprises step (i)(c) performed after step (i), wherein step (i) (c) comprises isolating the 5’-monophosphorylated fragments of mRNA from non-5’- monophosphorylated RNA.

By “non-5’-monophosphory!ated RNA”, we include an RNA in which the 5’ carbon of the 5’ nucleotide is not attached to one phosphate group. In one embodiment, the non-5’-monophosphorylated RNA is one or more RNA selected from the list consisting of: ribosomal RNA (rRNA); transfer RNA (tRNA); microRNA (miRNA); and small RNA.

One skilled in molecule biology would understand what is encompassed by the terms rRNA, tRNA, miRNA and small RNA. By “small RNA”, we include non-coding RNA, such as RNA that does not encode peptides or proteins.

In one embodiment, the isolating of the 5’-monophosphorylated fragments of mRNAfrom rRNA comprises rRNA hybridisation.

In one embodiment, the isolating of the 5’-monophosphorylated fragments of mRNAfrom rRNA and/or tRNA comprises selective degradation of the rRNA and/or tRNA or the complementary DNA (cDNA- sometimes referred to as copy DNA) produced from the rRNA and/or tRNA after reverse transcription. An exemplary method for the selective degradation of rRNA is shown in the Example, using the Ribozero rRNA removal kit produced by lllumina or by using rRNA DNA oligo depletion mixes.

In one embodiment, the method further comprises step (i)(d) performed after step (i), wherein step (i)(d) comprises producing one or more cDNAs from the one or more 5’- monophosphorylated fragments of mRNA, for example by reverse transcription.

The person skilled in molecular biology would understand how to produce cDNA from mRNA, such as how is described in the Example.

In a particular embodiment, the cDNA can be isolated from rRNA and/or tRNA by selective degradation of the rRNA and/or tRNA or by degradation of all of the RNA (for example, the degradation of all of the RNA can be mediated by a high pH, such as by the addition of NaOH).

A particularly preferred embodiment of the invention comprises:

• producing one or more cDNAs from the one or more 5’-monophosphorylated fragments of mRNA; and/or

• degradation of all of the RNA following the producing of the one or more cDNAs (for example, by a high pH, such as the addition of NaOH); and/or the selective degradation of the cDNA produced from rRNA.

Preferably, the rRNA degradation is mediated by an enzyme, such as a nuclease (for example, a duplex-specific nuclease, such as duplex-specific nuclease (DSN)).

In one embodiment, the determination in step (iii) is from the sequence information of the cDNA.

In one embodiment, the method further comprises step (i)(e) performed after step (i), wherein step (i)(e) comprises adding to the one or more 5’-monophosphorylated fragments of mRNA two of more oligonucleotides (preferably RNA oligonucleotide) that comprise a sequence identifier. The method of such a ligation would be known to one skilled in molecular biology, such as using a method described in the Example.

In one embodiment, each of the two or more oligonucleotides further comprises a same sequence of nucleotides.

In one embodiment, the adding comprises ligating the oligonucleotides to the one or more 5’- monophosphorylated fragments of mRNA.

In one embodiment, the ligating is mediated by a ligase, such as an RNA ligase, preferably T4 RNA ligase.

In one embodiment, the oligonucleotide comprises a moiety for isolating the 5’- monophosphorylated fragments of mRNA, optionally wherein the moiety comprises biotin.

In one embodiment, the method further comprises step (i)(f) performed after step (i), wherein step (i)(f) comprises removing the 5’-monophosphate from the 5’-monophosphorylated fragments of mRNA.

In one embodiment, the method further comprises step (i)(g) performed after step (i), wherein step (i)(g) comprises cutting the 5’-monophosphorylated fragments of mRNA to form one or more shorter 5’-monophosphorylated fragments of mRNA.

In one embodiment, the method further comprises step (i)(h) performed after step (i)(g), wherein step (i)(h) comprises phosphorylating the one or more shorter 5’-monophosphorylated fragments of mRNA. In one embodiment, the phosphorylating of the one or more shorter 5’-monophosphorylated fragments is mediated by a kinase, such as polynucleotide kinase (PNK).

In one embodiment, the determination of the sequence of the one or more 5’- monophosphorylated fragments of mRNA in step (ii) comprises using polymerase chain reaction and/or high-throughput sequencing and/or next-generation sequencing. In a particular embodiment, it is the sequence of the one or more complementary DMAs (cDNAs) produced from the one or more 5’-monophosphorylated fragments of mRNA that is determined, such as using polymerase chain reaction and/or high-throughput sequencing and/or next-generation sequencing.

One skilled in molecular biology would understand how to undertake methods involving polymerase chain reaction and/or high-throughput sequencing and/or next-generation sequencing, in order to obtain sequence information for nucleic acid molecules (such as the one or more 5’-monophosphorylated fragments of mRNA).

In one embodiment, the high-throughput sequencing is one or more technique selected from the group consisting of: sequencing by synthesis (for example, techniques from lllumina, pyrosequencing, and Ion Torrent); sequencing by ligation (for example, solid sequence); single-molecule imaging (for example, techniques from Pacific Biosciences); nanopore sequencing (for example, techniques from Oxford Nanopore); and direct RNA sequencing (for example, techniques from Oxford Nanopore). For example, the sequencing can be undertaken using NextSeq500 lllumina sequencer, such as is used in the Example.

In one embodiment, the method further comprises step (ii)(a) performed after step (ii), wherein step (ii) (a) comprises determining the position of a ribosome on the sequence of the one or more 5’-monophosphorylated fragments of mRNA, with that information being used for the determination in step (iii).

In one embodiment, the method further comprises step (ii)(b) performed after step (ii), wherein step (ii)(b) comprises determining the relative positions in the sequence of one of more of the following RNA structures: the 5’P; the stop codon; one or more codons; one or more single nucleotide polymorphism (SNP); the 5’ untranslated region; the 3’ untranslated region; RNA binding protein binding sites; RNA secondary structure (such as RNA hairpins); RNA modifications (such as alternative or chemically modified RNA bases), with that information being used for the determination in step (iii). In a preferred embodiment, the RNA structure is the 5’P.

In one embodiment, the determination in step (iii) further comprises analysing the relative positions of the one of more RNA structures, which optionally comprises determining an accumulation of an mRNA sequence(s) of a 5’-monophosphorylated fragment with one or more of the RNA structures defined herein. In a preferred embodiment, the accumulation of an mRNA sequence(s) of a 5’-monophosphorylated fragment is of an accumulation of the mRNA sequence(s) with a 5’P at the second nucleotide (i.e. F1) and/or a 5’P 11 nucleotides upstream of the start codon and/or a 5’P 11 nucleotides upstream of the stop codon and/or a 5’P 14 nucleotides upstream of the start codon and/or a 5’P 14 nucleotides upstream of the stop codon. For example, as described in the Example, B. subtilis can be characterised as having 5^'P reads at the second nucleotide (F1) and at 11 and 14 nucleotides upstream of start and stop codons.

By “determining an accumulation of a particular mRNA sequence”, we include that the specific mRNA sequence of the 5’-monophosphorylated fragment is found in a higher amount and/or concentration than the other mRNA sequences of 5’-monophosphorylated fragments determined by the method.

As discussed in the Example, the presence of relative positions in the sequence of particular RNA structures, and an accumulation of sequences with those structures, can often be characteristic of the various features that the methods described herein seek to identify, such as the particular type of prokaryotic cell, the effect of an environmental condition on a prokaryotic cell, the physiological state of the prokaryotic cell, and/or the effect of an agent (such as an antibiotic) on a prokaryotic cell. For example, and in a particular embodiment, the use of mupirocin (which is an inhibitor of the bacterial isoleucyl tRNA synthetase) will cause a pause relative to isoleucine codons, optionally this leads to an accumulation of mRNA sequences with 5’ P 14 nucleotides upstream of isoleucine codons (as exemplified in the Examples for L. plantarum and L reuteri).

In a particular preferred embodiment, the method further comprises step (ii)(c) performed after step (ii), wherein step (ii)(c) comprises identifying a three-nucleotide periodicity in the one or more 5’-monophosphorylated fragments of mRNA, with that information being used for the determination in step (iii). In one embodiment, the method further comprises step (iii) comprises analysing the three- nucleotide periodicity which optionally comprises determining an accumulation of a particular mRNA sequence of a 5’-monophosphorylated fragment with a particular three-nucleotide periodicity.

As discussed in the Example, the three-nucleotide (3-nt) periodicity is generated by the positioning of the ribosome on the 5’-monophosphorylated fragment of mRNA. This periodicity, and an accumulation of sequences with a particular periodicity, can often be characteristic of the various features that the methods described herein seek to identify, such as the particular type of prokaryotic cell, the effect of an environmental condition on a prokaryotic cell, the physiological state of the prokaryotic cell, and/or the effect of an agent (such as an antibiotic) on a prokaryotic cell.

In a particular embodiment, the method comprises step (ii)(a) described herein and/or step (ii)(b) described herein and/or step (ii)(c) described herein, preferably step (ii)(b) described herein and step (ii)(c) described herein.

In one embodiment, the method further comprises step (ii)(d) performed after step (ii), wherein step (ii)(d) comprises comparing the sequence of the one or more 5’-monophosphorylated fragments of mRNA with known prokaryotic mRNA sequences.

In one embodiment, the determination in step (iii) comprises comparing the sequence of the one or more 5’-monophosphorylated fragments of mRNA with the known prokaryotic mRNA sequences.

In one embodiment, the 5’-monophosphorylated fragments of mRNA comprises single- stranded 5’-monophosphorylated fragments of mRNA.

In one embodiment, the 5’-monophosphorylated fragments of mRNA comprises about 9 or more nucleotides in length, preferably about 15 or more nucleotides.

In a particular embodiment, the 5’-monophosphorylated fragments of mRNA comprises about 9 or more nucleotides in length; for example: about 10 or more nucleotides in length; about 11 or more nucleotides in length; about 12 or more nucleotides in length; about 13 or more nucleotides in length; about 14 or more nucleotides in length; about 15 or more nucleotides in length; about 16 or more nucleotides in length; about 17 or more nucleotides in length; about 18 or more nucleotides in length; about 19 or more nucleotides in length; about 20 or more nucleotides in length; about 25 or more nucleotides in length; about 30 or more nucleotides in length; about 35 or more nucleotides in length; about 40 or more nucleotides in length; about 45 or more nucleotides in length; about 50 or more nucleotides in length; about 55 or more nucleotides in length; about 60 or more nucleotides in length; about 65 or more nucleotides in length; about 70 or more nucleotides in length; about 75 or more nucleotides in length; about 80 or more nucleotides in length; about 85 or more nucleotides in length; about 90 or more nucleotides in length; about 95 or more nucleotides in length; about 100 or more nucleotides in length; about 110 or more nucleotides in length; about 120 or more nucleotides in length; about 130 or more nucleotides in length; about 140 or more nucleotides in length; about 150 or more nucleotides in length; about 160 or more nucleotides in length; about 170 or more nucleotides in length; about 180 or more nucleotides in length; about 190 or more nucleotides in length; about 200 or more nucleotides in length; about 250 or more nucleotides in length; about 300 or more nucleotides in length; about 350 or more nucleotides in length; about 400 or more nucleotides in length; about 450 or more nucleotides in length; about 500 or more nucleotides in length; about 550 or more nucleotides in length; about 600 or more nucleotides in length; about 650 or more nucleotides in length; about 700 or more nucleotides in length; about 750 or more nucleotides in length; about 800 or more nucleotides in length; about 850 or more nucleotides in length; about 900 or more nucleotides in length; about 950 or more nucleotides in length; about 1 ,000 or more nucleotides in length; about 1 ,500 or more nucleotides in length; about 2,000 or more nucleotides in length; about 2,500 or more nucleotides in length; about 3,000 or more nucleotides in length; about 3,500 or more nucleotides in length; about 4,000 or more nucleotides in length; about 4,500 or more nucleotides in length; about 5,000 or more nucleotides in length; about 5,500 or more nucleotides in length; about 6,000 or more nucleotides in length; about 6,500 or more nucleotides in length; about 7,000 or more nucleotides in length; about 7,500 or more nucleotides in length; about 8,000 or more nucleotides in length; about 8,500 or more nucleotides in length; about 9,000 or more nucleotides in length; about 9,500 or more nucleotides in length; about 10,000 or more nucleotides in length; about 10,500 or more nucleotides in length; about 11 ,000 or more nucleotides in length; about 11 ,500 or more nucleotides in length; about 12,000 or more nucleotides in length; about 12,500 or more nucleotides in length; about 13,000 or more nucleotides in length; about 13,500 or more nucleotides in length; about 14,000 or more nucleotides in length; about 14,500 or more nucleotides in length; and about 15,000 or more nucleotides in length.

Most preferably, the 5’-monophosphorylated fragments of mRNA comprises about 30 or more nucleotides in length. In an alternative preferable embodiment, the 5’-monophosphorylated fragments of mRNA comprises about 60 or more nucleotides in length. In one embodiment, the 5’-monophosphorylated fragments of mRNA comprises about 9 nucleotides to about 15,000 nucleotides in length, preferably about 15 nucleotides to about 15,000 nucleotides in length, most preferably about 300 nucleotides to about 400 nucleotides in length.

In a particular embodiment, the 5’-monophosphorylated fragments of mRNA comprises about

9 nucleotides to about 15,000 nucleotides in length; for example: about 10 nucleotides to about 15,000 nucleotides in length; about 20 nucleotides to about 15,000 nucleotides in length; about 30 nucleotides to about 15,000 nucleotides in length; about 40 nucleotides to about 15,000 nucleotides in length; about 50 nucleotides to about 15,000 nucleotides in length; about 60 nucleotides to about 15,000 nucleotides in length; about 70 nucleotides to about 15,000 nucleotides in length; about 80 nucleotides to about 15,000 nucleotides in length; about 90 nucleotides to about 15,000 nucleotides in length; about 100 nucleotides to about 15,000 nucleotides in length; about 150 nucleotides to about 15,000 nucleotides in length; about 10 nucleotides to about 400 nucleotides in length; about 20 nucleotides to about 400 nucleotides in length; about 30 nucleotides to about 400 nucleotides in length; about 40 nucleotides to about 400 nucleotides in length; about 50 nucleotides to about 400 nucleotides in length; about 60 nucleotides to about 400 nucleotides in length; about 70 nucleotides to about 400 nucleotides in length; about 80 nucleotides to about 400 nucleotides in length; about 90 nucleotides to about 400 nucleotides in length; about 100 nucleotides to about 400 nucleotides in length; about 10 nucleotides to about 350 nucleotides in length; about 10 nucleotides to about 300 nucleotides in length; about 10 nucleotides to about 250 nucleotides in length; about

10 nucleotides to about 200 nucleotides in length; about 10 nucleotides to about 400 nucleotides in length; about 60 nucleotides to about 150 nucleotides in length; about 60 nucleotides to about 140 nucleotides in length; about 60 nucleotides to about 130 nucleotides in length; about 60 nucleotides to about 120 nucleotides in length; about 60 nucleotides to about 110 nucleotides in length; and about 60 nucleotides to about 100 nucleotides in length.

Most preferably, the 5’-monophosphorylated fragments of mRNA comprises about 30 nucleotides to about 400 nucleotides in length. In an alternative preferable embodiment, the 5’-monophosphorylated fragments of mRNA comprises about 60 nucleotides to about 120 nucleotides in length.

It will be appreciated that the “shorter 5’-monophosphorylated fragments of mRNA” described herein are fewer nucleotides in length when compared to the 5’-monophosphorylated fragments. The specific lengths of the shorter 5’-monophosphorylated fragments of mRNA will depend on the length of the 5’-monophosphorylated fragments from which they are derived. However, it will be appreciated that shorter 5’-monophosphorylated fragments of mRNA can be any of the lengths of 5’-monophosphorylated fragments described herein.

In one embodiment, the one or more prokaryotic cell is one or more bacterial cell. In an alternative embodiment, the one or more prokaryotic cell is one or more archeal cell.

One skilled in microbiology would understand what is encompassed by the terms “bacterial cell” and “archeal cell” (for example, cells of organisms of the kingdom Archaea).

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a sample described herein.

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a phylum selected from the list consisting of: proteobacteria; firmicutes; actinobacteria; cyanobacteria; tenericutes; spirochaetales; chloroflexi; deinococcus-thermus; synergystetes; fusobacteria; and fibrobacteres. In a further embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from the phylum Euryarchaeota.

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a class selected from the list consisting of: alphaproteobacteria; deltaproteobacteria; epsilonproteobacteria; gammaproteobacterial; bacilli; and Clostridia.

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from an order selected from the list consisting of: bacillales; and lactobacillales.

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a species selected from the list consisting of: Bacillus subtilis; Akkermansia muciniphila; Alistipes fmegoldii; Alistipes obesi; Alistipes putredinis; Alistipes shahii; Atopobium vaginae; Bacillus amyloliquefaciens; Bacteroides caccae; Bacteroides cellulosilyticus; Bacteroides coprocola; Bacteroides coprophilus; Bacteroides faecichinchillae; Bacteroides fluxus; Bacteroides fragilis; Bacteroides ovatus; Bacteroides plebeius; Bacteroides salanitronis; Bacteroides salyersiae; Bacteroides stercoris; Bacteroides thetaiotaomicron; Bacteroides uniformis; Caulobacter vibrioides; Clostridium phoceensis; Collinsella aerofaciens; Enterococcus faecalis; Clostridium innocuum; Clostridium saccharogumia; Faecalibacterium prausnitzii; Gardnerella vaginalis; Synechocystis sp. PCC 6633; Intestinimonas b utyriciproducens; Lactobacillus plantarum; Lactobacillus reuteri; Lactobacillus crispatus; Lactobacillus iners; Lactobacillus fermentum; Listeria monocytogenes; Megasphaera genomosp. (Type 1); Parabacteroides distasonis; Parabacteroides goldsteinii; Prevotella amnii; Prevotella buccal is; Sneathia sanguinegens; Staphylococcus aureus; Streptococcus salivarius; Streptococcus thermophilus; and Ureaplasma parvum.

In one embodiment, the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is selected from the list consisting of: Abiotrophia defective, Acaricomes phytoseiuli, Acaryochloris marina, Acetivibrio cellulolyticus, Acetobacter aceti, Acetobacter malorum, Acetobacter nitrogenifigens, Acetobacter okinawensis, Acetobacter pasteurianus, Acetobacteraceae bacterium, Acetobacterium woodii, Acetohalobium arabaticum, Acetonema longum, Acholeplasma axanthum, Acholeplasma equifetale, Acholeplasma granularum, Acholeplasma hippikon, Acholeplasma laidlawii, Acholeplasma modicum, Acidaminococcus fermentans, Acidaminococcus intestini, Acidimicrobium ferrooxidans, Acidiphilium angustum, Acidiphilium cryptum, Acidobacteria bacterium, Acidobacteriaceae bacterium, Acidobacterium capsulatum, Acidobacterium sp., Acidocella facilis, Acidothermus cellulolyticus, Aciduliprofundum boonei, Aciduliprofundum sp., Actibacterium atlanticum, Actinoalloteichus cyanogriseus, actinobacterium acAMD-5, actinobacterium LLX17, Actinobaculum massiliae, Actinobaculum schaalii, Actinobaculum sp., Actinobaculum urinale, Actinocatenispora sera, Actinokineospora enzanensis, Actinokineospora sp., Actinomadura atramentaria, Actinomadura flavalba, Actinomadura madurae, Actinomyces cardiffensis, Actinomyces coleocanis, Actinomyces dentalis, Actinomyces europaeus, Actinomyces georgiae, Actinomyces graevenitzii, Actinomyces israelii, Actinomyces massiliensis, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces sp., Actinomyces suimastitidis, Actinomyces timonensis, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces vaccimaxillae, Actinomyces viscosus, Actinomycetospora chiangmaiensis, Actinoplanes friuliensis, Actinoplanes globisporus, Actinoplanes missouriensis, Actinoplanes sp., Actinoplanes subtropicus, Actinoplanes utahensis, Actinopolymorpha alba, Actinopolyspora erythraea, Actinopolyspora halophila, Actinopolyspora mortivallis, Actinosporangium sp., Actinosynnema mirum, Adlercreutzia equolifaciens, Aerococcus urinae, Aerococcus viridans, Aeromicrobium marinum, Aeromicrobium massiliense, Aestuariibacter salexigens, Aestuariimicrobium kwangyangense, Afifella pfennigii, Afipia birgiae, Afipia broomeae, Afipia felis, Afipia sp., Agrobacterium albertimagni, Agrobacterium fabrum, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium sp., Agrobacterium tumefaciens, Agrobacterium vitis, Agrococcus lahaulensis, Agromyces italicus, Agromyces subbeticus, Ahrensia sp., Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus herbarius, Alicyclobacillus macrosporangiidus, Alicyclobacillus pohliae, Alicyclobacillus pomorum, Aliihoeflea sp,, AHshewanella agri, AHshewanella jeotgali, Alkalibacterium sp., Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Alkaliphilus transvaalensis, Allobaculum stercoricanis, Allofustis seminis, Alloiococcus otitis, Allokutzneria albata, Alloscardovia omnicolens, alpha proteobacterium, Alteromonas australica, Alteromonas macleodii, Alteromonas sp., Aminiphilus circumscriptus, Aminobacter sp., Aminobacterium colombiense, Aminobacterium mobile, Aminomonas paucivorans, Ammonifex degensii, Amorphus coralli, Amphibacillus jilinensis, Amphibacillus xylan us, Amycolatopsis balhimycina, Amycolatopsis benzoatilytica, Amycolatopsis japonica, Amycolatopsis jejuensis, Amycolatopsis mediterranei, Amycolatopsis methanolica, Amycolatopsis nigrescens, Amycolatopsis orientalis, Amycolatopsis rifamycinica, Amycolatopsis vancoresmycina, Amycolicicoccus subflavus, Anabaena cylindrica, Anabaena sp., Anabaena variabilis, Anaeroarcus burkinensis, Anaerobaculum hydrogeniformans, Anaerobaculum mobile, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus prevotii, Anaerococcus senegalensis, Anaerococcus sp., Anaerococcus tetradius, Anaerococcus vaginalis, Anaerofustis stercorihominis, Anaeroglobus geminatus, Anaerolinea thermophila, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Anaerosalibacter sp., Anaerostipes caccae, Anaerotruncus colihominis, Anaerotruncus sp., Anaerovibrio lipolyticus, Anaerovibrio sp., Anaerovorax odorimutans, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Aneurinibacillus aneurinilyticus, Anoxybacillus flavithermus, Anoxybacillus gonensis, Anoxybacillus tepidamans, Aquamicrobium defluvii, Arcanobacterium haemolyticum, Arcanobacterium sp., Archaeoglobus sulfaticallidus, Arcobacter butzleri, Arcobacter cibarius, Arcobacter nitrofigilis, Arcobacter sp., Arsenicicoccus bolidensis, Arthrobacter arilaitensis, Arthrobacter aurescens, Arthrobacter castelli, Arthrobacter chlorophenolicus, Arthrobacter gangotriensis, Arthrobacter globiformis, Arthrobacter nicotinovorans, Arthrobacter phenanthrenivorans, Arthrobacter sanguinis, Arthrobacter sp., Arthrospira platensis, Asaia platycodi, Aster yellows, Asticcacaulis benevestitus, Asticcacaulis biprosthecum, Asticcacaulis excentricus, Asticcacaulis sp., Atopobacter phocae, Atopobium parvulum, Atopobium rimae, Atopobium sp., Atopobium vaginae, Atopococcus tabaci, Aureimonas ureilytica, Aureispira sp., Austwickia chelonae, Azorhizobium caulinodans, Azorhizobium doebereinerae, Azospirillum halopraeferens, Bacillus aidingensis, Bacillus akibai, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus atrophaeus, Bacillus bataviensis, Bacillus bogoriensis, Bacillus boroniphilus, Bacillus cellulosilyticus, Bacillus cereus, Bacillus chagannorensis, Bacillus cibi, Bacillus clausii, Bacillus coagulans, Bacillus coahuilensis, Bacillus cytotoxicus, Bacillus endophyticus, Bacillus firm us, Bacillus flexus, Bacillus fordii, Bacillus gaemokensis, Bacillus gelatini, Bacillus halodurans, Bacillus lehensis, Bacillus licheniformis, Bacillus macauensis, Bacillus manliponensis, Bacillus massilioanorexius, Bacillus massiliosenegalensis, Bacillus megaterium, Bacillus methanolicus, Bacillus mojavensis, Bacillus mycoides, Bacillus oceanisediminis, Bacillus okhensis, Bacillus pseudofirmus, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus selenitireducens, Bacillus simplex, Bacillus smithii, Bacillus sonorensis, Bacillus sp., Bacillus subtilis, Bacillus thermoamylovorans, Bacillus timonensis, Bacillus vietnamensis, Bacillus vireti, Bacillus weihenstephanensis, Bacillus xiamenensis, Bacteriovorax marinus, Bacteriovorax sp., bacterium JKG1, bacterium LF-3, bacterium MS4, bacterium OL-1, Bacteroides coprosuis, Bacteroides pectinophilus, Balneimonas flocculans, Barnesiella viscericola, Bartonella alsatica, Bartonella australis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis, Bartonella clarridgeiae, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae, Bartonella koehlerae, Bartonella melophagi, Bartonella quintana, Bartonella rattaustraliani, Bartonella rattimassiliensis,

Bartonella rochalimae, Bartonella schoenb uchensis, Bartonella senegalensis, Bartonella sp., Bartonella tamiae, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii, Bartonella washoensis, Bavariicoccus seileri, Beijerinckia indica, Beijerinckia mobilis, Belnapia moabensis, Beutenbergia cavernae, Bhargavaea cecembensis, Bifidobacterium actinocoloniiforme, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium biavatii, Bifidobacterium bifidum, Bifidobacterium bohemicum, Bifidobacterium bombi, Bifidobacterium breve, Bifidobacterium callitrichos, Bifidobacterium catenulatum, Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacterium crudilactis, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium gallinarum, Bifidobacterium longum, Bifidobacterium magnum, Bifidobacterium merycicum, Bifidobacterium minimum, Bifidobacterium mongoliense, Bifidobacterium moukalabense, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium psychraerophilum, Bifidobacterium pullorum, Bifidobacterium reuteri, Bifidobacterium ruminantium, Bifidobacterium scardovii,

Bifidobacterium sp., Bifidobacterium stellenboschense, Bifidobacterium subtile, Bifidobacterium thermophilum, Bifidobacterium tsurumiense, Bilophila wadsworthia, Blastococcus saxobsidens, Blastococcus sp., Blastomonas sp., Blautia hansenii, Blautia hydrogenotrophica, Blautia producta, Blautia wexlerae, Bosea sp., Brachybacterium faecium, Brachybacterium paraconglomeratum, Brachybacterium phenoliresistens, Brachybacterium squillarum, Brachyspira hampsonii, Brachyspira hyodysenteriae, Brachyspira intermedia, Brachyspira murdochii, Brachyspira pilosicoli, Bradyrhizobiaceae bacterium, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Bradyrhizobium genosp., Bradyrhizobium japonicum, Bradyrhizobium oligotrophicum, Bradyrhizobium sp., Brevibacillus borstelensis, Brevibacillus brevis, Brevibacillus laterosporus, Brevibacillus massiliensis, Brevibacillus panacihumi, Brevibacterium casei, Brevibacterium linens, Brevibacterium massiliense, Brevibacterium mcbrellneri, Brevibacterium senegalense, Brevibacterium sp., Brevundimonas aveniformis, Brevundimonas bacteroides, Brevundimonas diminuta, Brevundimonas naejangsanensis, Brevundimonas sp., Brevundimonas subvibrioides, Brucella melitensis, Bryobacter aggregatus, Bulleidia extructa, Butyricicoccus pullicaecorum, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Butyrivibrio proteoclasticus, Butyrivibrio sp., Caenispirillum salinarum, Caldalkalibacillus thermarum, Caldanaerobacter subterraneus, Caldanaerobius polysaccharolyticus, Caldicellulosiruptor bescii, Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor obsidiansis, Caldicellulosiruptor owensensis, Caldicellulosiruptor saccharolyticus, Caldicoprobacter oshimai, Caldilinea aerophila, Caldisericum exile, Calditerrivibrio nitroreducens, Caloramator australicus, Caloramator sp., Calothrix sp., Campylobacter coli, Campylobacter concisus, Campylobacter cuniculorum, Campylobacter fetus, Campylobacter gracilis, Campylobacter hominis, Campylobacter jejuni, Campylobacter lari, Campylobacter rectus, Campylobacter showae, Campylobacter sp., Campylobacter sputorum, Campylobacter upsaliensis, Campylobacter ureolyticus, Candidatus Aquiluna, Candidatus Arthromitus, Candidatus Atelocyanobacterium, Candidatus Blastococcus, Candidatus Caedibacter, Candidatus Clostridium, Candidatus Desulforudis, Candidatus Endolissoclinum, Candidatus Halobonum, Candidatus Hepatobacter, Candidatus Hepatoplasma, Candidatus Korarchaeum, Candidatus Koribacter, Candidatus Liberibacter, Candidatus Methylomirabilis, Candidatus Microthrix, Candidatus Mycoplasma, Candidatus Odyssella, Candidatus Paracaedibacter, Candidatus Pelagibacter, Candidatus Phytoplasma, Candidatus Puniceispirillum, Candidatus Rhodoluna, Candidatus Solibacter, Candidatus Stoquefichus, Candidatus Sulfuricurvum, Carboxydothermus hydrogenoformans, Carnimonas nigrificans, Carnobacterium alterfunditum, Carnobacterium divergens, Carnobacterium funditum, Carnobacterium gallinarum, Carnobacterium maltaromaticum, Carnobacterium mobile, Carnobacterium pleistocenium, Carnobacterium sp., Catellicoccus marimammalium, Catelliglobosispora koreensis, Catenulispora acidiphila, Catenuloplanes japonicus, Catonella morbi, Caulobacter crescentus, Caulobacter henricii, Caulobacter segnis, Caulobacter sp., Caulobacteraceae bacterium, Celeribacter baekdonensis, Cellulomonas fimi, Cellulomonas flavigena, Cellulomonas massiliensis, Cellulomonas sp., Cellulosimicrobium cellulans, Cellvibrio gilvus, Centipeda periodontii, Cetobacterium somerae, Chamaesiphon minutus, Chelativorans sp., Chelatococcus sp., Chitinivibrio alkaliphilus, Chloroflexus aggregans, Chloroflexus aurantiacus, Chloroflexus sp., Chlorogloeopsis fritschii, Chondromyces apiculatus, Chromohalobacter salexigens, Chroococcidiopsis thermalis, Chrysiogenes arsenatis, Chthonomonas calidirosea, Citreicella sp., Citricoccus sp., Citromicrobium bathyomarinum, Citromicrobium sp., Clavibacter michiganensis, Clostridiaceae bacterium, Clostridiales bacterium, Clostridiales genomosp., Clostridiisalibacter paucivorans, Clostridium acetobutylicum, Clostridium acidurici, Clostridium algidicarnis, Clostridium aminophilum, Clostridium arbusti, Clostridium baratii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium carboxidivorans, Clostridium celatum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridio forme, Clostridium colicanis, Clostridium drakei, Clostridium hiranonis, Clostridium hylemonae, Clostridium innocuum, Clostridium intestinale, Clostridium josui, Clostridium kluyveri, Clostridium lentocellum, Clostridium leptum, Clostridium litorale, Clostridium ljungdahlii, Clostridium lundense, Clostridium mangenotii, Clostridium methylpentosum, Clostridium novyi, Clostridium orbiscindens, Clostridium papyrosolvens, Clostridium paraputrificum, Clostridium pasteurianum, Clostridium perfringens, Clostridium saccharob utylicum, Clostridium saccharogumia, Clostridium saccharolyticum, Clostridium saccharoperb utylacetonicum, Clostridium scindens, Clostridium senegalense, Clostridium sordellii, Clostridium sp., Clostridium spiroforme, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium sticklandii, Clostridium sulfidigenes, Clostridium symbiosum, Clostridium termitidis, Clostridium tetanomorphum, Clostridium tyrobutyricum, Clostridium uitunense, Clostridium Wide, Cobetia crustatorum, Cobetia marina, Cohnella laeviribosi, Coleofasciculus chthonoplastes, Collinsella intestinaiis, Collinsella sp., Collinsella stercoris, Collinsella tanakaei, Commensalibacter intestini, Commensalibacter sp., Conexibacter woesei, Congregibacter litoralis, Coprobacillus sp., Coprococcus eutactus, Coprothermobacter platensis, Coprothermobacter proteolyticus, Corallococcus coralloides, Coriobacteriaceae bacterium, Coriobacterium glomerans, Corynebacterium accolens, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium argentoratense, Corynebacterium atypicum, Corynebacterium aurimucosum, Corynebacterium callunae, Corynebacterium capitovis, Corynebacterium casei,

Corynebacterium caspium, Corynebacterium ciconiae, Corynebacterium diphtheriae, Corynebacterium doosanense, Corynebacterium durum, Corynebacterium efficiens,

Corynebacterium falsenii, Corynebacterium freiburgense, Corynebacterium freneyi, Corynebacterium genitalium, Corynebacterium glucuronolyticum, Corynebacterium glutamicum, Corynebacterium glycinophilum, Corynebacterium halotolerans, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium lipophiloflavum, Corynebacterium lubricantis, Corynebacterium maris, Corynebacterium massiliense, Corynebacterium mastitidis, Corynebacterium matruchotii, Corynebacterium nuruki, Corynebacterium pilosum, Corynebacterium propinquum, Corynebacterium pseudogenitalium, Corynebacterium pseudotuberculosis, Corynebacterium pyruviciproducens, Corynebacterium resistens, Corynebacterium sp., Corynebacterium sputi, Corynebacterium striatum, Corynebacterium terpenotabidum, Corynebacterium timonense, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium ulceribovis, Corynebacterium urealyticum, Corynebacterium variabile, Corynebacterium vitaeruminis, Crinalium epipsammum, Cryobacterium roopkundense, Cryptobacterium curtum, Curtobacterium sp., Cyanobacterium aponinum, cyanobacterium PCC, Cyanobacterium stanieri, Cyanobium gracile, Cyanobium sp., Cyanothece sp., Cylindrospermopsis raciborskii, Cylindrospermum stagnale, Cystobacter fuscus, Dactylococcopsis salina, Dactylosporangium aurantiacum, Deferribacter desulfuricans, Deferrisoma camini, Defluviimonas sp., Dehalobacter sp., Dehalococcoides mccartyi, Dehalogenimonas lykanthroporepellens, Deinococcus apachensis, Deinococcus aquatilis, Deinococcus deserti, Deinococcus frigens, Deinococcus geothermalis, Deinococcus gobiensis, Deinococcus maricopensis, Deinococcus marmoris, Deinococcus misasensis, Deinococcus peraridilitoris, Deinococcus phoenicis, Deinococcus pimensis, Deinococcus proteolyticus, Deinococcus sp., Deinococcus wulumuqiensis, Demetria terragena, Denitrovibrio acetiphilus, Dermabactersp., Dermacoccus nishinomiyaensis, Dermacoccus sp., Desmospora sp., Desulfatibacillum alkenivorans, Desulfitobacterium dehalogenans, Desulfitobacterium dichloroeliminans, Desulfitobacterium hafniense, Desulfitobacterium metallireducens, Desulfobacca acetoxidans, Desulfobacter curvatus, Desulfobacter postgatei, Desulfobacterium autotrophicum, Desulfobacula sp., Desulfococcus multivorans, Desulfococcus oleovorans, Desulfocurvus vexinensis, Desulfohalobium retbaense, Desulfomicrobium baculatum, Desulfomonile tiedjei, Desulfonatronospira thiodismutans, Desulfonatronovibrio hydrogenovorans, Desulfonauticus sp., Desulforegula conservatrix, Desulfosarcina sp., Desulfospira joergensenii, Desulfosporosin us acidiphilus, Desulfosporosin us meridiei, Desulfosporosin us orientis, Desulfosporosin us sp., Desulfosporosin us youngiae, Desulfotomaculum acetoxidans, Desulfotomaculum alcoholivorax, Desulfotomaculum alkaliphilum, Desulfotomaculum carboxydivorans, Desulfotomaculum gibsoniae, Desulfotomaculum guttoideum, Desulfotomaculum hydrothermale, Desulfotomaculum kuznetsovii, Desulfotomaculum nigrificans, Desulfotomaculum reducens, Desulfotomaculum ruminis, Desulfotomaculum thermocistern urn, Desulfovibrio acrylicus, Desulfovibrio aespoeensis, Desulfovibrio africanus, Desulfovibrio alaskensis, Desulfovibrio alcoholivorans, Desulfovibrio alkalitolerans, Desulfovibrio aminophilus, Desulfovibrio basfmii, Desulfovibrio cf, Desulfovibrio desulfuricans, Desulfovibrio frigidus, Desulfovibrio fructosivorans, Desulfovibrio gigas, Desulfovibrio hydrothermalis, Desulfovibrio longus, Desulfovibrio magneticus, Desulfovibrio oxyclinae, Desulfovibrio piezophilus, Desulfovibrio piger, Desulfovibrio salexigens, Desulfovibrio sp., Desulfovibrio vulgaris, Desulfovirgula thermocuniculi, Desulfurella acetivorans, Desulfurispirillum indicum, Desulfurispora thermophila, Desulfuromonas sp., Dethiobacter alkaliphilus, Dethiosulfovibrio peptidovorans, Devosia sp., Dialister invisus, Dialister micraerophilus, Dialister microaerophilus, Dialister succinatiphilus, Dictyoglomus thermophilum, Dictyoglomus turgidum, Dietzia alimentaria, Dietzia sp., Dinoroseobacter shibae, Dolosigranulum pigrum, Donghicola xiamenensis, Dongia sp., Dorea formicigenerans, Dorea longicatena, Dorea sp., Eggerthella lenta, Eggerthella sp., Eggerthia catenaformis, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ruminantium, Ehrlichia sp., Elioraea tepidiphila, endosymbiont of, Enhygromyxa salina, Enorma massiliensis, Ensifer sojae, Ensifer sp., Enterococcus asini, Enterococcus avium, Enterococcus caccae, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus italicus, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, Enterococcus pallens, Enterococcus phoeniculicola, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus villorum, Enterorhabdus caecimuris, Enterorhabdus mucosicola, Eremococcus coleocola, Erysipelatoclostridium ramosum, Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum, Erysipelotrichaceae bacterium, Erythrobacter litoralis, Erythrobacter longus, Erythrobacter sp., Erythrobacter vulgaris, Ethanoligenens harbinense, Eubacterium acidaminophilum, Eubacterium brachy, Eubacterium cellulosolvens, Eubacterium desmolans, Eubacterium dolichum, Eubacterium eligens, Eubacterium hallii, Eubacterium infirmum, Eubacterium limosum, Eubacterium nodatum, Eubacterium plexicaudatum, Eubacterium ram ulus, Eubacterium saphenum, Eubacterium sp., Eubacterium sulci, Eubacterium ventriosum, Eubacterium xylanophilum, Eubacterium yurii, Exiguobacterium antarcticum, Exiguobacterium marinum, Exiguobacterium oxidotolerans, Exiguobacterium pavilionensis, Exiguobacterium sibiricum, Exiguobacterium sp., Exiguobacterium undae, Facklamia hominis, Facklamia ignava, Facklamia languida, Facklamia sourekii, Faecalibacterium cf, Faecalibacterium prausnitzii, Faecalicoccus pleomorphus, Falsirhodobacter sp., Ferrimicrobium acidiphilum, Fervidicoccus fontis, filamentous cyanobacterium, Filifactor alocis, Fimbriimonas ginsengisoli, Finegoldia magna, Firmicutes bacterium, Fischerella muscicola, Fischerella sp., Fischerella thermalis, Flexistipes sinusarabici, Fodinicurvata fenggangensis, Fodinicurvata sediminis, Frankia alni, Frankia sp., Frankia symbiont, Fructobacillus fructosus, Fulvimarina pelagi, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium perfoetens, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium sp., Fusobacterium ulcerans, Fusobacterium varium, gamma proteobacterium, Gardnerella vaginalis, Geitlerinema sp., Gemella bergeriae, Gemella cuniculi, Gemella haemolysans, Gemella morbillorum, Gemella sanguinis, Geminicoccus roseus, Geminocystis herdmanii, Gemmobacter nectariphilus, Geobacillus caldoxylosilyticus, Geobacillus kaustophilus, Geobacillus sp., Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus thermoglucosidasius, Geobacillus vulcani, Geobacter bemidjiensis, Geobacter daltonii, Geobacter lovleyi, Geobacter metallireducens, Geobacter sp., Geobacter sulfurreducens, Geodermatophilaceae bacterium, Geodermatophilus obscurus, Geoglobus acetivorans, Geomicrobium sp., Geopsychrobacter electrodiphilus, Geothrix fermentans, Geovibrio sp., Glaciecola arctica, Glaciecola lipolytica, Glaciecola nitratireducens, Glaciecola psychrophila, Glaciecola punicea, Glaciecola sp., Glaciibacter superstes, Gloeobacter kilaueensis, Gloeobacter violaceus, Gloeocapsa sp., Gluconacetobacter diazotrophicus, Gluconacetobacter rhaeticus, Gluconacetobacter xylinus, Gluconobacter frateurii, Gluconobacter morbifer, Gluconobacter oxydans, Glycomyces arizonensis, Glycomyces sp., Gordonia aichiensis, Gordonia alkanivorans, Gordonia amarae, Gordonia amicalis, Gordonia bronchialis, Gordonia effusa, Gordonia hirsute, Gordonia kroppenstedtii, Gordonia malaquae, Gordonia neofelifaecis, Gordonia paraffinivorans, Gordonia polyisoprenivorans, Gordonia rhizosphera, Gordonia shandongensis, Gordonia sihwensis, Gordonia soli, Gordonia sp., Gordonia sputi, Gordonia terrae, Gracilibacillus boraciitolerans, Gracilibacillus halophilus, Gracilibacillus lacisalsi, Granulibacter bethesdensis, Granulicatella adiacens, Granulicatella elegans, G ran u Heel la mallensis, Granulicella tundricola, Granulicoccus phenolivorans, Gryllotalpicola ginsengisoli, Gulosibacter molinativorax, Haematobacter massiliensis, Haladaptatus cibarius, Haladaptatus paucihalophilus, Halalkalibacillus halophilus, Halalkalicoccus jeotgali, Halanaerobium hydrogeniformans, Halanaerobium praevalens, Halanaerobium saccharolyticum, Halapricum salinum, Halarchaeum acidiphilum, haloarchaeon 3A1_DGR, Haloarcula hispanica, Haloarcula japonica, Haloarcula marismortui, Halobacillus dabanensis, Halobacillus halophilus, Halobacillus kuroshimensis, Halobacillus sp., Halobacterium sp., Halobacteroides halobius, Halobellus rufus, Halobiforma lacisalsi, Halobiforma nitratireducens, Halococcus hamelinensis, Halococcus morrhuae, Halococcus sp., Halococcus thailandensis, Haloferax mediterranei, Haloferax mucosum, Haloferax volcanii, Halogeometricum borinquense, Haloglycomyces a lb us, Halogranum salarium, Halomicrobium mukohataei, Halomonas alkaliantarctica, Halomonas anticariensis, Halomonas elongata, Halomonas halocynthiae, Halomonas jeotgali, Halomonas lutea, Halomonas salina, Halomonas sp., Halomonas xinjiangensis, Halomonas zhanjiangensis, Halonatronum saccharophilum, Halonotius sp., halophilic archaeon, Halopiger salifodinae, Halopiger sp., Halopiger xanaduensis, Haloplanus natans, Haloplasma contractile, Haloquadratum walsbyi, Halorhabdus tiamatea, Halorhabdus utahensis, Halorubrum ezzemoulense, Halorubrum halophilum, Halorubrum lacusprofundi, Halorubrum saccharovorum, Halosarcina pallida, Halosimplex carlsbadense, Halostagnicola larsenii, Haloterrigena limicola, Haloterrigena salina, Haloterrigena turkmenica, Halothece sp., Halothermothrix orenii, Halovivax ruber, Hamadaea tsunoensis, Helcococcus kunzii, Helcococcus sueciensis, Helicobacter acinonychis, Helicobacter bilis, Helicobacter bizzozeronii, Helicobacter canadensis, Helicobacter canis, Helicobacter cetorum, Helicobacter cinaedi, Helicobacter felis, Helicobacter fennelliae, Helicobacter hepaticus, Helicobacter macacae, Helicobacter mustelae, Helicobacter pametensis, Helicobacter pullorum, Helicobacter pylori, Helicobacter rodentium, Helicobacter sp., Helicobacter suis, Helicobacter trogontum, Helicobacter winghamensis, Heliobacterium modesticaldum, Hellea balneolensis, Henriciella marina, Herbidospora cretacea, Herpetosiphon aurantiacus, Hippea alviniae, Hippea jasoniae, Hippea maritime, Hippea sp., Hirschia baltica, Hirschia maritima, Hoeflea phototrophica, Hoeflea sp., Holdemanella biformis, Holdemania filiformis, Holdemania massiliensis, Holophaga foetida, Holospora obtusa, Humibacter a lb us, Hyphomicrobium denitrificans, Hyphomicrobium sp., Hyphomicrobium zavarzinii, Hyphomonas adhaerens, Hyphomonas jannaschiana, Hyphomonas johnsonii, Hyphomonas neptunium, Hyphomonas oceanitis, Hyphomonas polymorpha, Hyphomonas sp., Ilumatobacter coccineus, llyobacter polytropus, Intestinibacter bartlettii, Intestinimonas b utyriciproducens, Intrasporangiaceae bacterium, Intrasporangium calvum, Intrasporangium oryzae, Isoptericola variabilis, Janibacter hoylei, Janibacter sp., Jannaschia sp., Jeotgalicoccus marinus, Jeotgalicoccus psychrophilus, Jeotgalicoccus sp., Johnsonella ignava, Jonesia denitrificans, Jonesia quinghaiensis, Jonquetella anthropi, Kaistia granuli, Kallipyga massiliensis, Kamptonema formosum, Kandleria vitulina, Ketogulonicigenium vulgare, Kiloniella laminariae, Kineococcus radiotolerans, Kineosphaera limosa, Kineosporia aurantiaca, Kitasatospora arboriphila, Kitasatospora azatica, Kitasatospora cheerisanensis, Kitasatospora mediocidica, Kitasatospora setae, Knoellia aerolata, Knoellia flava, Knoellia sinensis, Knoellia subterranea, Kocuria marina, Kocuria polaris, Kocuria rhizophila, Komagataeibacter medellinensis, Kordiimonas gwangyangensis, Kozakia baliensis, Kribbella catacumbae, Kribbella flavida, Ktedonobacter racemifer, Kurthia huakuii, Kurthia massiliensis, Kurthia sp., Kushneria aurantia, Kutzneria albida, Kutzneria sp., Kyrpidia tusciae, Kytococcus sedentarius, Labrenzia aggregate, Laceyella sacchari, Lachnoanaerobaculum saburreum, Lachnoanaerobaculum sp., Lachnobacterium bovis, Lachnoclostridium phytofermentans, Lachnospira multipara, Lachnospiraceae bacterium, Lachnospiraceae oral, Lacticigenium naphtae, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apodemi, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus composti, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curieae, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus equicursoris, Lactobacillus fabifermentans, Lactobacillus farciminis, Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillus florum, Lactobacillus fructivorans, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hominis, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus malefermentans, Lactobacillus murinus, Lactobacillus namurensis, Lactobacillus nodensis, Lactobacillus oris, Lactobacillus oryzae, Lactobacillus otakiensis, Lactobacillus parabrevis, Lactobacillus parafarraginis, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus psittaci, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sanfranciscensis, Lactobacillus shenzhenensis, Lactobacillus sp., Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus ultunensis, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini, Lactobacillus zeae, Lactococcus garvieae, Lactococcus lactis, Lactococcus raffinolactis, Lawsonia intracellularis, Lebetimonas sp., Lechevalieria aerocolonigenes, Leifsonia aquatica, Leifsonia rubra, Leifsonia sp., Leifsonia xyli, Leisingera aquimarina, Leisingera caerulea, Leisingera daeponensis, Lentibacillus jeotgali, Lentzea albidocapillata, Leptolyngbya boryana, Leptolyngbya sp., Leptospirillum ferrooxidans, Leptotrichia buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii, Leptotrichia shahii, Leptotrichia sp., Leptotrichia trevisanii, Leptotrichia wadei, Leucobacter chironomi, Leucobacter chromiiresistens, Leucobacter salsicius, Leucobacter sp., Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc fallax, Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc kimchii, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leucothrix mucor, Liberibacter crescens, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, Listeria weihenstephanensis, Listeria welshimeri, Listeriaceae bacterium, Loktanella cinnabarina, Loktanella hongkongensis, Loktanella vestfoldensis, Longispora albida, Luminiphilus syltensis, Lutibaculum baratangense, Lyngbya sp., Lysinibacillus fusiformis, Lysinibacillus manganicus, Lysinibacillus massiliensis, Lysinibacillus odysseyi, Lysinibacillus sinduriensis, Lysinibacillus sp., Lysinibacillus sphaericus, Lysinibacillus varians, Lysinimicrobium mangrovi, Macrococcus caseolyticus, Magnetococcus marinus, Magnetospirillum gryphiswaldense, Magnetospirillum sp., Mahella australiensis, Maribius sp., Maricaulis maris, Maricaulis sp., marine actinobacterium, marine gamma, Marine Group, Marinithermus hydrothermalis, Marinobacter adhaerens, Marinobacterhydrocarbonoclasticus, Marinobacter lipolyticus, Marinobacter nanhaiticus, Marinobacter sp., Marinomonas posidonica, Marinomonas sp., Marinomonas ushuaiensis, Marinospirillum insulare, Marinospirillum minutulum, Mariprofundus ferrooxydans, Maritimibacter alkaliphilus, Marmoricola aequoreus, Marmoricola sp., Martelella mediterranea, Marvinbryantia formatexigens, Mastigocladopsis repens, Megamonas hypermegale, Megamonas rupellensis, Meganema perideroedes, Megasphaera elsdenii, Megasphaera genomosp., Megasphaera micronuciformis, Megasphaera sp., Meiothermus cerbereus, Meiothermus chliarophilus, Meiothermus ruber, Meiothermus rufus, Meiothermus si Ivan us, Meiothermus taiwanensis, Meiothermus timidus, Mesoplasma florum, Mesorhizobium amorphae, Mesorhizobium australicum, Mesorhizobium ciceri, Mesorhizobium loti, Mesorhizobium metallidurans, Mesorhizobium opportunistum, Mesorhizobium sp., Metascardovia criceti, Methanobacterium arcticum, Methanobacterium lacus, Methanobacterium paludis, Methanobacterium sp., Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter sp., Methanocaldococcus fervens, Methanocaldococcus infernus, Methanocaldococcus jannaschii, Methanocaldococcus sp., Methanocaldococcus vulcanius, Methanocella arvoryzae, Methanocella conradii, Methanocella paludicola, Methanococcus aeolicus, Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltae, Methanocorpusculum labreanum, Methanoculleus marisnigri, Methanolobus psychrophilus, Methanomicrobium mobile, Methanoplanus petrolearius, Methanoregula boonei, Methanoregula formicica, Methanosarcina barkeri, Methanosarcina mazei, Methanosphaera stadtmanae, Methanosphaerula palustris, Methanospirillum hungatei, Methanothermococcus okinawensis, Methanothermococcus thermolithotrophicus, Methylobacterium extorquens, Methylobacterium nodulans, Methylobacterium oryzae, Methylobacterium populi, Methylobacterium radiotolerans, Methylobacterium sp., Methylocapsa acidiphila, Methylocapsa aurea, Methylocella silvestris, Methylococcus capsulatus, Methylocystis parvus, Methylocystis sp., Methyloferula stellata, Methylophaga aminisulfidivorans, Methylophaga frappieri, Methylophaga lonarensis, Methylophaga nitratireducenticrescens, Methylopila sp., Methylosinus sp., Methylosinus trichosporium, Micavibrio aeruginosavorus, Microbacterium gubbeenense, Microbacterium indicum, Microbacterium luticocti, Microbacterium oleivorans, Microbacterium paraoxydans, Microbacterium profundi, Microbacterium sp., Microbacterium testaceum, Microbacterium yannicii, Microchaete sp., Micrococcus luteus, Microcoleus sp., Microcoleus vaginatus, Microcystis aeruginosa, Microlunatus phosphovorus, Micromonospora aurantiaca, Micromonospora chokoriensis, Micromonospora lupini, Micromonospora parva, Micromonospora sp., Microtetraspora glauca, Microvirga aerilata, Microvirga lotononidis, Microvirga lupini, Mitsuokella jalaludinii, Mitsuokella multacida, Mitsuokella sp., Mobilicoccus pelagius, Mobiluncus curtisii, Mobiluncus mulieris, Modestobacter marinus, Mogibacterium sp., Mollicutes bacterium, Moorea producens, Moorella thermoacetica, Mucispirillum schaedleri, Mycetocola saprophilus, Mycobacterium abscessus, Mycobacterium aromaticivorans, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium chubuense, Mycobacterium colombiense, Mycobacterium cosmeticum, Mycobacterium genavense, Mycobacterium gilvum, Mycobacterium hassiacum, Mycobacterium intracellulare, Mycobacterium iranicum, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium mageritense, Mycobacterium marinum, Mycobacterium neoaurum, Mycobacterium parascrofulaceum, Mycobacterium phlei, Mycobacterium rhodesiae, Mycobacterium rufum, Mycobacterium septicum, Mycobacterium smegmatis, Mycobacterium sp., Mycobacterium thermoresistibile, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium tusciae, Mycobacterium vaccae, Mycobacterium vanbaalenii, Mycobacterium vulneris, Mycoplasma agalactiae, Mycoplasma aikaiescens, Mycoplasma alligatoris, Mycoplasma anatis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma auris, Mycoplasma bovigenitalium, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasma californicum, Mycoplasma canis, Mycoplasma columbinum, Mycoplasma conjunctivae, Mycoplasma crocodyli, Mycoplasma cynos, Mycoplasma fermentans, Mycoplasma gallisepticum, Mycoplasma genitalium, Mycoplasma haemofelis, Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma iowae, Mycoplasma leachii, Mycoplasma mobile, Mycoplasma mycoides, Mycoplasma ovipneumoniae, Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma pulmonis, Mycoplasma putrefaciens, Mycoplasma sp., Mycoplasma synoviae, Mycoplasma yeatsii, Myxococcus fulvus, Myxococcus sp., Myxococcus stipitatus, Myxococcus xanthus, Myxosarcina sp., Nakamurella multi partita, Natranaerobius thermophilus, Natrialba asiatica, Natrialba magadii, Natrinema altunense, Natrinema pallidum, Natrinema pellirubrum, Natrinema sp., Natronobacterium gregoryi, Natronococcus amylolyticus, Natronococcus occultus, Natronolimnobius innermongolicus, Natronomonas moolapensis, Natronomonas pharaonis, Natronorubrum bangense, Natronorubrum sulfidifaciens, Natronorubrum tibetense, Nautilia profundicola, Neorhizobium galegae, Neorickettsia risticii, Neorickettsia sennetsu, Nesterenkonia alba, Nesterenkonia sp., Nitratifractor salsuginis, Nitratireductor aquibiodomus, Nitratireductor basaltis, Nitratireductor indicus, Nitratireductor pacificus, Nitratiruptor sp., Nitrobacter hamburgensis, Nitrobacter winogradskyi, Nocardia aobensis, Nocardia asteroides, Nocardia brasiliensis, Nocardia brevicatena, Nocardia carnea, Nocardia concava, Nocardia cyriacigeorgica, Nocardia exalbida, Nocardia farcinica, Nocardia higoensis, Nocardia jiangxiensis, Nocardia niigatensis, Nocardia nova, Nocardia otitidiscaviarum, Nocardia pneumoniae, Nocardia rhamnosiphila, Nocardia sp., Nocardia takedensis, Nocardia testacea, Nocardia transvalensis, Nocardia veterana, Nocardioidaceae bacterium, Nocardioides halotolerans, Nocardioides sp., Nocardiopsis alba, Nocardiopsis baichengensis, Nocardiopsis dassonvillei, Nocardiopsis ganjiahuensis, Nocardiopsis kunsanensis, Nocardiopsis potens, Nocardiopsis prasina, Nocardiopsis sp., Nocardiopsis valliformis, Nocardiopsis xinjiangensis, Nodosilinea nodulosa, Nodularia spumigena, Nonomuraea coxensis, Nosocomiicoccus sp., Nostoc azollae, Nostoc punctiforme, Nostoc sp., Novispirillum itersonii, Novosphingobium acidiphilum, Novosphingobium aromaticivorans, Novosphingobium lindaniclasticum, Novosphingobium nitrogenifigens, Novosphingobium pentaromativorans, Novosphingobium resinovorum, Novosphingobium sp., Novosphingobium tardaugens, Oceanibaculum indicum, Oceanibulbus indolifex, Oceanicaulis alexandrii, Oceanicaulis sp., Oceanicola batsensis, Oceanicola granulosus, Oceanicola nanhaiensis, Oceanicola sp., Oceaniovalibus guishaninsula, Oceanithermus profundus, Oceanobacillus iheyensis, Oceanobacillus kimchii, Oceanobacillus manasiensis, Oceanobacillus massiliensis, Oceanobacillus picturae, Oceanobacillus sp., Ochrobactrum anthropi, Ochrobactrum rhizosphaerae, Octadecabacter antarcticus, Octadecabacter arcticus, Oenococcus kitaharae, Oenococcus oeni, Oerskovia turbata, Oligotropha carboxidovorans, Olsenella profusa, Olsenella sp., Olsenella uli, Onion yellows, Orenia marismortui, Oribacterium parvum, Oribacterium sinus, Oribacterium sp., Orientia tsutsugamushi, Ornithinibacillus scapharcae, Ornithinimicrobium pekingense, Oscillatoria acuminata, Oscillatoria nigro-viridis, Oscillatoria sp., Oscillatoriales cyanobacterium, Oscillibacter ruminantium, Oscillibacter sp., Oscillibacter valericigenes, Oscillochloris trichoides, Paenibacillaceae bacterium, Paenibacillus alginolyticus, Paenibacillus alvei, Paenibacillus borealis, Paenibacillus curdlanolyticus, Paenibacillus daejeonensis, Paenibacillus darwinianus, Paenibacillus durus, Paenibacillus elgii, Paenibacillus fonticola, Paenibacillus ginsengihumi, Paenibacillus graminis, Paenibacillus lactis, Paenibacillus larvae, Paenibacillus massiliensis, Paenibacillus odorifer, Paenibacillus peoriae, Paenibacillus pini, Paenibacillus polymyxa, Paenibacillus sabinae, Paenibacillus sanguinis, Paenibacillus senegalensis, Paenibacillus sp., Paenibacillus terrae, Paenibacillus terrigena, Paenibacillus wynnii, Paenirhodobacter enshiensis, Paenisporosarcina sp., Palaeococcus pacificus, Pannonibacter phragmitetus, Paracoccus aminophilus, Paracoccus denitrificans, Paracoccus halophilus, Paracoccus pantotrophus, Paracoccus sp., Paracoccus versutus, Paracoccus yeei, Paracoccus zeaxanthinifaciens, Paraoerskovia marina, Parascardovia denticolens, Parvibaculum lavamentivorans, Parvimonas micra, Parvimonas sp., Parvularcula bermudensis, Parvularcula oceani, Patulibacter americanus, Patulibacter minatonensis, Paucisalibacillus sp., Peanut witches-broom, Pediococcus acidilactici, Pediococcus claussenii, Pediococcus pentosaceus, Pelagibaca bermudensis, Pelagibacterium halotolerans, Pelobacter carbinolicus, Pelobacter propionicus, Pelosinus fermentans, Pelosinus sp., Pelotomaculum thermopropionicum, Peptoclostridium difficile, Peptoniphilus duerdenii, Peptoniphilus grossensis, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus rhinitidis, Peptoniphilus senegalensis, Peptoniphilus sp., Peptoniphilus timonensis, Peptostreptococcaceae bacterium, Peptostreptococcus anaerobius, Peptostreptococcus sp., Peptostreptococcus stomatis, Phaeobacter gallaeciensis, Phaeobacter inhibens, Phaeospirillum fulvum, Phaeospirillum molischianum, Phascolarctobacterium sp., Phascolarctobacterium succinatutens, Phenylobacterium zucineum, Phycicoccus jejuensis, Phyllobacterium sp., Pimelobacter simplex, Planktomarina temperata, Planktothrix agardhii, Planococcus antarcticus, Planococcus donghaensis, Planococcus sp., Planomicrobium glaciei, Pleomorphomonas koreensis, Pleomorphomonas oryzae, Pleurocapsa sp., Polymorphum gilvum, Pontibacillus chungwhensis, Pontibacillus halophilus, Pontibacillus litoralis, Pontibacillus marinus, Pontibacillus yanchengensis, Ponticaulis koreensis, Porphyrobacter cryptus, Porphyrobacter sp., Prauserella rugosa, Prochlorococcus marinus, Prochlorococcus sp., Prochlorothrix hollandica, Prolixibacter bellariivorans, Promicromonospora kroppenstedtii, Propionibacteriaceae bacterium, Propionibacterium acidifaciens, Propionibacterium acidipropionici, Propionibacterium acnes, Propionibacterium acnes, Propionibacterium avidum, Propionibacterium freudenreichii, Propionibacterium granulosum, Propionibacterium jensenii, Propionibacterium propionicum, Propionibacterium sp., Propionibacterium thoenii, Propionimicrobium sp., Proteiniclasticum ruminis, Proteocatella sphenisci, Pseudaminobacter salicylatoxidans, Pseudanabaena sp., Pseudobacteroides cellulosolvens, Pseudobutyrivibrio ruminis, Pseudobutyrivibrio sp., Pseudoclavibacter soli, Pseudoflavonifractor capillosus, Pseudomonas pelagia, Pseudonocardia acaciae, Pseudonocardia asaccharolytica, Pseudonocardia autotrophica, Pseudonocardia dioxanivorans, Pseudophaeobacter arcticus, Pseudoramibacter alactolyticus, Pseudovibrio sp., Pyramidobacter piscolens, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Pyrococcus sp., Pyrococcus yayanosii, Raphidiopsis brookii, Rathayibacter toxicus, Renibacterium salmoninarum, Reyranella massiliensis, Rheinheimera baltica, Rheinheimera perlucida, Rheinheimera sp., Rhizobiales bacterium, Rhizobium alamii, Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium grahamii, Rhizobium larrymoorei, Rhizobium leguminosarum, Rhizobium leucaenae, Rhizobium mesoamericanum, Rhizobium mongolense, Rhizobium rubi, Rhizobium selenitireducens, Rhizobium sp., Rhizobium sullae, Rhizobium tropici, Rhizobium undicola, Rhizobium vignae, Rhodobacter capsulatus, Rhodobacter sp., Rhodobacter sphaeroides, Rhodobacteraceae bacterium, Rhodobacterales bacterium, Rhodococcus defluvii, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus fascians, Rhodococcus jostii, Rhodococcus opacus, Rhodococcus pyridinivorans, Rhodococcus rhodnii, Rhodococcus rhodochrous, Rhodococcus ruber, Rhodococcus sp., Rhodococcus triatomae, Rhodomicrobium vannielii, Rhodopseudomonas palustris, Rhodospirillales bacterium, Rhodospirillum centenum, Rhodospirillum photometricum, Rhodospirillum rubrum, Rhodothermus marinus, Rhodovibrio salinarum, Rhodovulum sp., Rhodovulum sulfidophilum, Richelia intracellularis, Rickettsia akari, Rickettsia australis, Rickettsia bellii, Rickettsia canadensis, Rickettsia felis, Rickettsia helvetica, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia sibirica, Rickettsia tamurae, Rickettsia typhi, Rickettsiales bacterium, Rivularia sp., Robiginitomaculum antarcticum, Robinsoniella peoriensis, Roseburia hominis, Roseburia inulinivorans, Roseibacterium elongatum, Roseibium sp., Roseiflexus castenholzii, Roseiflexus sp., Roseivivax halodurans, Roseivivax isoporae, Roseivivax sp., Roseobacter denitrificans, Roseobacter litoralis, Roseobacter sp., Roseomonas aerilata, Roseomonas gilardii, Roseomonas sp., Roseovarius mucosus, Roseovarius nubinhibens, Roseovarius sp., Rothia dentocariosa, Rothia mucilaginosa, Ruania albidiflava, Rubidibacter lacunae, Rubritepida flocculans, Rubrobacter radiotolerans, Rubrobacter xylanophilus, Ruegeria conchae, Ruegeria halocynthiae, Ruegeria pomeroyi, Ruegeria sp., Ruminiclostridium thermocellum, Ruminococcaceae bacterium, Ruminococcus alb us, Ruminococcus bicirculans, Ruminococcus callidus, Ruminococcus flavefaciens, Ruminococcus gauvreauii, Ruminococcus gnavus, Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus sp., Ruminococcus torques, Saccharibacillus kuerlensis, Saccharibacter floricola, Saccharibacter sp,, Saccharomonospora cyanea, Saccharomonospora glauca, Saccharomonospora marina, Saccharomonospora sp., Saccharomonospora viridis, Saccharomonospora xinjiangensis, Saccharopolyspora erythraea, Saccharopolyspora rectivirgula, Saccharopolyspora spinosa, Saccharospirillum impatiens, Saccharothrix espanaensis, Saccharothrix sp., Saccharothrix syringae, Sagittula stellata, Salimicrobium sp., Salinarchaeum sp., Salinarimonas rosea, Salinicoccus alb us, Salinicoccus carnicancri, Salinimonas chungwhensis, Salinispora arenicola, Salinispora pacifica, Salinispora tropica, Salipiger mucosus, Salsuginibacillus kocurii, Sandarakinorhabdus limnophila, Sandarakinorhabdus sp., Sanguibacter keddieii, SAR116 cluster, Scardovia inopinata, Scardovia wiggsiae, Sciscionella sp., Scytonema hofmanni, Sebaldella termitidis, Sedimentitalea nanhaiensis, Sediminimonas qiaohouensis, Segniliparus rotundus, Segniliparus rugosus, Selenomonas bovis, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, Selenomonas ruminantium, Selenomonas sp., Selenomonas sputigena, Senegalimassilia anaerobia, Serinicoccus marinus, Serinicoccus profundi, Sharpea azabuensis, Shewanella putrefaciens, Shewanella sediminis, Shimazuella kribbensis, Shinella sp., Shuttleworthia satelles, Shuttleworthia sp., Silicibacter lacuscaerulensis, Silicibacter sp., Sinorhizobium americanum, Sinorhizobium arboris, Sinorhizobium fredii, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium sp., Slackia exigua, Slackia heliotrinireducens, Slackia piriformis, Smaragdicoccus niigatensis, Solibacillus silvestris, Solirubrobacterales bacterium, Solobacterium moorei, Sorangium cellulosum, Sphaerobacter thermophilus, Sphingobium baderi, Sphingobium chlorophenolicum, Sphingobium herbicidovorans, Sphingobium japonicum, Sphingobium lactosutens, Sphingobium sp., Sphingobium ummariense, Sphingobium xenophagum, Sphingobium yanoikuyae, Sphingomonas astaxanthinifaciens, Sphingomonas echinoides, Sphingomonas elodea, Sphingomonas melonis, Sphingomonas parapaucimobilis, Sphingomonas paucimobilis, Sphingomonas phyllosphaerae, Sphingomonas sanxanigenens, Sphingomonas sp., Sphingomonas taxi, Sphingomonas wittichii, Sphingopyxis alaskensis, Sphingopyxis baekryungensis, Sphingopyxis sp., Spirillospora albida, Spiroplasma apis, Spiroplasma chrysopicola, Spiroplasma culicicola, Spiroplasma diminutum, Spiroplasma melliferum, Spiroplasma mirum, Spiroplasma sabaudiense, Spiroplasma syrphidicola, Spiroplasma taiwanense, Spirulina subsalsa, Sporichthya polymorpha, Sporolactobacillus laevolacticus, Sporolactobacillus terrae, Sporolactobacillus vineae, Sporomusa ovata, Sporosarcina newyorkensis, Sporosarcina sp., Sporosarcina ureae, Stackebrandtia nassauensis, Stanieria cyanosphaera, Staphylococcus agnetis, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus massiliensis, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus sp., Staphylococcus vitulinus, Staphylococcus warned, Staphylococcus xylosus, Starkeya novella, Stigmatella aurantiaca, Stomatobaculum longum, Streptacidiphilus a lb us, Streptacidiphilus carbonis, Streptacidiphilus jeojiense, Streptacidiphilus jiangxiensis, Streptacidiphilus neutrinimicus, Streptacidiphilus oryzae, Streptacidiphilus rugosus, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus australis, Streptococcus caballi, Streptococcus canis, Streptococcus castoreus, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus dentisani, Streptococcus devriesei, Streptococcus didelphis, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus entericus, Streptococcus equi, Streptococcus equinus, Streptococcus ferus, Streptococcus gordonii, Streptococcus henryi, Streptococcus ictaluri, Streptococcus infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedius, Streptococcus macacae, Streptococcus macedonicus, Streptococcus marimammalium, Streptococcus massiliensis, Streptococcus merionis, Streptococcus minor, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus orisratti, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcus peroris, Streptococcus plurextorum, Streptococcus pneumoniae, Streptococcus porci, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sp., Streptococcus suis, Streptococcus thermophilus, Streptococcus thoraltensis, Streptococcus tigurinus, Streptococcus uberis, Streptococcus urinalis, Streptococcus vestibularis, Streptomyces acidiscabies, Streptomyces alboflavus, Streptomyces albulus, Streptomyces alb us, Streptomyces atroolivaceus, Streptomyces auratus, Streptomyces aureocirculatus, Streptomyces aureofaciens, Streptomyces avermitilis, Streptomyces avicenniae, Streptomyces bicolor, Streptomyces bikiniensis, Streptomyces bingchenggensis, Streptomyces californicus, Streptomyces catenulae, Streptomyces cattleya, Streptomyces celluloflavus, Streptomyces cellulosae, Streptomyces chartreusis, Streptomyces clavuligerus, Streptomyces coelicolor, Streptomyces colli n us, Streptomyces davawensis, Streptomyces durhamensis, Streptomyces flavidovirens, Streptomyces flavochromogenes, Streptomyces flavovariabilis, Streptomyces fradiae, Streptomyces fulvissimus, Streptomyces fulvoviolaceus, Streptomyces gal bus, Streptomyces ghanaensis, Streptomyces glaucescens, Streptomyces griseoaurantiacus, Streptomyces griseoflavus, Streptomyces griseofuscus, Streptomyces griseoluteus, Streptomyces griseorubens, Streptomyces griseus, Streptomyces halstedii, Streptomyces hygroscopicus, Streptomyces iakyrus, Streptomyces katrae, Streptomyces lavenduligriseus, Streptomyces lydicus, Streptomyces megasporus, Streptomyces mobaraensis, Streptomyces mutabilis, Streptomyces niger, Streptomyces niveus, Streptomyces olindensis, Streptomyces olivaceus, Streptomyces pratensis, Streptomyces pristinaespiralis, Streptomyces purpeofuscus, Streptomyces purpureus, Streptomyces pyridomyceticus, Streptomyces rapamycinicus, Streptomyces resistomycificus, Streptomyces roseoverticillatus, Streptomyces scabiei, Streptomyces scabrisporus, Streptomyces sclerotialus, Streptomyces scopuliridis, Streptomyces seoulensis, Streptomyces somaliensis, Streptomyces sp., Streptomyces sulphurous, Streptomyces sviceus, Streptomyces thermolilacinus, Streptomyces toyocaensis, Streptomyces varsoviensis, Streptomyces venezuelae, Streptomyces violaceusniger, Streptomyces violens, Streptomyces virginiae, Streptomyces viridochromogenes, Streptomyces viridosporus, Streptomyces vitaminophilus, Streptomyces wedmorensis, Streptomyces xylophagus, Streptomyces yeochonensis, Streptosporangium amethystogenes, Streptosporangium roseum, Subdoligranulum sp., Subdoligranulum variabile, Succinispira mobilis, Sulfitobacter donghicola, Sulfitobacter guttiformis, Sulfitobacter mediterraneus, Sulfitobacter sp., Sulfobacillus acidophilus, Sulfolobus islandicus, Sulfuricurvum kujiense, Sulfuricurvum sp., Sulfurimonas autotrophica, Sulfurimonas denitrificans, Suifurimonas gotlandica, Sulfurimonas sp., Sulfurospirillum arcachonense, Sulfurospirillum barnesii, Sulfurospirillum deleyianum, Sulfurospirillum multivorans, Sulfurospirillum sp., Sulfurovum sp., Symbiobacterium thermophilum, Synechococcus elongatus, Synechococcus sp., Synechocystis sp., Synergistes jonesii, Synergistes sp., Syntrophobacter fumaroxidans, Syntrophobotulus glycolicus, Syntrophomonas wolfei, Syntrophotherm us lipocalidus, Tepidanaerobacter acetatoxydans, Terasakiella pusilla, Terracoccus sp., Terribacillus aidingensis, Terriglobus roseus, Terriglobus saanensis, Tetragenococcus halophilus, Tetragenococcus muriaticus, Tetrasphaera elongata, Thalassobacter arenae, Thalassobacter stenotrophicus, Thalassobium sp., Thalassolituus oleivorans, Thalassospira profundimaris, Thalassospira sp., Thalassospira xiamenensis, Thermacetogenium phaeum, Thermaerobacter marianensis, Thermaerobacter subterraneus, Thermanaerovibrio acidaminovorans, Thermanaerovibrio velox, Thermicanus aegyptius, Thermincola potens, Thermoactinomyces daqus, Thermoanaerobacter italicus, Thermoanaerobacter kivui, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter wiegelii,

Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium xylanolyticum, Thermobacillus composti, Thermobaculum terrenum, Thermobifida fusca, Thermobispora bispora, Thermobrachium celere, Thermococcus barophilus, Thermococcus gammatolerans, Thermococcus kodakarensis, Thermococcus nautili, Thermococcus onnurineus, Thermococcus sibiricus, Thermococcus sp., Thermococcus zilligii, Thermodesulfobium narugense, Thermofilum sp., Thermogemmatispora sp., Thermomicrobiales bacterium, Thermomicrobium roseum, Thermomonospora curvata, Thermosediminibacter oceani, Thermosinus carboxydivorans, Thermosynechococcus elongatus, Thermotoga hypogea, Thermotoga lettingae, Thermotoga maritima, Thermotoga neapolitana, Thermotoga petrophila, Thermotoga sp., Thermotoga thermarum, Thermovirga lienii, Therm us antranikianii, Therm us aquaticus, Therm us caliditerrae, Therm us igniterrae, Thermus islandicus, Therm us oshimai, Therm us scotoductus, Therm us sp., Thermus tengchongensis, Thermus thermophilus, Thioalkalimicrobium aerophilum, Thioalkalimicrobium cyclicum, Thioclava dalianensis, Thioclava pacifica, Thioclava sp., Thiomicrospira sp., Thiorhodovibrio sp., Timonella senegalensis, Tistrella mobilis, Tomitella biformata, Treponema primitia, Trichodesmium erythraeum, Tropheryma whipplei, Truepera radiovictrix, Tsukamurella paurometabola, Tsukamurella sp., Tuberibacillus calidus, Tumebacillus flagellatus, Turicella otitidis, Turicibacter sanguinis, Tyzzerella nexilis, Ureaplasma diversum, Ureaplasma parvum, Ureaplasma urealyticum, Ureibacillus thermosphaericus, Vagococcus lutrae, Varibaculum cambriense, Veillonella atypica, Veillonella dispar, Veillonella magna, Veillonella montpellierensis, Veillonella parvula, Veillonella ratti, Veillonella sp., Verrucosispora maris, Virgibacillus alimentarius, Virgibacillus halodenitrificans, Virgibacillus sp., Viridibacillus arenosi, Weissella cibaria, Weissella confusa, Weissella halotolerans, Weissella hellenica, Weissella koreensis, Weissella oryzae, Weissella paramesenteroides, Wenxinia marina, Wheat blue, Wolbachia endosymbiont, Wolinella succinogenes, Woodsholea maritima, Xanthobacter autotrophicus, Xanthobacter sp., Xanthobacteraceae bacterium, Xenococcus sp., Xylanimonas cellulosilytica, Yaniella halotolerans, Youngiibacter fragilis, Zetaproteobacteria bacterium, Zimmermannella faecalis, Zymomonas mobilis, and Zymophilus raffinosivorans.

In one embodiment, the one or more prokaryotic cell is one or more prokaryotic cell from the genus, species, or strain, comprising a genome as specified in Table 4.

In one embodiment, the one or more prokaryotic cell is one or more prokaryotic cell from the phylum, class order, family, genus, species, or strain, as specified in Table 5.

In one embodiment, the one or more prokaryotic cell is not from a phylum selected from the list consisting of: bacteroidetes; chlorobi; chlamydiae; verrucomicrobia; thermotogae; and aquificae.

In one embodiment, the one or more prokaryotic cell is not from the class betaproteobacteria. In one embodiment, the one or more prokaryotic cell is not from a species selected from the list consisting of: Escherichia coli; Pseudomonas aeruginosa; and Salmonella enterica.

In one embodiment, the one or more prokaryotic cell is not from the genus Bacillus, for example it is not from the species Bacillus subtilis.

In one embodiment, the method does not comprise determining endonucleolytic cleavage of the one or more 5’-monophosphorylated fragments of mRNA.

In one embodiment, the one or more 5’-monophosphorylaied fragments of mRNA do not arise from endonucleolytic cleavage of mRNA.

In a seventh aspect, the invention provides a kit of parts for any one of the methods as defined in the aspects of the inventions described herein, wherein the kit comprises instructions for any one of the methods as defined in the aspects of the inventions described herein

In one embodiment, the kit of part comprises one or more component from the list consisting of: one or more enzyme; one or more DNA oligonucleotide compatible sequencing platform; one or more RNA oligonucleotide compatible sequencing platform; one or more magnetic bead; one or more tube; and one or more pipette tip.

In one embodiment, the one or more enzyme is selected from the list consisting of: a T4 RNA ligase; a reverse transcriptase; a DNA polymerase (such as a themostable DNA polymerase); and a double-strand specific nuclease.

In one embodiment, the one or more magnetic bead is for polyethylene glycol (PEG) based DNA purification (for example, Agencourt Ampure XP and/or Magbio Highprep Beads).

It will be appreciated that the one or more DNA oligonucleotide compatible sequencing platform and one or more RNA oligonucleotide compatible sequencing platform can be selected from the high-throughput sequencing techniques and next-generation sequencing techniques described herein.

In an eight aspect, the invention provides a library of sequences obtained from and/or obtainable by any one of the methods of any one of the aspects of the invention. In a ninth aspect, the invention provides a use of one or more 5’-monophosphorylated fragment of mRNA for determining the identity of one or more rrsRNA sequence being translated in a prokaryotic cell, wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

The uses of the ninth aspect of the invention can comprise the features and/or steps and/or embodiments of any one of the aspects describing the methods of the invention.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The references discussed herein are incorporated in their entirety.

Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

The invention will now be described by reference to the following Figures and Examples. Preferred, non-limiting examples which embody certain aspects of the invention are described, with reference to the following figures:

Figure 1. 5'P mRNA sequencing can be used as a proxy for in vivo prokaryotic ribosome dynamics, a, Metacounts (5Pseq reads per Million) of 5’ mapping positions relative to translation initiation and termination codons of open reading frames (ORF’s) from Bacillus subtHis, [²⁰], Exponentially growing cells (No treatment, in solid line), Chloramphenicol treated (CAM, in dashed line) and randomly fragmented (in pointed line) are shown. Identified RNases are highlighted in the upper right corner (unidentified are in light grey). Original 5PSeq positions are reported (no P-site correction was applied), b, Fast Fourier Transform (FFT) for the observed periodicity, c, Relative 5PSeq frame protection for all codons

Figure 2. SPSeq reveals codon specific ribosome pauses patterns associated with stress and antibiotic treatment. Heatmaps for amino acid specific 5'P RNA coverage from dark grey (low) to light grey (high). Distance from specific amino acids is indicated as the number of nucleotides. Ribosomes paused at -14 indicates an A-site stall. Selected amino acids are shown also as line plots, a, Lactobacillus plantarum pauses after Chloramphenicol (CAM, dashed line) and Mupirocin (MUP, pointed line) treatment, b, Bacillus subtilis context specific Chloramphenicol pauses induced by second-to-last amino acid (of peptide-chain) shown at -8 position (CAM, dashed line), c, Lactobacillus plantarum pauses after stress treatment, d, Principal component analysis of ribosome protection phenotype (see methods) allows distinguishing stress and drug treatment.

Figure 3. 5PSeq enables the study of species- and codon-specific ribosome pauses in complex samples, a, Line plots showing 5PSeq metagene abundance in respect to Isioleucine (lie) codons for L reuteri (solid line) and L. plantarum (dotted line) mixed at different rations (from 1 :1 to 1 :10,000; L plantarum vs L. reuteri). Mupirocin treatment of L. plantarum (MUP, bottom) leads to clear lie pauses in respect to non-treated L reuteri (NT), b, 5PSeq analysis from frozen cell suspension from the ZymoBIOMICS Microbial Community Standard (intended for DNA analysis). Numbers of assigned RNA reads are marked in circles. Relative frame protection and Fast Fourier Transform (FFT) as in Fig.1.

Figure 4. Ribosome dynamic in vaginal microbiome samples, a, 5PSeq analysis from previously isolated vaginal microbiomes. Number of assigned reads to each species and patient are marked in circles. Relative frame protection and Fast Fourier Transform (FFT) as in Fig.1. b, Principal component analysis of ribosome protection phenotype across clusters of phylogenetically close species, c, Example of in vivo amino acid specific ribosome protection in vaginal microbiomes.

Figure 5. Co-translational mRNA decay is conserved across prokaryotes From inside to outside: Taxonomic tree of investigated species; inner circle which is predominantly light grey (number of assigned reads); darker grey bars extending from the inner circle (strength of 3-nt ribosome protection periodicity, protection frame preference (F0, F1 , F2); Presence of selected enzymes involved in RNA degradation at genus level (RNJA (Ribonuclease J1), RNJB (Ribonuclease J2), RNY (Ribonuclease Y), RNE (Ribonuclease E), RNAG (Ribonuclease G) and PNP (Polyribonucleotide nucleotidyltransferase). Overall, 46 genera present in samples from cultured bacteria and complex environments, including a Zymobiomics mixture, vaginal swabs, feces and compost. In complex samples, species with at least 1000 reads in the coding regions (300 in case of compost) were considered and respective genera were analyzed. See methods for details. Phyla depicted with sectors of different shade of grey and the first 2 letters (Fi, Pr, Ac, Ve, Cy, Fu, Te and Ba) and genera labelled with a number (1-16) and indicated in the figure.

Figure 6. 5'P 3-nt periodicity is associated with co-translation mRNA decay, a, Gene specific 3-nt protection for Bacillus subtilis as reported by fivepseq™. Each point corresponds to a gene and the proximity to the triangle boundaries (F0,F1 ,F2) their relative protection. Non- treated exponentially growing cells are shown in grey (labelled “No treatment”), polyribosome associated mRNA degradation intermediates in dark grey (labelled “Polysomes”), chloramphenicol treated cells in light grey (labelled “CAM”)and random fragmented (in most dark grey - labelled “Fragmented”), b, 5PSeq metagene analysis of Bacillus subtilis after polyribosome fraction isolation (As in Fig.1). Bar on the bottom of the agarose gel indicates fractions used for 5PSeq library generation.

Figure 7. Ribosome associated 3-nt periodicity can be found in multiple prokaryotic species. Metagene analysis for multiple species displaying metagene 5PSeq protection, Fast Fourier Transform (FFT), relative frame protection and identified RNases (as in Fig.1). a, Escherichia coli. b, Caulobacter crescentus. c, Lactobacillus plantarum. d, Lactobacillus reuteri, e, Bacillus amyloliquefaciens f, Synechocystis sp. PCC 6083.

Figure 8. Species- and codon-specific ribosome pauses in response to stress or antibiotic treatment. Line plots showing amino acid specific ribosome pauses as measured by 5PSeq. a, Isoleucine (lie) pause comparing no treatment (NT) and mupirocin treatment (MUP) in L. plantarum and L. reuteri, b, relative Alanine (Ala) and Serine (Ser) ribosome pause as measured by 5PSeq for multiple species in response to chloramphenicol treatment (CAM), c, relative Arginine (Arg) pauses in stationary phase growth for L. plantarum (27 hours) and B. subtilis (8 days).

Figure 9. Longer ribosome protected 5PSeq reads allows for better species-specific assignment in complex microbiomes. Shown are the relative number of uniquely mapped reads (scaled within each sample) as a function of read length.

Figure 10. Ribosome dynamic in fecal and compost microbiomes. a, 5PSeq analysis from fecal microbiomes. Number of assigned reads are marked in circles. Example of in vivo amino acid specific (Alanine) 3-nt ribosome protection periodicity for selected species, b, same for compost microbiome. c, Principal component analysis of ribosome protection phenotype across analyzed phyla (all the phyla from cultured and complex microbiome samples including 46 genera, with treatments).

Figure 11. Co-translational mRNA decay in species with relatively high coverage. From inside to outside: Taxonomic tree of investigated species; red bars (strength of 3-nt ribosome protection periodicity, protection frame preference (F0, F1 , F2); Presence of selected enzymes involved on RNA degradation at genus level (RNJA (Ribonuclease J1), RNJB (Ribonuclease J2), RNY (Ribonuclease Y), RNE (Ribonuclease E), RNAG (Ribonuclease G) and PNP (Polyribonucleotide nucleotidyltransferase)), with opacity indicating the fraction of species with identified enzyme in each genus. Overall, 11 genera including species with at least 100K reads in the coding regions, from samples of cultured bacteria and complex environments, including a Zymobiomics mixture, vaginal swabs, feces and compost are analyzed. See methods for details.

Example

Summary

The microbiome has revealed itself as a key player in health and disease¹. To better understand its role, in addition to microbial diversity, it is important to understand species- specific activity and gene expression. While metatranscriptomics investigates mRNA abundance², it does not inform about faster post-transcriptional regulation³. Although prokaryotic translation is a common target for antibiotics⁴, a direct measurement of microbiome ribosome dynamics remains inaccessible. Here we demonstrate that, contrary to expectation, co-translational mRNA degradation is common in prokaryotes, and that in vivo ribosome protection generates widespread 3-nt periodicity in 5^'P mRNA decay intermediates. Consequently, 5^'P sequencing allows the study of codon and gene specific ribosome stalling in response to stress and drug treatment at single nucleotide resolution. We validate its wide applicability by investigating in vivo species-specific ribosome footprints of clinical and environmental microbiomes and show that amino acid-specific ribosome protection patterns can be used to phenotype microbiome perturbations. Furthermore, we show that multiple RNase activities collaborate to generate in vivo ribosome footprints and that co-translational degradation is phylogenetically conserved across prokaryotes. This strategy opens the way for the study of the metatranslatome, and allows to investigate fast species-specific post- transcriptional responses to environmental and chemical perturbations in unculturable microbial communities.

Introduction

During the last decade the microbiome has revealed itself as a key player to understand health and disease^{1 5}. The variance of host and microbiome interactions translates into differential drug sensitivity and is associated with multiple disease states⁵·⁶. To understand how complex microbial communities interact between themselves and the host, it is necessary to understand not only its composition (metagenomics), but also how they respond to external stimuli. Metatranscriptomics, where microbial mRNA abundance is measured, is the current standard to investigate microbiome gene expression. However, it cannot inform about post- transcriptional regulation, which is essential to understand how produced mRNA translates into diverse phenotypes⁷. Metaproteomics is a useful approach, but it only offers a limited view of the species-specific proteomes⁸. Approaches like ribosome profiling have been crucial to understand genome-wide ribosome dynamics and posttranscriptional regulation⁹. However, this method cannot be directly applied to complex microbiome communities. Here we overcome these impediments by sequencing the 5^'monophosphorylated mRNA decay intermediates (5^'P) in microbial communities. We show that co-translational mRNA degradation is common among prokaryotes and that 5^'P RNA molecules serve as a good readout of ribosome dynamics in clinical and environmental microbiomes, opening thus the avenue for the study of the metatranslatome.

5^'-3^'co-translational mRNA degradation is common in prokaryotes. We and others have demonstrated that 5^'-3^'co-translational mRNA degradation is widespread in eukaryotic organisms, such as yeast¹⁰ and plants^11'13. In prokaryotes, mRNA degradation was primarily thought to initiate via endonucleolytic cleavage followed by 3^'-5^'degradation^14‘ ^1b. More recent studies have demonstrated that 5^'-3^'RNA exonucleolytic activity also occurs in prokaryotes. For example, ribonuclease J was identified in Bacillus subtiiis ¹⁷ and its homologues are present in many prokaryotes¹⁸. The direct interaction between the translation process and the 5^'-3^'exonucleolytic activity has nevertheless not been investigated experimentally across the prokaryotic tree of life. And thus, it is not well understood if 5^'- 3^'degradation is common among prokaryotes. To investigate its potential similarity to 5^'-3^'co- translational decay in eukaryotes^{10 19}, we studied the 5^'P mRNA degradome in cultured and complex bacterial communities using optimized 5PSeq¹⁰·^20'22 (Table 1 and 2).

Table 1 - Oligonucleotides used in this study. P1 and PE2 refer to oligos similar to the ones used for multiplexing and amplification of an lllumina library. oVP613 refers to the Human rRNA depletion mix, oVP670 to the rRNA depletion mix for Cultures and oVP676 to the rRNA depletion mix for Complex samples. lUPAC nucleotide codes are used to indicate oligonucleotides with degenerated bases; * refers to S-linkage between the two bases. Sequences are represented in 5’ to 3’orientation.

We first investigated the 5^'P degradome of open reading frames in B. subtilis (Fig. 1a). This revealed a clear 3-nt periodicity (Fig. 1b) as previously described for yeast¹⁰, with a clear 5^'P preference for the second nucleotide of each codon (F1) both at metagene and single gene levels (Fig. 1c and Fig 6). 5^'P degradation intermediates accumulate 11 and 14 nucleotides upstream of translation start and stop sites respectively, as expected from slow initiating and terminating ribosomes, analogous to the -14 nt from start and -17 nt from stop observed in budding yeast¹⁰·²². The smaller protection size can easily be explained by the known difference in ribosome size between eukaryotes and prokaryotes²³. To confirm the association of 5^'P decay intermediates to translating ribosomes, we performed 5PSeq in polyribosomal fractions of sucrose density gradients and observed similar patterns (Fig. 6b). Finally, to demonstrate its biological origin, we confirmed that in vitro fragmentation of the same RNA nearly eliminated the observed in vivo 3-nt periodicity and start/stop codon footprints (Fig. 1a).

To assess how widespread 5^'-3^'co-translational mRNA degradation is across prokaryotes, we investigated other species with and without predicted 5^'-3^' RNA exonucleolytic activity (Fig. 7)¹⁸. We investigated representative species from the phylum Firmicutes (Bacillus amyloloquefaciens, Lactobacillus plantarum, Lactobacillus reuteri), Cyanobacteria (Synechocystis sp PCC 6803) and Proteobacteria (Escherichia coli and Caulobacter crescentus). As expected, E. coli, lacking 5 ^'-3^' RNA exonuclease activity, failed to generate a clear ribosome associated 3-nt pattern (Fig. S2a), while a clear 3-nt pattern was observed in all other species (Fig. 7). In most cases we observed an accumulation of 5^'P reads at the second nucleotide (F1) and at 11 and 14 nt upstream of start and stop codons (Fig. 7), as in B. subtilis (Fig. 1a). In C. crescentus, although we detected a clear 3-nt pattern, we did not observe footprints associated with slow ribosomes at the start or stop codons (Fig. S2b). Interestingly, in Synechocystis, the ribosome protection pattern was displaced by one extra nucleotide (/.e. accumulation at -12 nt from start and -15 nt from stop) (Fig. S2f). This could be explained by a different ribosome protection size, conformation or presence of potential cofactors⁹²⁴.

To confirm the causative role of the ribosome shaping the distribution of 5^'P mRNA degradation intermediate, we perturbed ribosome dynamics using chloramphenicol (CAM), which is commonly used in prokaryotic ribosome profiling experiments²³. As expected from ribosomes stalled at elongation level, we observed a clear increase of the 3-nt ribosome protection pattern in B. subtilis (Fig.1), L plantarum, L reuteri and B. amyloliquefaciens (Fig. 7). We also observed a relative excess of 5^'P degradation intermediates in the 5^'regions of the open reading frames (ORFs) and a decrease of the protection at termination level, consistent with specific inhibition of translation elongation and not of initiation or termination²³²⁵. This 5^'P accumulation in the 5^'regions of the ORFs was also evident in E. coli which lacked clear 3-nt periodicity. All these demonstrate that ribosome protection commonly shapes the 5^'P mRNA degradome in prokaryotes.

5^'P serves as proxy for ribosome position and dynamics Once demonstrated that 5 ^'-3^' co-translational mRNA degradation is common among prokaryotes, we investigated up to what degree the abundance of 5^'P mRNA decay intermediates can serve as a proxy for codon-specific pauses and ribosome dynamics. By aligning 5^'P reads for each respective amino acid, we generated amino acid specific metagene profiles (Fig. 2 and Fig. 8). In L plantarum this showed a clear 5^'P accumulation associated with slow ribosomes at stop and Cysteine codons, likely associated with limited cysteine in the growth media (Fig. 2a). We then perturbed the translation process and investigated its consequences in 5^'P accumulation. First, we tested our ability to detect codon-specific pauses after 10 min treatment with Mupirocin (MUP). MUP is an antibiotic targeting isoleucyl t-RNA synthetase, and thus expected to stall ribosomes at isoleucine codons²³²⁵. As predicted, MUP treatment led to a clear accumulation of 5^'P reads 14 nucleotides upstream of isoleucine codons in L. plantarum and L reuteri (A-site stall, Fig. 2a and 8). This demonstrates the causal effect of ribosome position shaping the prokaryotic degradome. Then we investigated more subtle codon-specific pauses associated with 5 minutes CAM treatment in B. subtilis, L. plantarum, L reuteri and B. amyloliquefaciens (Fig. 2a, b, Fig. 8). In addition to a general inhibition of translation elongation, CAM also leads to context-specific accumulation of ribosomes²⁶. Using ribosome profiling it was previously shown in E. coll that CAM treatment led to artifactual pauses when Alanine (Ala), or less frequently Serine (Ser), Threonine or Glycine are positioned in the E site. Reassuringly, 5PSeq is also able to discover CAM induced ribosome stalling 8 nt upstream of Ala and Ser in the tested species (Fig. 2a, b and Fig. 8). This shows that previously described context-specific CAM pauses are conserved across prokaryotes.

Next, we set out to investigate environmentally regulated changes in ribosome position. We studied the 5^'P RNA degradation profiles under various stress conditions in B. subtilis and L plantarum (Fig. 2c, Fig. 8). We identified clear codon pauses in L plantarum at Tyrosine during heat shock and low nutrient conditions, and at Histidine and Arginine in stationary growth (Fig. 3c). As 5PSeq provides an easy overview of the ribosome-dependent accumulation of 5^'P degradation intermediates at all amino acid positions, we used this information to generate a general amino acid-specific “ribosome protection phenotype” for each sample (see Materials and Methods). Using this ribosome protection phenotype and principal component analysis (PCA), we could clearly separate control, stressed and drug treated samples (Fig. 2d). The first two principal components clearly separate cases of MUP and CAM treatments, stress conditions and time course measurements of stationary-phase B. subtilis (24 h, 48h and 8d) (Fig. 2e). This shows that codon-specific ribosome protection phenotypes can be leveraged to distinguish across drugs and stress conditions. In addition to global patterns, 5PSeq also provides information regarding gene-specific ribosome stalls. We measured the relative strength and 3-nt protection pattern across genes (gene-specific protection index, see methods for details). This provides a measure of the relative stalling of the last translating ribosome (i.e. comparing peaks and valleys) independent of mRNA abundance for each gene. Drug and stress treatments induce changes in the protection index of individual genes involved in bacterial responses. For example, salt stress induced decreased pausing in genes involved in cell wall organization and iron-sulfur cluster assembly, in agreement with previous proteome- level studies (Table 3)²⁷·²⁸. All these demonstrate that 5PSeq can inform about ribosome position and translation regulation during stress, while detecting fast (i.e. in minutes) phenotypical changes in the translation process in response to drugs. Table 3 - The list of genes with decreased ribosome pausing at the second nucleotide of each codon upon high salt stress in Bacillus subtilis (subsp. subtilis 168). It highlights the genes involved in cell wall organization and teichoic acid biosynthesis that are driving gene-set enrichment scores for those GO biological processes.

Notes on Table 3: Shown are genes with negative change in the second nucleotide (diff_F1) compared to untreated samples with q value less than 0.05. The topmost gene is involved in iron-sulfur cluster assembly. The genes involved in cell wall organization and teichoic acid biosynthesis (emboldened) are driving gene-set enrichment scores for those GO biological processes (GO:BP).

5PSeq allows the study of ribosome position in complex microbiome samples

Ribosome profiling, based on polyribosome fractionation followed by in vitro RNA footprinting and sequencing, is the current gold-standard for genome-wide measurement of ribosome positions²⁹. Despite its multiple advantages, ribosome profiling is not well-suited to the study of complex mixtures of species. Ribosome profiling can only be applied to samples from which polyribosome fractions can be cleanly isolated (i.e. culturable microorganisms or tissue samples) and the ribosome protection fragments it generates are generally short, 23-24nt²³³⁰. In contrast, 5PSeq investigates longer in vivo produced ribosome-protected fragments, which allows the distinction of closely related species within a complex sample (Fig. 9), does not require subcellular fractionation and can be applied to RNA previously isolated and stored for years¹⁰²⁰. As this would allow the investigation of ribosome positions in currently inaccessible microbiome samples (metatranslatome), we set to demonstrate the feasibility of this approach.

We first investigated 5^'P mRNA degradation intermediates in defined mixtures. We tested the ability of 5PSeq to detect species-specific perturbations in a mix of two closely related species, L reuteri and L plantarum (63-80% core nucleotide identity). We pooled different amounts of RNA from MUP-treated L. plantarum with untreated L reuteri (from 1 :1 to 1 :10000, Fig. 3a). Reassuringly, the described MUR associated Isoleucine pause can be clearly detected even at the 1 :10000 ratio (0.01% abundance). And this limit is simply driven by our sequencing depth (i.e. in the 1 :10000 mix only 82 reads were mapped to L. plantarum coding sequences). Once confirmed our ability to detect species-specific perturbations, we investigated the ribosome dynamics in more complex cellular mixtures. We set out to apply 5PSeq to RNA extracts of a defined microbial community standard for metagenomic analysis. We confirmed the existence of a clear 3-nt periodicity in Lactobacillus fermentum, Enterococcus faecalis, Staphylococcus aureus, Listeria monocytogenes and Bacillus subtilis from phylum Firmicutes, while it was absent in Pseudomonas aeruginosa, Escherichia coli and Salmonella enterica from phylum Proteobacteria (Fig. 3b). This demonstrates our ability to investigate ribosome dynamics in cells harvested for a different objective.

Metatranslatome analysis of clinical and environmental microbiomes

Having proven that 5PSeq is suitable for analysis of complex synthetic mixtures, we decided to investigate clinical and environmental microbiome samples. We first studied the metatranslatome of vaginal microbiomes using previously isolated RNA samples. In addition to RNA-based indication of species abundance, we obtained clear 3-nt profiles for most identified species (Fig. 4a). This data allows to investigate in vivo ribosome protection phenotype across species and patients. We can clearly observe sample clustering according to individual bacterial species, reflecting species-specific ribosome protection phenotypes (Fig. 4b). In addition to global profiles, we can also investigate species-specific in vivo ribosome footprints (Fig. 4). This opens the possibility to investigate species-specific ribosome dynamics in unculturable species, something that has not been possible until now.

Once demonstrated that 5PSeq can be applied to clinical microbiome samples, we expanded our study to more complex microbiomes³¹. We investigated the human fecal microbiome and identified clear 3-nt patterns in multiple species (e.g. across Firmicutes and Bacteroidetes) (Fig. S10a). Finally, we applied 5PSeq to investigate translational phenotypes of bacteria found in compost (Fig 10b), revealing the applicability of our method to environmental microbiomes. Similar to the findings of the vaginal microbiome, species-specific ribosome protection phenotypes of faeces and compost microbiomes show phyla-driven clustering (Fig 10c).

5PSeq can be used as proxy for ribosome position across many prokaryotes

Having investigated the metatranslatome of both cultured species, clinical and environmental microbiomes, we used the generated data to investigate the conservation of 5^'-3^'co- translational mRNA decay across the prokaryotic tree of life (Fig. 5 and Fig. 11). In total we obtained information for 84 species with relatively high coverage distributed across 46 genera (Table 4 and 5). We can observe a clear 3-nt periodicity across most genera (darker grey bars extending from the inner circle, in Figures 5 and 7). As previously described for Synechocystis (Fig. 7), different species present slightly different ribosome protection size. Protection in F1 is common across Firmicutes (e.g. Lactobacillus, Bacillus, Megasphaera), while protection in F2 is more common in Bacteroidetes (e.g. Parabacteroides, Alistipes, Bacteroides). This suggests that the size of the ribosome-protected footprint is similar across phylogenetically related species. Additionally, many species present more complex protection patterns. For example, Ureaplasma (Tenericutes), Akkermansia (Verucomicrobia) present a dual protection pattern in F1 and F2, while Caulobacter (Proteobacteria) and Prevotella (Bacteroidetes) in F1 and F0. However, it is important to note that ribosome-protected fragment size can also be influenced by environmental conditions or drug treatments⁹²⁴. Finally, as expected, a few species for which we do have good sequencing coverage (inner grey circle), such as E. coli (representative of class Gammaproteobacteria), do not present clear evidences of 5-3^'co-translational decay (Fig. 5).

Table 4 - All 5804 genomes: describes the list of 5804 prokaryotic genome assemblies retrieved from NCBI and used for alignment. Status of the genome as of January, 2020

Finally, we investigated the observed 5^'P mRNA degradome profiles in relation to the phylogenetic conservation of the prokaryotic mRNA degradation machinery (outer circle in Fig. 5) focusing on species with >100K reads (Fig. 11)³²·³³. We observed agreement of 3-nt periodicity and frame preference patterns with taxonomic classification. Genera containing the 5-3^'exonuclease Ribonuclease J, (coloured as indicated in the key of Fig. 11), such as Bacillus, Lactobacillus and Caulobacter, mostly present clear periodicities. While species more dependent on RNase E and G for mRNA decay (coloured as indicated in the key of Fig. 11) such as E. coli (Gammaproteobacteria) present little 3-nt periodicity. Interestingly many species without conserved RNase J, but with endonucleases RNase Y (RNY), RNase E/G (RNE, RNG) and 3-5^'exonuclease PNPase (PNP), like Bacteroidetes, also present clear 3-nt periodicity, albeit with a different ribosome protection pattern. All these suggest that in addition to the canonical 5^'-3^'RNA exonuclease activity, other prokaryotic nuclease activities are also able to shape the degradome in respect to the translation process.

Discussion

Here we have shown that co-translation mRNA degradation is widespread among prokaryotes and that measurement of 5^'P mRNA decay intermediates enables the study of ribosome dynamics. We have demonstrated the ability of 5PSeq to detect in vivo codon- and gene- specific ribosome dynamics without the need for drug treatment, subcellular fractionation or in vitro RNA degradation. We confirmed that it can easily detect environmentally triggered translational regulation in multiple species, both at codon- (Fig. 2) and gene-specific levels (Table 3). We also demonstrate that 5PSeq can easily detect fast (e.g. within 5 minutes) and specific perturbations of the translational process induced by drug treatments (Fig. 2). This opens new avenues to study environmental or chemical regulation of the translational process without the need for culture or subcellular fractionation. Additionally, studying faster post- transcriptional regulation may help to disentangle direct from indirect secondary effects. We show that 5PSeq can be widely applied to complex clinical and environmental microbiomes to obtain species-specific metatranslatome profiles. By eliminating the need for complex experimental protocols and bacterial culturing, we can now investigate ribosome protection phenotypes in uncultivable bacterial species, which are estimated to comprise more than half of the human bacterial communities³⁴. More importantly, we can investigate it without the need for treatments that might alter cellular physiology or other biological processes. We show that our approach can detect translational responses even in species present at 0.01% abundance in a bacterial community, and by using longer ribosome footprint reads we can analyze complex bacterial communities at single-species resolution. 5PSeq will allow for studies of post-transcriptional gene expression regulation in microbiomes, and will complement current multi-om/cs approaches. As changes in ribosome position do not require variations in mRNA accumulation, we expect 5PSeq to be able to identify changes faster than metatranscriptomic approaches, as we describe for CAM. Complete understanding of all molecular mechanisms shaping co-translational mRNA degradation across the prokaryotic tree of life will require extensive mechanistic dissection. However, our results reveal that mRNA degradation pathways involving the exonuclease RNase J or the endonuclease RNase G (preferentially in absence of RNase E) can shape 5^'P mRNA degradation intermediates in respect to ribosome position (Fig. 5). We expect that in the future metatranslatome analysis will enable the study of the unexplored post-transcriptional regulation in microbiome communities. This will bring new insights on how microbial communities interact with each other and the host, and respond to drugs and environmental challenges.

Methods and methods

Bacterial growth and microbiome samples.

Overnight bacterial Cultures (not exceeding ODeoo 1) were used to dilute main culture to a starting ODeoo of 0.03- 0.05. Culture was harvested by centrifugation when reached the logarithmic phase (ODeoo 0.4- 0.8) unless indicated otherwise. If not stated differently, bacterial cultures were grown at 37°C and rotating, using the recommended growth Media.

In detail, Lactobacillus plantarum (ATCC 8014) and Lactobacillus reuteri (DSM 17938) were grown in MRS Broth (Sigma-Aldrich). L plantarum stress treatments were carried out as follows: Stationary-phase cultures were grown for 27 hours post inoculation and harvested at ODeoo ~4.5. To generate samples for untreated control, heat shock and low nutrient, biological replicate cultures (40ml) were grown to mid-log phase (ODeoo 0.3-0.6), split (10ml for untreated control, 15ml for heat shock and 15ml of low nutrient sample) and cells were harvested by centrifugation. Untreated control pellet was flash frozen immediately after for RNA analysis. Prior to heat shock, cells were resuspended in prewarmed MRS broth and incubated in Thermomixer for 15 minutes at 60°C. Low nutrient cell pellets were washed excessively with 50ml 0.5xLB media, centrifuged and supernatant was completely removed. Cells were then resuspended in prewarmed O.SxLB media (Sigma) and harvested after a total incubation time of 15 minutes at 37°C.

Bacillus subtilis (strain 168 trpC2) was cultured in 2xYT (1.6% (wt/vol) Tryptone (Bacto), 1% (wt/vol) Yeast extract (Bacto) and 0.5% (wt/vol) NaCI ) For extended growth, we collected samples at mid-log, 24 hours, 48 hours and eight days post inoculation. Salt stress was performed by mixing equal Volume of 2M NaCI with mid-log phase grown B. subtilis (ODeoo 0.5-0.6) followed by 10 minutes incubation at room temperature and harvest by centrifugation. Bacillus amyloliquefaciens and Escherichia coli strain DH5a (Invitrogen, Cat. No. 18265-017) were grown in LB Media. Biological Replicate Cultures of L. plantarum (ATCC 8014), Lreuteri (DSM 17938), E.coli (DH5a Invitrogen) and Bacillus amyloliquefaciens were grown to log phase and split to generate samples for untreated control, random fragmented control, Chloramphenicol(CAM) and Mupirocin (Mup) (only for Lplantarum (ATCC 8014) and L.reuteri (DSM 17938)). Chloramphenicol (CAM) was added to mid-log phase grown cultures at a final concentration of 100μg/ml, incubated for 5 minutes at 37 ^°C and subsequent harvested on ice containing additional 100μg/ml CAM ¹⁰. Mupirocin treatment (final 65pg/m MUP, Sigma- Aldrich) of mid-log L. plantarum and L.reuteri was carried out for 10 minutes at 37 ^°C following centrifugation and flash freezing of pellet. Caulobacter crescentus (strain NA1000) was grown in PYE Media containing 0.2% (wt/vol) peptone (Bacto), 0.1% yeast extract (Bacto) at 30 ^°C to mid-log, followed by centrifugation and flash freezing of cell pellet. Synechocystis strain PCC6083 was cultured in BG11 growth Media at 30 ^°C with a light intensity of 30 mE and 1% of atmospheric C02and harvested at mid-log phase³⁵.

RNA extraction

RNA was extracted (if not stated otherwise) as described in³⁶ with minor modifications In brief, cell pellets were resuspended in equal volume of LET (25 mM Tris pH 8.0, 100 mM LiCI, 20 mM EDTA) and water saturated Phenol pH 6.6 (Thermo Fisher). Cells were lysed with acid washed glass beads (Sigma-Aldrich) by vortexing for three minutes in MultiMixer. Following the addition of equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water, lysis was extended by additional two-minute vortexing followed by centrifugation. Resulting aqueous phase was purified in two steps using phenol-chloroform isoamyl alcohol (25:24:1) followed by chloroform. After centrifugation, the clean aqueous phase was precipitated with sodium acetate-ethanol. For Lactobacillus mixtures (Fig3A), Microbial RNA extracted from L. plantarum (Untreated and Mupirocin treated) were mixed prior to RNA ligation step of 5PSeq Library protocol at different ratios with RNA extracted of L.reuteri (Untreated).

Technical replicates of the Microbial Community Standard (Zymobiomics Cat#D6300, Lot# ZRC 190633) consisting of eight deactivated bacterial strains, were generated by extracting RNA from 75-125mI thawed cell suspension.

Vaginal swab samples were mechanically lysed using beads in 10OOmI of DNA/RNA shield (ZymoResearch) and lysate was stored at -80°C for 2 months before use. Lysate was thawed and 250mI was used to extract microbial RNA.

Feces from a healthy donor was collected and transported in 40% Glycerol. Technical replicates of RNA were extracted on the same day as stated earlier with minor modifications listed as follows. In brief, 500mI Feces-Glycerol suspension was mixed with equal volume of LET Buffer containing SDS (25 mM Tris pH 8.0, 100 mM LiCI, 20 mM EDTA, 10% SDS) and water saturated Phenol pH 6.6 (Thermo Fisher). Lyses was done by vortexing and carbide beads. Lyses duration was extended to 10 minutes after the addition of equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water. All subsequent steps were performed as already indicated.

Compost RNA was extracted with 2g starting material (from Sundbyberg, Sweden) using RNeasy PowerSoil Total RNA Kit (Qiagen) as recommended in manufacturer’s guidelines.

For all extractions, quality of RNA was assessed by either loading 1 pg of total RNA on a 1.2% Agarose Gel or 12ng on a BioAnalyzer using an RNA Nano 6000 chip (Agilent Technologies).

Polyribosome fractionation

Was performed as described in Huch et aF with minor modifications. In brief, B. subtilis (168trpC2) was cultured in LB Media to mid-log phase at 37 ^°C and harvested on ice, containing 100μg/ml Chloramphenicol, by five-minute centrifugation. Resulting pellet was lysed in 1xTN (50mM TRIS/HCI pH 7.4,150mM NaCI, 1mM DTT, 100μg/ml CAM and Complete EDTA Free Protease inhibitor tablet) using glass beads and vortexing for 2 minutes, following a 5 min incubation on ice. Lysis and incubation procedure was repeated twice. Cell debris was cleared by centrifugation at 1500g for 5 minutes at 4°C and supernatant was loaded on to a 15-50% sucrose gradient having an 80% cushion. After ultracentrifugation at 36,000 rpm for 90 minutes Abs₂₅₄ was monitored and fractionated. Subsequently, RNA was extracted from sucrose fractions by adding equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water followed by two-minute vortexing and centrifugation. Aqueous phase was further cleaned up by the addition of Chloroform, vortexing and centrifugation. Resulting aqueous phase was sodium acetate-ethanol precipitated.

Preparation of 5P Sequencing Libraries

5PSeq libraries were prepared as previously described²⁰ with minor modifications using 150- 9000ng total RNA as an input. To prepare random fragmented samples (negative controls), ribosomal RNA was depleted from DNA-free RNA and subsequent fragmented by incubating five minutes at 80 ^°C in fragmentation buffer (40mM Tris Acetate pH 8.1 , 100mM KOAc and 30mM MgOAc). Reaction was purified using 2 volumes of RNACleanXP beads (Beckman Coulter) as recommended by the manufacturer. Free 5ΌH sites were re-phosphorylated using 5 Units of T4 Polynucleotide kinase (PNK, NEB) and incubated at 37 ^°C for 60 minutes as recommended by the manufacturer. Re-phosphorylated fragmented RNA was purified using Phenol:Chloroform: Isoamyl Alcohol (24:25:1), followed by sodium acetate-ethanol precipitation. From this step forward, procedures for random fragmented and standard 5PSeq library preparation merge²⁰. RNAwas Ligated to either rP5_RND or rP5_RNA oligo (specified in Table 2) containing unique molecular identifiers. Ribosomal RNAwas depleted using Ribozero rRNA removal kit (lllumina) suitable for Bacteria, Yeast and Human samples. Ribosomal RNA depleted sample was purified using 1 8V of Ampure beads (Abeam) and fragmented with heat (80°C) for 5 min in 5x Fragmentation Buffer (200mM Tris Acetate pH 8.1 ,500mM KOAc,150mM MgOAc). Subsequent samples were reverse transcribed using random hexamers to prime. Resulting cDNA was bound to streptavidin beads (M-280), subjected to enzymatic reactions of DNA end repair, fill-in of adenine to 5’ protruding ends of DNA fragments using Klenow Fragment (NEB). Common adaptor (P7-MPX) was ligated and 5PSeq Libraries were amplified by PCR (15-17 cycles), purified using 1.8V of Ampure beads (Abeam) and quantified using Qubit (Thermo Fisher. Library size was estimated from bioanalyzer traces. 5PSeq Libraries were pooled by mixing equal amounts of each sample, following enrichment of 300-500nt size fragments.

HT-5PSeq Libraries were generated as recently described²². In brief, DNA- free RNA was ligated with RNA oligos containing unique molecular identifiers. Ligated RNA was reverse transcribed priming with oligos containing a random hexamer and an lllumina compatible region. RNA was eliminated by addition of NaOH. Ribosomal RNA was depleted by adding in- house rRNA DNA oligo depletion mixes (Table 1) to the cDNA and performing a duplex-specific nuclease (DSN, Evrogen) treatment. rRNA depleted cDNA was PCR amplified (15-17 cycles). Depletion of ribosomal RNA with Ribozero lllumina (for bacteria and yeast) was done after the single-stranded RNA ligation step. Ribosomal depleted RNA was purified and reverse transcribed using the same oligos as stated above, and then amplified by PCR. Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequence using a NextSeqSOO lllumina sequencer.

Data availability

Sequencing data is deposited on GEO under accession number GSE153497. Clinical sample information will be deposited in dbGaP under access control.

Sequence data pre-processing and mapping

Demultiplexing and fastq generation of sequencing bcl image files was performed using bcl2fastq (version 2) with default options. Adapter and quality trimming was performed with the bbduk program of the BBTools suite (https://sourceforqe.net/proiects/bbmapA, with options {qtrim=r, ktrim=r, hdist=3, hdist2=2, K=20, mink=14, trimq=16, minlen=30, maq=16}, using BBTools default adapter set and polyG or polyA sequences for short reads. In order to reduce computational time, reads with both identical unique molecular identifier (UMI) and insert sequence were de-duplicated prior to mapping using the dedupe program of the BBTools suite with its default parameters. UMI sequences found in the first eight bases of each read were extracted using UMI-tools (version 1) with default options (using --bc-pattern NNNNNNNN).

Bacterial genomes were downloaded from the National Center for Biotechnology Information Assembly database (https://www.ncbi. nlm.nih.gov/assemblv/) with the search terms: "bacteria"[Filter] AND (latest[fi Iter] AND ("representative genome"[filter] OR "reference genome"[filter]) AND (a 11 [filter] NOT "derived from surveillance project"[filter] AND all[fi Iter] NOT anomalous[filter])) on March 21^st, 2019. The list was further filtered to include only one strain per species, giving priority to genomes marked as “reference”. The resulting 5804 genomes (Table 4) were used to build the reference index. The index was built with the bbmap program of the BBTools suite, with default options (and k=10). Besides the reference index containing the 5804 bacterial genomes, separate indexes were built for individually cultured species and genus-level groups. The genomes for the latter groups were chosen from the initial set of 5804 genomes. Alignment was performed with the bbmap program of the BBTools suite, with the {32bit=t -da -eoom k=11 strictmaxindel=10 intronlen=0 t=16 trd=t minid=0.94 nzo=t} parameters. Alignment files were sorted and indexed with SAMtools “Deduplication based on UMIs was then performed with UMI-tools (version 1)³⁹ with options { -soft-clip-threshold 1 - edit-distance 2 -method unique}. The BAM files were then processed to count the number of reads in each species. We have used a pre-stored dictionary of chromosome and species names and used a custom script to perform counts in each species. Distribution of counts between genes coding for rRNA, tRNA, mRNA and other RNA types was computed with bedtools [https://bedtools.readthedocs.io/en/latest/ ]. The counts at mRNA coding genes were used to select top species in the complex samples as described below.

Individually cultured species were directly mapped to their reference indexes. All Zymobiomics mixtures, vaginal, fecal and compost microbiome samples were aligned to the bacterial reference index including 5804 species. We chose species with at least 1000 reads in the coding regions in all the samples except for compost, where we relaxed the selection to 300 reads, as there were less species with high counts. In total, 83 bacterial species with specified coverage belonging to 46 genuses were identified in all the samples. Reference indexes were built for those 46 genera (species were chosen from the preselected list of 5804), and all the complex fecal and compost samples were separately aligned to those references.

Fivepseq and ribosome dynamics analysis

Deduplicated alignment files, along with genome sequence and annotation files, were provided as input to our recently developed fivepseq package ²¹ for analysis and visualization of 5’ endpoint distribution of reads with default options applied. Fivepseq provides information regarding presence of 3-nt periodicity (FFT, Fast Fourier transform), distribution of 5’ counts relative to CDS start/stop or to nucleotides within each codon (translational frames), and codon and amino acid specific protection patterns. Fivepseq analyzes only one genome per run. Thus, alignment files for complex samples were used as input for fivepseq for each genome separately. For genus-level analysis, sequence and annotation files for individual species were concatenated into one.

To generate a "ribosome protection phenotype" we took the sum of counts positioned 30 to 1 nucleotide upstream of each amino acid and concatenated the per amino acid scaled counts to obtain a vector that describes ribosome protection in each sample. These vectors were used as input for principal component analysis (RCA) performed with the prcomp function of the R package stats (v 3.6.1). The PCA plots were generated with the autoplotly package (vO.1.2) in R.

In addition to the global pattern of ribosome pausing in either of the nucleotides in each codon (F0, F1 or F2), we have also computed gene-specific changes in the protection index upon different treatments. For this, we considered the counts in each reading frame, excluding the first two and the last codon of each transcript. A likelihood ratio test of independence was used to estimate differences in frame preference in each gene in untreated and treated conditions. The p- values were derived from X distribution of the log-likelihood statistic. Multiple testing correction was performed with Benjamini-Hochberg procedure (implemented in statsmodels package vO.11.1)⁴⁰. The effect size was reported using Cramer’s V metric, adjusted for directionality of the change. The adjusted effect size metric was used as input for gene set enrichment analysis (via Webgestalt (http://www.webqestalt.org I) along with reference functional annotation set obtained from the Uniprot database (https://www.uniprot.orq/).

Taxonomic analysis

Taxonomic trees were generated with the graphlan tool (vO.9.7) (https://huttenhower.sph.harvard.edu/qraphlan). The taxonomic lineage information for all the 84 bacterial species identified in our samples was downloaded from the NCBI Taxonomy database with the efetch program from NCBI e-utilities. Trees were annotated with information about the library size, 3-nt periodicity, preferred ribosome protection frame and presence of enzyme annotations for each genus. The library size equaled the maximum number of rrsRNA reads per species per sample, brought to the range of 0 to 1 (>1M reads) reads.

The 3-nt periodicity was computed taking into account the absolute value of Fast Fourier transform (FFT) signal for the 3-nt periodicity wave and the preference for ribosome protection frame, as computed by the fivepseq package. For FFT, the maximum of the signals for transcripts aligned either at the start or at the end was taken. The preference for ribosome protection frame was assessed based on the value of frame protection index (FPI), computed by the fivepseq package as 2 - ¾ , for each frame F,. The frame with maximum absolute FPI value was regarded as (mis) preferred, and the significance of the preference was assessed based on t-test p value comparing counts in the given frame with the other two combined (the FPI and p values are found in the frame_stats.txt file of the fivepseq output). A positive FPI value means that one of the nucleotides in each codon on average has higher counts (is preferred), while a negative FPI value means that one of the nucleotides on average has low counts (is misprefered), while the other two nucleotides receive more counts. E.g. if Fi is preferred, it will have positive FPI value and will be highlighted in the tree as a single preferred frame of protection, while if say F₂ is (mis)preferred (has a negative FPI value), the tree will highlight F₀ and Fi as the frames of preference. The FFT and FPI values were brought to the range of 0 to 1 , and the maximum of the two values was taken to describe the strength of 3nt-periodicity.

Enzyme annotations were obtained from the EggNOG database (v5.0)³².The presence of each enzyme in each genus was counted as a number between 0 and 1 , depending on the fraction of species within the genus annotated with the enzyme. The tree highlights these values with corresponding opacity.

References for the Example and figure legends

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Additional definition of the present invention

The invention is additionally defined by the following numbered paragraphs:

1. A method for determining the identity of one or more mRNA sequence being translated in a prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the identity of one or more mRNA sequence being translated in the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

2. A method for determining the presence and/or identity of one or more prokaryotic cell in a sample, comprising the steps of:

(i) providing a sample;

3. The method of Paragraph 2, wherein the method comprises determining the presence and/or identity of two or more prokaryotic cells in the sample, wherein the method further comprises the step of: (vi) identifying, from the information in step (iii), whether the two or more prokaryotic cells belong to the same genus and/or species and/or strain or two or more different genus and/or species and/or strain.

4. A method for the diagnosis and/or prognosis of a condition in a patient, comprising the steps of:

(i) providing a sample from the patient;

5. The method of Paragraph 4, further comprising the step of:

(v) from the diagnosis and/or prognosis in step (iv), treating the condition.

6. A method for determining the effect of an agent on the mRNA sequences being translated in a prokaryotic cell, comprising the steps of:

(i) contacting the agent with one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the effect of the agent on the mRNA sequences being translated in the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

7. The method of Paragraph 6, wherein: step (i) further comprises providing one or more prokaryotic cell which has not been contacted with the agent; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not contacted with the agent, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) comprises determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) for the one or more prokaryotic cell contacted with the agent with the sequence information in step (ii) for the one or more prokaryotic cell not contact with the agent.

8. The method of Paragraph 6 or 7, wherein step (iii) comprises determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) with a control for the effect of the agent on the one or more prokaryotic cell.

9. A method for determining the physiological status of one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the physiological status of the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs. 10. The method of Paragraph 9, wherein step (iii) comprises determining the physiological status by comparing the sequence information in step (ii) with a control for the physiological status of the one or more prokaryotic cell.

11. A method for determining the effect of an environmental condition on one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the effect of an environmental condition on the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs.

12. The method of Paragraph 11 , wherein step (i) further comprises providing the one or more prokaryotic cell in the environmental condition or isolated from the environmental condition.

13. The method of Paragraph 11 or 12, wherein the environmental condition is one or more selected from the list consisting of: a mixture comprising about two or more prokaryotic species, including the one or more prokaryotic cell; a mixture comprising the one or more prokaryotic cell and one or more eukaryotic cells; a mixture comprising the one or more prokaryotic cell and one or more virus; a mixture comprising the one or more prokaryotic cell and one or more prions; a sample; heat; cold; a temperature range; a pH; a pH range; radiation; a level of radiation; a level of a contaminant; a change in nutrition; stationary phase; and a level of one or more heavy metal.

14. The method of any one of Paragraphs 1 or 4-10, wherein in step (i) the one or more prokaryotic cell is in a sample.

15. The method of any one of Paragraphs 2, 3, 13 or 14, wherein the sample is one or more selected from the list consisting of: a sample from a patient; an environmental sample; and a laboratory sample. 16. The method of any one of Paragraphs 4, 5 or 15, wherein the sample from the patient is one or more selected from the list consisting of: a biopsy; skin; a skin swab; mucus; vomit; faeces; blood; tissue; urine; sweat; bodily fluid; semen; vaginal discharge; a vaginal swab; a mouth swab; buccal cells; a buccal swab; an eye swab; an ear swab; a nose swab; a sample from under one or more nail; a gastric washing; oral fluid; a throat swab; a wound swab; a cough swab; a sinus washout; and saliva.

17. The method of Paragraph 15, wherein the environmental sample is one or more selected from the list consisting of: water; fresh water; salt water; tap water; drinking water; a liquid; a plant; soil; compost; a sample from a bioreactor; a sample from a landfill site; fertiliser; a swab or sample from a non-patient surface; a non-patient swab; and a food stuff.

18. The method of any one of the preceding paragraphs, wherein mRNA is being degraded by 5’-3’ co-translational degradation.

19. The method of any one of the preceding paragraphs, wherein the co-translational degradation of mRNA is exonucleolytic co-translational degradation.

20. The method of any one of the preceding paragraphs, wherein the co-translational degradation of mRNA is not mediated by 5’ to 3’ decay.

21. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell comprises one or more exonuclease comprising 5’ to 3’ exonuclease activity.

22. The method of any one of the preceding paragraphs, wherein the co-translational degradation of mRNA is mediated by one or more exonuclease comprising 5’ to 3’ exonuclease activity.

23. The method of Paragraph 21 or 22, wherein the exonuclease is an exonuclease stimulated by 5'-monophosphate.

24. The method of any one of Paragraphs 21-23, wherein the exonuclease is one or more selected from the list consisting of: an RNase J; an analogue of RNase J; a homologue of RNase J; a paralog of RNase J; an orthologue of RNase J; an analogue of XRN1 ; a homologue of XRN 1 ; a paralog of XRN1 ; an orthologue of XRN1 ; an analogue of XRN2; a homologue of XRN2; a paralog of XRN2; and an orthologue of XRN2. 25. The method of Paragraph 24, wherein RNase J is RNase J1 and/or RNase J2.

26. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell are at least two prokaryotic cells, and wherein the at least two prokaryotic cells belong to the same genus and/or species or two or more different genus and/or species.

27. The method of any one of the preceding paragraphs, further comprising step (i)(a) performed after step (i), wherein step (i)(a) comprises subjecting the one or more prokaryotic cell with an agent and/or environmental condition that modifies ribosome function.

28. The method of Paragraph 27, wherein: step (i) further comprises providing one or more prokaryotic cell, wherein the one or more prokaryotic cell has not been subjected to the agent and/or environmental condition that modifies ribosome function; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not subjected to the agent and/or environmental condition that modifies ribosome function, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) further comprises providing the determination by comparing the sequence information in step (ii) for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function with the sequence information in step (ii) for the one or more prokaryotic cell not subjected to agent and/or environmental condition that modifies ribosome function.

29. The method of Paragraph 27 or 28, wherein step (iii) further comprises providing the determination by comparing the sequence information in step (ii) with a known standard for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function.

30. The method of any one of Paragraphs 27-29, wherein the agent that modifies ribosome function is an antibiotic.

31. The method of Paragraph 30, wherein the antibiotic is one or more antibiotic selected from the list consisting of: mupirocin; and chloramphenicol. 32. The method of Paragraph 30, wherein the environmental condition is as defined in Paragraph 13.

33. The method of any one of the preceding paragraphs, further comprising step (i)(b) performed after step (i), wherein step (i)(b) comprises isolating, from the one or more prokaryotic cell, one or more 5’-monophosphorylaied fragment of mRNA.

34. The method of Paragraph 33, wherein the isolating comprises purifying, from the one or more prokaryotic cell, one or more 5’-monophosphorylated fragment of mRNA.

35. The method of Paragraph 34, wherein the purifying comprises purifying the 5’- monophosphorylated fragment of mRNA from one or more of the following: proteins; lipids; small molecule metabolites; and salts.

36. The method of any one of the preceding paragraphs, further comprising step (i)(c) performed after step (i), wherein step (i)(c) comprises isolating the 5’-monophosphorylated fragments of mRNA from non-5’-monophosphorylated RNA.

37. The method of Paragraph 36, wherein the non-5’-monophosphorylated RNA is one or more RNA selected from the list consisting of: ribosomal RNA (rRNA); transfer RNA (tRNA); microRNA (miRNA); and small RNA.

38. The method of Paragraph 37, wherein isolating the 5’-monophosphorylated fragments of mRNA from rRNA comprises rRNA hybridisation.

39. The method of any one of Paragraphs 33-38, isolating the 5’-monophosphorylated fragments of mRNA from rRNA and/or tRNA comprises selective degradation of the rRNA and/or tRNA.

40. The method of any one of the preceding paragraphs, further comprising step (i)(d) performed after step (i), wherein step (i)(d) comprises producing one or more complementary DNAs (cDNAs) from the one or more 5’-monophosphorylated fragments of mRNA, for example by reverse transcription.

41. The method of Paragraph 40, wherein the determination in step (iii) is from the sequence information of the cDNA. 42. The method of any one of the preceding paragraphs, further comprising step (i)(e) performed after step (i), wherein step (i)(e) comprises adding to the one or more 5’- monophosphorylated fragments of mRNA two of more oligonucleotides that comprise a sequence identifier.

43. The method of Paragraph 42, wherein each of the two or more oligonucleotides further comprises a same sequence of nucleotides.

44. The method of Paragraph 42 and 43, wherein the adding comprises ligating the oligonucleotides to the one or more 5’-monophosphorylated fragments of mRNA.

45. The method of Paragraph 44, wherein the ligating is mediated by a ligase, such as an RNA ligase, preferably T4 RNA ligase.

46. The method of any one of Paragraphs 43-45, wherein the oligonucleotide comprises a moiety for isolating the 5’-monophosphorylated fragments of mRNA, optionally wherein the moiety comprises biotin.

47. The method of any one of the preceding paragraphs, further comprising step (i)(f) performed after step (i), wherein step (i)(f) comprises removing the 5’-monophosphate from the 5’-monophosphorylated fragments of mRNA.

48. The method of any one of the preceding paragraphs, further comprising step (i)(g) performed after step (i), wherein step (i)(g) comprises cutting the 5’-monophosphorylated fragments of mRNA to form one or more shorter 5’-monophosphorylated fragments of mRNA.

49. The method of Paragraph 48, further comprising step (i)(h) performed after step (i) (g), wherein step (i)(h) comprises phosphorylating the one or more shorter 5’-monophosphorylated fragments of mRNA.

50. The method of Paragraph 49, wherein the phosphorylating of the one or more shorter 5’- monophosphorylated fragments is mediated by a kinase, such as polynucleotide kinase (PNK).

51. The method of any one of the preceding paragraphs, wherein the determination of the sequence of the one or more 5’-monophosphorylated fragments of mRNA in step (ii) comprises using polymerase chain reaction and/or high-throughput sequencing and/or next-generation sequencing. 52. The method of any one of the preceding paragraphs, further comprising step (ii)(a) performed after step (ii), wherein step (ii) (a) comprises determining the position of a ribosome on the sequence of the one or more 5’-monophosphorylated fragments of mRNA.

53. The method of Paragraph 52, wherein the ribosome is the most 5’ molecule on the sequence.

54. The method of Paragraph 52, wherein the ribosome is the most 3’ molecule on the sequence.

55. The method of any one of the preceding paragraphs, further comprising step (ii)(b) performed after step (ii), wherein step (ii)(b) comprises determining the relative positions in the sequence of one of more of the following RNA structures: the 5’P; one or more codons; one or more single nucleotide polymorphism (SNP); the 5’ untranslated region; the 3’ untranslated region; RNA binding protein binding sites; RNA secondary structure (such as RNA hairpins); RNA modifications (such as alternative or chemically modified RNA bases).

56. The method of Paragraph 55, wherein the determination in step (iii) comprises analysing the relative positions of the one of more RNA structures.

57. The method of any one of the preceding paragraphs, further comprising step (ii)(c) performed after step (ii), wherein step (ii)(c) comprises identifying a three-nucleotide periodicity in the one or more 5’-monophosphorylated fragments of mRNA.

58. The method of Paragraph 57, wherein the determination in step (iii) comprises analysing the three-nucleotide periodicity.

59. The method of any one of the preceding paragraphs, further comprising step (ii) (d) performed after step (ii), wherein step (ii) (d) comprises comparing the sequence of the one or more 5’-monophosphorylated fragments of mRNA with known prokaryotic mRNA sequences.

60. The method of Paragraph 59, wherein the determination in step (iii) comprises comparing the sequence of the one or more 5’-monophosphorylated fragments of mRNA with the known prokaryotic mRNA sequences. 61. The method of any one of the preceding paragraphs, wherein the 5’-monophosphorylated fragments of mRNA are single-stranded 5’-monophosphorylated fragments of mRNA.

62. The method of any one of the preceding paragraphs, wherein the 5’-monophosphorylated fragments of mRNA are about 9 or more nucleotides in length, preferably about 15 or more nucleotides.

63. The method of any one of the preceding paragraphs, wherein the 5’-monophosphorylated fragments of mRNA are about 9 nucleotides to about 15,000 nucleotides in length, preferably about 15 nucleotides to about 15,000 nucleotides in length, most preferably about 300 nucleotides to about 400 nucleotides in length.

64. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell is one or more bacterial cell.

65. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a phylum selected from the list consisting of: proteobacteria; firmicutes; actinobacteria; cyanobacteria; tenericutes; spirochaetales; chloroflexi; synergystetes; deinococcus-thermus; fusobacteria; and fibrobacteres.

66. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a class selected from the list consisting of: alphaproteobacteria; deltaproteobacteria; epsilonproteobacteria; gammaproteobacterial; bacilli; and Clostridia.

67. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from an order selected from the list consisting of: bacillales; and lactobacillales.

68. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell in which 5’-3’ co-translational degradation of mRNA occurs is from a species selected from the list consisting of: Bacillus subtilis; Akkermansia muciniphila; Alistipes finegoldii; Alistipes obesi; Alistipes putredinis; Alistipes shahii; Atopobium vaginae; Bacillus amyloliquefaciens; Bacteroides caccae; Bacteroides cellulosilyticus; Bacteroides coprocola; Bacteroides coprophilus; Bacteroides faecichinchillae; Bacteroides fluxus; Bacteroides fragilis; Bacteroides ovatus; Bacteroides plebeius; Bacteroides salanitronis; Bacteroides salyersiae; Bacteroides stercoris; Bacteroides thetaiotaomicron; Bacteroides uniformis; Caulobacter vibrioides; Clostridium phoceensis; ColHnsella aerofaciens; Enterococcus faecalis; Clostridium innocuum; Clostridium saccharogumia; Faecalibacterium prausnitzii; Gardnerella vaginalis; Synechocystis sp. PCC 6633; Intestinimonas b utyriciproducens; Lactobacillus plantarum; Lactobacillus reuteri; Lactobacillus crispatus; Lactobacillus iners; Lactobacillus fermentum; Listeria monocytogenes; Megasphaera genomosp. (Type 1); Parabacteroides distasonis; Parabacteroides goldsteinii; Prevotella amnii; Prevotella buccalis; Sneathia sanguinegens; Staphylococcus aureus; Streptococcus salivarius; Streptococcus thermophilus; and Ureaplasma parvum.

69. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell is not from a phylum selected from the list consisting of: bacteroidetes; chlorobi; chlamydiae; verrucomicrobia; thermotogae; and aquificae.

70. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell is not from the class betaproteobacteria.

71. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell is not from a species selected from the list consisting of: Escherichia coli; Pseudomonas aeruginosa; and Salmonella enterica.

72. The method of any one of the preceding paragraphs, wherein the one or more prokaryotic cell is not from the genus Bacillus, for example it is not from the species Bacillus subtilis.

73. The method of any one of the preceding paragraphs, wherein the method does not comprise determining endonucleolytic cleavage of the one or more 5’-monophosphorylated fragments of mRNA.

74. The method of any one of the preceding paragraphs, wherein the one or more 5 monophosphorylated fragments of mRNA do not arise from endonucleolytic cleavage of mRNA.

75. A kit of parts for any one of the method as defined in any one of the preceding paragraphs, wherein the kit comprises instructions for the method as defined in any one of the preceding paragraphs. 76. A library of sequences obtained from and/or obtainable by any one of the methods as defined in any one of the preceding paragraphs.

77. A use of one or more 5’-monophosphorylated fragment of mRNA for determining the identity of one or more mRNA sequence being translated in a prokaryotic cell, wherein the prokaryotic cell is one in which 5’-3’ co-translational degradation of mRNA occurs. 78. A kit or method or library or use, as described herein with reference to the description and/or figures.

Claims

Claims:

(i) providing one or more prokaryotic cell;

(i) providing a sample;

3. The method of Claim 2, wherein the method comprises determining the presence and/or identity of two or more prokaryotic cells in the sample, wherein the method further comprises the step of:

(iv) identifying, from the information in step (iii), whether the two or more prokaryotic cells belong to the same genus and/or species and/or strain or two or more different genus and/or species and/or strain.

(i) providing a sample from the patient;

(ii) determining, from the sample, the sequence of one or more 5’-monophosphorylated fragments of rrsRNA;

5. The method of Claim 4, further comprising the step of:

(v) from the diagnosis and/or prognosis in step (iv), treating the condition.

(i) contacting the agent with one or more prokaryotic cell;

7. The method of Claim 6, wherein: step (i) further comprises providing one or more prokaryotic cell which has not been contacted with the agent; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not contacted with the agent, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) comprises determining the effect of the agent on the mRNA sequences by comparing the sequence information in step (ii) for the one or more prokaryotic cell contacted with the agent with the sequence information in step (ii) for the one or more prokaryotic cell not contact with the agent.

8. A method for determining the physiological status of one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

(iii) determining, from the sequence information in step (ii), the physiological status of the one or more prokaryotic cell; wherein the prokaryotic cell is one in which 5 3 co-translational degradation of mRNA occurs.

9. A method for determining the effect of an environmental condition on one or more prokaryotic cell, comprising the steps of:

(i) providing one or more prokaryotic cell;

10. The method of Claim 9, wherein the environmental condition is one or more selected from the list consisting of: a mixture comprising about two or more prokaryotic species, including the one or more prokaryotic cell; a mixture comprising the one or more prokaryotic cell and one or more eukaryotic cells; a mixture comprising the one or more prokaryotic cell and one or more virus; a mixture comprising the one or more prokaryotic cell and one or more prions; a sample; heat; cold; a temperature range; a pH; a pH range; radiation; a level of radiation; a level of a contaminant; a change in nutrition; stationary phase; and a level of one or more heavy metal.

11. The method of any one of Claims 2, 3, or 10, wherein the sample is one or more selected from the list consisting of: a sample from a patient; an environmental sample; and a laboratory sample.

12. The method of any one of the preceding claims, wherein the co-translational degradation of mRNA is exonucleolytic co-translational degradation.

13. The method of any one of the preceding claims, wherein the one or more prokaryotic cell comprises one or more exonuclease comprising 5’ to 3’ exonuclease activity.

14. The method of any one of the preceding claims, further comprising step (i)(a) performed after step (i), wherein step (i)(a) comprises subjecting the one or more prokaryotic cell with an agent and/or environmental condition that modifies ribosome function.

15. The method of Claim 14, wherein: step (i) further comprises providing one or more prokaryotic cell, wherein the one or more prokaryotic cell has not been subjected to the agent and/or environmental condition that modifies ribosome function; and/or step (ii) further comprises determining, from the one or more prokaryotic cell not subjected to the agent and/or environmental condition that modifies ribosome function, the sequence of the one or more 5’-monophosphorylated fragments of mRNA; and/or step (iii) further comprises providing the determination by comparing the sequence information in step (ii) for the one or more prokaryotic cell subjected to the agent and/or environmental condition that modifies ribosome function with the sequence information in step (ii) for the one or more prokaryotic cell not subjected to agent and/or environmental condition that modifies ribosome function.

16. The method of any one of the preceding claims, further comprising step (i)(b) performed after step (i), wherein step (i)(b) comprises isolating, from the one or more prokaryotic cell, one or more 5’-monophosphorylated fragment of mRNA.

17. The method of any one of the preceding claims, wherein the determination of the sequence of the one or more 5’-monophosphorylated fragments of mRNA in step (ii) comprises using polymerase chain reaction and/or high-throughput sequencing and/or next-generation sequencing.

18. The method of any one of the preceding claims, further comprising step (ii)(a) performed after step (ii), wherein step (ii)(a) comprises determining the position of a ribosome on the sequence of the one or more 5’-monophosphorylated fragments of mRNA.

19. The method of any one of the preceding claims, further comprising step (ii)(b) performed after step (ii), wherein step (ii)(b) comprises determining the relative positions in the sequence of one of more of the following RNA structures: the 5’P; one or more codons; one or more single nucleotide polymorphism (SNR); the 5’ untranslated region; the 3’ untranslated region; RNA binding protein binding sites; RNA secondary structure (such as RNA hairpins); RNA modifications (such as alternative or chemically modified RNA bases).

20. The method of any one of the preceding claims, further comprising step (ii)(c) performed after step (ii), wherein step (ii)(c) comprises identifying a three-nucleotide periodicity in the one or more 5’-monophosphorylated fragments of mRNA.

21. The method of any one of the preceding claims, wherein the 5’-monophosphorylated fragments of mRNA are about 9 or more nucleotides in length, preferably about 15 or more nucleotides.

22. The method of any one of the preceding claims, wherein the one or more prokaryotic cell is one or more bacterial cell.

23. The method of any one of the preceding claims, wherein the one or more prokaryotic cell in which 5 3 co-translational degradation of mRNA occurs is from a species selected from the list consisting of: Bacillus subtilis; Akkermansia muciniphila; Alistipes finegoldii; Alistipes obesi; Alistipes putredinis; Alistipes shahii; Atopobium vaginae; Bacillus amyloliquefaciens; Bacteroides caccae; Bacteroides cellulosilyticus; Bacteroides coprocola; Bacteroides coprophilus; Bacteroides faecichinchillae; Bacteroides fluxus; Bacteroides fragilis; Bacteroides ovatus; Bacteroides plebeius; Bacteroides salanitronis; Bacteroides salyersiae; Bacteroides stercoris; Bacteroides thetaiotaomicron; Bacteroides uniformis; Caulobacter vibrioides; Clostridium phoceensis; Collinsella aerofaciens; Enterococcus faecalis; Clostridium innocuum; Clostridium saccharogumia; Faecalibacterium prausnitzii; Gardnerella vaginalis; Synechocystis sp. PCC 6633; Intestinimonas b utyriciproducens; Lactobacillus plantarum; Lactobacillus reuteri; Lactobacillus crispatus; Lactobacillus iners; Lactobacillus fermentum; Listeria monocytogenes; Megasphaera genomosp. (Type 1); Parabacteroides distasonis; Parabacteroides goldsteinii; Prevotella amnii; Prevotella buccalis; Sneathia sanguinegens; Staphylococcus aureus; Streptococcus salivarius; Streptococcus thermophilus; and Ureaplasma parvum.

24. A kit of parts for any one of the method as defined in any one of the preceding claims, wherein the kit comprises instructions for the method as defined in any one of the preceding claims.

25. A library of sequences obtained from and/or obtainable by any one of the methods as defined in any one of the preceding claims.