GB2505819A - Methods for identifying genes mediating antibiotic sensitivity or resistance using transposons with different promoters - Google Patents

Abstract

A method is disclosed for identifying genes mediating antibiotic sensitivity or resistance, wherein the method utilises transposons with three different promoters of varying strengths to generate mutant bacteria. The method comprises the steps of generating a pool of mutant bacteria by transposon mutagenesis of Gram-negative bacterium with a plurality of activating transposons (TnA), each transposon comprising an outward-facing promoter (TnP); growing bacteria from the mutant pool in the presence of different amounts of said antibiotic; and comparing the distribution of TnA insertions between test cultures to identify a gene which mediates antibiotic sensitivity or resistance in said bacterium; wherein said plurality of activating transposons comprises three outward-facing promoters TnP1, TnP2, TnP3, and wherein the strength of said promoters is TnP1> TnP2> TnP3; such that transposon insertion into bacterial DNA generates a pool of mutant bacteria in which one or more genes are transcribed from TnP1, one or more genes are transcribed from TnP2 and one or more genes are transcribed from TnP3.

Description

METHOD FOR IDENTIFYING GENES INVOLVED IN ANTIBIOTIC RESISTANCE AND

SENSITIVITY IN GRAM-NEGATIVE BACTERIA

Field of the Invention

The present invention relates to methods for identifying antibiotic targets in bacteria, to methods for identifying antibiotics and to processes for producing antibiotics and pharmaceutical compositions comprising said antibiotics.

Backaround to the Invention

There is an urgent need for new antibiotics to counter the emergence of new pathogens and resistance to existing antimicrobial drugs. The identification of the targets of candidate antibiotics is critical, since such information can provide access to a large number of functionally related novel drug families. For example, the discovery of the penicillin-binding proteins as targets of penicillin led to the development of a large family of antibiotics, including multiple generations of cephalosporins, penicillins and carbapenems (see Schmid (2006) Nature Biotechnology 24(4): 419-420).

Transposon directed insertion-site sequencing (TraDIS -see Langridge et 8/. (2009) Genome Research 19: 2308-2316) has recently been described and used to identify: (a) essential genes; (b) genes advantageous (but not essential) for growth; (c) genes disadvantageous for growth under particular conditions; and (d) genes involved in conferring tolerance to certain conditions ("niche-specific" essential genes). Similar techniques have been described in e.g. Gawronski et at (2009) PNAS 106: 16422-16427; Goodman at at (2009) Cell Host Microbe 6: 279-289; van Opijnen at at (2009) Nat.

Methods 6: 767-772 and Gallagher at a/. (2011) mBio 2(1):e00315-10, and such techniques are now collectively dubbed "Tn-seq' methods.

However, an important class of antibiotic targets are gene products involved in cellular processes essential for viability in the growth conditions used. Such targets cannot be identified by Tn-seq (including TraDIS), since transposon insertions into essential genes (including those serving as antibiotic targets) are not significantly represented in the initial mutant pool. Thus, differences in transposon distribution after growth of the mutant pool with or without (or with varying amounts of) antibiotic would not arise, with the result that Tn-seq cannot distinguish between an essential gene and an essential gene serving as an antibiotic target.

There is therefore a need for high-throughput functional screens for antibiotic targets which are capable of identifying essential genes serving as antibiotic targets.

W020121150432 describes a method for identifying an essential gene which serves as an antibiotic target in a bacterium comprising the steps of: (a) generating a pool of mutant bacteria by transposon mutagenesis with an activating transposon (Tn4, wherein the TnA comprises a promoter such that transposon insertion into bacterial DNA increases the transcription of a gene at or near the insertion site; (b) growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and (c) comparing the distribution of TnA insertions between test cultures to identify a putative essential gene serving as a target of said antibiotic in said bacterium.

It has now been discovered that the quality of the quantitative sequencing data obtained via such methods is greatly improved and enriched by probing the bacterial genome with a mixture of different activating transposons which have outward facing promoters of different strengths. In particular, it has been found that a broader range of genes involved in antibiotic resistance and/or sensitivity are recovered if a mixture of activating transposons with at least three different promoters of progressively decreasing strength are employed to generate the mutant pool.

Summary of the Invention

According to an aspect of the present invention, there is provided a method for identifying a gene which mediates antibiotic sensitivity or resistance in a Gram-negative bacterium, the method comprising the steps of: (a) generating a pool of mutant bacteria by transposon mutagenesis of said Gram-negative bacterium with a plurality of activating transposons (Tn4, wherein each TnA comprises an outward-facing promoter (Tn) such that transposon insertion into DNA of the bacterium results in transcription of a gene at or near the insertion site from said Tn promoter; (b) growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and (c) comparing the distribution of mA insertions between test cultures to identify a gene which mediates antibiotic sensitivity or resistance in said bacterium; wherein in step (a) said plurality of activating transposons comprises: (i) an activating transposon comprising an outward-facing first promoter Tnp1; (ii) an activating transposon comprising an outward-facing second promoter Tn2; and (Hi) an activating transposon comprising an outward-facing third promoter Tn3, wherein the relative strength of said promoters is: Tnp1> Tnp2> Tnp3; such that transposon insertion into bacterial DNA generates a pool of mutant bacteria in which one or more genes are transcribed from Tn1, one or more genes are transcribed from Tn2 and one or more genes are transcribed from Tnp3.

The use of a plurality of activating transposon with promoters of varying strength ensures that transposon inseitions into substantially all genes involved in mediated antibiotic sensitivity or resistance are represented in the initial mutant pool, since transposon insertion can now result in gene activation to yield an appropriate level of transcription (neither too high, nor too low). Thus, the effect of the presence of antibiotic during subsequent culture of the mutant pool on transposon distribution can be studied (and the identity of the relevant gene target(s) thereby determined).

The information generated from the use of the invention enables very early triage of antibiotic hits allowing focused chemical resource on appropriate scaffolds. This not only focuses chemistry, but allows selection of antibiotic hits which using conventional triage methods would be overlooked. The data also allows rational optimization and selection of antibiotics with a low probability of resistance.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art: Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," aie to be read to indicate the inclusion of any lecited integer (e.g. a feature, element, characteristic, propeity, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

The term gene is a term describing a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome or plasmid and determines a particular characteristic in an organism. A gene may determine a characteristic of an organism by specifying a polypeptide chain that forms a protein or part of a protein (structural gene); or encode an RNA molecule; or regulate the operation of other genes or repress such operation; or affect phenotype by some other as yet undefined mechanism.

The terms genomic DNA is a term of art used herein to define chromosomal DNA as distinct from extrachromosomally-maintained plasmid DNA.

The term genome is a term of art used herein to define the entire genetic complement of an organism, and so includes chromosomal, plasmid, prophage and any other DNA.

The term Gram-negative bacterium is a term of art defining a particular class of bacteria that are grouped together on the basis of certain cell wall staining characteristics.

Examples of Gram-negative bacterial genera include Kiebsie/la, Acinetobacter, Escherichia, Pseudomonas, Enterobacter and Neisseria.

As used herein, the term essential gene" is a term of art defining a particular class of genes the products of which are necessary for viability, either under all conditions or under the conditions of growth used. An important subclass of essential gene are those encoding products (e.g. proteins, peptides and regulatory polynucleotides) which contribute to metabolic processes essential for viability under important growth conditions (for example, and in the case of pathogenic bacteria, under conditions which prevail during infection or multiplication in the host).

Antibiotics and antibiotic targets The antibiotic used to produce the test cultures of the invention is typically a novel investigational antibiotic (anti-bacterial chemotherapeutic agent), the mechanism of action (and hence biological target(s)) of which are unknown. In many applications, the antibiotic is selected from combinatorial libraries, natural product libraries, defined chemical entities, peptides, peptide mimetics and oligonucleotides.

The antibiotic target identified according to the invention is an essential gene/gene product, and may therefore be involved in one or more of the following biological processes in the bacterial host: (a) cell division; (b) DNA replication (including polymerization and supercoiling); (c) transcription (including priming, elongation and termination); (d) translation (including ribosome components, initiation, elongation and release); (e) biosynthetic pathways (including peptidoglycan and fatty acids); (f) plasmid addiction; (g) cell wall assembly; and/or (h) bacterial cell integrity.

Bacteria for use in the methods of the invention The methods of the invention may be applied to identify an antibiotic target in any Gram-negative bacterium.

The methods of the invention find particular application in the identification of an antibiotic target in a bacterium selected from Kiebsiella pneumoniae, Acinetobacter baumanil, Escherichia co/i (including ST131) and Pseudomonas aeruginosa.

Mutant pools The methods of the invention involve generating a pool of mutant bacteria by transposon mutagenesis. The size of the mutant pool affects the resolution of the method: as the pool size increases, more and more different genes with TnA insertions will be represented (and so effectively assayed). As the pool size decreases, the resolution of the method reduces, genes will be less effectively assayed, and more and more genes will not be assayed at all.

Ideally, the mutant pool generated in the methods of the invention is comprehensive, in the sense that insertions into every gene are represented. The number of TnA insertion mutants (i.e. the mutant pool size) required to achieve this depends on various factors, including: (a) the size of the bacterial genome; (b) the average size of the genes; and (c) any TnA insertion site bias.

With regard to the latter, some areas of bacterial genomes attract a low frequency of insertion (especially GC-rich regions). Thus, insertion frequencies and pool sizes large enough to ensure that insertions into insertion-refractory regions are preferred.

In general, a minimum insertion rate of one transposon per 25bp is required to achieve a comprehensive pool/library, which typically entails a minimum pool size for bacteria having a genome size of 4 to 7Mb of 0.5 x io to 1 x io, for example 5x105, preferably at least about 1x106 mutants. In many cases, 1x106 mutants will allow identification of -300,000 different insertion sites and correspond to 1 transposon insertion every 13 to 23 bp (or about 40-70 different insertion sites per gene).

However, the methods of the invention do not necessarily require a comprehensive mutant pool (in the sense defined above) in order to return useful information as to the identity of genes involved in resistance or sensitivity to antibiotic drugs. Rather, pool sizes less than the ideal comprehensive pool may be used, provided that a reduction in resolution (and attendant failure to assay certain genes) can be tolerated. This may be the case, for example, where the method is designed to be run iteratively until the target is identified: in such embodiments the effective pool size grows with each iteration of the method.

Transposon mutacienesis Transposons, sometimes called transposable elements, are polynucleotides capable of inserting copies of themselves into other polynucleotides. The term transposon is well known to those skilled in the art and includes classes of transposons that can be distinguished on the basis of sequence organisation, for example short inverted repeats at each end; directly repeated long terminal repeats (LTR5) at the ends; and polyA at 3'ends of RNA transcripts with 5' ends often truncated.

Transposomes are transposase-transposon complexes wherein the transposon does not encode transposase. Thus, once inserted the transposon is stable. Preferably, in order to ensure mutant pool stability, the transposon does not encode transposase and is provided in the form of a transposome (i.e. as a complex with transposase enzyme), as described below.

As used herein, the term activating transposon" (hereinafter abbreviated "TnA") defines a transposon which comprises a promoter such that transposon insertion increases the transcription of a gene at or near the insertion site (i.e. an outward-facing promoter functional in the host bacterium). Examples of such transposons are described in Troeschel SaL (2010) Methods Mol Biol. 668:117-39 and Kim SaL (2008) Curr Microbiol.

57(4): 391-394.

The activating transposon/transposome can be introduced into the bacterial genome (including chromosomal and/or plasmid DNA) by any of a wide variety of standard procedures which are well-known to those skilled in the art. For example, TnA transposomes can be introduced by electroporation (or any other suitable transformation method).

Preferably, the transformation method generates 1x103 to 5x103 transformants/ng DNA, and such transformation efficiencies are generally achievable using electroporation.

Alternatively, transposon mutagenesis using TnA may be performed in vitro and recombinant molecules transformed/transfected into bacterial cells. In such embodiments, transposomes can be prepared according to a standard protocol by mixing commercially available transposase enzyme with the transposon DNA fragment. The resulting transposomes are then mixed with plasmid DNA of the plasmid of interest to allow transposition, then the DNA introduced into a host bacterial strain using electrotransformation to generate a pool of plasmid transposon mutants.

In embodiments where mutagenesis is performed in vitro, it is possible to mix transposomes with genomic DNA in vitro and then introduce the mutagenized DNA (optionally, after fragmentation and/or circularization) into the host bacterial strain (e.g. by electroporation) whereupon endogenous recombination machinery incorporates it into the genome. Such an approach may be particularly useful in the case of bacteria which are naturally competent (e.g. Acinetobacterspp.) and/or can incorporate DNA via homologous crossover (e.g. double crossover) recombination events.

Activating transposons for use in the methods of the invention Any suitable activating transposon may be used in the methods of the invention. Suitable transposons include those based on Tn3 and the Tn3-like (Class II) transposons including yO(Tnl000), Tn501, Tn2501, Tn21, Tn9l7and their relatives. Also TnlO, Tn5, TnphoA, Tn903, bacteriophage Mu and related transposable bacteriophages. A variety of suitable transposons are also available commercially, including for example the EZTn5TM c R6Ky0riIKAN-2> transposon.

Preferred transposons are those which carry antibiotic resistance genes (which may be useful in identifying mutants which carry a transposon) including Tn5, Tn 10 and TnphoA.

For example, Tn10 carries a tetracycline resistance gene between its IS elements while Tn5 carries genes encoding polypeptides conferring resistance to kanamycin, streptomycin and bleomycin. Other suitable resistance genes include those including chioramphenicol acetyltransferase (conferring resistance to chioramphenicol).

It is of course possible to generate new transposons by inserting different combinations of antibiotic resistance genes between IS elements, or by inserting combinations of antibiotic resistance genes between transposon mosaic ends (preferred), or by altering the polynucleotide sequence of the transposon, for example by making a redundant base substitution or any other type of base substitution that does not affect the transposition or the antibiotic resistance characteristics of the transposon, in the coding region of an antibiotic resistance gene or elsewhere in the transposon. Such transposons are included within the scope of the invention.

In many embodiments, a single transposon is used to generate the mutant pool. However, as explained above, the number of Tn insertion mutants (i.e. the mutant pool size) required to achieve a comprehensive pool or library depends inter a/ia on any Tn insertion site bias.

Thus, in cases where the transposon insertion site bias occurs, two or more different transposons may be used in order to reduce or eliminate insertion site bias. For example, a combination of two different transposons based on Tn5 and TnlO may be employed.

Promoters for use in activating transposons A wide variety of promoters may be used according to the present invention provided that at least three different promoters are used wherein the relative strength of said promoters is: Tnp1> Tnp2> Tnp3; such that transposon insertion into bacterial DNA generates a pool of mutant bacteria in which one or more genes are transcribed from Tnp1, one or more genes are transcribed from Tn2 and one or more genes are transcribed from Tn3.

Preferably, Tnp1 is a strong promoter, Tnp2 a medium-strength promoter and Tnp3 a weak promoter in the mutagenized Gram-negative bacteria under the conditions used for growth of the mutant pool in the presence of different amounts of the test antibiotic (i.e. step (b) of the method of the invention). In some embodiments, the relative transcription initiation rate of Tn1 is at least 3 times, at least 100 times, at least 1000 times or at least 10000 times higher than that of Tnp3 under these conditions.

Each promoter includes: (a) a Pribnow box (-10 element); (b) a -35 element and (c) an UP element. Those skilled in the art are able to readily identify promoters having the required relative strengths by sequence analysis and/or in vitro or in vivo assays using expression constructs.

For example, suitable promoters can be engineered or selected as described in Rhodium etal. (2011) Nucleic Acids Research: 1-16.

Moreover, the rapid application of next generation sequencing to RNA-seq is now providing a wealth of high-resolution information of transcript start sites at a genomic level, which greatly simplifies the identification of promoter sequences in any given Gram-negative bacterium. This permits the construction of descriptive promoter models for entire genomes. RNA-seq also provides quantitative information on transcript abundance and hence promoter strength, which enables the construction of promoter strength models that can then be used for predictive promoter strength rankings (see Rhodium et al. (2011) Nucleic Acids Research: 1-18).

Suitable promoters can also be identified by assay. For example, a series of plasmids based on that shown in Figure 1 can be used to test promoter strength empirically. Briefly, the promoter to be tested is placed upstream of an antibiotic resistance gene (in this case Kanamycin) and then transformed into the relevant bacteria. General cloning assembly and plasmid amplification can be carried out in E.co/i (facilitated by the ampicillin resistance gene and the pBR322 on) and the activity of the promoter in the target bacterium can then be assayed by generating a killing curve with Kanamycin -a very high level promoter gives more KmR expression and therefore survival at a higher antibiotic concentration. The plasmid series is designed to be modular so that the origin of replication, resistance gene(s) and promoter can be easily switched.

Suitable promoters include the E. CO/i rp/J (large ribosomal subunit protein; moderate strength promoter); tac (artificial /ac/trp hybrid; strong promoter) and rrnB (ribosomal RNA gene promoter; very strong promoter) promoters.

As used herein, the terms P1ij and Pu-nB specifically refer to the E. coli promoters for the SOS ribosomal subunit protein L10 and 16S ribosomal RNA genes, respectively.

Orthologues of these (and other) F. co/i promoters from other Gram-negative bacteria can also be used, including in particular the orthologous Pseudomonas aeruginosa or Acinetobacter baumannil promoters.

For example, the orthologous Acinetobacterbaumannil gene corresponding to the E. coli rrnB PIInB has the gene symbol A1S_r12 and encodes the Acinetobacterbaumannh/ 165 ribosomal RNA gene, so that the corresponding orthologous promoter is herein designated P(A1s 112). Thus, when the method is applied to Acinetobacterbaumannll, Tn1 may be P(A1s12).

Similarly, when the method of the invention is applied to Pseudomonas aerug/nosa, Tn2 may be the 16S ribosomal RNA gene promoter from P. aeruginosa (i.e. PS.PirnB) while Tnp3 may be selected from the rpsj (small (30S) ribosomal subunit 510 protein) gene promoter from P. aeruginosa (i.e. Ps.P1) and the E. co/i PB.

Determining the distribution of TnA insertions The distribution of transposon insertions is preferably determined by sequencing bacterial DNA adjacent or near (5' and/or 3') the TnA insertion site (e.g. by sequencing DNA which comprises TnA -genomic DNA junctions). Typically, bacterial DNA flanking or adjacent to one or both ends of the TnA is sequenced.

The length of adjacent DNA sequenced need not be extensive, and is preferably relatively short (for example, less than 200 base pairs).

Various methods can be used to determine the TnA insertion distribution using DNA sequencing: such methods have recently been dubbed Tn-seq procedures (van Opijnen et aL (2009) Nat. Methods 6: 767-772). For example, Tn-seq procedures include affinity purification of amplified Tn junctions (Gawronski eta/. (2009) PNAS 106: 16422-16427); ligation of adaptors into genome sequences distal to the end of the transposon using a specialized restriction site (Goodman eta!. (2009) Cell Host Microbe 6: 279-289; van Opijnen et al. (2009) Nat. Methods 6: 767-772); selective amplification (Langridge et aL (2009) Genome Research 19: 2308-2316) and the generation of single-stranded DNA circles bearing Tn junctions, which serve as templates for amplification and sequencing after elimination of genomic DNA by exonuclease digestion (Gallagher et aL (2011) mBio 2(1):e00315-10).

Any suitable high-throughput sequencing technique can be used, and there are many commercially available sequencing platforms that are suitable for use in the methods of the invention. Sequencing-by-synthesis (SBS)-based sequencing platforms are particularly suitable for use in the methods of the invention: for example, the llluminaTM system is generates millions of relatively short sequence reads (54, 75 or lOObp) and is particularly preferred.

Other suitable techniques include methods based on reversible dye-terminators. Here, DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). Four types of ddNTPs are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labelled nucleotides then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing a next cycle.

Other systems capable of short sequence reads include SOLiDTM and Ion Torrent technologies (both sold by Applied BiosystemsTM). SOLiDTM technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labelled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing.

Ion Torrent Systems Inc. have developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerisation of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

Functional assessment of putative essential genes The putative essential gene identified by comparing the distribution of TnA insertions between test cultures may be further characterized by various techniques which directly or indirectly assess its function. In this way, an essential function may be definitively assigned to said putative essential gene.

Suitable techniques include bioinformatics, where the (full or partial) sequence of the putative essential gene is used to interrogate sequence databases containing information from the bacterium assayed and/or other species in order to identify genes (e.g. orthologous genes in other species) for which essential biochemical function(s) have already been assigned and/or which have been shown to be essential.

Suitable bioinformatics programs are well known to those skilled in the art and include the Basic Local Alignment Search Tool (BLAST) piogiam (Altschul et a/. (1990) J. Mol. Biol.

215: 403-410 and Altschul eta/. (1997) NucI. Acids Res. 25: 3389-3402). Suitable databases include, for example, EMBL, GENBANK, TIGR, EBI, SWISS-FROT and trEMBL.

Alternatively, or in addition, the (full or partial) sequence of the putative essential gene is used to interrogate a sequence database containing information as to the identity of essential genes which has been previously constructed using the conventional Tn-seq methods described in the prior art (e.g. as described in Gawronski ot al. (2009) PNAS 106: 16422-16427; Goodman et a!. (2009) Cell Host Microbe 6: 279-289; van Opijnen et a/.

(2009) Nat. Methods 6: 767-772; Langridge eta/. (2009) Genome Research 19: 2308- 2316; Gallagher at aL (2011) mBio 2(1):e00315-10) and/or the techniques described in WO 01/07651 (the contents of which are hereby incorporated by reference).

Alternatively, or in addition, essentiality can be imputed by eliminating the possibility that a putative essential gene acts as an antibiotic lesistance gene. For example, the (full or partial) sequence of the putative essential gene is used to interrogate sequence databases containing sequence information of genes previously identified as antibiotic resistance genes using the Tn-seq methods described in e.g. Gawronski et a!. (2009) PNAS 106: 16422-16427; Goodman et a!. (2009) Cell Host Microbe 6: 279-289; Langridge et al. (2009) Genome Research 19: 2308-2316 or Gallagher et a/. (2011) mBio 2(1):e00315-10.

Antibiotic resistance genes may be identified in such methods as a class of niche-specific/conditionally essential genes.

Despite the presence of a promoter within the inserted sequence, many TnA insertions will disrupt gene/DNA function and allow identification of essential/important DNA regions, as in standard Tn-seq (including TraDIS). However, some transposons will be positioned appropriately with respect to specific important DNA regions, whereby transcription of those specific regions, driven by the inserted promoter, is enhanced significantly compared to endogenous transcription. By growing the mutant pool in increasing antibiotic concentrations and repeating the sequencing it is possible to observe changes in the number of reads, indicating not only which DNA region contributes to antibiotic survival, but also the relative contribution. The higher levels of specific antibiotic target transcription (driven by the transposon-inserted promoters) will favour bacterial survival in antibiotic and link insertion site to DNA region by proximity.

To identify the specific antibiotic target(s), the position of the inserted promoter can be assessed with respect to its contribution to increased transcription of relevant downstream DNA sequences. A mathematically/technically straightforward bioinformatics component of this technique permits recognition of the contribution of the inserted promoter sequence to transcription of the putative antibiotic target gene relative to data generated in the absence of the antibiotic. For example, transcription of the antibiotic target partial gene product may be enough to confer antibiotic resistance and bioinformatic analysis would allow an explanation. This information is observed in the number of specific mutants providing this advantage being greater than those observed in the absence of the antibiotic. In addition, the partial gene transcript may still encode enough information to allow translation of a truncated, but functional essential protein. Bioinformatics would allow the effects of transcriptional read through on genes downstream of the gene adjacent to the inserted transposon to be considered, where there is there no defined RNA transcription termination sequence.

For example, a transposon/promoter upstream of genes A, B and C may generate a polycistronic transcript of all three genes (A-C), upstream of B a polycistronic transcript of genes B and C and upstream of C just gene C. If the reads for the first two transposons were high and the third low in antibiotic then the antibiotic target would be gene B. ExemQlification The invention will now be described with reference to specific Examples. These are merely exemplaly and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

Preparation of bacteria for electroporation Bacteria are grown in 2 x TY broth to an OD0 of 0.3-0.5. Cells are then harvested and washed three times in 1/2 original culture volume 10% glycerol and resuspended in 1/1000 original culture volume 10% glycerol and stored at -80°C.

Prepalation of trans,osomes Transposon DNA (a derivative of EZ-Tn5TM < R6Kyori/KAN-2> possessing an internal lac promoter was amplified using oligonucleotides 5'-CTGTCTCTTATACACATCTCCCT and 5'-CTGTCTCTTATACACATCTCTTC with Pfu Ultra Fusion II DNA polymerase (Stratagene). As an alternative, the internal lac promoter can be replaced with a tac promoter (as described supra). The resultant amplicon was then phosphorylated using T4 polynucleotide kinase (New England Biolabs). Two hundred nanograms of this DNA were then incubated with EZ-Tn5TM transposase (Epicenter Biotechnologies) at 37°C for 1 h then stored at -20°C at a DNA concentration of 2OngIpl.

Generation of mutant bacterial pools Sixty microliteis of cells (previously stored at -80°C are mixed with O.2p1 (4ng) of transposomes and 1 p1 (20g) complementing plasmid comprising essential genes and electrotransformed in a 2-mm electrode gap cuvette using a Bio-Rad GenePulser II set to 2.4 kV, 25 pF, and 200 0. Cells are resuspended in 1 mL of Soc medium (Invitrogen) and incubated at 37°C for 2 h then spread on [-agar bacteroiological growth medium supplemented with an appropriate concentration of kanamycin. The concentration of kanamycin used is strain dependent and determined empirically After incubation overnight at 37°C, the number of colonies on several plates is estimated by counting a proportion of them, and from this the total number of colonies on all plates is estimated conservatively. Kanamycin resistant colonies are harvested by resuspension in sterilized deionized water using a bacteriological spreader. Resuspended cells from several electroporations are then pooled to create mutant library mixtures estimated to include over 1 million mutants.

Identifying antibiotic target gene(s) Eight cultures of 100 ml broth medium are prepared, six of which are supplemented, in duplicate, with the test antibiotic at a concentrations 0.5, 1 and 2 x MIC. Any required promoter inducer is also be added to the medium at this time to ensure active transcription directed into the chromosomal DNA from the transposon sequence.

Assuming a transposon mutant pool of 1 million mutants, ion-lO9cfu of the pool are used to inoculate each culture. Cultures are grown to stationary phase and cells harvested for genomic DNA extraction. Fresh cultures are also prepared and inoculated with W6-lO9cfu from the first cultures. These are grown to stationary phase and cells harvested for extraction of genomic DNA.

Genomic DNA is sequenced using the llluminaTM platform incorporating the TraDIS modification to obtain sequence reads initiated from the transposon insertion sites.

Sequence reads are then mapped to the bacterial genome sequence and compared with the genome annotation to determine the number of sequence reads that map to each gene for the 8 cultures (6 test and 2 control). Comparison of the control data sets with each other and of test data sets with each other indicates the degree of experimental variation.

Comparison of control data with test data sets shows experimental reproducibility and indicates gene(s) targeted by the antibiotic. llluminaTM sequence reads from transposon insertion within the essential gene antibiotic target gene(s) increase in cells grown with antibiotic, where the promoter caused an increase in this specific gene transcription.

Moreover, the relative read count from the target gene(s) increase with concentration of antibiotic used.

Exclusion of antibiotic resistance genes Conventional transposon directed insertion-site sequencing (TraDIS -see Langridge et aL (2009) Genonie Fesearch 19: 2308-2316) can be used to identify antibiotic resistance genes which are not essential to growth under normal conditions but which confer tolerance to the antibiotic (i.e. a class of the "niche-specific" essential genes discussed in Langridge et at. (2009)). This permits the elimination of antibiotic resistance genes from candidate antibiotic target genes, as described below.

The MIC of the antibiotic to be tested is determined for the bacterium of interest. Four cultures of 100 ml broth medium are prepared, two of which are supplemented with the antibiotic at a concentration 0.5 to 0.75 x MIC (i.e. just below Mb). Assuming a transposon mutant pool of 1 million mutants, ion-lO9cfu of the pool are used to inoculate each culture. Cultures are grown to stationary phase and cells harvested for genomic DNA extraction. Fresh cultures are also prepaled and inoculated with io lO9cfu from the first cultures. These are grown to stationary phase and cells harvested for extraction of genomic DNA. Genomic DNA is sequenced using the llluminaTM platform incorporating the TraDIS modification to obtain sequence reads initiated from the transposon insertion sites.

Sequence reads are then mapped to the bacterial genome sequence and compared with the genome annotation to determine the number of sequence reads that map to each gene for the 4 cultures (2 test and 2 control).

Comparison of the control data sets with each other and of test data sets with each other indicates the degree of experimental variation. Comparison of control data (minus antibiotic) with test data (plus antibiotic) sets shows experimental reproducibility and indicates target antibiotic genes..

Figure 2 shows the results of a study to identify the molecular target genes of a novel antibiotic in Escherichia coil Ki 2. The graph includes data for every gene in the bacterium's genome. Three promoter transposon insertion libraries were grown in 4 conditions: 2 control cultures (no antibiotic) and 2 cultures with antibiotic at levels which kill wild type Escherichia ccii K12. Each point on the graph shown represents a gene and the positions of that point on the X and Y axis are log2 of the total number of transposon insertions directly upstream of that gene. The three circled points are genes where an increase in transcription of those genes was expected in the presence of the novel antibiotic relative to no antibiotic and therefore represented potential target genes of the antibiotic used.

Equivalents The foregoing description details presently preferred embodiments of the present invention.

Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims (21)

1. CLAIMS: 1. A method for identifying a gene which mediates antibiotic sensitivity or resistance in a Gram-negative bacterium, the method comprising the steps of: (a) generating a pool of mutant bacteria by transposon mutagenesis of said Gram-negative bacterium with a plurality of activating transposons (TnA), wherein each lilA comprises an outward-facing promoter (Tn) such that transposon insertion into DNA of the bacterium results in transcription of a gene at or near the insertion site from said Tnp promoter; (b) growing bacteria from the mutant pool in the presence of different amounts of said antibiotic to produce two or more test cultures; and (c) comparing the distribution of mA insertions between test cultures to identify a gene which mediates antibiotic sensitivity or resistance in said bacterium; wherein in step (a) said plurality of activating transposons comprises: (i) an activating transposon comprising an outward-facing first promoter Tnp1; (ii) an activating transposon comprising an outward-facing second promoter Tn2; and (Hi) an activating transposon comprising an outward-facing third promoter Tnp3, whelein the lelative strength of said promoters is: Tn1> Tn2> Tn3; such that transposon insertion into bacterial DNA generates a pool of mutant bacteria in which one or more genes are transchbed from Tnp1, one or more genes are transcribed from Tnp2 and one or more genes are transcribed from Tn3.
2. 2. The method of claim 1 wherein the Gram-negative bacterium is selected from Escherichia co/i, KISs/ella pneumonia, Pseudomonas aeruginosa and Acinetobacter baumannhl.
3. 3. The method of claim 2 wherein the Gram-negative bacterium is Escherichia co/i and Tnp1 is P1, Tnp2 is P00 and Tnp3 is PrrnB.
4. 4. The method of claim 2 wherein the Gram-negative bacterium is Kiebsiella pneumon/ae and Tn1 is P1ij, Tn2 is Ptac and Tn3 is PirnB.
5. 5. The method of claim 2 wherein the Gram-negative bacterium is Pseudomonas aeruginosa and Tn1 is P30, Tn2 is Ps.P119 and Tn3 is selected from Ps.P1 and PrrnB.
6. 6. The method of claim 5 wherein Tn1 is Fiac, Tn2 is F5.Prrn6 and Tn3 is F5.Prpsj further comprising a fourth promoter, Tnp4, being PirnB.
7. 7. The method of claim 2 wherein the Gram-negative bacterium is Acinetobacter baumannii and Tn1 is P(Als r12), Tn2 is P116 and Tn3 is Prpij.
8. 8. The method of any one of the preceding claims wherein the pool of mutant bacteria comprises: (a) at least 0.5 x i05 mutants, for example at least 1 x i05 mutants; (b) at least 5x105 mutants; (c) at least lxi 06 mutants; (d) 0.5 x106 to 2 x106 mutants; (e) about 1 x106 mutants; (f) about 2 x106 mutants; or (g) about 10 x106 mutants.
9. 9. The method of any one of the preceding claims wherein the transposon mutagenesis step (a) yields an insertion rate of: (a) at least one transposon per 50 base pairs of bacterial DNA; (b) at least one transposon per 30 base pairs of bacterial DNA; (c) at least one transposon per 25 base pairs of bacterial DNA; (d) at least one transposon per 15 base pairs of bacterial DNA; (e) at least one transposon per 10 base pairs of bacterial DNA; (f) at least one transposon per 5 base pairs of bacterial DNA; or (g) at least one transposon every base pair of bacterial DNA.
10. 10. The method of any one of the preceding claims wherein in step (a) said DNA of the bacterium is chromosomal (genomic) DNA.
11. ii. The method of any one of claims ito 9 wherein in step (a) said DNA of the bacterium is plasmid DNA or a mixture of chromosomal (genomic) and plasmid DNA.
12. 12. The method of any one of the preceding claims wherein the transposon mutagenesis of step (a) occurs in vivo.
13. 13. The method of any one of claims 1 to 11 wherein the transposon mutagenesis of step (a) occurs in vitro.
14. 14. The method of any one of the preceding claims wherein bacteria are grown from the mutant pool in step (b) by inoculating growth medium with i07 to i09, for example about i08, cfu from the mutant pool.
15. 15. The method of any one of the preceding claims wherein bacteria are grown from the mutant pool in step (b) in the presence of antibiotic at a concentration of about 0.5, about 1 and about 2 x MIC to produce at least three test cultures.
16. 16. The method of any one of the preceding claims wherein the distribution of TnA insertions between test cultures is compared by identifying: (a) the insertion position in the genome, and (b) the abundance of each insertion in the genome.
17. 17. The method of any one of the preceding claims wherein the distribution of TnA insertions between test cultures is compared by a method comprising sequencing DNA adjacent or near the insertion site of the TnA.
18. 18. The method of claim 17 wherein the sequencing of DNA adjacent or near the insertion site of the TnA comprises selective amplification of transposon-bacterial DNA junctions.
19. 19. The method of claim 17 or claim 18 wherein the sequencing comprises sequencing-by-synthesis (SBS) biochemistry.
20. 20. The method of any one of claims 17 to 19 wherein about 25, 50, 75, 100 or greater than 100 base pairs of DNA adjacent or near the TnA insertion site are sequenced.
21. 21. The method of any one of claims 17 to 20 wherein the sequenced DNA is 5' and/or 3' to the TnA insertion site.