WO2023275328A1

WO2023275328A1 - Recombinant bacteria resistant to horizontal gene transfer and phage infection

Info

Publication number: WO2023275328A1
Application number: PCT/EP2022/068202
Authority: WO
Inventors: Melanie BLOKESCH; Milena JASKOLSKA; David William Adams
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2021-07-02
Filing date: 2022-06-30
Publication date: 2023-01-05

Abstract

The invention relates to an expression vector encoding a DNA defence system comprising polypeptides derived from Vibrio cholerae, or equivalent functional variants or homologues, conferring resistance to the maintenance of extra-genomic DNA to a recipient bacterial cell. The invention further relates to methods for obtaining an isolated bacterium, or a bacterial population capable of eliminating extra-genomic DNA, or of protecting against phage infection, and an isolated bacterium, or a product comprising an expression vector according to the invention.

Description

Recombinant bacteria resistant to horizontal gene transfer and phage infection

The present invention relates to methods for modulating sensitivity to gene transfer in bacteria, and expression systems useful in the methods of the invention. Methods and expression systems disclosed herein employ the DdmD and DdmE proteins of Vibrio cholerae. Other related methods and expression systems disclosed herein employ the DdmA, DdmB and DdmC proteins of Vibrio cholerae, or combinations of all mentioned Ddm proteins.

The present application claims benefit of the priority of European patent application no. 21183501 .2 filed July 22021 , fully incorporated herein by reference.

Background of the Invention

Horizontal gene transfer (HGT) through mobile genetic elements, in particular bacteriophages and plasmids, underlies the adaptability of bacteria to local conditions, and has been harnessed for targeted modifications of bacteria in industry and research. However, in some settings, acquisition of heterogeneity through HGT can be detrimental, as mobile genetic elements can introduce biosafety risks by introducing antibiotic resistance, or impose added transcriptional requirements which reduce fitness or recombinant protein production in large scale industrial fermentation processes.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to obtain bacteria with reduced ability to acquire or maintain introduced nucleic acids in a circular, double stranded (ds) DNA form (plasmids and bacteriophages). This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

Summary of the Invention

The invention as described herein provides the means and methods to obtain an expression vector which encodes a multigene DNA elimination system (DES) system conferring resistance to horizontal gene transfer events mediated by double stranded (ds) DNA in a circular form. Expression of said functional DES proteins in a bacterium clears circular vectors, such as plasmid, or bacteriophage from a recipient cell.

These features may be desirable in an industrial setting to prevent problems caused by bacteriophage infection or plasmid uptake, such as deletions, genetic rearrangements, undesirable activation, or down- regulation or inactivation of neighbouring gene expression in commercially relevant recombinant protein expression systems, or acquisition of undesirable traits such as antibiotic resistance in the context of pure bacterial cultures or pathogenic organisms.

A first aspect of the invention relates to an expression vector, or expression system, encoding a functional DNA elimination system (DES) comprising a V. cholerae DdmD protein, ora functional variant, or homologue thereof, and a V. cholerae DdmE protein, or a functional variant, or homologue thereof. A second aspect of the invention relates to an expression vector or expression system encoding a functional DNA elimination system (DES) comprising the functional V. cholerae polypeptides DdmA, DdmB, and DdmC, or functional variants, or homologues thereof.

The invention further encompasses methods to obtain a bacterial preparation (e.g. an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium) capable of reducing, or eliminating plasmids, to reduce or eliminate intracellular plasmid from such a bacterial preparation, or to protect such a bacterial preparation against plasmid acquisition and maintenance or its effects, particularly plasmid-encoded antibiotic resistance. These methods employ expression vectors or expression systems according to the first aspect of the invention.

Further aspects of the invention relate to methods to obtain a bacterial preparation (e.g. an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium) capable of reducing, or eliminating bacteriophages, to reduce or eliminate intracellular bacteriophages from such a bacterial preparation, orto protect such a bacterial preparation against bacteriophages maintenance or its effects, such as cell lysis. These methods employ expression vectors or expression systems according to the second aspect of the invention.

Additional aspects of the invention relate to an isolated bacterium obtained by a method according to the invention, or expressing a functional DES from an expression vector according to the invention, and also commercial products comprising said isolated bacterium.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e. , contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et at, Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et at, Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids, that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein, and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3^rd ed. p. 21). Sequences are written left to right in the direction from the amino to the carboxy terminus.

The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

The terms gene expression or expression, or alternatively the term gene product, may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products. The term gene expression may also be applied to the transcription and processing of an RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.

The term variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in its primary amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring, or it may be a variant that is not known to occur naturally.

The term DNA elimination system, or DES in the context of the present specification relates to a functional, multi-gene or multi-protein system, which confers the ability, or an increased ability to eliminate extra-genomic, double stranded DNA from a cell expressing said system, particularly extra- genomic DNA in the form of plasmids, and/or bacteriophages which replicate and/or propagate by means of a circular intermediate or a plasmid-like state. A functional DES comprises specific combinations of DNA defence molecules, polypeptides which may be expressed from a single, or multiple transcription sites.

The term DNA defence molecule, or Ddm in the context of the present specification relates to the constituent polypeptides which perform the multiple DNA elimination functions of a DES system. The term Ddm in the context of the present specification relates to the previously uncharacterised hypothetical proteins derived from Vibrio cholerae (V. choierae) pathogenicity island 2 (VPI-2) and the Vibrio seventh pandemic island II (VSP-II), from which 7^th pandemic strains obtain the capacity to eliminate extra-genomic DNA. A non-limiting annotated example of each of the following Ddm polypeptides according to the invention (with reference genome locus tag in parentheses) is provided for the representative A1552 V. cholera strain.

DdmA relates to (A1552VC_00256) NCBI: AWB73039.1 ;

DdmB relates to (A1552VC_00255) NCBI: AWB73038.1 ;

DdmC relates to (A1552VC_00254) NCBI: AWB73037.1 ;

DdmD relates to (A1552VC_01554), NCBI: AWB74290.1 ;

DdmE relates to (A1552VC_01553) NCBI: AWB74289.1 ;

These Ddm terms further encompass natural homologues and recombinant variants, with overlapping sequence identify and a shared biological function to the polypeptides above.

The ability of a functional DES according to the invention to eliminate plasmids, or bacteriophages from a cell depends on its polypeptide components. A functional DES comprises at least one of the following combinations of polypeptides:

DdmD and DdmE;

DdmA, DdmB and DdmC; or

DdmD and DdmE, DdmA, DdmB and DdmC.

The expression of DdmD and DdmE polypeptides together in a cell is required to eliminate plasmid, while the DdmA, DdmB and DdmC polypeptides mediate elimination of bacteriophage. The latter also enhance the anti-plasmid function of DdmD and DdmE when expressed together at low, physiological levels, for example, as they occur naturally in V. cholerae.

Homologues of the Ddm polypeptide sequences provided herein are similarly encompassed by the invention, and may have significantly lower sequence similarity, while maintaining essential functional domains and associated functions. Naturally occurring homologues, variants, or modified recombinant polypeptide sequences similar (e.g., at least about 70% sequence similarity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence similarity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

In the context of the present specification, the terms, sequence similarity, sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. Unless stated otherwise, sequence similarity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

The term expression vector in the context of the present specification relates to a polynucleotide, for example a plasmid, a linear DNA strand, a transposon, or a viral genome, which is used to transform (in case of a plasmid), transpose (in case of a transposon), or transduce (in case of a viral genome) a target cell with a certain gene, or genes of interest. In the case of a DNA expression vector, or expression construct, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell. Thus, the gene of interest is transcribed either constitutively or in response to a stimulus, or dependent on the cell’s status. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.

Typically, an expression vector comprises cis-regulatory sequences, such as transcription and translation initiation and termination sequences. In certain embodiments, the expression vector comprises a plurality of cloning sites for inserting sequences encoding the polypeptides according to the invention within operable distance of the regulatory sequences described above. In particular embodiments, all Ddm components of a functional DES are inserted together in a single operon. In particular embodiments, selection markers are incorporated into the expression vector according to the invention in order to isolate cells which have acquired the desired function.

The term plasmids in the context of the present specification relates to autonomously replicating, extra- chromosomal DNA molecules, which are common vehicles for horizontal gene transfer (HGT), and are ubiquitous throughout the bacteria, conferring a wide range of adaptive and niche-specific traits. Plasmids are circular, double-stranded (ds) DNA molecules, naturally present in many bacteria, and can also be generated artificially. Replicative plasmids comprise an origin of replication, or Ori site and usually at least one gene. Some plasmids encode a conjugative apparatus, or transfer genes to allow transfer of genetic material between cells, for example by means of a mating pilus, or membrane fusion, which can be adjusted to permit transfer to, for example, gram-negative or gram-positive recipients.

The term having substantially the same biological function, or biological activity in the context of the present invention relates to either one, or both possible functions of a DES, and depends on the constituent polypeptides it comprises according to the various aspects and embodiments on the invention. A first biological function is plasmid elimination, meaning removal of intracellular plasmid (mediated by a DES comprising DdmD and DdmE, in some settings enhanced by co-expression of DdmA, DdmB and DdmC). A second, distinct biological function is elimination of bacteriophages from a bacterial population (mediated by a DES comprising DdmA, DdmB and DdmC polypeptides). Together, these two functions lead to a broad resistance to the maintenance of extra-genomic DNA in a bacterium expressing a functional DES comprising both systems. A biological activity of an expression vector comprising all DdmA, B, C, D, and E polypeptides must meet the threshold provided for both functions, plasmid elimination, and phage elimination. Measures of biological function, or activity can be assessed over successive generations of bacterial replications. Generation time varies based on multiple features, for example the bacterial species which is the recipient of the DES system, or the conditions of growth (e.g., growth medium, aeration, temperature etc). Taking E. coli as a representative example, generation times are estimated to range from approximately 15 minutes to 1 hour under laboratory conditions, and up to 12-24 hours in the gastrointestinal tract. It is understood that where the term “having substantially the same biological function” is applied to a variant of a particular genetic construct, it is the function of the original genetic construct that the comparison refers to.

The term plasmid elimination or plasmid reduction refers to the function mediated by any DES comprising at least a functional DdmD and DdmE polypeptide. Plasmid elimination may be measured with an assay that determines the relative, or absolute amount of an intracellular plasmid in a bacterial cell, or population of cells over time, or relative to an identical cell, or cell population lacking said DES system. This may apply to both episomal plasmids, and plasmids which incorporate into the genome, before such a genomic integration event occurs. Measures of plasmid elimination are demonstrated in the examples, and include, without being limited to, use of plasmid-specific primers in a quantitative real time polymerase chain reaction (qPCR) assay to quantify the amount of plasmid DNA present in a sample, compared to a DNA standard. Reduced indirect measures of a functional characteristic not present in a host bacterium, such as antibiotic resistance, a metabolic capacity, or genetic stability, may also be used to measure the amount of plasmid present in a sample, as demonstrated in the methods in the Plasmid stability assay, explained further under Assay to measure the biological function of plasmid elimination. Indirectly labelled plasmid can also be visualised and quantified using light microscopy (Fig. 10).

In some embodiments, expression of a functional DES confers at least a 30% reduction in the amount of plasmid in daughter bacteria cells/a bacterial preparation within 50 generations (cell replication cycles) compared to a bacterial cell of identical genetic configuration but lacking the DES. In particular embodiments, a 30% reduction in plasmid is achieved within 10 bacterial generations. This is equivalent to approximately one third of the almost complete elimination of plasmid achieved by expression of a DES comprising a DdmD and DdmE polypeptide, in either V. cholerae, or E. coli in the examples within 10 bacterial generations (where the DES were expressed under arabinose-inducible promoters, with the addition of arabinose as an inducing factor). This plasmid-elimination function was effective against a vast majority of tested plasmids. One caveat demonstrated in the examples is that plasmids which carry a trait absolutely required in the relevant conditions, such as antibiotic resistance, may be maintained longer in a population while the plasmid confers a selective advantage. For example, a subset of antibiotic-sensitive bacterial cells may retain a plasmid carrying antibiotic resistance while said antibiotic is present, despite expression of a functional DES. In this case, it is understood that removal of the plasmid from the bacterial population requires suspension of exposure to the antibiotic, as demonstrated in Fig. 1.

Assay to measure the biological function of plasmid elimination

The expression of at least functional DdmD and DdmE polypeptides together in a cell is required to eliminate plasmids. The inventors demonstrate that transformation of E. coli with a plasmid carrying nucleic acid sequences encoding the DdmD (SEQ ID NO 001) and DdmE (SEQ ID NO 002) polypeptides under control of a conditional promoter operable in E. coli (the PBAD promoter permitting dose-dependent inducible gene activation in response to arabinose), results in the elimination of the plasmid from >99% of cells, and results in a similar reduction in plasmid yield from plasmid DNA extractions performed on the recipient bacteria. The inventors further demonstrate that a chromosomally integrated construct with nucleic acid sequences encoding the DdmD (SEQ ID NO 001) and DdmE (SEQ ID NO 002) polypeptides, under the control of a conditional promoter operable in E. coli (the PBAD promoter permitting dose-dependent inducible gene activation in response to arabinose), is sufficient to eliminate plasmids encoding a variety of different origins of replication from >90-99% of cells. In addition, variant DdmD and DdmE polypeptides, or homologue proteins are encompassed by the invention, with the proviso that at least 30% of the biological function of a functional operon encoding both DdmD SEQ ID NO 001 , and DdmE SEQ ID NO 002 is retained. In other words, a candidate DdmD polypeptide can be at least 70% identical to SEQ ID NO 001 , or can be a homologue of SEQ ID NO 001 , and/or a candidate DdmE polypeptide can be at least 70% identical to SEQ ID NO 002, or a homologue of DdmE, as long as expression of both together confers at least a 30% reduction in plasmid in a recipient bacterial population. The following assay is provided to measure the biological function of a candidate DdmD or DdmE polypeptide.

In brief, a test plasmid carrying an antibiotic resistance gene is used to determine plasmid stability in an E. coli strain with a chromosomally integrated construct containing both 1) and 2) under the operational control of a conditional promoter such as the PBAD promoter:

1) a nucleic acid encoding: a V. cholerae DdmD polypeptide having the sequence SEQ ID NO 001 , or a variant polypeptide at least 70% identical to SEQ ID NO 001 ; or a homologue of said V. cholerae DdmD polypeptide, or a variant polypeptide at least 70% identical to the homologue of said V. cholerae DdmD polypeptide; and 2) a nucleic acid encoding: a V. cholerae DdmE polypeptide having the sequence SEQ ID NO 002, a variant polypeptide at least 70% identical to SEQ ID NO 002; or a homologue of said V. cholerae DdmE polypeptide, or a variant polypeptide at least 70% identical to said homologue of said V. cholerae DdmE polypeptide.

Using standard genetic methods, the test plasmid is introduced into a culture of the suitable E. coli strain MG1655 with a chromosomally integrated construct containing both 1) and 2) under the operational control of the conditional PBAD promoter, followed by antibiotic selection for the transformed recipients. Persistence of the test plasmid in the E. coli strain is then evaluated after growth for 10 generations in the absence of antibiotic selection, either with or without the addition of 0.2% arabinose to induce the expression of 1) and 2). Plasmid stability is calculated as the percentage of antibiotic resistant (i.e. plasmid-carrying) clones, as determined by the number of individual bacterial colonies on replica agar plates with and without the relevant antibiotic to which the plasmid confers resistance. Sufficient biological function of plasmid elimination a DdmD or DdmE candidate is confirmed if either the plasmid stability obtained from transformed E. coli grown in the presence of arabinose, is at least 30% lower than the plasmid stability of matched cultures grown without arabinose.

A second biological function carried out by a functional DES according to the invention, is referred to by the terms bacteriophage elimination, bacteriophage reduction, phage elimination, or anti-phage defence. Bacteriophage elimination can be defined as a reduction within a bacterium, or a bacterial population, in a measure of phage genetic material, or by a reduction of a measure of phage function, such as lysis in the case of a lytic phage. The term bacteriophage elimination, or bacteriophage resistance refers to an ability to destroy bacteriophage DNA once it has entered the cell, or replication inhibition of the phage’s genetic material (e.g., abortive infection) resulting in reduced incorporation of phage genetic material into the cell, and/or reduced bacteriophage-mediated lysis of the bacterium, or the bacterial population, in the case of a lytic bacteriophage. Increased genetic stability may be another feasible measure of anti-bacteriophage function.

One representative method for measuring bacteriophage elimination is demonstrated in the Bacteriophage plaque assay described in the examples, which can detect the reduced lysis of DES- carrying bacteria infected with lytic bacteriophages. The data in the examples demonstrates that a 1000- fold reduction of lysis by P1 or lambda bacteriophages, is achieved upon expression of a DES comprising DdmA, DdmB and DdmC polypeptides in E. coli (Fig. 4 expressed under control of an arabinose-inducible promoter, in the presence of arabinose). 10% of this effect, i.e., a 100-fold reduction in plaque formation may be of use in an industrial setting. Thus, one threshold for sufficient antibacteriophage function of a DES measured by this method, is at least a 100-fold reduction in plaque formation in a bacterial preparation comprising lytic bacteriophages, when compared to a bacterial preparation of identical genetic configuration, but lacking a DES comprising functional DdmA, DdmB, and DdmC polypeptides. In particular embodiments of the DES mediating resistance to bacteriophages according to the invention, a DES comprising a recombinant variant, or homologue Ddm polypeptide sequence, can be classified as retaining the biological function of a DES comprising V. cholera polypeptides as tested in the examples, if expression of the DES confers a 100-fold or more reduction in plaque formation on a bacterial population exposed to lytic bacteriophages.

Assay to measure the biological function of abortion of bacteriophage infection

The biological function of a DdmA, DdmB and DdmC polypeptide expressed together is primarily the abortion of bacteriophage infection. An assay to measure the biological function of a candidate DdmA, DdmB or DdmC polypeptide according to the invention is as follows. In brief, the efficiency of bacteriophage infection is determined using an E. coli strain with a chromosomally integrated construct containing nucleic acid sequences encoding a 1) DdmA, 2) DdmB and 3) DdmC polypeptide under the operational control of a conditional promoter such as the PBAD promoter, permitting dose-dependent inducible gene activation in response to arabinose, as follows:

1) a nucleic acid encoding: a V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003, or a variant polypeptide at least 70% identical to SEQ ID NO 003; or a homologue of said V. cholerae DdmA polypeptide, or a variant polypeptide at least 70% identical to said homologue of the V. cholerae DdmA polypeptide; and

2) a nucleic acid encoding: a V. cholerae DdmB polypeptide having the sequence SEQ ID NO 004, a variant polypeptide at least 70% identical to SEQ ID NO 004; or a homologue of said V. cholerae DdmB polypeptide; or a variant polypeptide at least 70% identical to said homologue of V. cholerae DdmB polypeptide, and

3) a nucleic acid encoding: a V. cholerae DdmC polypeptide having the sequence SEQ ID NO 005, or a variant polypeptide at least 70% identical to SEQ ID NO 005; or a homologue of said V. cholerae DdmC polypeptide, or a variant polypeptide at least 70% identical to said homologue of the V. cholerae DdmC polypeptide.

Using standard genetic methods, a chromosomally integrated construct containing nucleic acid sequences encoding 1), 2) and 3) under the operational control of the conditional PBAD promoter, is first introduced into the E. coli strain MG1655, before being used to assess protection against bacteriophage infection using a plaque assay. Prey E. coli are grown at 37°C with shaking for 2 hours in Lysogeny Broth (LB) medium, in either the absence or presence of arabinose. Exponentially growing cultures are then diluted 1 :40 in a molten top agar (LB + 0.5% agar supplemented with 5 mM CaCL, 5 mM MgCL - /+ 0.2% arabinose), poured on top of a bottom layer of pre-solidified LB + 1 .5% agar, and allowed to dry for 1 h. Protection against bacteriophage infection is then determined by preparing a dilution series for a panel of the E. coli bacteriophages P1 in LB + 5 mM CaCL, 5 mM MgCL and spotting 5 pi of each of the serial dilutions onto the seeded plates. Plates are then incubated overnight (~ 16-18 h) at 37°C, and the fold-protection determined by counting the number of plaques present on cultures of prey E. coli grown with and without induction of 1), 2), and 3), at a dilution where individual plaques can be clearly distinguished. Fold protection is calculated as the ratio of the number of bacteriophage plaques formed on the non-induced prey E. coli control (no arabinose) to the number of bacteriophage plaques formed on the prey E. coli grown with inducer (arabinose) expressing 1), 2) and 3). The biological function of bacteriophage protection is confirmed if the fold-protection is determined to be at least 100-fold.

Detailed Description of the Invention

DNA defence molecule (Ddm) components of DN A elimination systems (DES)

A first aspect of the invention is an expression vector comprising a polynucleotide that encodes a functional DES, said DES comprising a functional DdmD polypeptide, and a functional DdmE polypeptide. According to this aspect of the invention, the function of the DES component polypeptides DdmD and DdmE when co-expressed in a bacterial cell, is the reduction, or elimination of plasmid from said cell.

A second aspect of the invention is an expression vector comprising a polynucleotide which encodes a functional DES, said DES comprising a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide. Co-expression of these three polypeptides in a bacterial cell provides a DES which confers the ability to reduce, or eliminate bacteriophages from a bacterial cell, or bacterial population, expressing said DES.

In certain embodiments, the expression vector encodes a functional DES, comprising a DdmD and DdmE polypeptide as specified above, and additionally comprises a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide. The addition of the latter was demonstrated to enhance the function of DdmD, and DdmE expressed at low, physiological levels, and also conferred an additional anti-bacteriophage function. The function of a DES comprising these five polypeptides co-expressed in a bacterium, or a bacterial population, is the reduction, or elimination, of a broader selection of extra-genomic DNA forms, encompassing both plasmids (targeted by DES comprising a DdmD polypeptide and a DdmE polypeptide but also DdmA, DdmB, and DdmC when produced at higherthan physiological levels in certain bacteria), and bacteriophages (eliminated by DES comprising a DdmA, DdmB, and DdmC polypeptides).

In some embodiments of the invention, the expression vector encodes a DES comprising a functional DdmA, DdmB, DdmC, DdmD or DdmE polypeptide derived from Vibrio cholerae (V. choierae), as present in V. cholerae strains characterised by the presence of a complete, non-truncated Vibrio pathogenicity island 2 and a Vibrio seventh pandemic island II ( VPI-2 and VSP-II). Within the currently known examples of the Vibrio genus, both ddmDE and ddmABC are present in the 7th pandemic V. cholerae strains of the 01 El Tor biotype. Non-limiting examples of 01 El Tor V. cholerae strains comprising genes encoding the Ddm that provide a functional DES include, but are not limited to, the representative V. cholerae strains A1552, N 16961 , C6706, E7946, P27459, or DRC-193A V. cholerae strains demonstrated to express these polypeptides in the examples. In other embodiments, the vector encodes a DES comprising a functional artificial, or natural variant of the V. cholerae DdmA, DdmB, DdmC, DdmD or DdmE polypeptide sequences, having a sequence similarity of (>) 70%, >75%, >80%, >85% compared to the V. cholerae sequence to which it is related. In particular embodiments, the variant polypeptide is >90% similar. In more particularly embodiments, the variant has a sequence identity of >95% compared to one of said V. cholerae polypeptides. According to such embodiments, amino acids have been exchanged, deleted, or inserted, with the proviso that the variant protein retains at least of 30% of the function of clearing plasmids and/or at least 10% reduction bacteriophage achieved by the equivalent V. cholerae- derived Ddm polypeptide sequences, when co-expressed with the specified Ddm partner polypeptides from an expression vector in a bacterial cell.

In certain embodiments, the expression vector encodes a DES comprising a naturally occurring homologue of a V. cholerae DdmA, DdmB, DdmC, DdmD or DdmE polypeptide.

The term homologue in the context of the present specification relates to naturally occurring Ddm polypeptide present in a bacterial species other than V. cholerae, having a similar sequence, for example at least 70% sequence identity, and equivalent biological activity. In some cases, sequence identity of less than 70% might lead to the same biological activity in a homologue, if amino acids with similar properties are exchanged in the same region of the protein sequence, particularly if key functional domains are conserved. Alternatively, a homologous protein might be identified by a shared gene arrangement and regulatory structure, such as the DdmDE operon in certain Lactobacillales species, or on shared motifs, for example the domain of unknown function (DUF) DUF3732, broadly present among Proteobacteria. Homologues may be identified, for example, by using a V. cholerae Ddm protein sequence (for example DdmD: SEQ ID NO 001 , DdmE: SEQ ID NO 002, DdmA: SEQ ID NO 003, DdmB SEQ ID NO 004, or DdmC: SEQ ID NO 005) to query a freely available sequence similarity online search tool, as in the methods section headed Bioinformatic Analysis. Databases available at - Basic Local Alignment Search Tool = BLAST (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi), Pfam (http://pfam.xfam.org, Mistry J. et at. 2020 Nucleic Ac. Res. doi: 10.1093/nar/gkaa913), or STRING (https://string-db.org/cgi/about) may be used to identify conserved motifs in naturally occurring Ddm homologues.

Ddm Polypeptide Functional Domains

To identify homologues of Ddm proteins suitable for incorporation into a DES according to the invention using homology databases such as Pfam, the presence of canonical domains, motifs, or gene placement in operons may be used to identify proteins with a conserved function, as demonstrated in Fig. 11. In the nomenclature used herein, an x denotes any amino acid, an h denotes any hydrophobic amino acid, and amino acids use the single letter code: glycine (G), lysine (K), alanine (A), threonine (T), Aspartic acid (D), glutamic acid (E), glutamine (Q), arginine (R), proline (P).

Naturally occurring homologues of V. cholerae DdmD and DdmE polypeptides are often encoded together in an operon of 2 or more genes. DdmD polypeptides contain a predicted N-terminal helicase domain (or domains), containing motifs consistent with a Superfamily 2 helicase designation (i.e. Motif I; a Walker A “GxGKT”-type motif (SEQ ID NO 006), Motif II; a Walker B “h₄DExD”-type motif (SEQ ID NO 007), Motif III; a SAT motif and Motif VI; an arginine finger, such as a “QxxGRxxR”-type motif (SEQ ID NO 008), in addition to a C-terminal domain containing a “PD-(D/E)xK”-type nuclease motif (SEQ ID NO 009). In some embodiments, a ddmE gene is located downstream of a ddmD gene. In some embodiments, the DES encodes a DdmE protein that exhibits predicted structural similarity to Argonaute proteins.

The following features may aid identification of a naturally occurring DdmA, DdmB and/or DdmC polypeptide according to the invention. DdmA, DdmB, and DdmC are often encoded together in an operon of 3 or more genes. DdmA polypeptides contain an N-terminal domain with similarity to a DUF4297 domain, and also a nuclease domain comprising a “PD-(D/E)xK”-type motif (SEQ ID NO 009). DdmC polypeptides exhibit predicted structural similarity to structural maintenance of chromosome (SMC) proteins, containing an N-terminal SMC_N like domain with a Walker A “Gx4GKS/T”-type motif (SEQ ID NO 010), and a C-terminal domain containing a DUF3732 domain, which is characterised by a predicted inactive Walker B “tuDQ’-like motif (SEQ ID NO 011). The two domains are separated by >2 predicted coiled-coil regions.

The inventors show that a DES comprising DdmD variants generated to inactivate either predicted DdmD helicase activity (for example either of the Walker A; K55A, or Walker B; E273A mutations tested on the DdmD polypeptide in the Fig. 11), or nuclease activity (for example, the mutation of the “PD- (D/E)xK” motif (SEQ ID NO 009); K1102A) fail to mediate plasmid elimination. Likewise, A DES comprising DdmA variants generated to have perturbed activity of a predicted nuclease region (for example, disturbing the “PD-(D/E)xK” motif (SEQ ID NO 009) with a substitution of K to A) or DdmC variants lacking a predicted ATP-binding function (for example, bearing a substitution inactivating walker A, such as the K40A substitution shown in Fig. 11) cannot mediate bacteriophage protection according to the invention.

The term catalytic applied to an amino acid residue as used in the current specification refers to an amino acid residue demonstrated to be essential for a protein’s, or a canonical domain’s, predicted function. This residue is located within a motif which characterises said canonical domain or protein, and can be identified using standard methods of homology matching. The most common motifs that characterise a domain, or protein type, are listed in this specification for the purpose of informing a homology search which may identify a Ddm homologue according to this invention. One skilled in the art will recognise that similar, rarer motifs, may also comprise the catalytic residue according to the invention, and that certain motifs are more common in specific protein families. For example, SMC-like proteins generally comprise a Walker A domain with a “Gx4GKS/T”-like motif (SEQ ID NO 010), where the last amino acid residue may be wither S or T, whereas superfamily II helicase domains more commonly contain a similar Walker A “GxGKT”-like motif (SEQ ID NO 006) terminating in a T. In addition, conservative amino acid substitutions to the amino acids which surround the catalytic residue will provide similar motifs, which may also identify a catalytic residue according the invention.

In some embodiments of the expression vector according to the invention, it encodes a DES comprising a DdmD polypeptide which comprises a functional superfamily II helicase domain, and in addition, a functional nuclease domain. In some embodiments, the DdmD functional superfamily II helicase domain comprises both a functional Walker A motif, and a functional Walker B motif. In particular embodiments, the helicase domain is positioned N-terminal relative to the functional nuclease domain.

In some embodiments of the invention, the functional Walker A motif comprised within the DdmD helicase domain is a Walker A motif typical of superfamily II helicase domains, comprising a catalytic K residue. In particular embodiments, the Walker A motif is a “GxGKT”-type motif (SEQ ID NO 006). The presence of such a functional motif within the DdmD polypeptide can be confirmed, for example by disturbance of the catalytic K with an amino acid mutation, for example, by substitution of the catalytic K in the Walker A motif with an A. In particular embodiments of the DES according to the invention, performing a K to A mutation within a Walker A motif within the DdmD polypeptide, is able to abolish the capacity to eliminate plasmids from a cell expressing such a mutant DES.

In some embodiments, the functional superfamily II helicase within the DdmD polypeptide comprises a functional Walker B motif typical of superfamily II helicase domains, comprising a catalytic E residue. In particularly embodiments, the Walker B motif is a “huDExD ype motif (SEQ ID NO 007). In more particular embodiments, performing a substitution mutation in the DdmD polypeptide to exchange the catalytic E in the functional Walker B motif with an A, abolishes the function of plasmid elimination from a cell expressing such a mutant DES.

In other embodiments of the expression vector encoding a DES according to the invention, the DES comprises a DdmD polypeptide, in which the functional superfamily II helicase comprises an “SAT” motif. In some embodiments, the DdmD polypeptide is characterised by an arginine finger structure comprising a “QxxGRxxR’-type motif. (SEQ ID NO 008)

In further embodiments of the expression vector encoding a DES according to the invention, the DES comprises a DdmD polypeptide, in which the functional nuclease domain comprises a catalytic K residue located within a “PD-(D/E)xK”-type motif (SEQ ID NO 009). In such embodiments, performing a substitution mutation of said catalytic K in the functional nuclease domain to replace it with an A, abolishes the capacity to clear plasmid from a cell expressing said mutant DES.

In other embodiments of the expression vector encoding a DES according to the invention, the vector encodes a DdmA polypeptide which comprises a functional nuclease domain. In particular embodiments, the functional nuclease domain of the DdmA polypeptide comprises a “PD-(D/E)xK”-type motif (SEQ ID NO 009). In such embodiments, a substitution of the catalytic K in the “PD-(D/E)xK”-type motif (SEQ ID NO 009) with an A, abolishes the function of a DES comprising said mutant DdmA when expressed in a bacterial cell, either enhancement of plasmid clearance, or particularly, protection from bacteriophages.

In further embodiments of the vector encoding a functional DES, it encodes a DdmC polypeptide which is characterised by structural similarity to the ATP-binding SMC family of proteins. In particular embodiments, the DdmC polypeptide comprises both a functional Walker A domain, and a DUF3732 domain, wherein the functional Walker A domain and the DUF3732 domain are located on either side of a coiled-coil-containing region. In certain embodiments, the DdmC polypeptide comprises a functional Walker A domain typical of the SMC family of proteins. In particular embodiments, the DdmC polypeptide comprises a “GxxxxGK-(S/T)”-type Walker A motif (SEQ ID NO 010), where substitution of a catalytic K in the functional Walker A domain with an A, abolishes the ability of the encoded DES to protect a cell against bacteriophages. In other embodiments, the DdmC polypeptide comprises a DUF3732 domain containing an inactive Walker B domain characterised by a “huDCT-type motif (SEQ ID NO 011).

Bacterial Ddm polypeptides

In certain embodiments of the expression vector according to the invention, it encodes a functional DES comprising a Ddm polypeptide derived from a member of class Gamma proteobacteria, such as a member of the order Vibrio nates.

In other embodiments, the vector encodes a functional DES comprising a DdmD, or DdmE polypeptide derived from a member of Firmicutes, such as members of the order LactobaciHales.

In certain embodiments, the expression vector encodes a DES comprising a naturally occurring homologue of V. cholerae DdmD. In particular embodiments, the DES comprises a homologue derived from Vibrionaceae, Lactobacillaceae, Enterococcaceae, or is a polypeptide sharing at least 70% sequence similarity with said homologues.

In particular embodiments, the DdmD polypeptide, and/or DdmE polypeptide sequence is derived from a member of Lactobacillaceae, such as Lactobacillus casei, or Lactobacillus rhamnosus, as in these species, the genes encoding DdmD and DdmE homologues are present in a similar arrangement to the related V. cholerae genes, leading the inventors to surmise they are highly likely to retain the same function (Fig. 8 and 9).

In other embodiments, the functional DES comprises a DdmD or DdmE polypeptide which is a homologue of V. cholerae DdmD or DdmE, derived from a species of the order Vibrionales (for example Ddm polypeptides present in Vibrio vulnificus (strain YJ016), Vibrio coralliilyticus, or Aliivibrio iogei). In alternative embodiments, the Ddm polypeptide is derived from a member of the order LactobaciHales as shown in Table 1 (such as those present in Lactobacillus hammesii strain DSM 16381).

In certain embodiments, the expression vector encodes a DES comprising a naturally occurring homologue of V cholerae Ddm, derived from Vibrionales, Alteromonadales, Oceanospirillales, Pseudomonadales, or Clostridiales, or is a polypeptide sharing at least 70% sequence identity with said homologues.

In particular embodiments, the expression vector encodes a DES comprising a functional DdmD, and/or DdmE polypeptide sequence derived from Vibrionaceae, or Lactobacillaceae, where the homologous gene arrangement resembles the V cholerae operons, suggesting they are highly likely to retain the same function. The taxonomic analysis in Fig. 8, and Fig. 9 of the examples, illustrates homologues which closely resemble the sequence of the functional DdmE and DdmD polypeptides of V. cholerae. Among these, references to annotated homologues are provided for representative species in Table. 1 .

In some embodiments of the expression vector according to the invention, it encodes a DES comprising a functional Ddm polypeptide which is a naturally occurring homologue of a V. cholerae Ddm polypeptide. In particular embodiments, it encodes a DES comprising a Ddm homologue derived from the taxonomic groups expressing DdmA, DdmB or DdmC identified in the examples: • Gamma proteobacteria, particularly a member of Gamma proteobacteria selected from a member of Pseudomonadales, Vibrionales, Enterobacterales, Xanthomonadales, Pasteurellales, Alteromonadales, Thiotrichales, Methylococcales, Oceanospirillales, or Aeromonadales ;

• Alpha proteobacteria, particularly a member of Alpha proteobacteria selected from Rhizobiales, Rhodobacterales, Rhodospirillales, Sphingomonadales, or Caulobacterales ;

• Betaproteobacteria, particularly a member of Betaproteobacteria selected from Burkholderiales, Rhodocyclales, or Nitrosomonadales,

• Deltaproteobacteria,

• Epsilon proteobacteria;

• Bacilli;

• Clostridia;

• Actinobacteria, particularly a member of Actinobacteria selected from Streptomycetales, Micrococcales, Cornynbacteriales, Propionibacteriales, Micromonosporales, or Pseudonocardiales ;

• Bacteroidia,

• Flavobacteriia,

• Sphingobacteriia,

• Cytophagia or

• Chitinophagia

In other embodiments of the expression vector encoding a DES, the DES comprises a functional DdmA, DdmB and/or DdmC polypeptide encoded by a gene present in a member of the order Enterobacterales, for example, Salmonella enterica. For example, Table 1 provides references to the annotated homologues of the DdmA, DdmB, and DdmC polypeptides present in subspecies enterica serovar Tennessee strain TXSC_TXSC08-19.

In other embodiments, the expression vector encodes a DES comprising a functional DdmA, DdmB and/or DdmC polypeptide, derived from a naturally occurring polypeptide present in a member of the Pseudomonadales order, for example, Acinetobacter baumannii. Table 1 of the examples provides, for example, references to the annotated homologues of the DdmA, DdmB, and DdmC polypeptides present in the strain 1525283.

In other embodiments, the expression vector encodes a DES comprising a functional DdmA, DdmB and/or DdmC polypeptide encoded by a naturally occurring plasmid, for example a plasmid found in a member of the Rhizobiales order, including, but not limited to, the homologues present in plasmids of Agrobacterium tumefaciens in the Rhizobiaceae family.

In other embodiments, the expression vector encodes a DES comprising a functional DdmA, DdmB, DdmC, DdmE or DdmE polypeptide with a sequence at least 70% similar to a naturally occurring homologue derived from a taxonomical group as specified above, or a species noted in the bioinformatic analysis identifying homologues in the examples. In other embodiments, the functional DES according to the invention comprises a functional, artificial, or recombinant variant of a naturally occurring homologue of a V. cholerae Ddm polypeptide, having an sequence similarity of at least (>) 70%, >75%, >80%, >85% to the homologue of the V. cholerae Ddm polypeptide from which it is derived. In particular embodiments, the sequence similarity of the variant is >90%. In more particular embodiments, the variant has a sequence similarity of >95%, compared to said homologue of a V. cholerae Ddm polypeptide.

In particular embodiments, the expression vector encodes a DES comprising a functional DdmA, DdmB, DdmC, DdmD, and/or DdmE polypeptide sequence, which is derived from a naturally occurring protein in a member of genus Vibrio. In particular embodiments, the DES comprises a Ddm polypeptide occurring naturally in V. cholerae. In more particular embodiments, the DES comprises a Ddm polypeptide occurring naturally in a V. cholerae 01 El Tor strain, including, but not limited to, homologues listed in Table 1 and/or the bioinformatics analysis in the Examples.

In particular embodiments of the expression vector encoding a functional DES according to the invention, the DES comprises a DdmD polypeptide with the sequence SEQ ID NO 001. In other particular embodiments, the DES comprises a DdmE polypeptide of the sequence SEQ ID NO 002. In other particular embodiments, the DES comprises a DdmA polypeptide of the sequence SEQ ID NO 003. In further particular embodiments, the DS comprises a DdmB polypeptide of the sequence SEQ ID NO 004. In further particular embodiments, the DES comprises a DdmC polypeptide of the sequence SEQ ID NO 005. In still more particular embodiments, the DES comprises the two polypeptides designated SEQ ID NO 001 and SEQ ID NO 002. In other particular embodiments, the DES comprises the three polypeptides designated SEQ ID NO 003, SEQ ID NO 004, and SEQ ID NO 005. In further particular embodiments, the DES comprises the five polypeptides designated SEQ ID NO 001 , SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, and SEQ ID NO 005.

In other embodiments of the expression vector according to the invention, it encodes a DES comprising a Ddm polypeptide which is a variant of one of the V. cholerae- derived Ddm polypeptides specified in the paragraph above, having a sequence >70%, >75%, >80%, >85% or even >90%, similar to said V. cholerae protein. In particular embodiments, said variant is >95% similar to at one or more polypeptides selected from SEQ ID NO 001 , SEQ ID NO 002, SEQ ID NO 003, SEQ ID NO 004, or SEQ ID NO 005.

In some embodiments of the expression vector encoding a DES, it comprises Ddm polypeptides which are all naturally occurring V. cholerae proteins.

In other embodiments, the expression vector encodes a DES comprising at least one Ddm polypeptide with >70% sequence identity of a V. cholerae Ddm polypeptide.

In further embodiments, the expression vector encodes a DES comprising at least one homologue of a naturally occurring V. cholerae Ddm polypeptide derived from another bacterial species.

In further embodiments, the expression vector encodes a DES comprising at least one Ddm polypeptide with >70% identical sequence to a V. cholerae Ddm polypeptide, or a naturally occurring homologue of a V. cholerae Ddm polypeptide. In particular embodiments of the expression vector encoding a functional DES according to the invention, different embodiments of functional DdmA, DdmB, DdmC, DdmD, and DdmE polypeptides listed in the section Bacterial Ddm polypeptides are combined, to provide a DES which has >30%, particularly >40%, more particularly >50% of the function of reducing plasmid from a bacterial cell expressing the respective V. cholerae Ddm polypeptides.

Thus, in embodiments that relate to an expression vector comprising both a functional DdmD, and a functional DdmE polypeptide, (and optionally functional DdmA, DdmB and DdmC polypeptides which may enhance DdmD and DdmE function), a bacterial cell co-expressing any chosen homologues, or variant Ddm polypeptides, has >30% of the biological function of a bacterial cell expressing V. cholerae derived equivalents. In particular embodiments, the cell has >40% of the biological function of a cell expressing a DES comprising V. cholerae Ddm polypeptides. In more particular embodiments, the cell has >50% of the biological function. The biological function according to these embodiments, is the capacity to induce a complete, or almost complete reduction in the amount of plasmid in daughter bacteria cells, ora bacterial preparation within 50 generations, each generation comprising a cell division event. In particular embodiments, plasmid is removed from the bacterial population within 10 generations. The data in the examples demonstrates that recombinant expression of a DES comprising a functional DdmD and a DdmE polypeptide, can significantly reduce the concentration of a range of plasmids characterised by a variety of Ori sites of replication.

Fig. 4 to Fig. 6 of the examples demonstrate that an E. coli bacterial population expressing a DES encoding V. cholerae DdmD and DdmE polypeptides eliminates small plasmids harbouring a variety of different RNA- or replication protein-based plasmid replication origin sequences. These data demonstrate non-limiting examples of origin of replication sequences sensitive to DES comprising a DdmD and DdmE polypeptide.

In further particular embodiments of a functional DES according to the invention, functional DdmA, DdmB, and DdmC polypeptides from differing embodiments listed under Bacterial Ddm polypeptides are combined to provide a DES which has >5%, of the function of reducing bacteriophages from a bacterial cell, or population, compared to a cell, or population of cells expressing the respective V. cholerae Ddm polypeptides. In particular embodiments, this threshold is >10%, in other words, expression of the DES confers at least a 100-fold reduction in the amount of bacteriophage in a bacterial preparation. In more particular embodiments, the threshold is >20% of the biological function of a DES comprising V. cholera Ddm polypeptides. This function is particularly relevant to bacteriophages which have a phase of their intracellular life cycle in which they are present in a circularised dsDNA form, as these plasmid-like forms are demonstrably vulnerable to elimination and/or abortive infection within cells expressing a DdmABC operon.

In particular embodiments, the expression vector encodes a DES comprising a functional DdmA, DdmB and DdmC polypeptide, and the DES induces elimination of P1 or lambda bacteriophage in a recipient cell, as demonstrated with E. coli expressing a DdmABC operon in Fig. 4 of the examples.

In certain embodiments, the expression vector encodes a DES comprising both a functional DdmA polypeptide, a functional DdmB polypeptide and a functional DdmC polypeptide, and additionally, a functional DdmD, and a functional DdmE polypeptide. A recipient bacterium expressing said DES will have broad DNA defence capacity against maintaining genetic material encoded on either plasmids, or bacteriophages.

In other embodiments, the vector is a plasmid which encodes a DES comprising a functional DdmD polypeptide, and a functional DdmE polypeptide, to provide a self-curing plasmid. The induction of expression of said functional DdmD and DdmE polypeptides from a DES-sensitive plasmid introduced into the cell, leads to elimination of the self-curing plasmid vector encoding said DES.

Expression vectors

The expression vector according to the invention encodes a DES comprising functional, multi-gene Ddm polypeptides combinations, comprised within a mobile genetic element suitable for integration and replication of the DES components within a bacterial cell. Examples of such mobile genetic elements include, without being limited to, a plasmid, a viral vector, a transposon, or in the case of a competent cell under amenable conditions (as demonstrated using V. cholerae cultures in the examples), linear DNA that can be recombined into the bacterial genome. Transfer of DNA to facilitate expression of the nucleic acids encoding recombinant Ddm may be achieved by standard molecular biology means known in the art, such as transformation of a cell with a plasmid, or infection with a virus comprising genetic material derived from the genome of a donor bacterial organism (general transduction), through suicide plasmid based recombineering, or transfer of a double strand of DNA by natural competence (the latter applicable to organisms amenable to said method, for example, B. subtilis).

In particular embodiments, the nucleic acid sequence encoding said functional DES within an expression vector is placed within operable distance of a cis-acting regulatory element enabling expression of the polypeptides components of the functional DES in a bacterial cell. The choice of cis-acting regulatory elements is not particularly limited according to the invention, and in terms of promoters, relates generally to those which enable expression in the recipient. DES expressed from known, broad-host promoters will permit functional DES expression in members of Gamma-proteobacteria, and are likely to be similarly active more broadly, for example, in members of Proteobacteria. Promoters may also be selected in order to optimize expression for gram-positive or gram-negative recipients, as the presence of homologues of both DES systems identified in both bacterial classes in the examples suggests the multigene system will be functional in most recipient bacterial species.

In particular embodiments, the expression vector comprises a nucleic acid sequence which encodes a functional DES, or DES component, within operable distance of a constitutive promoter.

In alternative embodiments, the expression vector comprises a nucleic acid sequence encoding a DES, or DES component, within operable distance of a conditional, or inducible promoter, activated by the presence of a exogenous compound, for example, the arabinose-induced promoter used in the examples. This may be particularly desirable to regulate promoter strength for vectors encoding an expression vector comprising functional DdmA, DdmB, and DdmC polypeptides, expression of which can lead to toxicity if expressed at high levels in a recipient cell in the presence of intracellular plasmid. As demonstrated in the examples, toxicity can be ameliorated by selecting a weaker promoter to drive DES expression, for example a natural promoter, or exposing a recipient cell to a lower dose of an inducible promoter activator.

In some embodiments of the expression vector according to the invention, it comprises a polynucleotide encoding said functional DES, comprised within a transmissible, self-replicating genetic element. In some embodiments, the expression vector is a plasmid. In alternative embodiments, the expression vector is a bacteriophage.

In some embodiments, the expression vector permits genomic integration of the DES into a recipient bacterium, by means oftransposons, or other standard microbiological methods mediating insertion into the genome. In some such embodiments, the vector encoding said DES comprises integrative mobile genetic elements enabling insertion into the genome, such as a transposon, or integron. In alternative embodiments, the DES is expressed from an extra-genomic expression vector. The examples included herein demonstrate the feasibility of competent uptake of linear DNA, plasmids, or plasmid comprising integrative transposon elements by V. cholerae, and E. coli recipients.

The data presented in the examples demonstrates that the polypeptide components of a functional DES need to work in concert (DdmD, and DdmE or DdmA, DdmB, and DdmC), as the systems no longer work if one of the genes of the two, orthree gene operons associated with plasmid, and/or bacteriophage clearance, are missing. However inducible expression of single genes shows that most genes can also be expressed in different order, for example, DdmC (VC0490) expressed from a transposon in a DdmC- knockout V. cholerae strain.

In particular embodiments of the expression vector according to the invention, DdmD, and DdmE polypeptides are encoded within a single DdmDE operon. In other embodiments, the DdmA, DdmB, and DdmC polypeptides are encoded within a single DdmABC operon (i.e. wherein the multiple genes encoding the Ddm comprised within each DES are transcribed downstream of a single promoter).

In alternative embodiments of the expression vector encoding a DES, the individual Ddm polypeptide components of the DES are expressed from more than one nucleic acid construct, optionally under control of different cis- regulatory regions. In further alternative embodiments, the individual Ddm genes of the expression vector encoding a DES are expressed from a combination of episomal and genomic locations, under control of different cis-regulatory regions.

The data in the examples demonstrates expression of a functional DES under control of a standard PBAD promoter, permitting dose-dependent inducible gene activation in response to arabinose. The gene complementation experiments in Fig. 2 of the examples demonstrate the individual genes of the DES may be expressed from individual constructs, or from a combination of episomal and genomic locations.

In certain embodiments, the polynucleotide encoding the functional DES is comprised within a transmissible genetic element. In particular embodiments, the DES is expressed from a self-replicating genetic element (e.g. a self-replicating plasmid). In particular embodiments, the DES is expressed from a mobile genetic element (e.g. a plasmid or transposon). Promoters

Promoters controlling expression of a functional DES according to the invention include constitutive and inducible promotors known to a microbiologist or geneticist for use in prokaryote transgene expression. These include inducible araBAD promotor commonly found in pBAD vectors, 77 derived from the 77 bacteriophage driving constitutive expression in the presence of T7 RNA polymerase, T7lac a synthetic 77 promotor including lac operators as found in pET vectors, Sp6 derived from the Sp6 bacteriophage driving constitutive expression in the presence of SP6 RNA polymerase, the trp promoter derived from E.coli driving gene repression in the presence of tryptophan, the constitutive lac promoter derived from the lac operon driving constitutive expression in the absence of lac repressors ( lac orlaclq) and inducible by IPTG or lactose, Ptac a synthetic hybrid of the lac and top promoter, pL derived from lambda bacteriophage driving temperature sensitive expression, or 73 derived from the T3 bacteriophage driving constitutive expression in the presence of T3 RNA polymerase.

In particular embodiments of the expression vector encoding a functional DES according to the invention, the expression of the DES is under control of a commonly used genetic engineering promotor, particularly a promoter selected from the list consisting of 77, T7lac, Sp6, araBAD, trp, lac, pL rpoS, PrhaBAD, tetA, tac, tacM, cspA, cspA, phyL, NBP2510, P43, Pspac, P170, and Pgrac.

Methods of obtaining a bacterium resistant to plasmid and bacteriophages

A next aspect of the invention is a method comprising contacting an isolated bacterial cell, a bacterial culture, a bacterial population, or a bacterial consortium, with a composition comprising an expression vector encoding a functional DES, said functional DES comprising a functional DdmD polypeptide and a functional DdmE polypeptide (and optionally further comprising DdmA, DdmB and DdmC polypeptides). The term contacting here refers to incubating a bacterium, or bacterial population together with said expression vector, in a manner permitting the uptake of the genetic material comprised within said vector. This contact occurs under conditions facilitating uptake of the expression vector into said isolated bacterial cell, or cells, and leads to expression of the functional DES, capable of reducing plasmid maintenance. This method may be of use to protect a bacterium or bacterial population from future exposure to an undesired plasmid, or to reduce, or eliminate an undesired plasmid that is already present. The data in the examples shows that this method protects against the maintenance of genetic material encoded in a plasmid which has been introduced by horizontal gene transfer.

In some embodiments of the method, it provides an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium capable of reducing, or eliminating intracellular plasmid. This is desirable, for example, in the context of a commercial bacterial isolate which will be used to seed a bioreactor for recombinant protein expression. In other words, to obtain a bacterial starter culture with improved genetic stability. In other embodiments, this method provides an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium, protected against future exposure to a plasmid, by preventing the maintenance of any plasmid entering the recipient cell.

In other embodiments, the method according to this aspect of the invention reduces, or eliminates intracellular plasmid from an isolated bacterium or its descendants, or from a bacterial starter culture, a bacterial population, or a bacterial consortium. This method may be desirable, for example, to reduce maintenance of, or eliminate an undesired plasmid, sometimes referred to as “parasitic plasmid”. Parasitic plasmid can reduce protein production in a commercial bioreactor bacterial culture. This method may be of use to increase the yield of a recombinant protein in an industrial fermentation setting.

In other embodiments, the method according to this aspect of the invention reduces, or eliminates plasmid-encoded antibiotic resistance in an isolated bacterium or its descendants, bacterial starter culture, bacterial population, or bacterial consortium, with the proviso that the antibiotic must be absent for the DES comprising at least a DdmD and DdmE polypeptide to remove plasmid from the cell within 10, or 50 generations.

Another aspect of the invention is a method comprising contacting an isolated bacterial cell, a bacterial culture, a bacterial population, or a bacterial consortium with a composition comprising an expression vector encoding a functional DES comprising a functional DdmA, DdmB, and a DdmC polypeptide, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, or cells. This leads to expression of a functional DES capable of protecting the cell, or cell population, against bacteriophage predation. This method offers protection from the persistence, and/or replication, lysis, or other undesired effects of bacteriophage infection on the population level.

In some embodiments of the method to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium capable of reducing, or eliminating intracellular bacteriophage, the expression of the functional DES facilitates at least 100-fold reduction in the amount of bacteriophage in a cell, or population of cells, compared to a control cell, or population lacking the functional DES. In some embodiments of this aspect of the invention, the method is used to protect an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium from bacteriophage maintenance, orto reduce bacteriophage replication. In some embodiments of this aspect of the invention, the method provides an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium resistant to bacteriophage-mediated lysis.

Another aspect of the invention is a method to create a recombinant bacteria capable of eliminating extra-genomic, circular dsDNA. This method comprises contacting an isolated bacterial cell, a bacterial culture, a bacterial population, or a bacterial consortium, with a composition comprising an expression vector (e.g., a self-replicating plasmid or a transposon-delivery vehicle) encoding a functional DES comprising both a functional DdmD and DdmE polypeptide and functional DdmA, DdmB, and a DdmC polypeptides, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, or cells. This leads to expression of a functional DES broadly capable of defence against extra- genomic dsDNA, reducing intracellular bacteriophage and plasmid maintenance.

In some embodiments of methods using an expression vector encoding a functional DES according to the invention, the bacterial recipient, or recipients of said expression vector does/do not naturally express an endogenous functional DES. In alternative embodiments of methods according to the invention, the recipient bacterium, or bacterial population does naturally express an endogenous DES comprising functional Ddm polypeptides, and transgenic expression of a DES enhances the expression level of functional Ddm polypeptides, or their activity, above their natural level. Another aspect of the invention is a method of protecting a recipient bacterium from extra-genomic, circular dsDNA, by means of transfer of an expression vector comprising a DES, from a donor bacterium, to said recipient bacterium. The method according to this aspect comprises contacting said recipient bacterium with a donor bacterium, said donor bacterium expressing an engineered transmissible genetic element encoding a functional DES, in order to transmit the transmissible genetic element from the donor to the recipient bacterium. The term contacting in reference to this aspect of the invention refers to incubating a bacterium, or bacterial population, together with a second (and sometimes, a third) bacterium or bacterial culture, so that there is contact between cells, permitting the exchange of genetic material, also known as conjugation, or more generally, horizontal gene transfer. Expression of the functional DES in the recipient bacterium will subsequently eliminate extra-genomic, circular dsDNA. In some embodiments, the recipient bacterium and the donor bacterium are not identical strains. In alternative embodiments of the aspect of the invention specified above, the recipient bacterium and the donor bacterium are identical strains.

In alternative embodiments, the expression vector encoding a function DES is transferred from one bacterium, to another, by means of triparental mating known in the art. According to this embodiment, a first bacterial helper strain is present which contains a conjugative plasmid encoding a transposase. Said transposase assists the transfer of a mobilizable, transferable transposon from a second donor bacterial strain, into a third recipient bacterial strain, as demonstrated in Fig 4 of the examples herein. The three bacterial strains may be the same, or different strains.

In particular embodiments of the method according to this aspect of the invention, the DES comprises a functional DdmD polypeptide and a functional DdmE polypeptide, and said recipient bacterium acquires an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES. In other particular embodiments of this aspect of the invention, the DES comprises a functional DdmA polypeptide, a functional DdmB polypeptide and a functional DdmC polypeptide, and said recipient bacterium acquires an improved capacity to eliminate bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES. In further particular embodiments of this aspect of the invention, the DES comprises both a functional DdmD polypeptide and a functional DdmE polypeptide, and a functional DdmA polypeptide, a functional DdmB polypeptide and a functional DdmC polypeptide, and said recipient bacterium acquires an improved capacity to eliminate bacteriophage and plasmid compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES comprising all five genes.

Isolated bacteria expressing a recombinant DES

The capacity of the Ddm systems according to the invention to deplete extra-genomic DNA from bacterial cytoplasm means that an extra-genomic plasmid or phage conferring expression of the genes is necessarily “self-curing”, i.e. removed once the functional Ddm is expressed. In industrial settings, for example, a lactic acid fermenting bacteria in a food manufacturing environment, or a genetically modified recombinant bacteria expressing for example, insulin, chromosomal incorporation of the Ddm polypeptides according to the invention can obtain stable DNA-defense function. Thus, recombinant isolated bacterium expressing a functional DES, encompassing commercially available bacteria characterized by chromosomal incorporation of a recombinant polynucleotide encoding a functional DES, and in compliance with safety and containment limitations placed on organisms classed as genetically modified organisms for industrial or medical applications are encompassed by the current invention.

Another aspect of the invention relates to an isolated bacterium, genetically modified to express a functional DES, in other words a recombinant bacterium intentionally modified to express a functional DES not present naturally in said bacterium. In other words, a transgene encoding said DES, a foreign polynucleotide not naturally present in a bacterial cell of that species. For example, an isolated bacterium comprising a transmissible genetic element (e.g., a plasmid, ortransposon) encoding a functional DES, or having said functional DES inserted into its genome. In some embodiments, said isolated bacterium comprises recombinant Ddm polypeptides, but no longer comprises the polynucleotide from which these functional DES polypeptides were expressed, such as, for example, an isolated bacterium which has acquired a functional DES by means of a self-curing plasmid, after which said self-curing plasmid has subsequently been eliminated from the cell.

Intentionally recombinant bacteria comprising a synthetic plasmid comprising a polynucleotide encoding a functional DES can be distinguished from a DES acquired by horizontal gene transfer by a skilled practitioner, by the possession of a synthetic plasmid DNA backbone comprising cloning restriction endonuclease sites that does not occur naturally in said bacterium. In particular embodiments, the polynucleotide encoding the DES is within a plasmid backbone comprising one or more antibiotic resistance genes under the control of non-natural promoters such as those listed in the section Promoters, In more particular embodiments, the polynucleotide encoding the DES is codon optimized for expression in said isolated recombinant bacterium. In still more particular embodiments, the plasmid encoding a DES in a recombinant isolated bacterium is characterized by all of the features listed above.

In particular embodiments of intentionally recombinant bacteria according to the invention, they are characterized by a chromosomally integrated polynucleotide encoding a DES. In more particular embodiments, the polynucleotides is placed at an isolated neutral locus e.g. glmS in E. coli, which does not naturally contain DES in the recipient bacterium. In further particular embodiments, polynucleotide encoding the DES is placed at a locus that does not naturally contain a DES operon in any known bacterium.

In some embodiments, the polynucleotide encoding the DES in an isolated bacterium according to the invention is controlled by a synthetic promoter. In some particular embodiments, said promoter is engineered to confer constitutive expression of said DES. In other embodiments, a synthetic promoter engineered to be conditional on the presence of a specific inducer controls expression of said DES. In particular embodiments, this promoter is not naturally associated a DES listed in the examples.

The presence of promoter sequences commonly used to induce recombinant expression of proteins can identify intentional recombinant bacterium expressing a functional Ddm system according to the invention, as they are distinct from promotors operationally linked to Ddm systems as they occur in nature. In particular embodiments of the isolated recombinant bacteria according to the invention, the polynucleotide encoding the DES is within operational control of a synthetic promotor conferring constitutive expression of the DES. In other particular embodiments of the isolated recombinant bacteria according to the invention, the polynucleotide encoding the DES is within operational control of a synthetic promotor conferring inducible expression of the DES. Examples of such synthetic promotors are listed in the section entitled Promoters. In some embodiments, the isolated bacterium expresses a recombinant DES comprising a functional DdmD polypeptide, and a functional DdmE polypeptide, and said isolated bacterium is characterised by an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration, but lacking the recombinant DES. In further particular embodiments of recombinant isolated bacteria according to the invention, said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium does not comprise or consist of a species within the class Gamma Proteobacteria, in particular embodiments of said isolated bacterium expressing a recombinant DES, it is not a species within the order Vibrionales. In further particular embodiments of said isolated bacterium, it is not a species within the family Vibrionaceae. In still further particular embodiments of said isolated bacterium, it is not a species within the genus Vibrio In other embodiments, the isolated bacterium is not a species within Lactobacillaceae. In other particular embodiments, the isolated bacterium characterised by a transgene encoding a DES comprising a DdmD and a DdmE polypeptide is not a species within Lactobacillaceae or Vibrionaceae. In other embodiments, the isolated bacterium expresses a recombinant DES which comprises a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide, and said isolated bacterium is characterised by an improved capacity to eliminate bacteriophage compared to a bacterium of identical genetic configuration, but lacking the recombinant DES.

Some embodiments of this aspect of the invention relate to an isolated bacterium expressing a recombinant DES which comprises both a functional DdmD polypeptide, and a functional DdmE polypeptide, and a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide. In these embodiments, said recipient bacterium is characterised by an improved capacity to eliminate both plasmid and bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the recombinant DES.

The isolated bacterium according to the invention may belong to any species within the Kingdom Bacteria. The bioinformatics analysis in the examples demonstrates that DdmD and DdmE homologues can be identified in numerous gram-negative or gram-positive bacterial species, and DdmA, DdmB, and DdmC homologues are present in even more taxonomic groups, including, but not limited to Gamma- proteobacteria, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes. The inventors thus deem it reasonable that this system is widely adaptable to perform its biological function in all kinds of bacteria. Standard genetic engineering methods known in the art allow optimisation of cis-regulatory elements, facilitating low or high expression levels, or to incorporate specific conditions under which expression would occur in any such taxonomic class. Furthermore, the data in the examples demonstrates expression of transgenes encoding functional DES enabled increased clearance of plasmids in V. cholerae, and both plasmids and bacteriophages in E. coli.

In some embodiments, the isolated bacterium or the recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium according to the invention, comprises or consists of species within the phylum Proteobacteria. In particular embodiments, it is a member of Gamma proteobacteria. In more particular embodiments, it is a species or strain within Enterobacterales. In still more particular embodiments, it is a member of the family Enterobacteriaceae. In still more particular embodiments, the isolated bacterium, or bacterial population comprises or consists of Escherichia coli.

In other embodiments, the isolated recombinant bacterium is a species amenable to industrial recombinant protein expression, for example a member of the class Bacilli such as Bacillus subtilis. In particular embodiments, the isolated recombinant bacterium is a species of B. subtilis comprising nucleic acids encoding a DdmD and a DdmE polypeptide only. In other particular embodiments, the isolated recombinant bacterium is B. subtilis and comprises nucleic acids encoding a DdmD and a DdmE polypeptide and a DdmA, DdmB and DdmC polypeptide. In other particular embodiments, the isolated recombinant bacterium is a species of B. subtilis comprising nucleic acids encoding DdmA, DdmB and DdmC polypeptide only. In other embodiments, the isolated recombinant bacterium is a species amenable to food production or fermentation, for example lactic acid bacteria such as genera Carnobacterium, Lactobacillus, Lactococcus, Streptococcus and Weissella. In particular embodiments, the isolated recombinant bacterium is a species of Lactobacillus comprising nucleic acids encoding a DdmD and a DdmE polypeptide only. In particular embodiments, the isolated recombinant bacterium is a species of Lactobacillus comprising nucleic acids encoding a DdmA, DdmB and a DdmC polypeptide only. In other embodiments, said isolated bacterium is Lactobacillus comprising transgenes encoding each of a DdmA, DdmB, DdmC, DdmD, and DdmE polypeptide.

A further aspect of the invention relates to products such as biotechnology kits or reagents, foods, food additives, feed, nutritional supplements, probiotic supplements, personal care products, health care products, or veterinary compositions, which comprise an expression vector encoding a DES, or an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium genetically modified to express a DES according to the methods provided herein.

The invention further encompasses the following items:

A. An expression vector comprising a polynucleotide encoding a functional DNA elimination system (DES), said DES comprising: a functional DNA defence molecule (Ddm) D polypeptide, and a functional DdmE polypeptide, wherein the DdmD polypeptide is selected from: a. a Vibrio cholerae (V. cholerae) DdmD polypeptide, or a polypeptide sequence having an identity of at least (>) 70%, >75%, >80%, >85%, particularly >90%, more particularly an identity of >95% compared to said V. cholerae DdmD polypeptide, wherein said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmD polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of said V. cholerae DdmD polypeptide in a bacterial cell comprising said DdmE polypeptide; or b. a homologue of the V. cholerae DdmD polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmD polypeptide, wherein said homologue of the V. cholerae DdmD polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmD polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of said V. cholerae DdmD polypeptide in a bacterial cell comprising said DdmE polypeptide; particularly wherein the polypeptide sequence of the DdmD polypeptide, or the homologue of the DdmD polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae or Lactobacillaceae, particularly a member of genus Vibrio, particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmD polypeptide has the sequence SEQ ID NO 001 ; and wherein the DdmE polypeptide is selected from: c. a V. cholerae DdmE polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmE polypeptide, wherein recombinant expression of said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmE polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of said V. cholerae DdmE polypeptide in a bacterial cell expressing said DdmD polypeptide; or d. a homologue of the V. cholerae DdmE polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmE polypeptide, wherein said homologue of the V. cholerae DdmE polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmE polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of said V. cholerae DdmE polypeptide in a bacterial cell expressing said DdmD polypeptide; particularly wherein the polypeptide sequence of the DdmE polypeptide, or the homologue of the DdmE polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae or Lactobacillaceae, particularly a member of genus Vibrio, particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmE polypeptide has the sequence SEQ ID NO 002.

B. The expression vector according to item A, wherein said DES further comprises a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide, wherein the DdmA polypeptide is selected from: • a V. cholerae DdmA polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmA polypeptide, wherein said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmA polypeptide in a bacterial cell expressing said DdmD polypeptide, DdmE polypeptide as specified in item A, and said DdmB polypeptide and DdmC polypeptide; or

• a homologue of the V. cholerae DdmA polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmA polypeptide, wherein said homologue of the V. cholerae DdmA polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmA polypeptide in a bacterial cell expressing said DdmD polypeptide, DdmE polypeptide, DdmB polypeptide and DdmC polypeptide; particularly wherein the polypeptide sequence of the DdmA polypeptide, or the homologue of the DdmA polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmA polypeptide has the sequence SEQ ID NO 003; and wherein the DdmB polypeptide is selected from:

• a V. cholerae DdmB polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmB polypeptide, wherein said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmB polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmB polypeptide in a bacterial cell expressing said DdmD polypeptide, DdmE polypeptide, DdmA polypeptide and DdmC polypeptide; or

• a homologue of the V. cholerae DdmB polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmB polypeptide, wherein said homologue of the V. cholerae DdmB polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmB polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmB polypeptide in a bacterial cell expressing said DdmD polypeptide, DdmE polypeptide, DdmA polypeptide and DdmC polypeptide; particularly wherein the polypeptide sequence of the DdmB polypeptide, or the homologue of the DdmB polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmB polypeptide has the sequence SEQ ID NO 004; and wherein the DdmC polypeptide is selected from

• a V. cholerae DdmC polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmC polypeptide, wherein said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmC polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmC polypeptide in a cell expressing said DdmD polypeptide, DdmE polypeptide, DdmA polypeptide and DdmB polypeptide; or

• a homologue of the V. cholerae DdmC polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmC polypeptide, wherein said homologue of the V. cholerae DdmC polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmC polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of said V. cholerae DdmC polypeptide in a cell expressing said DdmD polypeptide, DdmE polypeptide, DdmA polypeptide and DdmB polypeptide; particularly wherein the polypeptide sequence of the DdmC polypeptide, or the homologue of the DdmC polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmC polypeptide has the sequence SEQ ID NO 005.

C. An expression vector comprising a polynucleotide encoding a functional DES, said DES comprising: a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide, wherein the DdmA polypeptide is selected from: a. a V. cholerae DdmA polypeptide, ora polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmA polypeptide, wherein said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmA polypeptide in a bacterial cell expressing said DdmB polypeptide and DdmC polypeptide; or b. a homologue of the V. cholerae DdmA polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmA polypeptide, wherein homologue of the V. cholerae DdmA polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmA polypeptide in a bacterial cell expressing said DdmB polypeptide and DdmC polypeptide; particularly wherein the polypeptide sequence of the DdmA polypeptide, or the homologue of the DdmA polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmA polypeptide has the sequence SEQ ID NO 003; and wherein the DdmB polypeptide is selected from: c. a V. cholerae DdmB polypeptide, ora polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmB polypeptide, wherein recombinant expression of said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmB polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmB polypeptide in a bacterial cell expressing said DdmA polypeptide and DdmC polypeptide; or d. a homologue of the V. cholerae DdmB polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmB polypeptide, wherein recombinant expression of said homologue of the V. cholerae DdmB polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmB polypeptide, has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmB polypeptide, in a bacterial cell expressing said DdmA polypeptide and DdmC polypeptide when expressed in said recombinant bacterial cell; particularly wherein the polypeptide sequence of the DdmB polypeptide, or the homologue of the DdmB polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmB polypeptide has the sequence SEQ ID NO 004; and wherein the DdmC polypeptide is selected from e. a V. cholerae DdmC polypeptide, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmC polypeptide, wherein recombinant expression of said polypeptide sequence having an identity of >70% compared to said V. cholerae DdmC polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmC polypeptide in a bacterial cell expressing said DdmA polypeptide and DdmB polypeptide; or f. a homologue of the V. cholerae DdmC polypeptide, or a polypeptide sequence having an identity of >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmC polypeptide, wherein recombinant expression of said homologue of the V. cholerae DdmC polypeptide, or polypeptide sequence having an identity of >85% compared to said homologue of the V. cholerae DdmC polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of said V. cholerae DdmC polypeptide in a cell expressing said DdmA polypeptide and DdmB polypeptide; particularly wherein the polypeptide sequence of the DdmC polypeptide, or the homologue of the DdmC polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the DdmC polypeptide has the sequence SEQ ID NO 005.

CC. An expression vector comprising a polynucleotide encoding a functional DES, said DES comprising: a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide, wherein the DdmA polypeptide is selected from:

• a V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003; or • a homologue of a V. cholerae DdmA polypeptide, wherein the V. cholerae DdmA polypeptide has the sequence SEQ ID NO 003, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmA polypeptide, wherein the DES comprising the polypeptide sequence having an identity of >70% compared to SEQ ID NO 003, said homologue of the V. cholerae DdmA polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of a DES comprising a V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003, a DdmB polypeptide having the sequence SEQ ID NO 004, and a DdmC polypeptide having the sequence SEQ ID NO 005 in a bacterial cell; particularly wherein the homologue of the DdmA polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue of the DdmA polypeptide is a DdmA polypeptide listed in Table 1 ; and wherein the DdmB polypeptide is selected from:

• a V. cholerae DdmB polypeptide having the sequence SEQ ID NO 004, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmB polypeptide having the sequence SEQ ID NO 004; or

• a homologue of a V. cholerae DdmB polypeptide , wherein the V. cholerae DdmB polypeptide has the sequence SEQ ID NO 004, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmB polypeptide, wherein a DES comprising said polypeptide sequence having an identity of >70% compared to the sequence SEQ ID NO 004, or said homologue of the V. cholerae DdmB polypeptide, or polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmB polypeptide, has >10%, particularly >20%, more particularly >30% of the biological function of the DES comprising the DdmA polypeptide having a sequence SEQ ID NO 003, a V. cholerae DdmB polypeptide having the sequence SEQ ID NO 004, and a DdmC polypeptide having the sequence SEQ ID NO 005, in a bacterial cell; particularly wherein the homologue of the DdmB polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue of the DdmB polypeptide is a DdmB polypeptide listed in Table 1 ; and wherein the DdmC polypeptide is selected from

• a V. cholerae DdmC polypeptide having the sequence SEQ ID NO 005, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmC polypeptide having the sequence SEQ ID NO 005; or

• a homologue of a V. cholerae DdmC polypeptide, wherein the V. cholerae DdmC polypeptide has the sequence SEQ ID NO 005, or a polypeptide sequence having an identity of >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmC polypeptide, wherein a DES comprising said polypeptide sequence having an identity of >70% to the sequence SEQ ID NO 005, or said homologue of the V. cholerae DdmC polypeptide, or said polypeptide sequence having an identity of >85% compared to said homologue of the V. cholerae DdmC polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of a DES comprising a DdmA polypeptide having the sequence SEQ ID NO 003, a DdmB polypeptide having the sequence SEQ ID NO 004, and a V. cholerae DdmC polypeptide having the sequence SEQ ID NO 005 in a bacterial cell; particularly wherein the homologue of the DdmC polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue of the DdmC polypeptide is a DdmC polypeptide listed in Table 1.

D. The expression vector according to any one of the items A or B, wherein the DdmD polypeptide comprises: a. a functional superfamily II helicase domain comprising: i. a functional Walker A motif; and ii. a functional Walker B motif; and b. a functional nuclease domain.

E. The expression vector according to any one of the items B to CC, wherein the DdmA polypeptide comprises a functional nuclease domain, and/or wherein the DdmC polypeptide comprises: a. a functional Walker A motif; and b. a DUF3732 domain; and wherein the functional Walker A domain and the DUF3732 domain are located on either side of a coiled-coil-containing region.

F. The expression vector according to any one of the items A to E, wherein the polynucleotide encoding said functional DES is within operable distance of a cis-acting regulatory element enabling expression of said functional DES in a bacterial cell.

G. The expression vector according to any one of the items A to F, wherein said polynucleotide encoding a functional DES is comprised within a transmissible genetic element, particularly a transmissible genetic element selected from a self-replicating genetic element, or a mobile genetic element, more particularly wherein the expression vector is a plasmid.

GG. The expression vector according any one of the items A to G, wherein said polynucleotide encoding a functional DES is within operable distance from a constitutive promoter.

GGG. The expression vector according any one of the items A to G, wherein said polynucleotide encoding a functional DES is within operable distance from an inducible promoter, wherein said functional DES is expressed in the presence of at least one inducer, particularly an inducible promoter in which gene expression occurs in the presence of an inducer selected from arabinose, lactose, IPTG, tryptophan, a lantibiotic peptide or a functional analogue thereof.

GGGG. The expression vector according to any one of the items A to GGG, wherein the polynucleotide encoding a functional DES is within operable distance of a synthetic promoter, particularly a promoter selected form the list consisting of araBAD, T7, T7lac, Sp6, Trp, lac, Ptac, pL, cspB, NBP3510, Pgrac, Pspac, P43, tetA, PrhaBAD, rpoS, pac, npr, Ipp, syn, P₁₇o or 3.

H. A method: to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium capable of reducing, or eliminating plasmids; to reduce or eliminate intracellular plasmids from an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium; to protect an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium against plasmid maintenance; or to reduce or eliminate plasmid-encoded antibiotic resistance in an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium; wherein the method comprises contacting said isolated bacterial cell, bacterial culture, bacterial population, or bacterial consortium with a composition comprising the expression vector encoding a functional DES as specified in any one of the items A or B, or D to GGGG, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium, leading to expression of the functional DES. I. A method: to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium capable of reducing, or eliminating extra-genomic, circular dsDNA; to protect an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium from maintenance of extra-genomic, circular dsDNA; or to eliminate or reduce extra-genomic, circular dsDNA in an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium; particularly wherein said extra-genomic, circular dsDNA comprises a plasmid, and/or a bacteriophage, and wherein the method comprises contacting said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium with a composition comprising an expression vector encoding a functional DES as specified in any one of the items A to GGGG under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial culture, or bacterial consortium, leading to expression of the functional DES.

J. A method: to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium capable of reducing or eliminating bacteriophage; to eliminate or reduce intracellular bacteriophage within an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium; to protect an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium from bacteriophage maintenance; or to protect an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium from bacteriophage-mediated lysis; wherein the method comprises contacting said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium with a composition comprising an expression vector encoding a functional DES as specified in any one of the items B to GGGG, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium, leading to expression of the functional DES.

K. A method of protecting a recipient bacterium from extra-genomic, circular dsDNA wherein the method comprises contacting said recipient bacterium with a donor bacterium expressing the expression vector encoding a functional DES as specified in any one of items A to CC, to provide a recipient bacterium which eliminates extra-genomic, circular dsDNA; particularly wherein: a. the DES comprises a functional DdmD polypeptide and a functional DdmE polypeptide as specified in item A or B, and said recipient bacterium is characterised by an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES, and/or b. the DES comprises a functional DdmA polypeptide, a functional DdmB polypeptide and a functional DdmC polypeptide as specified in item B, C or CC, and said recipient bacterium is characterised by an improved capacity to eliminate bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES, and wherein optionally, the recipient bacterium and the donor bacterium are not identical.

L. An isolated bacterium genetically modified to express a functional DES, said DES comprising: a. a functional DdmD polypeptide, and a functional DdmE polypeptide as specified in item A, wherein said isolated bacterium is characterised by an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration, but lacking the functional DES; and/or b. a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide as specified in item B, C or CC, wherein said recipient bacterium is characterised by an improved capacity to eliminate bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES.

LL. An isolated bacterium characterised by expression of a recombinant (transgenic) functional DES, said DES comprising: a functional DdmD polypeptide, a functional DdmE polypeptide, a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide as specified in item B, wherein said recipient bacterium is characterised by an improved capacity to eliminate plasmid, and protect against bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES particularly wherein said functional DES consist of said functional DdmA, DdmB and DdmC polypeptides.

LLL. An isolated bacterium characterised by expression of a recombinant (transgenic) functional DES, said recombinant functional DES comprising: a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide as specified in item C or CC, wherein said recipient bacterium is characterised by an improved capacity to eliminate bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the recombinant functional DES.

M. The method according to any one of the items H to K, or the isolated bacterium according to any one of claims L to LLL, wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium comprises or consists of species within the phylum Proteobacteria, particularly within Gamma proteobacteria, more particularly within Enterobacterales, still more particularly within family Enterobacteriaceae, still more particularly wherein the isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium is Escherichia coli.

MM. The method according to any one of the items H to K, or M, or the isolated bacterium according to any one of the items L to M, wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium does not comprise or consist of species within the within the class Gamma proteobacteria, particularly within order Vibrionales or Lactobacillaceae, more particularly within the family Vibrionaceae, still more particularly species within the genus Vibrio.

MMM. The method according to any one of the items H to K, M, or MM, or the isolated bacterium according to any one of the items L to MM, wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium comprises or consists of species within a genus selected from the list consisting of Carnobacterium, Lactobacillus, Lactococcus, Streptococcus, Weissella, or Bacillus

N. The method according to any one of the items I to K, or M to MMM, or the isolated bacterium according to one of items L to MMM, wherein said bacteriophage circularises its genome upon cell entry, and/or replicates and/or propagates by means of a circular intermediate or plasmidlike state, particularly wherein the bacteriophage is a P1 phage, or a lambda phage.

O. A product selected from a food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, or veterinary composition, said product comprising an expression vector according to any one of the items A to GGGG, an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium obtained by a method according to any one of the items H to K, M to N, or an isolated bacterium genetically modified to express a DES according to any one of the items L to N.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Fipures

Fig. 1 shows that plasmids are unstable in V. cholerae 01 El Tor. a, Time course of pBAD and pSa5Y (pSa5Y-Amp) stability in the seventh pandemic 01 El Tor strain A1552. Strains were grown in the absence (-Amp) and presence (+Amp) of selection, with sampling after growth for 0 and approx. 10, 30 and 50 generations b, Morphology of exponentially growing cells carrying either pBAD or pSa5Y in the absence and presence of ampicillin (100 pg/ml), as indicated. Scale bar = 5 pm. c, Retention of pSa5Y (pSa5Y-Kan) in various 01 , 0139 and non-01/non- 0139 (environmental strains and a toxigenic 037 strain ATCC25872) strains after growth for approx. 50 generations without antibiotic selection d, Retention of plasmids with various origins of replication in A1552 and three Californian non-01/non-0139 (SL5Y, SP6G, SA3G) strains aftergrowth for approx. 50 generations without antibiotic selection. The different origins of replication were cloned into a neutral plasmid backbone containing a conditional origin of replication inactive in the tested strains. Bar charts represent the mean value from three independent experiments (individual dots) with error bars specify the standard deviation.

Fig. 2 shows that in V. cholerae 01 El Tor contains a DNA defence system that is split between two pathogenicity islands a, Schematic showing the organisation of Vibrio pathogenicity island 2 (VPI-2) with the region missing in strain MO10 highlighted b, pSa5Y-Amp stability in strains A1552 (01 serogroup), MO10 (0139 serogroup), A1552 transformant with the 01 serogroup exchanged to 0139 (01>0139) and A1552 strains with the 01 serogroup cluster partially exchanged by the 0139 region (AIIIHSGC#4 and #5). Strain AIIIHSGC#4 additionally cotransferred the truncated version of VPI-2 (AVC1760-VC1788). c, Effect of deletion of VPI-2 segments missing in MO10 (D#1-#5, marked in a) on plasmid stability in strain A1552. d, Effect of individual deletions of VC1770-VC1772 on plasmid stability in strain A1552. e, Expression of VC1770 and VC1771 complements the respective deletion but does not complement the deletion of VC1770-72. f, Schematic indicating the positions of OTCf, Vibrio pathogenicity island 1 and 2 (VPI-1 and -2), and Vibrio seventh pandemic island I and II (VSPI-I and -II) on the large chromosome of A1552. g, Schematic showing the organisation of VSP-II and the positions of the deleted segments. LAT-1 - Latin America specific VSP-II variant with an insertion between VC0510 and VC0516. h, pSa5Y-Amp stability in strain A1552 deleted for VPI-1 , VSP-I and VSP-II. i, Effect of the deletion of VSP-II segments (D#1-#5, marked in g) on plasmid stability in strain A1552. j, Effect of individual deletions of VC0490-VC0493 on plasmid stability in strain A1552. k, Expression of VC0490 and VC0492 complements the respective deletion but does not complement the deletion of VC0490-VC0493. VC1770, VC1771 , VC0490, VC0491 and VC0492 were expressed from an arabinose-inducible promoter P_BAD within the transposon (Tn) constructs that were site-specifically integrated into the chromosome. For all panels, plasmid retention was evaluated after growth for approx. 50 generations in the absence of antibiotic selection. For panels (e) and (k), strains were grown in LB medium supplemented with 0.2% arabinose to induce the expression of the gene encoded in the transposon. Bar charts represent the mean value from three independent experiments (individual dots) with error bars specifying the standard deviation.

Fig. 3 shows mode of action of plasmid elimination, a, Schematic organisation of TnddmDE and TnddmABC constructs. Putative domains identified in the encoded proteins are indicated (more details on domains is given in Fig. 11). b, Inducible expression of ddmDE or ddmABC leads to plasmid destabilisation independent of the other part of the system. Plasmid retention was evaluated after growth for approx. 50 generations in the absence of antibiotic selection. 0.2% arabinose was added to the medium to induce the expression of the genes encoded in the transposon, except for strains containing TnddmABC, for which 0.02% arabinose was used due to toxicity, as indicated by #. c, Effect of DdmDE and DdmABC production on plasmid localisation and retention in either the respective operon deletions or in a double deletion background. Cells were imaged after growth for approx. 10 generations in the absence of antibiotic selection. + Ara - 0.2% arabinose was used to induce TnddmDE and 0.02% arabinose was used to induce TnddmABC. Scale bar = 5 pm. Bar charts below images represent quantification of the percentage of cells retaining plasmids. ND - not determined. Bar charts represent the mean value from three independent experiments (individual dots) with error bars specifying the standard deviation. Panel (c) show representative images from three independent experiments.

Fig. 4 shows heterologous expression in E. coli of ddmDE leads to plasmid elimination and ddmABC provides anti-phage defence, a, Stability of a conditionally replicating plasmid carrying either an empty transposon or a transposon with inducible ddmDE, ddmD or ddmE. Cultures were evaluated after growth for approx. 10 generations in the absence of selection in E. coli strain S17-1Ap/r, without (left, light grey) or with (right, dark grey) addition of 0.2% arabinose to induce the expression from the plasmid-encoded constructs. The gel picture above the graph shows the plasmid extraction yield from the same cultures b, Stability of plasmids with various origins of replication in the E. coli strain MG1655 bearing either the empty transposon (Tn- empty), TnddmDE or TnddmABC integrated in the chromosome. Plasmid retention was evaluated after growth for approx. 10 generations in the absence of antibiotic selection. Arabinose was added to the medium to induce the expression of the genes encoded in the transposon: 0.2% forTn-empty and TnddmDE ; 0.02% forTncfcfmABC. The different origins of replication were cloned into a neutral plasmid backbone containing a conditional origin of replication inactive in the tested strains c, Plaque assays with serial dilutions of phages l and P1 applied to bacterial lawns of MG1655 strains bearing either Tn-empty, TnddmDE or TnddmABC. Expression from the transposon constructs was induced by 0.02% or 0.2% arabinose. Pfu - plaque forming units. Bar charts represent the mean value from three independent experiments (individual dots) with error bars specifying the standard deviation. All images are representative from three independent experiments.

Fig. 5 shows characterisation of plasmids from an environmental Californian V. cholerae population a, Schematic comparing the pB1067 minimal origin region (dashed lines) with the homologous region of pSa5Y. Sequences encoding equivalents of the experimentally verified RNA I and RNA II from pB1067 are indicated by arrows along with a predicted transcriptional terminator (inverted triangles) b, Validation of the pSa5Y origin of replication ( oh ). The putative pSa5Y ori and ori sequences from plasmids pB1067 (MRB ori ), pBAD (ColE1 ori ) and pACYC177 (p15A ori ) were cloned into a conditionally replicating plasmid (pMJ174) containing the pir- dependent R6K ori. The resulting plasmids were introduced into pir- and pir+ E. coli strains and spotted on LB+Kan plates.

Fig. 6 shows a, conservation of the plasmid stability phenotype dependent on VC1771-70 and VC0492-90. Retention of plasmids with various origins of replication in strain A1552 and the AVC1770 and AVC0490 derivatives after growth for approx. 50 generations without antibiotic selection. The different origins of replication were cloned into a neutral plasmid backbone containing a conditional origin of replication inactive in the tested strains b, Comparison of the stability of various plasmids between V. cholerae A1552 (WT) and a AddmABCAddmDE double operon mutant (Addm), after growth for approx. 50 generations without antibiotic selection. Bar charts represent the mean value from two independent experiments (individual dots), error bars specify the standard deviation. Plasmids tested: pSa5Y (derived from environmental V. cholerae strain Sa5Y), pBAD (pBAD/Myc-HisA; common plasmid used in bacterial genetics; Invitrogen), pE7G2 (derived from environmental V. cholerae strain E7G), pSIO (derived from environmental V. cholerae strain SIO); pES213 (derived from V. fischeri strain ES213). All plasmids contain a genetically added antibiotic resistance marker ( aph ) encoding kanamycin resistance.

Fig. 7 shows bioinformatics analysis of DdmD (panel a), DdmE (panel b), and DdmC (panel c). Taxonomic distribution of putative homologues of derived from homology search using PHMMER. The numbers in parentheses at each node indicate the relative number of search hits within each taxonomic group. 685/728 (94%) of the significant matches to DdmC contain the same C-terminal DUF3732.

Fig. 8 shows the presence of intact 2-gene ddmDE operons and 3-gene ddmABC operons found within (a) Lactobacilalles and (b) Rhizobiaceae. Operons were detected by examining the genomic loci of PHMMER hits to DdmD(VC1771) and DdmC(VC0490) within the indicated taxonomic group (number in parentheses below each gene represent the size of the encoded protein (aa)). The presence of ddmABC on a plasmid is based either on the sequence annotation or on the proximity (*) to plasmid-specific genes ( repAB and tra).

Fig. 9 shows growth of strains carrying arabinose inducible ddmABC f TnddmABC ) was evaluated on plates either without additions or supplemented with 0.02% and 0.2% arabinose, as indicated, in the absence and presence of plasmid pSa5Y-Amp.

Fig. 10 shows production of DdmDE but not DdmABC leads to rapid plasmid loss a, Quantification and imaging of pSa5Y parS^MT1 localisation in single cells of the indicated stains of cells with plasmid foci in the indicated strains after growth for approx. 10 generations without antibiotic selection. Barcharts represent the mean value from three independent experiments (individual dots) with error bars specifying the standard deviation. Mean values are shown above the bars b and c, Time-course of pSa5Y-parS^MT1 localisation following TnddmDE (b) or TnddmABC (c) expression in a yGFP-ParB^MT1 ddmDE MdmABC background. Cultures were sampled every hour for 6 hours. 0.2% arabinose was used to induce the expression of TnddmDE and 0.02% was used for TnddmABC due to plasmid-related toxicity. For panels (b) and (c) representative images from three independent experiments are shown. Scale bars = 2.5 pm.

Fig. 11 shows the essential role of conserved domains identified in DdmDE and DdmABC in both plasmid elimination and anti-bacteriophage activity a, Schematic showing putative conserved domains identified in DdmDE and DdmABC. Underlined residues represent key catalytic residues predicted to be required for function for which variants with amino acid substitutions [following a common nomenclature: key residue at position XXX changed to the indicated amino acid] were created by site-directed mutagenesis b-c, Comparison of pSa5Y plasmid stability between V. cholerae A1552 (WT) and derivatives encoding site-directed variants of DdmD, DdmA and DdmC, after growth for approx. 50 generations without antibiotic selection. Bar charts represent the mean value from three independent experiments (individual dots), error bars specify the standard deviation (b) The results show that both DdmD helicase activity (either Walker A; K55A or Walker B; E273A) and nuclease activity (PD-(D/E)xK (SEQ ID NO 009); K1102A) are required to mediate plasmid elimination (c), Similarly, the results show that DdmA nuclease activity (PD-(D/E)xK (SEQ ID NO 009); K57A) and DdmC ATP- binding (Walker A; K40A) are required to mediate plasmid elimination d-e, Plaque assays with serial dilutions of bacteriophages l (d) and P1 (e) applied to bacterial lawns of E. coli strain MG1655 bearing either Tn-empty (No system), TnddmABC[WT] or TnddmABC derivatives encoding variants of either DdmA[K57A] or DdmC[K40A] Expression from the transposon constructs was induced by 0.2% arabinose. Pfu; plaque forming units. The results show that both DdmA nuclease activity (PD-(D/E)xK (SEQ ID NO 009); K57A) and DdmC ATP-binding (Walker A; K40A) are required to mediate anti-bacteriophage activity.

Fig. 12 shows the mode of action of DdmABC anti-bacteriophage activity. Charts show a plate reader assay comparing the growth kinetics of E. coli strain MG1655 bearing either Tn-empty (No system) or TnddmABC (+ DdmABC), in the absence (No Phage) and presence of an increasing multiplicity of infection (MOI; 0.2, 2, 5, 10) of bacteriophage P1. All cultures were grown in medium supplemented with 0.2% arabinose to induce expression from the transposon constructs. The results show that DdmABC production provides robust protection against phage-mediated culture collapse at lower MOI and at higher MOI suppresses growth of phage infected cells, consistent with an abortive infection mode of action.

Table 1. Reference genome locus tag numbers, and protein in parentheses for a selection of representative examples of DdmABC and DdmDE polypeptides.

Table 2. Summary of domain bioinformatics analysis. Gene locus tags in brackets according to the reference genome of strain N16961 by Heidelberg etal. 2000 Nat. 406: 477 (Protein sequence accession number for the V. cholerae strain A1552). Remote Homology detection by Hhpred. Structural prediction, Phyre2/iTasser produced similar results, except for DdmD, which was only predicted by Phyre2, and DdmE, which was only predicted by iTasser. Putative domains were predicted by PFAM or inferred from homology prediction.

Table 3. Plasmid strains used in the Examples.

Table 4. Bacteriophages used in the Examples.

Examples

Example 1: Environmental strains yield a model plasmid suitable for V. cholerae studies Plasmids are abundant throughout the Vibrionaceae and play important roles in the ecology and pathogenesis of several species. While plasmids are common in environmental strains of V. cholerae, they are absent from most current pandemic strains (7^th pandemic 01 El Tor clade; 7PET). The majority of these El Tor pandemic strains lack known homologues of well-characterised plasmid defence systems such as CRISPR-Cas and prokaryotic Argonautes, thus the inventors hypothesised that additional mechanisms of resistance to HGT may exist in 7PET V. cholerae strains.

Plasmid stability was measured in the representative 7PET strain A1552 during growth for approximately 50 generations, diluted approximately every 10 generations (~8 hours), with and without selection. 7PET cells carrying the experimental Escherichia coli ColE1/pMB1/pBR322/pUC-family plasmid pBAD/Myc- HisA (pBAD) derivatives were examined, however pBAD was poorly tolerated, unstable and resulted in gross morphological defects during exponential growth under selection (Fig. 1a, b). As this non-native plasmid appeared to be strongly affected by the Ddm modules, the inventors sought to identify a natural V. cholerae plasmid to use as a model. As expected, none were detected in the best-studied pandemic strains (N1691 , C6706 or E7946). In contrast, 5/15 strains from a collection of diverse Californian environmental isolates were found to contain small cryptic plasmids (Sa5Y, SL4G, S05Y, SA7G, E7G).

One 3.5-kb plasmid, pSa5Y, was present at c.a. 4-5 copies per genome, and carries 5 ORFs of unknown function including a gene encoding a putative adhesin. It is non-conjugative and lacks a recognisable origin of transfer. Notably, sequencing revealed characteristic genes involved in plasmid maintenance and partitioning such as toxin-antitoxin or parAB are also absent, and it could readily be cured. pSa5Y replicates using a Marine RNA-based (MRB) origin of replication, which, like the well-characterised ColE1 origin uses an RNA primer to initiate replication and an anti-sense RNA to control copy number (Fig. 5a, b). MRB-based plasmids such as pSa5Y are prevalent throughout the Vibrionaceae and are highly stable in their host species. In contrast to pBAD, pSa5Y derivatives with selectable antibiotic resistance markers were stable and well tolerated (Fig. 1a, b). However, in the absence of selection pSa5Y was still gradually lost (Fig. 1a). pSa5Y stability was examined in a diverse selection of environmental, clinical and pandemic V. cholerae strains. pSa5Y was highly stable in all tested strains, except those of the 7PET clade (Fig. 1c). Importantly, this phenotype was not specific to pSa5Y, since a neutral plasmid backbone containing the origins of replication from various plasmids behaved similarly (Fig. 1d).

0139 epidemic strains are thought to have emerged after an 01 El Tor ancestor acquired the genes encoding 0139-antigen synthesis via HGT. Converting the O-antigen region of strain A1552 from 01 to 0139 by natural transformation had no effect on plasmid stability (Fig. 2a, b). 0139 strains carry a truncated version of the 57-kb island VPI-2 (Fig. 2a). A hybrid transformant of A1552 that has undergone partial serogroup conversion, but that has also co-transferred this deletion, exhibits complete plasmid stabilisation (Fig. 2b). A deletion series of the 28 genes absent from 0139 identified a region containing genes VC1770-72 (Fig. 2c). Individual deletions of either VC1770 or VC1771 both resulted in complete plasmid stabilisation, whereas deletion of VC1772 had no effect (Fig. 2d). Furthermore, while both VC1770 and VC1771 could complement their respective individual deletions, neither was capable of complementing the whole region deletion, suggesting that they work together, consistent with their organisation in an operon (Fig. 2e).

Defence systems are often enriched on genomic islands. To test whether any of the other pathogenicity islands present in the 7PET are required for plasmid destabilisation VPI-1 , VSP-I and VSP-II were deleted (Fig. 2f, g). Interestingly, while the deletion of either VPI-1 or VSP-I had no effect, cells lacking VSP-II - a 26.9-kb island of mostly unknown function - exhibited a striking increase in plasmid stability, with more than half the population now retaining plasmids (Fig. 2h). Using the same deletion series approach outlined above, the region containing genes VC0490-93 was responsible for this phenotype (Fig. 2i). Individual deletions revealed that deletion of VC0490, VC0491 or VC0492, but not VC0493, exhibited a similar increased stability phenotype (Fig. 2j). As expected, VC0490 and VC0492 could both complement their individual deletions, though in the case of VC0491 complementation was poor, likely due to the gene’s small size and the overlapping structure of the operon (Fig. 2k). These three genes likely work together since none could complement the deletion of the whole region (Fig. 2k).

To exclude the possibility that these phenotypes are specific to the model plasmid, plasmids carrying various origins of replication were retested. Importantly, disruption of either VC1770 or VC0490 also resulted in plasmid stabilities similar to those seen with pSa5Y (Fig. 6a). Curiously, the p15A origin plasmid was stabilised only by the deletion of VC0490 (Fig. 6a), suggesting that some degree of specificity is present. The loss of either VC1770 or VC1771 results in near complete plasmid stabilisation, whereas the effect of VC0490, VC0491 and VC0492 is intermediate (Fig. 2d, j). These data suggest a model whereby these two discrete systems act together in the same pathway, with VC0492- 90 acting at a step upstream of VC1771-70. Hereafter, VC0492-90 and VC1771-70 are referred to as DNA-defence modules DdmABC and DdmDE, respectively. The plasmid elimination phenotype mediated by DdmABC and DdmDE was conserved across a variety of naturally occurring plasmids from V. cholerae and Vibrio fischeri as well as the E. coli derived plasmid pBAD/Myc-HisA, which is a commonly used vector for bacterial genetics (Fig. 6b).

These operons are not part of a known defence system. All 5 genes are annotated as encoding hypothetical proteins. Bioinformatics analysis of DdmD revealed that it contains a putative N-terminal helicase domain, with recognisable Walker A and B motifs (Table 2). In contrast, although DdmE comprised no known domains, structural modelling indicated a possible relationship to the Argonaute family. Prokaryotic Argonautes (pAgo) function as nucleic acid guided endonucleases, and can defend prokaryotes against plasmids and bacteriophages. DdmE lacks equivalents of the canonical Ago domains (e.g. PIWI), and was not identified in a pAgos screen (Ryazansky, S. et ai., 2018, mBio. 9: e01935-01918). Nevertheless, since many pAgos lack nuclease activity and instead function with a partner nuclease, one possibility is that DdmE functions as an Ago-like protein, involved in DNA- recognition, with DdmD acting as an effector protein. In addition to being found throughout the Vibrionaceae, two-gene operons encoding homologues of DdmDE were also found in the Lactobacillales (Fig. 7, Fig. 8, Table 1).

Bioinformatics analysis of DdmABC predicted DdmC is a Structural Maintenance of Chromosomes (SMC) family protein (Table 2). SMCs such as MukB form homo-dimeric complexes with the non-SMC protein MukE and the kleisin MukF, and by entrapping DNA, function as condensins to promote proper chromosome organisation. The SMC-like family Wadjet has been implicated in plasmid defence. Wadjets (e.g. jetABCD) are four-gene operons that resemble the mukFEB operon but notably, also contain a fourth gene encoding a predicted topoisomerase. Several features differentiate DdmABC from Wadjet. It lacks the fourth gene. Although, ddmABC is organised similarly to the mukFEB operon, DdmA and DdmB are not equivalent to MukF and MukE, as DdmA contains a distinct domain of unknown function, DUF4297. DdmC (653 aa) and its homologues are on average much smaller than either JetC or other condensins (c.a. 1000-1500 aa). And finally, while DdmC has an N-terminal Walker A motif, in contrast to all known SMC proteins, it lacks the canonical Walker B motif in the C-terminal domain, which instead contains the DUF3732. Notably, similar three-gene operons encoding a DdmC-like DUF3732 containing protein are widespread throughout the Proteobacteria and intriguingly, are also found on plasmids of the Rhizobiaceae including the Agrobacterium tumefaciens tumour-inducing plasmid (Figs. 7 and 8, Table. 1).

To test whether DdmABC enhances the activity of DdmDE, ectopically integrated arabinose-inducible versions of each operon were created (i.e. TnddmABC and TnddmDE, Fig. 3a) and examined for their effect on plasmid stability (Fig. 3). As expected, both were capable of complementing the deletions of their respective operons. However, in a double deletion background, although DdmABC production led to significant plasmid loss, this was incomplete. In contrast, DdmDE production resulted in complete plasmid elimination. Thus, both systems can function independently when overexpressed. One caveat is that under full induction TnddmABC produced a strong growth defect, and caused multiple abnormalities in chromosome structure and segregation, a common phenotype of SMC overproduction (Fig. 9). Unusually, however, this toxicity was plasmid-dependent and enhanced by the absence of ddmDE, suggesting that plasmid DNA renders DdmABC active. Consequently, ddmABC expression could only be evaluated at lower levels of induction.

To investigate the mechanisms underlying the activity of these two operons visualisation of pSa5Y was performed using the well-characterised MT 1 ParB IparS fluorescent labelling system (Nielsen et al. 2006. Mol. Microbiol. 62:331-338.). Cells carrying pSa5Y-parS^MT1 contained multiple highly mobile foci, consistent with pSa5Y being a low multi-copy number plasmid. In the absence of selection <1% cells of the parental control retained plasmid foci, compared to >99% of cells deleted for both operons ( ddmDE & ddmABC ). Some plasmid loss was apparent by 10 generations, but the most prominent difference was that a subset of cells contained intense clusters of plasmids that were largely absent from cells lacking ddmABC (Fig. 10a).

To examine plasmid elimination more directly, pSa5Y was visualised in cells overexpressing either ddmDE or ddmABC. In cells bearing individual deletions in either operon, the results were similar. Thus, compared to the uninduced controls, production of either DdmDE, or DdmABC led to rapid plasmid elimination, though DdmABC was noticeably less efficient and cells frequently retained bright plasmid clusters (Fig. 3c). Next, to separate the relative contribution of each system strain deleted for both operons was developed. Remarkably, DdmDE production was sufficient to eliminate plasmids from the majority of cells within only 10 generations (Fig. 10b). In contrast, most cells producing DdmABC retained plasmids, though they appeared to be trapped in large clusters (Fig. 10c). Thus, the two independent activities of these operons likely result from plasmid degradation by DdmDE and/or clustering by DdmABC, followed by cell division-mediated dilution. Time-course experiments revealed that compared to DdmABC, DdmDE triggers plasmid elimination within only a few generations (Fig. 10b, c). Altogether, these data support a simple model whereby under physiological expression levels, the clustering activity of DdmABC fosters either the recruitment and/or the activity of DdmDE, which then acts to degrade plasmid DNA, either directly or indirectly. The activity of DdmDE was tested in a heterologous organism by expressing it from a plasmid in E. coli. In agreement with the results in V. cholerae, production of DdmDE, but not either protein individually, was sufficient to remove the plasmid from >99% of cells within only 10 generations (Fig. 4a). Next, an arabinose-inducible TnddmDE and TnddmABC, whereby the respective operons were carried on a transposon, was integrated into the E. coli chromosome and tested for activity against plasmids carrying a variety of RNA- and Rep protein-based origins, which were otherwise stable in the control strain (Fig. 4b). Notably, DdmDE production was again sufficient to eliminate plasmids from most cells within only 10 generations, demonstrating that its activity is not dependent on the mode of plasmid replication initiation. In contrast, plasmid stability in strains producing DdmABC was almost unaffected within the time frame of the experiment (Fig. 4b). Finally, since some defence systems can exhibit dual activity, cells expressing each operon were challenged with a panel of 5 well-characterised E. coli bacteriophages (T4, T6, T7, l and P1). DdmABC conferred clear protection against both phage l and phage P1 , mediating a >3-log decrease in the efficiency of plaque formation (Fig. 4c). Importantly, this was not due to an indirect effect on cell growth as in the absence of plasmid DNA ddmABC induction had no obvious effect on E. coli growth (Fig. 9).

To interrogate the predicted functional domains present in DdmD (helicase, and nuclease domains), DdmA (nuclease) and DdmC (ATP-binding) (Fig. 11 a), site-directed mutagenesis was used to create variants encoding substitutions in key residues of well-characterised motifs that are known to be important for function. DdmD helicase activity (either Walker A; K55A or Walker B; E273A) and nuclease activity (PD-(D/E)xK [SEQ ID NO 009]; K1102A) are required to mediate plasmid elimination (Fig. 11 b). Similarly, DdmA nuclease activity (PD-(D/E)xK [SEQ ID NO 009]; K57A) and DdmC ATP-binding (Walker A; K40A) are required to mediate both plasmid elimination (Fig. 11c) and anti-bacteriophage activity (Fig. 11 d, e).

To investigate how DdmABC mediates anti-bacteriophage activity, the growth kinetics of E. coli cultures were assessed in the absence, and presence of DdmABC expression, when challenged with an increasing multiplicity of infection (MOI) of bacteriophage P1. As shown in Fig. 12, in the absence of DdmABC, P1 infection leads to a rapid culture collapse, the timing of which exhibits a dose-dependent response to MOI. In contrast, the results show DdmABC production provides robust protection against phage-mediated culture collapse at lower MOI, and at high and very high MOI, suppresses the growth of phage-infected cells. This phenotype is consistent with an abortive infection mode of action, wherein the system acts against the infected host cell before the bacteriophage can complete its life cycle and thus prevents bacteriophage proliferation to provide population level protection.

DdmDE clearly acts to rapidly degrade plasmid DNA, and an attractive possibility is that it does so in a manner analogous to prokaryotic Argonautes. The DdmABC module is an SMC-like system that can cluster plasmids and defend against phage. Notably, it has anti-phage activity function against P1 and l, which circularise their genomes upon entry. Example 2: Sequences

Methods

Bacterial strains and growth

The V. cholerae 7PET strain A1552, is a fully sequenced toxigenic 01 El Tor (Inaba) representative of the ongoing 7^th cholera pandemic (Matthey, N. et al., 2018, Microbiol. Resour. Announc. 7, doi: 10.1128/MRA.01574-18). Experiments in E. coli were performed using the E. coli K-12 strain MG1655. Cloning was done using E. coli strains DH5a, TOP10, and where appropriate, strains S17- Ilr/rand MFDp/rwere used for the propagation of plasmids with the conditional R6K origin of replication and for bacterial mating. Bacteria were cultured either in Lysogeny Broth (LB-Miller; 10 g/l NaCI, Carl Roth, Switzerland) or on LB agar plates at 30 or 37°C, as required. Where appropriate, antibiotic selection was done with ampicillin (Amp, 50 or 100 pg/ml), kanamycin (Kan, 75 pg/ml for V. cholerae ; 50 pg/ml for E. coli), gentamicin (Gent, 50 pg/ml), streptomycin (Strep, 100 pg/ml), rifampicin (Rif, 100 pg/ml) and zeocin (Zeo, 100 pg/ml). Counter-selection of E. coli following mating into V. cholerae was performed using thiosulfate citrate bile salts sucrose (TCBS; Sigma-Aldrich) agar supplemented with antibiotics. Mating with MFD pir derivatives to introduce the mini-Tn7 transposon into E. coli strain MG1655 was performed on agar supplemented with 0.3 mM diaminopimelic acid (DAP; Sigma-Aldrich) followed by selection of transconjugants on agar supplemented with gentamicin. SacB-based counterselection was done on NaCI-free media supplemented with 10% sucrose. For strain construction by natural transformation, V. cholerae was grown on chitin flakes in 0.5x defined artificial seawater (DASW) supplemented with 50 mM HEPES and vitamins (MEM, Gibco) (Marvig, R. L. and Blokesch M., 2010, BMC microbiol. 10:155). To induce expression from the PBAD promoter, growth media were supplemented with L-arabinose.

Strain construction

DNA manipulations and molecular cloning were performed using standard methods (Sambrook, J. 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press). Genetic engineering of V. cholerae was done using either natural transformation and FLP-recombination (TransFLP)(Marvig and Blokesch M., 2010, BMC Microbiol. 10:155) or by allelic exchange using the counter-selectable plasmid pGP704-Sac28 delivered via bi-parental mating from E. coli (Meibom K. L. et al., 2004, PNAS 101 :2524). Constructs were verified by colony PCR and Sanger sequencing (Microsynth AG, Switzerland). A mini-Tn7 transposon carrying araC and the gene(s) of interest under control of the PBAD promoter was integrated into a neutral chromosomal locus downstream of glmS in V. cholerae and E. coli by tri- pa rental mating (Bao, Y. et al., 1991 Gene 109:167).

Plasmids

Plasmid inserts were constructed as indicated in Tab. 3, and verified by sequencing (Microsynth AG, Switzerland). Plasmid DNA was isolated using either a GenElute HP Plasmid Miniprep Kit (Sigma- Aldrich) or a PureYield™ Plasmid Miniprep System (Promega). Where required, plasmids were introduced into electro-competent cells of V. cholerae by electroporation at 1 .6 kV and into chemically competent cells of E. coli using a standard heat shock protocol. The sequence of pSa5Y was obtained from a preliminary genome sequence of strain Sa5Y, and was confirmed by Sanger sequencing. Plasmids pSL4G and pS05Y, from strains SL4G and S05Y, were identified as pSa5Y-like by PCR and were sequenced using the same primers as for pSa5Y. To obtain the sequence of the two non-pSa5Y- like plasmids pSA7G3 and pE7G2, from strains SA7G and E7G, plasmid DNA was linearized with EcoRV, 5’end phosphate groups were removed with Calf Intestinal Alkaline Phosphatase (New England Biolabs), and the linearized fragments inserted into pCR-Blunt ll-TOPO™ vector (ThermoFisher

Scientific). Plasmid DNA from positive clones was isolated and the inserted fragments sequenced using vector-specific primers. The remainder of the plasmids were sequenced with primers annealing to the parts identified in the first round. pSa5Y was cured from strain Sa5Y by integrating a counter-selectable allele of the a subunit of phenylalanyl-tRNA synthetase, p/?eS*[A294G/T251A], which renders cells sensitive to 4-chloro-phenylalanine (c-Phe; Sigma Aldrich). A PCR fragment containing FRT-aph-pheS*- FRT as well as flanking regions homologous to sequences either side of the adhesin gene was integrated into pSa5Y in its native strain by natural transformation on chitin. An overnight culture of the resulting strain was diluted 10-fold, spread on LB plates containing 20 mM c-Phe, and incubated overnight at 30°C. The resulting colonies were screened for kanamycin sensitivity and the loss of pSa5Y was confirmed by PCR with plasmid-specific primers and by agarose gel electrophoresis of plasmid DNA extractions.

Plasmid copy number

The copy number of pSa5Y was determined by quantitative PCR (qPCR) using the ratio of plasmid- specific amplification to the amplification of a genome-specific fragment (Lee, C. et at., 2006 J. Biotechnol. 123:273). Cultures of strain Sa5Y were grown to either exponential (Oϋboo ~ 0.4-0.5) or stationary phase (overnight culture) and total DNA extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer’s instructions. DNA was diluted to a concentration of 1 ng/mI and used for qPCR runs (LightCycler Nano, Roche) with primer pairs annealing within the plasmid and to the genomic gyrA sequence. The standard curve was prepared using a serial dilution (10³-10⁷ copies/pl) of the calibrator plasmid pCal-Sa5Y-1 , which contains cloned fragments of the regions of pSa5Y and gyrA (1 :1 amplified fragment ratio) used as targets for qPCR.

Identification of the pSa5Y origin of replication

BLAST-N analysis of the full-length pSa5Y sequence (3494 bp) was used to probe for conserved regions that could be indicative of an origin of replication. A ~500 bp region of pSa5Y was found to be widely conserved in plasmids of the Vibrionaceae, including numerous known members of the Marine RNA- based (MRB) plasmid family. Comparison of this region with the minimal origin region of the prototype MRB plasmid pB1067 from Vibrio nigripulchritudo (Le Roux, F. etai., 2011 Nucleic Acids Res.) revealed a high degree of sequence identity (79%), as well as sequences encoding equivalents of the RNA I and RNA II species required for proper origin function (Fig. 5a). The functionality of the identified origin region was confirmed by its ability to impart p/r-independent replication to an otherwise conditionally replicating plasmid containing the pir- dependent R6K origin of replication (Fig. 5b).

Plasmid stability assay

For pSa5Y stability, Amp^R and Kan^R derivatives, pSa5Y-Amp and pSa5Y-Kan were used interchangeably, as needed. Overnight cultures grown in the presence of antibiotic selection (generation 0) were back-diluted to an Oϋboo of ~ 0.0025 in fresh LB medium, in the absence and presence of selection, as indicated, and grown with shaking at 30°C for approximately 10 generations, as estimated by optical density. To achieve growth for approximately 50 generations, cultures were back-diluted every 10 generations. At the desired end-point, cultures were serially diluted in PBS (phosphate buffered saline), spread on non-selective LB agar plates, and grown overnight at 30°C. The next day, 100 colonies were picked at random, patched onto selective media containing the appropriate antibiotic, and grown overnight at 30°C. Plasmid stability was calculated as the percentage of antibiotic resistant (i.e. plasmid-carrying) clones.

Quantitative Reverse Transcription PCR (qRT-PCR)

Overnight cultures were back-diluted 1 :100 in LB medium and grown at 30°C with agitation for 3 hours. After standard RNA purification and cDNA synthesis and quantitative PCR were performed with a LightCycler Nano. Transcript levels are presented relative to the mRNA levels of the reference gene gyrA. The data were analysed with the LightCycler Nano software package (Roche) using the standard curve method.

Bioinformatic analysis

Sequence similarity searches for homologues of the protein sequences of V. cholerae strain A1552 DdmA, DdmB, DdmC, DdmD and DdmE were carried out with BLAST-P and with the PHMMER tool of the HMMER Webserver (ebi.ac.uk/Tools/hmmer/) using the ensemblgenomes (v.44) database. Remote homology detection was done with the HHpred server (toolkit.tuebingen.mpg.de/tools/hhpred) using PDB_mmCIF70_Oct14 as the target database. Structural prediction was done with either the Phyre2.0 (sbg.bio.ic.ac.uk/phyre2) or i-TASSER (zhanglab.ccmb.med.umich.edu/l-TASSER) webservers, using the default settings. To predict the presence of an origin of transfer site ( oriT) plasmid DNA sequences were analysed using the OriTfinder Webserver (bioinfo-mml.sjtu.edu.cn/oriTfinder/).

Plasmid transformation frequency assay

Overnight cultures were back-diluted 1 :100 in LB medium and grown at 30°C with shaking for 3 hours to OD6OO ~ 1 . Cells were harvested, washed twice with ice-cold 2 mM CaCL, once with 10% glycerol and the pellets resuspended in ice-cold 10% glycerol. Aliquots of 50 pi were flash frozen in a dry ice/ethanol bath and stored at -80°C for 24 hours. Aliquots of electro-competent cells were thawed on ice, 150 ng of plasmid pSa5Y-Amp added (three technical replicates), and electroporation performed at 1.6 kV. Following electroporation, 900 mI of 2xYT medium was added and samples incubated at 37°C for 3 hours. Bacteria were enumerated following overnight growth on LB media in the absence and presence of ampicillin. Plasmid transformation frequency was calculated as the number of ampicillin-resistant transformants divided by the total number of bacteria, from three independent biological repeats, each with three technical replicates.

Microscopy

Cells were immobilised on slides coated with 1 .2% w/v agarose in PBS, covered with a N°1 coverslip and examined using a Zeiss Axio Imager M2 epi-fluorescence microscope attached to an AxioCam MRm camera, controlled by Zeiss Zen software. To stain bacterial nucleoids, cultures were incubated with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich) at 5 pg/ml for 30 min at RT, before washing once with PBS.

Plasmid labelling and quantification

Plasmids were visualised by a ParB -pars labelling using strains constitutively producing yGFP- pMT1A23ParB and carrying pSa5Y-Kan variants harbouring the pMT1 pars site. The lacZ region of the V. cholerae E7946 strain TND1379 ( A/acZ::P,_ac-CFP-parB^P1, yGFP-parB^MT1 Zeo^R) (Dalia, A. B. and Dalia, T. N., 2019, Cell 179:1499) was amplified by PCR and moved into V. cholerae A1552 by natural transformation. A pair of oligonucleotides harbouring a 148 bp fragment containing the pMT1 pars site was used to introduce the pars site into pSa5Y-Kan directly by PCR. To verify that ParB labelling did not affect plasmid stability, cultures were grown overnight with selection before being back-diluted to OD6OO ~ 0.0025 in fresh media and grown for approximately 50 generations, with and without selection. Cells were then processed for plasmid stability determination by plating, and fluorescence microscopy. To quantify plasmid stability, approximately 1600 cells were counted per strain, per condition, per repeat, and manually scored as either plasmid positive (>1 yGFP-ParB^MT1 focus) or plasmid negative (yGFP- ParB^MT1 diffuse). To examine the effect of DdmDE and DdmABC overproduction on plasmid localisation and stability, cells were imaged after growth without selection for approximately 10 generations, in the absence and presence of inducer, as indicated. Plasmid stability was determined by microscopy, as described above, by counting approximately 700 cells per strain, per condition, per repeat.

Bacteriophage plaque assay

The E. coli phages used in this study were obtained as active cultures from the German Collection of Microorganisms and Cell Cultures (DSMZ). Prey E. coli strains were grown at 37°C with shaking for 2 hours in LB medium, in the absence and presence of arabinose, as indicated. Exponentially growing cultures were then diluted 1 :40 in a molten top agar (LB + 0.5% agar supplemented with 5 mM CaCL, 5 mM MgCL and where appropriate arabinose), poured on top of a bottom layer of pre-solid ified LB + 1 .5% agar, and allowed to dry for 1 h. Phage were serially diluted in LB + 5 mM CaCL, 5 mM MgCL and 5 pi of each of the dilutions spotted onto the seeded plates. Plates were imaged after incubation overnight (~ 16-18 h) at 37°C.

Statistical analysis

GraphPad Prism software v. 8.4.3 was used for statistical analysis. Significant differences were determined by one-way ANOVA. Individual P values were determined by applying Dunnett’s multiple comparisons test. P values >0.05 were considered not statistically significant.

Table 1.

#start codon misannotated resulting in 1220 amino acid-containing protein instead of only 1190 amino acids.

Table 2.

#corrected length according to AWB74290.1

DExD motif (SEQ ID NO 013) , PD-(D/E)xK motif (SEQ ID NO 009). Table 3.

Table 4.

Claims

1 . An expression vector comprising a polynucleotide encoding a functional DNA elimination system (DES), said DES comprising: a functional DNA defence molecule (Ddm) D polypeptide, and a functional DdmE polypeptide, wherein the DdmD polypeptide is selected from:

• a Vibrio cholerae (V. cholerae) DdmD polypeptide having the sequence SEQ ID NO 001 , or a polypeptide sequence having an identity of at least (>) 70%, >75%, >80%, >85%, particularly >90%, more particularly an identity of >95%, compared to said V. cholerae DdmD polypeptide having the sequence SEQ ID NO 001 ; or

• a homologue of a V. cholerae DdmD polypeptide, wherein the V. cholerae DdmD polypeptide has the sequence SEQ ID NO 001 , or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmD polypeptide; wherein said DES comprising the polypeptide sequence having an identity of >70% to SEQ ID NO 001 , said homologue of the V. cholerae DdmD polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmD polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of a DES comprising a DdmD polypeptide having the sequence SEQ ID NO 001 and a DdmE polypeptide having the sequence SEQ ID NO 002 in a bacterial cell; particularly wherein the homologue of the DdmD polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae or Lactobacillaceae, particularly a member of genus Vibrio, particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue of the DdmD polypeptide is a DdmD polypeptide listed in Table 1 ; and wherein the DdmE polypeptide is selected from:

• a V. cholerae DdmE polypeptide having the sequence SEQ ID NO 002, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmE polypeptide having the sequence SEQ ID NO 002; or

• a homologue of a V. cholerae DdmE polypeptide, wherein the V. cholerae DdmE polypeptide has the sequence SEQ ID NO 002, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmE polypeptide; wherein said DES comprising the polypeptide sequence having an identity of >70% compared to the sequence SEQ ID NO 002, or said homologue of the V. cholerae DdmE polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmE polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of a DES comprising a DdmD polypeptide having the sequence SEQ ID NO 001 and a DdmE polypeptide having the sequence SEQ ID NO 002 in a bacterial cell; particularly wherein the homologue of the DdmE polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae or Lactobacillaceae, particularly a member of genus Vibrio, particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue the DdmE polypeptide is a DdmE polypeptide listed in Table 1.

2. The expression vector according to claim 1 , wherein said DES further comprises a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide, wherein the DdmA polypeptide is selected from:

• a V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said V. cholerae DdmA polypeptide having the sequence SEQ ID NO 003; or

• a homologue of a V. cholerae DdmA polypeptide, wherein the V. cholerae DdmA polypeptide has the sequence SEQ ID NO 003, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmA polypeptide; wherein a DES comprising said polypeptide sequence having an identity of >70 compared to the sequence SEQ ID NO 003, or said homologue of the V. cholerae DdmA polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmA polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of a DES comprising a DdmD polypeptide having the sequence SEQ ID NO 001 , a DdmE polypeptide having the sequence SEQ ID NO 002, a DdmA polypeptide having the sequence SEQ ID NO 003, a DdmB polypeptide having the sequence SEQ ID NO 004, and a DdmC polypeptide having the sequence SEQ ID NO 005 in a bacterial cell; particularly wherein the homologue of the DdmA polypeptide, is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein the homologue of the DdmA polypeptide is a DdmA polypeptide listed in Table 1 ; and wherein the DdmB polypeptide is selected from:

• a homologue of a V. cholerae DdmB polypeptide, wherein the V. cholerae DdmB polypeptide has the sequence SEQ ID NO 004, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmB polypeptide; wherein a DES comprising said polypeptide sequence having an identity of >70% compared to the sequence SEQ ID NO 004, said homologue of the V. cholerae DdmB polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmB polypeptide has >10%, particularly >20%, more particularly >30% of the biological function of a DES comprising a DdmD polypeptide having the sequence SEQ ID NO 001 , a DdmE polypeptide having the sequence SEQ ID NO 002, a DdmA polypeptide having the sequence SEQ ID NO 003, a DdmB polypeptide having the sequence SEQ ID NO 004, and a DdmC polypeptide having the sequence SEQ ID NO 005 in a bacterial cell; particularly wherein the homologue of the DdmB polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein homologue of the DdmB polypeptide s a DdmB polypeptide listed in Table 1 ; and wherein the DdmC polypeptide is selected from

• a homologue of a V. cholerae DdmC polypeptide, wherein the V. cholerae DdmC polypeptide has the sequence SEQ ID NO 005, or a polypeptide sequence having an identity of >70%, >75%, >80%, >85%, particularly >90%, more particularly >95% compared to said homologue of the V. cholerae DdmC polypeptide; wherein a DES comprising said polypeptide sequence having an identity of >70% compared to the sequence SEQ ID NO 005, or said homologue of the V. cholerae DdmC polypeptide, or said polypeptide sequence having an identity of >70% compared to said homologue of the V. cholerae DdmC polypeptide has >30%, particularly >40%, more particularly >50% of the biological function of a DES comprising a DdmD polypeptide having the sequence SEQ ID NO 001 , a DdmE polypeptide having the sequence SEQ ID NO 002, a DdmA polypeptide having the sequence SEQ ID NO 003, a DdmB polypeptide having the sequence SEQ ID NO 004, and a DdmC polypeptide having the sequence SEQ ID NO 005 in a bacterial cell ; particularly wherein the homologue of the DdmC polypeptide is a naturally occurring polypeptide sequence present in a member of family Vibrionaceae, more particularly a member of genus Vibrio, still more particularly a species of V. cholerae, more particularly a V. cholerae 01 El Tor strain, most particularly wherein said homologue of the DdmC polypeptide is a DdmC polypeptide listed in table 1 .

3. The expression vector according to any one of the claims 1 or 2, wherein the DdmD polypeptide comprises: a. a functional superfamily II helicase domain comprising: i. a functional Walker A motif; and ii. a functional Walker B motif; and b. a functional nuclease domain.

4. The expression vector according to claim 2 or 3, wherein the DdmA polypeptide comprises a functional nuclease domain, and/or wherein the DdmC polypeptide comprises:

• a functional Walker A motif; and

• a DUF3732 domain; and wherein the functional Walker A domain and the DUF3732 domain are located on either side of a coiled-coil-containing region.

5. The expression vector according to any one of the claims 1 to 4, wherein the polynucleotide encoding said functional DES is within operable distance of a cis-acting regulatory element enabling expression of said functional DES in a bacterial cell.

6. The expression vector according to any one of the claims 1 to 5, wherein said polynucleotide encoding a functional DES is comprised within a transmissible genetic element, particularly a transmissible genetic element selected from a self-replicating genetic element, or a mobile genetic element, more particularly wherein the expression vector is a plasmid.

7. A method to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium capable of reducing, or eliminating plasmids; wherein the method comprises contacting said isolated bacterial cell, bacterial culture, bacterial population, or bacterial consortium with a composition comprising the expression vector encoding a functional DES as specified in any one of the claims 1 to 6, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium, leading to expression of the functional DES.

8. The method according to claim 7, wherein the method results in the reduction or elimination of intracellular plasmids from an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium.

9. The method according to claim 7 or 8, wherein the method protects an isolated bacterium, bacterial starter culture, bacterial population, or a bacterial consortium against plasmid maintenance.

10. The method according to any one of the claims 7 to 9, wherein the method results in the reduction or elimination of plasmid-encoded antibiotic resistance in an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium.

11. A method to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium capable of reducing, or eliminating extra-genomic, circular dsDNA; wherein the method comprises contacting said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium with a composition comprising an expression vector encoding a functional DES as specified in any one of the claims 1 to 6 under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial culture, or bacterial consortium, leading to expression of the functional DES; particularly wherein said extra-genomic, circular dsDNA comprises a plasmid, and/or a bacteriophage.

12. A method according to claim 11 , wherein the method protects an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium from maintenance of extra- genomic, circular dsDNA.

13. A method according to claim 11 or 12, wherein the method eliminates or reduces extra- genomic, circular dsDNA in an isolated bacterium, bacterial starter culture, bacterial population, or bacterial consortium.

14. A method to obtain an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium protected from bacteriophage infection; wherein the method comprises contacting said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium with a composition comprising an expression vector encoding a functional DES as specified in any one of the claims 2 to 6, under conditions facilitating uptake of the expression vector into said isolated bacterial cell, bacterial starter culture, bacterial population, or bacterial consortium, leading to expression of the functional DES.

15. A method according to claim 14, wherein the method eliminates or reduces intracellular bacteriophage within an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium.

16. A method according to claim 14 or 15, wherein the method prevents bacteriophage maintenance in an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium.

17. A method according to any one of the claims 14 to 16, wherein the method protects an isolated bacterium, bacterial population, bacterial starter culture, or bacterial consortium from bacteriophage-mediated lysis.

18. A method of protecting a recipient bacterium from extra-genomic, circular dsDNA; wherein the method comprises contacting said recipient bacterium with a donor bacterium expressing the expression vector encoding a functional DES as specified in any one of the claims 1 to 6, to provide a recipient bacterium which eliminates extra-genomic, circular dsDNA; particularly wherein:

• the DES comprises a functional DdmD polypeptide and a functional DdmE polypeptide as specified in claim 1 or 2, and said recipient bacterium is characterised by an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES, or

• the DES comprises a functional DdmD polypeptide and a functional DdmE polypeptide, a functional DdmA polypeptide, a functional DdmB polypeptide and a functional DdmC polypeptide as specified in claim 2, and said recipient bacterium is characterised by an improved protection against plasmids and bacteriophage infection compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the functional DES, and wherein optionally, the recipient bacterium and the donor bacterium are not identical.

19. An isolated bacterium characterised by expression of a recombinant (transgenic) functional DES, said DES comprising: a functional DdmD polypeptide, and a functional DdmE polypeptide as specified in claim 1 or 2, wherein said isolated bacterium is characterised by an improved capacity to eliminate plasmid compared to a bacterium of identical genetic configuration, but lacking the recombinant functional DES.

20. The isolated bacterium characterised by expression of a recombinant (transgenic) functional DES according to claim 19, said DES comprising: a functional DdmD polypeptide, and a functional DdmE polypeptide, a functional DdmA polypeptide, a functional DdmB polypeptide, and a functional DdmC polypeptide as specified in claim 2, wherein said recipient bacterium is characterised by an improved capacity to eliminate plasmid and bacteriophage compared to a bacterium of identical genetic configuration to the recipient bacterium, but lacking the recombinant functional DES.

21 . The expression vector according any one of the claims 1 to 6, the method according to any one of the claims 7 to 18, or the isolated bacterium according to claim 19 or 20, wherein said polynucleotide encoding a functional DES is within operable distance from: a constitutive promoter; or an inducible promoter, wherein said functional DES is expressed in the presence of at least one inducer, particularly an inducible promoter in which gene expression occurs in the presence of an inducer selected from arabinose, lactose, IPTG, tryptophan, a lantibiotic peptide or a functional analogue thereof.

22. The expression vector according to any one of the claims 1 to 6, or 21 , the method according to any one of the claims 7 to 18, or 21 , or the isolated recombinant bacterium according to any one of the claims 19 to 21 , wherein the polynucleotide encoding a functional DES is within operable distance of a synthetic promoter, particularly a promoter selected form the list consisting of araBAD, T7, T7lac, Sp6, Trp, lac, Ptac, pL, cspB, NBP3510, Pgrac, Pspac, P43, tetA, PrhaBAD, rpoS, pac, npr, Ipp, syn, Puo or 73.

23. The method according to any one of the claims 7 to 18, 21 or 22, or the isolated bacterium according to any one of the claims 19 to 22 wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium comprises or consists of species within the phylum Proteobacteria, particularly within Gamma proteobacteria, more particularly within Enterobacterales, still more particularly within family Enterobacteriaceae, still more particularly wherein the isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium is Escherichia coli.

24. The method according to any one of the claims 7 to 18, or 21 to 23, or the isolated bacterium according to any one of the claims 19 to 23, wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium does not comprise or consist of species within the within the class Gamma proteobacteria, particularly within order Vibrionales or Lactobacillaceae, more particularly within the family Vibrionaceae, still more particularly species within the genus Vibrio.

25. The method according to any one of the claims 7 to 18, or 21 to 24, or the isolated bacterium according to any one of the claims 19 to 24, wherein said isolated bacterium, recipient bacterium, bacterial starter culture, bacterial population, or bacterial consortium comprises or consists of species within a genus selected from the list consisting of Carnobacterium, Lactobacillus, Lactococcus, Streptococcus, Weissella, or Bacillus.

26. The method according to any one of the claims 7 to 18, or 21 to 25, or the isolated bacterium according to any one of the claims 20 to 25, wherein said bacteriophage circularises its genome upon cell entry, and/or replicates and/or propagates by means of a circular intermediate or plasmid-like state, particularly wherein the bacteriophage is a P1 phage, or a lambda phage.

27. A product selected from a food, food additive, feed, nutritional supplement, probiotic supplement, personal care product, health care product, or veterinary composition, said product comprising an expression vector according to any one of the claims 1 to 6, an isolated bacterium, bacterial starter culture, bacterial population, or bacteria consortium obtained by a method according to any one of the claims 7 to 18, or 21 to 26, or an isolated bacterium genetically modified to express a DES according to any one of the claims 19 to 26.