TnpB homologues exapted from transposons are RNA-guided transcription factors.

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage.

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

Novel Viruses That Lyse Plant and Human Strains of Kosakonia cowanii.

Kosakonia cowanii (syn. Enterobacter cowanii) is a highly competitive bacterium that lives with plant, insect, fish, bird, and human organisms. It is pathogenic on some plants and an opportunistic pathogen of human. Nine novel viruses that lyse plant pathogenic strains and/or human strains of K. cowanii were isolated, sequenced, and characterized. Kc166A is a novel kayfunavirus, Kc261 is a novel bonnellvirus, and Kc318 is a new cronosvirus (all Autographiviridae). Kc237 is a new sortsnevirus, but Kc166B and Kc283 are members of new genera within Podoviridae. Kc304 is a new winklervirus, and Kc263 and Kc305 are new myoviruses. The viruses differ in host specificity, plaque phenotype, and lysis kinetics. Some of them should be suitable also as pathogen control agents.

Genetic Modification of Sodalis Species by DNA Transduction.

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing, and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram-negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms.IMPORTANCE A large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherent nature of these associations, insect endosymbionts cannot be usually isolated in pure culture or genetically manipulated. Here we use a broad-host-range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad-host-range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

Factores de riesgo para infección del tracto urinario por microorganismos productores de betalactamasas de espectro extendido en niños en Huancayo, Perú / Risk factors for urinary tract infection by microorganisms producing extended spectrum beta-lactamase in children of Huancayo, Peru

Introducción: La infección del tracto urinario en los niños es una de las infecciones bacterianas más frecuentes con una alta tasa de recurrencia. Objetivo: Determinar los factores de riesgo para infección del tracto urinario adquirida en la comunidad por microorganismos productores de betalactamasas de espectro extendido en niños en Huancayo, Perú. Métodos: Estudio de tipo analítico con diseño de casos y controles. Se estudiaron 220 niños entre el mes de nacido hasta 13 años de edad, ingresados en el hospital nacional Ramiro Priale Priale con el diagnóstico de infección del tracto urinario durante el año 2019. Se distribuyeron en dos grupos (40 casos y 80 controles). Para cada paciente se llenó un cuestionario con las variables de interés y se realizó la comparación entre los grupos. Se realizó el análisis multivariado considerando significativo un valor de p< 0,05. Resultados: La frecuencia de infección del tracto urinario causada por microorganismos productores de betalactamasas de espectro extendido es de 18,18 por ciento. En los casos la edad predominante está entre 1 y 3 años con 42,5 por ciento, sexo femenino con 62,5 por ciento, la bacteria predominante es: Escherichia coli en 85,0 por ciento. Durante el análisis multivariado la presencia de infección del tracto urinario complicada tuvo OR 18,62 y p= 0,000 y la recurrente OR 12,98 y p= 0,004, ambas estadísticamente significativas para el desenlace de esta infección en los niños. Conclusión: Los factores de riesgo para infección del tracto urinario adquirida en la comunidad por microorganismos productores de betalactamasas de espectro extendido en niños son: infección del tracto urinario complicada y la recurrente(AU)

Introduction: Urinary tract infection in children is one of the most frequent bacterial infections with a high rate of recurrence. Objective: Determine the risk factors for community-acquired urinary tract infection by microorganisms producing extended-spectrum beta-lactamases in children of Huancayo, Peru. Methods: Analytical study with case-control design. 220 children from one month to 13 years of age were studied, whom were admitted to Ramiro Priale Priale National Hospital with the diagnosis of urinary tract infection during the year 2019. They were distributed in two groups (40 cases and 80 controls). For each patient, a questionnaire was completed with the variables of interest, and the comparison between the groups was made. The multivariate analysis was performed considering significant a value of p< 0.05. Results: The frequency of urinary tract infection caused by microorganisms producing extended-spectrum beta-lactamases is 18.18 percent. In the cases, the predominant age is between 1 and 3 years with 42.5 percent, female sex with 62.5 percent, the predominant bacterium is: Escherichia coli in 85.0 percent. During the multivariate analysis, the presence of complicated urinary tract infection had OR 18.62 and p= 0.000 and recurrent OR 12.98 and p= 0.004, both statistically significant for the outcome of this infection in children. Conclusion: The risk factors for community-acquired urinary tract infection by microorganisms producing extended-spectrum beta-lactamases in children are complicated and recurrent urinary tract infections(AU)

Complete genome sequence of Buttiauxella phage vB_ButM_GuL6.

We present here the results of the analysis of the complete genome sequence of a lytic bacteriophage, vB_ButM_GuL6, which is the first virus isolated from Buttiauxella. Electron microscopy revealed that vB_ButM_GuL6 belongs to the family Myoviridae, order Caudovirales. The genome of vB_ButM_GuL6 is a linear, circularly permuted 178,039-bp dsDNA molecule with a GC content of 43.4%. It has been predicted to contain 282 protein-coding genes and two tRNA genes, tRNA-Met and tRNA-Gly. Using bioinformatics approaches, 99 (36%) of the vB_ButM_GuL6 genes were assigned a putative function. Genome-wide comparisons and phylogenetic analysis indicated that vB_ButM_GuL6 represents a new species of the subfamily Tevenvirinae and is most closely related to Escherichia virus RB43. These phages, together with Cronobacter phages Miller, CfP1, and IME-CF2, likely form a new genus within the subfamily Tevenvirinae.

Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

Bioinformatic analyses of a potential Salmonella-virus-FelixO1 biocontrol phage BPS15S6 and the characterisation and anti-Enterobacteriaceae-pathogen activity of its endolysin LyS15S6.

Foodborne Enterobacteriaceae pathogens, especially Salmonella, still seriously threaten food safety. To establish a foundation for further developing phage- and endolysin-based methods combating these pathogens, in this study, the newly isolated Salmonella-virus-FelixO1 phage BPS15S6 for biocontrol purposes was characterised by genomic bioinformatic analysis, and then its endolysin LyS15S6 was obtained using a prokaryotic expression system, characterised in vitro and evaluated in the antibacterial efficacy. It was shown that BPS15S6 had an 87,609-bp genome with 130 open reading frames and does not appear to carry known lysogeny-associated genes and other damaging genetic determinants and is unlikely to perform generalised transduction. Furthermore, LyS15S6 was determined to possess the high enzymatic activity of 1,001,000 U mg-1 and the broad spectrum of lysing 56 tested Gram-negative strains. The assays of thermostability and optimum pH revealed that LyS15S6 was stable up to 40 °C and more active at pH 7. Notably, we demonstrate that edible ε-poly-L-lysine (EPL) can be used as an outer-membrane permeabiliser to improve the antibacterial performance of endolysins. When combined with 1 µg ml-1 EPL, 2 µM LyS15S6 could cause 3-4 log viable cell reductions of the three tested Enterobacteriaceae pathogens in vitro after 2 h of reaction at 25 °C and 2.56 and 3.14 log reductions of Salmonella ATCC13076 after 15 min of reaction at 25 °C and 2 h of reaction at 8 °C respectively. A new strategy, the combined application of endolysins and edible EPL for combating Enterobacteriaceae pathogens in food, is thus presented in this work.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Variations on a protective theme: Hamiltonella defensa infections in aphids variably impact parasitoid success.

Protective mutualisms are common in nature and include insect infections with cryptic symbionts that defend against pathogens and parasites. An archetypal defensive symbiont, Hamiltonella defensa protects aphids against parasitoids by disabling wasp development. Successful defense requires H. defensa infection with bacteriophages (APSEs), which play other key roles in mutualism maintenance. Genomes of H. defensa strains are highly similar in gene inventories, varying primarily in mobile element content. Protective phenotypes are highly variable across aphid models depending on H. defensa/APSE, aphid and wasp genotypes. Infection frequencies of H. defensa are highly dynamic in field populations, influenced by a variety of selective and non-selective factors confounding biological control implications. Overall, H. defensa infections likely represent a global aphid protection network with effects radiating outward from focal interactions.

Bacteriophage acquisition restores protective mutualism.

Insects are frequently infected with inherited facultative symbionts known to provide a range of conditionally beneficial services, including host protection. Pea aphids (Acyrthosiphon pisum) often harbour the bacterium Hamiltonella defensa, which together with its associated bacteriophage A. pisum secondary endosymbiont (APSE) confer protection against an important natural enemy, the parasitic wasp Aphidius ervi. Previous studies showed that spontaneous loss of phage APSE resulted in the complete loss of the protective phenotype. Here, we demonstrate that APSEs can be experimentally transferred into phage-free (i.e. non-protecting) Hamiltonella strains. Unexpectedly, trials using injections of phage particles alone failed, with successful transfer occurring only when APSE and Hamiltonella were simultaneously injected. After transfer, stable establishment of APSE fully restored anti-parasitoid defenses. Thus, phages associated with heritable bacterial symbionts can move horizontally among symbiont strains facilitating the rapid transfer of ecologically important traits although natural barriers may preclude regular exchange.

Mechanisms of Type I-E and I-F CRISPR-Cas Systems in Enterobacteriaceae.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative Enterobacteriaceae family, especially Escherichia coli and Pectobacterium atrosepticum, have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the Enterobacteriaceae. We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.

Comparative analysis and characterization of Enterobacteria phage SSL-2009a and 'HK578likevirus' bacteriophages.

Enterobacteria phage SSL-2009a is a virulent bacteriophage with strong and abroad lytic ability against lots of engineering E. coli strains. In this study, we re-sequenced its whole genome and made a detail analysis on its genomic and proteomic characteristics according to the updated genomic sequence. The genome of SSL-2009a is a circular double-stranded DNA of 44,899 base pairs in length, with a 54.67% G + C content. A total of 67 open reading frames were predicted as protein coding sequences, 24 of which encode products highly homologous to known phage proteins. There are 10 promoters and 22 terminators identified in the genome of SSL-2009a, but no tRNA is found. SSL-2009a belongs to the 'HK578likevirus' genus of Siphoviridae. Comparative analyses indicated that other twelve phages share high homology with SSL-2009a at nucleotide and amino acid levels and also should be clustered into the same genus. In-depth analysis was performed to reveal the genomic, proteomic, and morphological features of these 'HK578likevirus' phages, which may promote our understanding of Enterobacteria phage SSL-2009a and the 'HK578likevirus' genus, even the biodiversity and evolution of bacteriophages.

Unlocking the Potential of 46 New Bacteriophages for Biocontrol of Dickeya Solani.

Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.

Characterization of a Salmonella Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria.

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.

Characterization of two related Erwinia myoviruses that are distant relatives of the PhiKZ-like Jumbo phages.

Bacteriophages are a major force in the evolution of bacteria due to their sheer abundance as well as their ability to infect and kill their hosts and to transfer genetic material. Bacteriophages that infect the Enterobacteriaceae family are of particular interest because this bacterial family contains dangerous animal and plant pathogens. Herein we report the isolation and characterization of two jumbo myovirus Erwinia phages, RisingSun and Joad, collected from apple trees. These two genomes are nearly identical with Joad harboring two additional putative gene products. Despite mass spectrometry data that support the putative annotation, 43% of their gene products have no significant BLASTP hit. These phages are also more closely related to Pseudomonas and Vibrio phages than to published Enterobacteriaceae phages. Of the 140 gene products with a BLASTP hit, 81% and 63% of the closest hits correspond to gene products from Pseudomonas and Vibrio phages, respectively. This relatedness may reflect their ecological niche, rather than the evolutionary history of their host. Despite the presence of over 800 Enterobacteriaceae phages on NCBI, the uniqueness of these two phages highlights the diversity of Enterobacteriaceae phages still to be discovered.

Identification and characterization of new broad host-range rV5-like coliphages C203 and P206 directed against enterobacteria.

We isolated and characterized two novel rV5-like lytic bacteriophages from independently collected food samples. Nucleotide sequence analysis revealed that these phages have linear double-stranded DNA genomes comprising 138,073 bp with 213 CDS and 5 tRNA genes. The two genomes contain completely identical nucleotide sequence, albeit there is a 10,718 bp-long shift in the sequence. The GC content of the phage genomes was 43.7% and they showed high general homology to rV5-like phages. The new phages were termed C203 and P206. The genome of both phages contains a unique ORF that encodes for a putative phage homing endonuclease. The phage produced clear plaques with a burst size of approx. 1000 viral particles and a latent period of 60 min. Morphological investigation indicated that the new phages are members of the family Myoviridae with an approximate head length of 85 nm, tail length of 75 nm, and a head width of 96 nm. C203 and P206 exhibit a broad and uniform host range, which included enterohemorrhagic Escherichia coli strains of serogroup O157, multi drug resistant (MDR) E. coli strains of various sero- and pathotypes, and both Shigella sonnei and S. dysenteriae strains. C203 and P206 both effectively reduced the number of living EHEC O157:H7 Sakai in experimentally inoculated minced meat. The same broad host range, the lack of any virulence related genes, the stability and its short latent period suggest that these newly found phages could be suitable candidates as a bio-control agents against food-borne pathogenic Enterobacteria.

The lytic Myoviridae of Enterobacteriaceae form tight recombining assemblages separated by discontinuities in genome average nucleotide identity and lateral gene flow.

In Bacteria, a working consensus of species circumscription may have been reached and one of the most prominent criteria is high average nucleotide identity (ANI). ANI in effect groups strains that may recombine more or less frequently, depending on their biology, as opposed to rare interspecies gene transfer. For bacteriophages, which show various lifestyles, the nature of the fundamental natural unit, if it exists in a biological sense, is not well understood and defined. The approaches based on dot-plots are useful to group similar bacteriophages, yet are not quantitative and use arbitrarily set cut-offs. Here, we focus on lytic Myoviridae and test the ANI metric for group delineation. We show that ANI-based groups are in agreement with the International Committee on Taxonomy of Viruses (ICTV) classification and already established dot-plot groups, which are occasionally further refined owing to higher resolution of ANI analysis. Furthermore, these groups are separated among themselves by clear ANI discontinuities. Their members readily exchange core genes with each other while they do not with bacteriophages of other ANI groups, not even with the most similar. Thus, ANI-delineated groups may represent the natural units in lytic Myoviridae evolution with features that resemble those encountered in bacterial species.

Culture-Facilitated Comparative Genomics of the Facultative Symbiont Hamiltonella defensa.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.