Hypervariation and phase variation in the bacteriophage 'resistome'

Most bacteria encode proteins for defence against infection by bacteriophages. The mechanisms that bring about phage defence are extremely diverse, suggesting frequent independent evolution of novel processes. Phage defence determinants are often plasmid or phage-encoded and many that are chromosomal show evidence of lateral transfer. Recent studies on restriction-modification (R-M) systems show that these genes are amongst the most rapidly evolving. Some bacteria have contingency genes that encode alternative target specificity determinants for Type I or Type III R-M systems, thus expanding the range of phages against which the host population is immune. The most counter-intuitive observation, however, is the prevalence of phase variation in many restriction systems, but recent arguments suggest that switching off expression of R-M systems can aid phage defence.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.     

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.     

Regulation of the HpyII restriction–modification system of Helicobacter pylori by gene deletion and horizontal reconstitution

Helicobacter pylori, Gram-negative, curved bacteria colonizing the human stomach, possess strain-specific complements of functional restriction-modification (R-M) systems. Restriction-modification systems have been identified in most bacterial species studied and are believed to have evolved to protect the host genome from invasion by foreign DNA. The large number of R-Ms homologous to those in other bacterial species and their strain-specificity suggest that H. pylori may have horizontally acquired these genes. A type IIs restriction-modification system, hpyIIRM, was active in two out of the six H. pylori strains studied. We demonstrate now that in most strains lacking M.HpyII function, there is complete absence of the R-M system. Direct DNA repeats of 80 bp flanking the hpyIIRM system allow its deletion, resulting in an "empty-site" genotype. We show that strains possessing this empty-site genotype and strains with a full but inactive hpyIIRM can reacquire the hpyIIRM cassette and functional activity through natural transformation by DNA from the parental R-M+ strain. Identical isolates divergent for the presence of an active HpyII R-M pose different restriction barriers to transformation by foreign DNA. That H. pylori can lose HpyII R-M function through deletion or mutation, and can horizontally reacquire the hpyIIRM cassette, is, in composite, a novel mechanism for R-M regulation, supporting the general hypothesis that H. pylori populations use mutation and transformation to regulate gene function.     

Combinational variation of restriction modification specificities in Lactococcus lactis

Three genes coding for a type I R-M system related to the class C enzymes have been identified on the chromosome of Lactococcus lactis strain IL1403. In addition, plasmids were found that encode only the HsdS subunit that directs R-M specificity. The presence of these plasmids in IL1403 conferred a new R-M phenotype on the host, indicating that the plasmid-encoded HsdS is able to interact with the chromosomally encoded HsdR and HsdM subunits. Such combinational variation of type I R-M systems may facilitate the evolution of their specificity and thus reinforce bacterial resistance against invasive foreign unmethylated DNA.     

Phase variable type III restriction-modification systems of host-adapted bacterial pathogens

Phase variation, the high-frequency on/off switching of gene expression, is a common feature of host-adapted bacterial pathogens. Restriction-modification (R-M) systems, which are ubiquitous among bacteria, are classically assigned the role of cellular defence against invasion of foreign DNA. These enzymes are not obvious candidates for phase variable expression, a characteristic usually associated with surface-expressed molecules subject to host immune selection. Despite this, numerous type III R-M systems in bacterial pathogens contain repetitive DNA motifs that suggest the potential for phase variation. Several roles have been proposed for phase variable R-M systems based on DNA restriction function. However, there is now evidence in several important human pathogens, including Haemophilus influenzae, Neisseria meningitidis and Neisseria gonorrhoeae, that these systems are 'phasevarions' (phase variable regulons) controlling expression of multiple genes via a novel epigenetic mechanism.     

Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.     

Identification of Restriction-Modification Systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.     

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.     

Context-dependent conservation of DNA methyltransferases in bacteria

DNA methytransferases (MTs) in bacteria are best understood in the context of restriction-modification (R-M) systems, which act as bacterial immune systems against incoming DNA including phages, but have also been described as selfish elements. But several orphan MTs, which are not associated with any restriction enzyme, have also been characterized and may protect against parasitism by R-M systems. The occurrence of MTs in these two contexts, namely as part of R-M systems or as orphans, is poorly understood. Here we report the results of a comparative genomic survey of DNA MTs across ～1000 bacterial genomes. We show that orphan MTs overwhelm R-M systems in their occurrence. In general, R-M MTs are poorly conserved, whereas orphans are nearly as conserved within a genus as any average gene. However, oligonucleotide usage and conservation patterns across genera suggest that both forms of MTs might have been horizontally acquired. We suggest that many orphan MTs might be 'degradation' products of R-M systems, based on the properties of orphan MTs encoded adjacent to highly diverged REs. In addition, several fully degraded R-M systems exist in which both the MT and the RE are highly divergent from their corresponding reference R-M pair. Despite their sporadic occurrence, conserved R-M systems are present in strength in two highly transformable genera, in which they may contribute to selection against integration of foreign DNA.     

A new algorithm for cluster analysis of genomic methylation: the Helicobacter pylori case

Motivation: The genomic methylation analysis is useful to typebacteria that have a high number of expressed type II methyltransferases.Methyltransferases are usually committed to Restriction andModification (R-M) systems, in which the restriction endonucleaseimposes high pressure on the expression of the cognate methyltransferasethat hinder R-M system loss. Conventional cluster methods donot reflect this tendency. An algorithm was developed for dendrogramconstruction reflecting the propensity for conservation of R-MType II systems. Results: The new algorithm was applied to 52 Helicobacter pyloristrains from different geographical regions and compared withconventional clustering methods. The algorithm works by firstgrouping strains that share a common minimum set of R-M systemsand gradually adds strains according to the number of the R-Msystems acquired. Dendrograms revealed a cluster of Africanstrains, which suggest that R-M systems are present in H.pylorigenome since its human host migrates from Africa. Availability: The software files are available at http://www.ff.ul.pt/paginas/jvitor/Bioinformatics/MCRM_algorithm.zip Contact: filipavale{at}fe.ucp.pt Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Martin Bishop     

Genomic methylation: a tool for typing Helicobacter pylori isolates

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.     

A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.