Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

Background

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.

Results

Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.

Conclusions

SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-831) contains supplementary material, which is available to authorized users.     

A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.     

Restriction-modification systems and bacteriophage invasion: Who wins?

The success of a phage that infects a bacterial cell possessing a restriction-modification (R-M) system depends on the activities of the host methyltransferase and restriction endonuclease, and the number of susceptible sites in the phage genome. However, there is no model describing this dependency and linking it to observable parameters such as the fraction of surviving cells under excess phage, or probability of plating at low amount of phages. We model the phage infection of a cell with a R-M system as a pure birth process with a killing state. We calculate the transitional probabilities and the stationary distribution for this process. We generalize the model developed for a single cell to the case of multiple identical cells invaded by a Poisson-distributed number of phages. The R-M enzyme activities are assumed to be constant, time-dependent, or random. The obtained results are used to estimate the ratio of the methyltransferase and endonuclease activities from the observed fraction of surviving cells.     

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.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.     

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.     

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.     

Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

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.     

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10(-1)-10(-2)). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study.     

Genetic analysis of the pnp-deaD genetic region reveals membrane lipoprotein NlpI as an independent participant in cold acclimatization of Salmonella enterica serovar Typhimurium

Plasmid pAMI7 of the methylotrophic bacterium Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) encodes a functional type II restriction-modification (R-M) system designated PamI. Homologous systems were identified in the genomes of distinct taxonomic groups of Bacteria and Archaea, which provides evidence that horizontal gene transfer has contributed to the wide dissemination of R-M modules - even between domains. Analysis of the cleavage specificity of the R.PamI endonuclease revealed that this protein is an isoschizomer of restriction enzyme NcoI. Interestingly, bioinformatic analyses suggest that R.PamI and NcoI are accompanied by methyltransferases of different methylation specificities (C5-methylcytosine and N4-methylcytosine methyltransferases, respectively), which possibly exemplifies recombinational shuffling of genes coding for individual components of R-M systems. The PamI system can stabilize plasmid pAMI7 in a bacterial population, most probably at the postsegregational level. Therefore, it functions in an analogous manner to plasmid-encoded toxin-antitoxin (TA) systems. Since the TA system of pAMI7 is nonfunctional, it is highly probable that this lack is compensated by the stabilizing activity of PamI. This indicates the crucial role of the analyzed R-M system in the stable maintenance of pAMI7, which is, to our knowledge, the first report of 'symbiosis' between a R-M system and a plasmid in the Alphaproteobacteria.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.     

Molecular evolution of bacteriophages: evidence of selection against the recognition sites of host restriction enzymes

Restriction enzymes produced by bacteria serve as a defense against invading bacteriophages, and so phages without other protection would be expected to undergo selection to eliminate recognition sites for these enzymes from their genomes. The observed frequencies of all restriction sites in the genomes of all completely sequenced DNA phages (T7, lambda, phi X174, G4, M13, f1, fd, and IKe) have been compared to expected frequencies derived from trinucleotide frequencies. Attention was focused on 6-base palindromes since they comprise the typical recognition sites for type II restriction enzymes. All of these coliphages, with the exception of lambda and G4, exhibit significant avoidance of the particular sequences that are enterobacterial restriction sites. As expected, the sequenced fraction of the genome of phi 29, a Bacillus subtilis phage, lacks Bacillus restriction sites. By contrast, the RNA phage MS2, several viruses that infect eukaryotes (EBV, adenovirus, papilloma, and SV40), and three mitochondrial genomes (human, mouse, and cow) were found not to lack restriction sites. Because the particular palindromes avoided correspond closely with the recognition sites for host enzymes and because other viruses and small genomes do not show this avoidance, it is concluded that the effect indeed results from natural selection.