T4-like Escherichia coli phages from the environment carry blaCTX-M.

The resistance determinant blaCTX-M has many variants and has been the most commonly reported gene in clinical isolates of extended spectrum beta-lactamase producing Escherichia coli. Phages have been speculated as potential reservoirs of resistance genes and efficient vehicles for horizontal gene transfer. The objective of the study was to determine the prevalence and characterize bacteriophages that harbour the resistance determinant blaCTX-M . Escherichia coli specific bacteriophages were isolated from 15 samples including soil and water across Mangaluru, India using bacterial hosts that were sensitive to ß-lactams. Phenotypic and genotypic characterization based on plaque morphology, host range, restriction fragment length polymorphism (RFLP), presence of blaCTX-M and electron microscopy was performed. Of 36 phages isolated, seven were positive for Group 1 of blaCTX-M . Based on host range and RFLP pattern, the seven phages were classified into four distinct groups, each harbouring a variant of blaCTX-M . Five phages were T4-like Myoviridae by electron microscopy which was further confirmed by polymerase chain reaction (PCR) for T4 specific gp14. Generalized transduction of the CTX-M gene from these phages was also observed. The high prevalence (20%) of this gene blaCTX-M in the phage pool confirms the significant role of Myoviridae members, specifically T4-like phages in the dissemination of this resistance gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The CTX-M gene that confers resistance to Beta-lactam class of drugs is widespread and diverse. Understanding mechanisms of antimicrobial resistance transfer is a key to devise methods for controlling it. Few studies indicate that bacteriophages are involved in the transfer of this gene but the type of phages involved and the degree of involvement remains to be explored. Our work has been able to identify the class of phages and the magnitude of involvement in the dissemination of this gene.

[Isolation and phylogenetic analysis of major capsid gene (g23) of bacteriophages infecting Sinorhizobium meliloti].

Objective: In order to provide scientific data for studying the ecology of phage infecting Sinorhizobium meliloti, we examined morphological characteristics of rhizobiophages and their phylogenetic status of the major captain protein g23. Methods: Rhizobiophages were isolated by the double-layer plate method with host Sinorhizobium meliloti USDA1002T. The morphological characteristic of rhizobiophages were studied by transmission electron microscope. Meanwhile, rhizobiophage DNA was extracted, and the g23 that encodes the major capsid protein of bacteriophages was chosen as objective gene in PCR amplification. Results: Three rhizobiophages were isolated, all had an icosahedral head with approximately 81 to 86 nm in diameter and a long contractile tail with 54 to 70 nm in length. Basic local alignment search tool searches in website of national center for biotechnology information (NCBI) revealed that the g23 amino acid sequences obtained in this study had high identity with each other, but had very lower identity with those from T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens. Phylogenetic analysis showed that the isolated g23 sequences formed a unique clade with those clones obtained from different ecosystem. Conclusion: All results indicated that the isolated rhizobiophages belong to family Myoviridae, a new group of T4 phages, which had lower identity with the g23 clones obtained in different environment.

Escherichia coli O157:H7 typing phage V7 is a T4-like virus.

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.

Characterization of the major capsid genes (g23) of T4-type bacteriophages in the wetlands of northeast China.

To obtain genetic information and to evaluate the composition of T4-type bacteriophage (phage) communities in wetlands, environmental soil and water DNAs were obtained from two natural wetlands dominated by Carex lasiocarpa and Deyeuxia angustifolia plant species, and a neighboring paddy field in Sanjiang plain of northeast China. The biomarker gene of g23, which encodes the major capsid protein of T4-type phages, was amplified with primers MZIA1bis and MZIA6, and the PCR products were cloned and sequenced. In total, 96 and 50 different g23 clones were obtained from natural wetlands and a paddy field, respectively. A larger number of clones with low levels of identity to known sequences were found in water than in soil both in the natural wetlands and the paddy field, suggesting that many of T4-type phages in wetland water and paddy floodwater in Sanjiang plain are uncharacterized. Phylogenetic analyses showed that the g23 clones in natural wetlands, irrespective of water and soil, were distinctly different from those in marine waters, lake waters, and upland black soils, but were similar to those in paddy fields. The UniFrac analysis of g23 assemblages indicated that T4-type phage community compositions were different between soils and waters, and also were different between the natural wetlands and the paddy field. In general, the global analysis of g23 clone assemblages demonstrated that T4-type phage community compositions were different among natural wetlands, marines, lakes, paddy fields, and upland black soils.

Clamp loader ATPases and the evolution of DNA replication machinery.

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.

Phylogenomics of T4 cyanophages: lateral gene transfer in the 'core' and origins of host genes.

The last two decades have revealed that phages (viruses that infect bacteria) are abundant and play fundamental roles in the Earth System, with the T4-like myoviruses (herein T4-like phages) emerging as a dominant 'signal' in wild populations. Here we examine 27 T4-like phage genomes, with a focus on 17 that infect ocean picocyanobacteria (cyanophages), to evaluate lateral gene transfer (LGT) in this group. First, we establish a reference tree by evaluating concatenated core gene supertrees and whole genome gene content trees. Next, we evaluate what fraction of these 'core genes' shared by all 17 cyanophages appear prone to LGT. Most (47 out of 57 core genes) were vertically transferred as inferred from tree tests and genomic synteny. Of those 10 core genes that failed the tree tests, the bulk (8 of 10) remain syntenic in the genomes with only a few (3 of the 10) having identifiable signatures of mobile elements. Notably, only one of these 10 is shared not only by the 17 cyanophages, but also by all 27 T4-like phages (thymidylate synthase); its evolutionary history suggests cyanophages may be the origin of these genes to Prochlorococcus. Next, we examined intragenic recombination among the core genes and found that it did occur, even among these core genes, but that the rate was significantly higher between closely related phages, perhaps reducing any detectable LGT signal and leading to taxon cohesion. Finally, among 18 auxiliary metabolic genes (AMGs, a.k.a. 'host' genes), we found that half originated from their immediate hosts, in some cases multiple times (e.g. psbA, psbD, pstS), while the remaining have less clear evolutionary origins ranging from cyanobacteria (4 genes) or microbes (5 genes), with particular diversity among viral TalC and Hsp20 sequences. Together, these findings highlight the patterns and limits of vertical evolution, as well as the ecological and evolutionary roles of LGT in shaping T4-like phage genomes.

Persistence of bacteriophage T4 in a starved Escherichia coli culture: evidence for the presence of phage subpopulations.

Bacteriophage T4 is able to adjust its development to the growth parameters of the host cell. Here, we present evidence for the production of two different subpopulations of phage particles, which differ in their ability to infect starved Escherichia coli cells. The ability of phage T4 to produce a fraction of virions unable to infect starved cells is linked to the functions of genes rI and rIII, as well as rIIA. This may represent the adaptation of phage T4 in order to persist in unfavourable environmental conditions.

[Genetic diversity of major capsid genes (g23) of T4-type bacteriophages in natural environments--a review].

During the past two decades, the genetic diversities of bacterial and fungal communities and their relationship with the inhabited environments were intensively studied with the development of molecular biological techniques. However, although bacteriophages are ubiquitous and more abundant than their hosts in biosphere, their diversity is still little known. In this paper, we targeted the major capsid genes (g23) of T4-type bacteriophages and reviewed the recent progress on their genetic diversity. The distribution of g23 of T4-type bacteriophages was distinctly different among natural environments of marines, lakes and paddy fields, and the majority of g23 were grouped into several novel clusters according to their obtained environments. In addition, several research tips and future research tendencies for the study of environmental g23 were also addressed.

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

Low-temperature T4-like coliphages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20.

Bacteriophages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20, showing an unusual low-temperature plating profile and producing constantly growing plaques, were isolated from aquatic environments of Lithuania. Although vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20 resembled phage T4 both in their genome size and in their major structural protein (gp23) pattern, physiological properties of all three phages tested differed significantly from those of T4. With an optimum temperature for plating around 24 degrees C and a high efficiency of plating in the range 7-30 degrees C, bacteriophages vB_EcoM-VR7 and vB_EcoM-VR20 failed to plate at 37 degrees C, whereas phage vB_EcoM-VR5 could not be plated at 40 degrees C. Sequence analysis of diagnostic g23 PCR products revealed that g23 of vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20 differed from the corresponding T4 g23 DNA sequence by 21, 21 and 20%, respectively.

Isolation and Characterization T4- and T7-Like Phages that Infect the Bacterial Plant Pathogen Agrobacterium tumefaciens.

In the rhizosphere, bacteria-phage interactions are likely to have important impacts on the ecology of microbial communities and microbe-plant interactions. To better understand the dynamics of Agrobacteria-phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacteriumtumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04-a T4-like phage belonging to the Myoviridae family-was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.

The immense journey of bacteriophage T4--from d'Hérelle to Delbrück and then to Darwin and beyond.

In spite of their importance, the genomics, diversity and evolution of phages and their impact on the biosphere have remained largely unexplored research domains in microbiology. Here, we report on some recent studies with the T4 phage superfamily that shed some new light on these topics.

Assessing the diversity of the g23 gene of T4-like bacteriophages from Lake Baikal with high-throughput sequencing.

Based on second generation sequencing (MiSeq platform, Illumina), we determined the genetic diversity of T4-like bacteriophages of the family Myoviridae by analysing fragments of the major capsid protein gene g23 in the plankton of Lake Baikal. The sampling depth in our study was significantly higher than in those obtained by the Sanger method before. We obtained 33 701 sequences of the g23 gene fragments, 141 operational taxonomic units (OTUs) of which were identified. 86 OTUs (60.9%) had the closest relatives from lakes Bourget and Annecy, and 28 OTUs (19.8%) had the highest identity with the Baikal g23 clones, which had been previously identified in the northern and southern basins of the lake by the Sanger method. The remaining OTUs were similar to the clones from other ecosystems. We showed a high genetic diversity of T4-type bacteriophages and a genetic difference with the phage communities from other ecosystems.

Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli.

Numerous T4-like Escherichia coli phages were isolated from human stool and environmental wastewater samples in Bangladesh and Switzerland. The sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages T4, RB69, RB49, and JS98. Thus, JS98 defines a new major subgroup of E. coli T4-like phages. We conducted an analysis of the 169-kb JS98 genome sequence. Overall, 198 of the 266 JS98 open reading frames (ORFs) shared amino acid sequence identity with the reference T4 phage, 41 shared identity with other T4-like phages, and 27 ORFs lacked any database matches. Genes on the plus strand encoded virion proteins, which showed moderate to high sequence identity with T4 proteins. The right genome half of JS98 showed a higher degree of sequence conservation with T4 and RB69, even for the nonstructural genes, than did the left genome half, containing exclusively nonstructural genes. Most of the JS98-specific genes were found in the left genome half. Two came as a hypervariability cluster, but most represented isolated genes, suggesting that they were acquired separately in multiple acquisition events. No evidence for DNA exchange between JS98 phage and the E. coli host genome or coliphages other than T4 was observed. No undesired genes which could compromise its medical use were detected in the JS98 genome sequence.

The sequences and activities of RegB endoribonucleases of T4-related bacteriophages.

The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine-Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression. Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB. The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart. Functional studies using plasmid-phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations. We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme.

The diversity and evolution of the T4-type bacteriophages.

Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses.

The genome, proteome and phylogenetic analysis of Sinorhizobium meliloti phage ΦM12, the founder of a new group of T4-superfamily phages.

Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12's novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria.

Genetic insertions and diversification of the PolB-type DNA polymerase (gp43) of T4-related phages.

In Escherichia coli phage T4 and many of its phylogenetic relatives, gene 43 consists of a single cistron that encodes a PolB family (PolB-type) DNA polymerase. We describe the divergence of this phage gene and its protein product (gp43) (gene product 43) among 26 phylogenetic relatives of T4 and discuss our observations in the context of diversity among the widely distributed PolB enzymes in nature. In two T4 relatives that grow in Aeromonas salmonicida phages 44RR and 25, gene 43 is fragmented by different combinations of three distinct types of DNA insertion elements: (a) a short intercistronic untranslated sequence (IC-UTS) that splits the polymerase gene into two cistrons, 43A and 43B, corresponding to N-terminal (gp43A) and C-terminal (gp43B) protein products; (b) a freestanding homing endonuclease gene (HEG) inserted between the IC-UTS and the 43B cistron; and (c) a group I intron in the 43B cistron. Phage 25 has all three elements, whereas phage 44RR has only the IC-UTS. We present evidence that (a) the split gene of phage 44RR encodes a split DNA polymerase consisting of a complex between gp43A and gp43B subunits; (b) the putative HEG encodes a double-stranded DNA endonuclease that specifically cleaves intron-free homologues of the intron-bearing 43B site; and (c) the group I intron is a self-splicing RNA. Our results suggest that some freestanding HEGs can mediate the homing of introns that do not encode their own homing enzymes. The results also suggest that different insertion elements can converge on a polB gene and evolve into a single integrated system for lateral transfer of polB genetic material. We discuss the possible pathways for the importation of such insertion elements into the genomes of T4-related phages.

Characterization of a novel T4-type Stenotrophomonas maltophilia virulent phage Smp14.

Stenotrophomonas maltophilia (Sm), with most of the isolates being resistant to multidrugs, is an opportunistic bacterium causing nosocomial infections. In this study, a novel virulent Sm phage, Smp14, was characterized. Electron microscopy showed that Smp14 resembled members of Myoviridae and adsorbed to poles of the host cells during infection. It lysed 37 of 87 clinical Sm isolates in spot test, displayed a latent period of ca. 20 min, and had a burst size of ca. 150. Its genome (estimated to be 160 kb by PFGE), containing m4C and two unknown modified bases other than m5C and m6A as identified by HPLC, resisted to digestion with many restriction endonucleases except MseI. These properties indicate that it is a novel Sm phage distinct from the previously reported phiSMA5 which has a genome of 250 kb digestible with various restriction enzymes. Sequencing of a 16 kb region revealed 12 ORFs encoding structural proteins sharing 15-45% identities with the homologues from T4-type phages. SDS-PAGE displayed 20 virion proteins, with the most abundant one being the 39 kDa major capsid protein (gp23), which had the N-terminal 52 amino acids removed. Phylogenetic analysis based on gp23 classified Smp14 into a novel single-membered T4-type subgroup.

Hoc protein regulates the biological effects of T4 phage in mammals.

We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours.