Andrey V. Letarov

Phages in nature.

Phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as ‘phages’ is debatable, but they are included as such in the scope this article. Phage have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools, and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together recently to provide unparalleled insights into these small but vital constituents of the microbial world.

The Genome of S-PM2, a “Photosynthetic” T4-Type Bacteriophage That Infects Marine Synechococcus Strains

Bacteriophage S-PM2 infects several strains of the abundant and ecologically important marine cyanobacterium Synechococcus. A large lytic phage with an isometric icosahedral head, S-PM2 has a contractile tail and by this criterion is classified as a myovirus (1). The linear, circularly permuted, 196,280-bp double-stranded DNA genome of S-PM2 contains 37.8% G+C residues. It encodes 239 open reading frames (ORFs) and 25 tRNAs. Of these ORFs, 19 appear to encode proteins associated with the cell envelope, including a putative S-layer-associated protein. Twenty additional S-PM2 ORFs have homologues in the genomes of their cyanobacterial hosts. There is a group I self-splicing intron within the gene encoding the D1 protein. A total of 40 ORFs, organized into discrete clusters, encode homologues of T4 proteins involved in virion morphogenesis, nucleotide metabolism, gene regulation, and DNA replication and repair. The S-PM2 genome encodes a few surprisingly large (e.g., 3,779 amino acids) ORFs of unknown function. Our analysis of the S-PM2 genome suggests that many of the unknown S-PM2 functions may be involved in the adaptation of the metabolism of the host cell to the requirements of phage infection. This hypothesis originates from the identification of multiple phage-mediated modifications of the hosts photosynthetic apparatus that appear to be essential for maintaining energy production during the lytic cycle.

Isolation and characterization of a novel indigenous intestinal N4-related coliphage vB_EcoP_G7C

Lytic coliphage vB_EcoP_G7C and several other highly related isolates were obtained repeatedly from the samples of horse feces held in the same stable thus representing a component of the normal indigenous intestinal communities in this population of animals. The genome of G7C consists of 71,759 bp with terminal repeats of about 1160 bp, yielding approximately 73 kbp packed DNA size. Seventy-eight potential open reading frames, most of them unique to N4-like viruses, were identified and annotated. The overall layout of functional gene groups was close to that of the original N4 phage, with some important changes in late gene area including new tail fiber proteins containing hydrolytic domains. Structural proteome analysis confirmed all the predicted subunits of the viral particle. Unlike N4 itself, phage G7C did not exhibit a lysis-inhibited phenotype.

The diversity of coliphages and coliforms in horse feces reveals a complex pattern of ecological interactions

ABSTRACT The diversity of coliphages and indigenous coliform strains (ICSs) simultaneously present in horse feces was investigated by culture-based and molecular methods. The richness of coliforms (as estimated by the Chao1 method) is about 1,000 individual ICSs distinguishable by genomic fingerprinting present in a single sample of feces. This unexpectedly high value indicates that some factor limits the competition of coliform bacteria in the horse gut microbial system. In contrast, the diversity of phages active against any selected ICS is generally limited to one to three viral genotypes present in the sample. The sensitivities of different ICSs to simultaneously present coliphages overlap only slightly; the phages isolated from the same sample on different ICSs are usually unrelated. As a result, the titers of phages in fecal extract as determined for different Escherichia coli strains and ICSs may differ by several orders of magnitude. Summarizing all the data, we propose that coliphage infection may provide a selection pressure that maintains the high level of coliform diversity, restricting the possibility of a few best competitors outgrowing other ICSs. We also observed high-magnitude temporal variations of coliphage titers as determined using an E. coli C600 test culture in the same animal during a 16-day period of monitoring. No correlation with total coliform count was observed. These results are in good agreement with our hypothesis.

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Significance The discovery of a novel modification system that inserts 7-deazaguanine derivatives in DNA, modifications thought until now to occur only in RNA, is an excellent illustration of the power of biological evolution to alter the ultimate function not only of the distinct proteins but also of entire metabolic pathways. The extensive lateral transfer of the gene cluster responsible for this modification highlights its significance as a previously unrecognized foreign DNA defense system that bacteria and phages use to protect their genomes. The characterization of these DNA modification pathways also opens the door to novel tools to manipulate nucleic acids. The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ0 and 2’-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2’-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.

Genomic Sequencing and Biological Characteristics of a Novel Escherichia Coli Bacteriophage 9g, a Putative Representative of a New Siphoviridae Genus

Bacteriophage 9g was isolated from horse feces using Escherichia coli C600 as a host strain. Phage 9g has a slightly elongated capsid 62 × 76 nm in diameter and a non-contractile tail about 185 nm long. The complete genome sequence of this bacteriophage consists of 56,703 bp encoding 70 predicted open reading frames. The closest relative of phage 9g is phage PhiJL001 infecting marine alpha-proteobacterium associated with Ircinia strobilina sponge, sharing with phage 9g 51% of amino acid identity in the main capsid protein sequence. The DNA of 9g is resistant to most restriction endonucleases tested, indicating the presence of hypermodified bases. The gene cluster encoding a biosynthesis pathway similar to biosynthesis of the unusual nucleoside queuosine was detected in the phage 9g genome. The genomic map organization is somewhat similar to the typical temperate phage gene layout but no integrase gene was detected. Phage 9g efficiently forms stable associations with its host that continues to produce the phage over multiple passages, but the phage can be easily eliminated via viricide treatment indicating that no true lysogens are formed. Since the sequence, genomic organization and biological properties of bacteriophage 9g are clearly distinct from other known Enterobacteriaceae phages, we propose to consider it as the representative of a novel genus of the Siphoviridae family.

Variations in O-Antigen Biosynthesis and O-Acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages.

Function of bacteriophage G7C esterase tailspike in host cell adsorption

Bacteriophages recognize and bind to their hosts with the help of receptor‐binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C – tailspikes gp63.1 and gp66 – are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N‐terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1‐gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1‐gp66 interaction is widespread among all three families of tailed bacteriophages.

Diversity and dynamics of bacteriophages in horse feces

The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5–11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (103-107 plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain.