3D Structure of the Tail Complex of E. coli Bacteriophage DT57C

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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.The work of the laboratory in the Winogradsky Institute was partially supported by Russian Science Foundation (RSF) grant #15–15‐0013

Novel genomic island modifies DNA with 7-deazaguanine derivatives

The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ[subscript 0]) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ[subscript 0] 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 preQ[subscript 0] 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.Deutsche ForschungsgemeinschaftSingapore-MIT Alliance in Research and Technology (SMART

The Burden of Survivors: How Can Phage Infection Impact Non-Infected Bacteria?

The contemporary understanding of complex interactions in natural microbial communities and the numerous mechanisms of bacterial communication challenge the classical concept of bacteria as unicellular organisms. Microbial populations, especially those in densely populated habitats, appear to behave cooperatively, coordinating their reactions in response to different stimuli and behaving as a quasi-tissue. The reaction of such systems to viral infection is likely to go beyond each cell or species tackling the phage attack independently. Bacteriophage infection of a fraction of the microbial community may also exert an influence on the physiological state and/or phenotypic features of those cells that have not yet had direct contact with the virus or are even intrinsically unable to become infected by the particular virus. These effects may be mediated by sensing the chemical signals released by lysing or by infected cells as well as by more indirect mechanisms

New Effective Method of Lactococcus Genome Editing Using Guide RNA-Directed Transposition

Lactococcus lactis is an important industrial microorganism and a widely used model object for research in the field of lactic acid bacteria (LAB) biology. The development of new L. lactis and related LAB strains with improved properties, including phage-resistant strains for dairy fermentation, LAB-based vaccines or strains with altered genotypes for research purposes, are hindered by the lack of genome-editing tools that allow for the easy and straightforward incorporation of a significant amount of the novel genetic material, such as large genes or operons, into the chromosomes of these bacteria. We recently employed a suggested system based on the CRISPR–Cas-associated transposon for the editing of the L. lactis genome. After the in-depth redesign of the system, we were able to achieve the stable incorporation of the fragments that were sized up to 10 kbp into the L. lactis beta-galactosidase gene. The efficiency of editing under the optimized conditions were 2 × 10−4 and 4 × 10−5 for 1 kbp and 10 kbp, respectively, which are sufficient for fast and easy modifications if a positive selection marker can be used

RB49-like Bacteriophages Recognize O Antigens as One of the Alternative Primary Receptors

The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer

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

Complete genome sequences of T5-related Escherichia coli bacteriophages DT57C and DT571/2 isolated from horse feces

We report the complete genome sequencing of two Escherichia coli T5-related bacteriophages, DT57C and DT571/2, isolated from the same specimen of horse feces. These two isolates share 96 % nucleotide sequence identity and can thus be considered representatives of the same novel species within the genus T5likevirus. The observed variation in the ltfA gene of these phages, resulting from a recent recombination event, may explain the observed host-range differences, suggesting that a modular mechanism makes a significant contribution to the short-term evolution (or adaptation) of T5-like phage genomes in the intestinal ecosystem. Comparison of our isolates to their closest relative, coliphage T5, revealed high overall synteny of the genomes and high conservation of the sequences of almost all structural proteins as well as of the other proteins with identified functions. At the same time, numerous alterations and non-orthologous replacements of non-structural protein genes (mostly of those with unknown functions) as well as substantial differences in tail fiber locus organization support the conclusion that DT57C and DT571/2 form a species-level group clearly distinct from bacteriophage T5

Branched Lateral Tail Fiber Organization in T5-Like Bacteriophages DT57C and DT571/2 is Revealed by Genetic and Functional Analysis

The T5-like siphoviruses DT57C and DT571/2, isolated from horse feces, are very closely related to each other, and most of their structural proteins are also nearly identical to T5 phage. Their LTFs (L-shaped tail fibers), however, are composed of two proteins, LtfA and LtfB, instead of the single Ltf of bacteriophage T5. In silico and mutant analysis suggests a possible branched structure of DT57C and DT571/2 LTFs, where the LtfB protein is connected to the phage tail via the LtfA protein and with both proteins carrying receptor recognition domains. Such adhesin arrangement has not been previously recognized in siphoviruses. The LtfA proteins of our phages are found to recognize different host O-antigen types: E. coli O22-like for DT57C phage and E. coli O87 for DT571/2. LtfB proteins are identical in both phages and recognize another host receptor, most probably lipopolysaccharide (LPS) of E. coli O81 type. In these two bacteriophages, LTF function is essential to penetrate the shield of the host's O-antigens. We also demonstrate that LTF-mediated adsorption becomes superfluous when the non-specific cell protection by O-antigen is missing, allowing the phages to bind directly to their common secondary receptor, the outer membrane protein BtuB. The LTF independent adsorption was also demonstrated on an O22-like host mutant missing O-antigen O-acetylation, thus showing the biological value of this O-antigen modification for cell protection against phages