RegB endoribonuclease is involved in the processing of transcripts from mobD gene cluster of bacteriophage T4

12 Bacteriophage T4 encoded endoribonuclease RegB introduces cuts in the middle of GGAG/U motifs of early phage mRNAs. Analysis of DNA sequence revealed 18 potential cleavage sites for T4 RegB nuclease in genes mobD.5-mobD comprising the mobD gene cluster. Primer extension sequencing of the transcripts from this genomic region showed the existence of seven 5' ends generated by RegB-dependent cleavage. All but one of them occurred in the middle of the GGAG tetranucleotide. Three newly identified RegB nuclease targets are located in the SD sequences of genes mobD.5, mobD.2 and mobD.1, while the other four are situated within the coding sequences of transcripts for genes mobD.4, mobD.1 and mobD. The primer extension analysis also indicates that most of GGAU motifs escape the processing by RegB. The results show that RegB nuclease efficiently cleaves the transcripts from the mobD gene cluster and thus plays a substantial role in the degradation of transcripts from this genomic region

Low-Temperature Virus vB_EcoM_VR26 Shows Potential in Biocontrol of STEC O26:H11

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB_EcoM_VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers (p < 0.01) by > 2 log10 at 22 °C, and ~3 log10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log10 CFU/piece higher than on phage-treated ones (p < 0.02). This, and the ability of VR26 to survive over a pH range of 3–11, indicates that VR26 could be used to control STEC O26:H11 in the food industry

Interaction between phage T4 protein RIII and host ribosomal protein S1 inhibits endoribonuclease RegB activation

Lytic viruses of bacteria (bacteriophages, phages) are intracellular parasites that take over hosts’ biosynthetic processes for their propagation. Most of the knowledge on the host hijacking mechanisms has come from the studies of the lytic phage T4, which infects Escherichia coli. The integrity of T4 development is achieved by strict control over the host and phage processes and by adjusting them to the changing infection conditions. In this study, using in vitro and in vivo biochemical methods, we detected the direct interaction between the T4 protein RIII and ribosomal protein S1 of the host. Protein RIII is known as a cytoplasmic antiholin, which plays a role in the lysis inhibition function of T4. However, our results show that RIII also acts as a viral effector protein mainly targeting S1 RNA-binding domains that are central for all the activities of this multifunctional protein. We confirm that the S1–RIII interaction prevents the S1-dependent activation of endoribonuclease RegB. In addition, we propose that by modulating the multiple processes mediated by S1, RIII could act as a regulator of all stages of T4 infection including the lysis inhibition state

Incomplete LPS core-specific Felix01-like virus vB_EcoM_VpaE1

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics

Pantoea agglomerans-Infecting Bacteriophage vB_PagS_AAS21: A Cold-Adapted Virus Representing a Novel Genus within the Family Siphoviridae

A novel cold-adapted siphovirus, vB_PagS_AAS21 (AAS21), was isolated in Lithuania using Pantoea agglomerans as the host for phage propagation. AAS21 has an isometric head (~85 nm in diameter) and a non-contractile flexible tail (~174 × 10 nm). With a genome size of 116,649 bp, bacteriophage AAS21 is the largest Pantoea-infecting siphovirus sequenced to date. The genome of AAS21 has a G+C content of 39.0% and contains 213 putative protein-encoding genes and 29 genes for tRNAs. A comparative sequence analysis revealed that 89 AAS21 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 63 AAS21 ORFs were functionally annotated, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. Proteomic analysis led to the experimental identification of 19 virion proteins, including 11 that were predicted by bioinformatics approaches. Based on comparative phylogenetic analysis, AAS21 cannot be assigned to any genus currently recognized by ICTV and may represents a new branch of viruses within the family Siphoviridae

<i>Pantoea</i> Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of Archaeosine

A novel low-temperature siphovirus, vB_PagS_Vid5 (Vid5), was isolated in Lithuania using Pantoea agglomerans isolate for the phage propagation. The 61,437 bp genome of Vid5 has a G&#8315;C content of 48.8% and contains 99 probable protein encoding genes and one gene for tRNASer. A comparative sequence analysis revealed that 46 out of 99 Vid5 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 33 Vid5 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a cluster of genes possibly involved in the biosynthesis of 7-deazaguanine derivatives was identified. Notably, one of these genes encodes a putative preQ0/preQ1 transporter, which has never been detected in bacteriophages to date. A proteomic analysis led to the experimental identification of 11 virion proteins, including nine that were predicted by bioinformatics approaches. Based on the phylogenetic analysis, Vid5 cannot be assigned to any genus currently recognized by ICTV, and may represent a new one within the family of Siphoviridae

<i>Klebsiella</i> Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family <i>Myoviridae</i>

<div><p>At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest <i>Klebsiella</i> infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the <i>Myoviridae</i> family.</p></div

Neighbor-joining tree based on the alignment of the tail spike/tail fiber proteins from various phage genomes.

<p>The bootstrap values indicated.</p