Prophages carrying Zot toxins on different Vibrio genomes: A comprehensive assessment using multilayer networks.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria.

The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector.

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities.

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Isolation and characterization of vibriophage vB_Vc_SrVc9: an effective agent in preventing Vibrio campbellii infections in brine shrimp nauplii (Artemia franciscana).

AIMS: This study describes the physicochemical and genomic characterization of phage vB_Vc_SrVc9 and its potential for phage therapy application against a pathogenic Vibrio campbellii strain. METHODS AND RESULTS: A lytic phage vB_Vc_SrVc9 against V. campbellii was isolated from shrimp farm sediment, and characterized physicochemical and genomically. The use of vB_Vc_SrVc9 phage increased the survival in brine shrimp Artemia franciscana and reduced presumptive V. campbellii to nondetectable numbers. Genomic analysis showed a genome with a single contig of 43·15 kb, with 49 predicted genes and no tRNAs, capable of recognizing and generating complete inhibition zones of three Vibrio sp. CONCLUSIONS: To our knowledge vB_Vc_SrVc9 is a lytic phage that could be used against Vibrio infections, reducing vibrio presence without any apparent impact over the natural microbiota at the family level in 28 libraries tested. SIGNIFICANCE AND IMPACT OF THE STUDY: vB_Vc_SrVC9 is a novel phage and ecofriendly alternative for therapeutic applications and biotechnological purposes because is stable at different environmental conditions, has the potential to eliminate several strains, and has a short latent period with a good burst size. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Use of Bacteriophages to Control Vibrio Contamination of Microalgae Used as a Food Source for Oyster Larvae During Hatchery Culture.

Cultured microalgae are the primary food source for oyster larvae during hatchery culture and are a potential vector for Vibrio spp. infection of larval cultures. Bacteriophages have shown potential for controlling contamination of Vibrio spp. in aquaculture systems and their application could be an effective biological control method to eliminate such bacterial contamination of microalgae. This study investigated whether Vibrio-free microalgae sources could be ensured via the application of Vibrio specific phages. As a first step, four different Vibrio bacteriophages (belonging to the Myoviridae viral family) were isolated from marine waters in Queensland, Australia and used in challenge tests against a Vibrio host species, previously isolated from New South Wales oyster hatchery and found to be closely related to V. alginolyticus (ATCC 17749). The genome sequence of one of the four isolated bacteriophages, Vibrio Φ-2, that displayed strongest virulence against the host was determined. The 242446 bp genome of this bacteriophage was predicted to encode 217 proteins with an average GC content of 43.91%, containing putative thymidine kinases and a lysin enzyme. Application of these bacteriophages to pathogenic Vibrio spp. contaminating microalgae suspensions resulted in significant decreases in their numbers within 2 h. Findings indicated that direct application of bacteriophages to microalgae suspensions could be an effective method of reducing the occurrence of vibriosis in oyster hatcheries.

Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens.

The fast-growing marine bacterium Vibrio natriegens represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of V. natriegens ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the Siphoviridae family into the culture supernatant. Freeing V. natriegens from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches.IMPORTANCE The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of V. natriegens Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.

Complete genomic sequence of bacteriophage P23: a novel Vibrio phage isolated from the Yellow Sea, China.

A novel Vibrio phage, P23, belonging to the family Siphoviridae was isolated from the surface water of the Yellow Sea, China. The complete genome of this phage was determined. A one-step growth curve showed that the latent period was approximately 30 min, the burst size was 24 PFU/cell, and the rise period was 20 min. The phage is host specific and is stable over a range of pH (5-10) and temperatures (4-65 °C). Transmission electron microscopy showed that phage P23 can be categorized into the Siphoviridae family, with an icosahedral head of 60 nm and a long noncontractile tail of 144 nm. The genome consisted of a linear, double-stranded 40.063 kb DNA molecule with 42.5% G+C content and 72 putative open reading frames (ORFs) without tRNA. The predicted ORFs were classified into six functional groups, including DNA replication, regulation and nucleotide metabolism, transcription, phage packaging, phage structure, lysis, and hypothetical proteins. The Vibrio phage P23 genome is a new marine Siphoviridae-family phage genome that provides basic information for further molecular research on interaction mechanisms between bacteriophages and their hosts.

Genome Analysis of Two Novel Lytic Vibrio maritimus Phages Isolated from the Coastal Surface Seawater of Qingdao, China.

Two novel Vibrio phages, LP.1 and LP.2 that infected Vibrio maritimus R-40493, were isolated from surface seawater in Qingdao coastal area by the double-agar layer method. Morphological analysis by transmission electron microscope showed that the two phages displayed head-tail structures with icosahedral heads of 62.37 and 54.00 nm in diameter and long non-contractile tails of 119.00 and 105.20 nm in length, respectively, and can be grouped into the Siphoviridae family. Thermal and pH sensitivity tests exhibited that LP.1 was stable at temperature ranging from - 20 to 65 °C and at pH ranging from 5 to 12, and LP.2 showed vitality over a wider range of temperature (- 20-75 °C) and pH (3-12). Both LP.1 and LP.2 contained linear and double-stranded DNA genomes with a length of 46,791-bp and 37,128-bp, respectively. The genome of both phages can be classified into four functional groups, including DNA replication and regulation, phage packaging, phage structure, and additional function. The bioinformatic analysis demonstrated that the Vibrio phages LP.1 and LP.2 are novel phages. By conducting morphological, biochemical, and genomic analysis, our study provides useful information for further research on the interaction between Vibrio phages and their host.

Application of the bacteriophage pVco-14 to prevent Vibrio coralliilyticus infection in Pacific oyster (Crassostrea gigas) larvae.

Vibrio coralliilyticus infects a variety of shellfish larvae, including Pacific oyster (Crassostrea gigas) larvae worldwide, and remains a major constraint in marine bivalve aquaculture practice, especially in artificial seed production facilities. In this study, we isolated and characterized the bacteriophage (phage) that specifically infects V. coralliilyticus. The phage was designated pVco-14 and classified as Siphoviridae. We also investigated the potential efficacy of the isolated phage against V. coralliilyticus infection. We conducted a survey to replace the overuse of antibiotics, which generate multi-antibiotic-resistant strains and causes environmental pollution. The latent period of pVco-14 was estimated to be approximately 30 min, whereas the burst size was 13.3 PFU/cell. The phage was found to infect four strains of tested V. coralliilyticus. pVco-14 was stable at wide temperature (4-37 °C) and pH (5.0-9.0) ranges. Eighty-one percent of oyster larvae died in an immersion challenge at a dose 1.32 × 105 CFU/ml of virulent V. coralliilyticus (strain 58) within 24 h. When oyster larvae were pre-treated with the phage before the bacterial challenge (bacterial conc.: 1.32 × 104 and 1.32 × 105 CFU/ml), mortality of the phage-treated oyster larvae was lower than that of the untreated control. These results suggest that pVco-14 has potential to be used as a prophylactic agent for preventing V. coralliilyticus infection in marine bivalve hatcheries and can reduce the overuse of antibiotics.

The structure of temperate phage-bacteria infection networks changes with the phylogenetic distance of the host bacteria.

With their ability to integrate into the bacterial chromosome and thereby transfer virulence or drug-resistance genes across bacterial species, temperate phage play a key role in bacterial evolution. Thus, it is paramount to understand who infects whom to be able to predict the movement of DNA across the prokaryotic world and ultimately the emergence of novel (drug-resistant) pathogens. We empirically investigated lytic infection patterns among Vibrio spp. from distinct phylogenetic clades and their derived temperate phage. We found that across distantly related clades, infections occur preferentially within modules of the same clade. However, when the genetic distance of the host bacteria decreases, these clade-specific infections disappear. This indicates that the structure of temperate phage-bacteria infection networks changes with the phylogenetic distance of the host bacteria.

Tripartite species interaction: eukaryotic hosts suffer more from phage susceptible than from phage resistant bacteria.

BACKGROUND: Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 × 75 phage-bacteria infection matrix. RESULTS: According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria. CONCLUSION: These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution.

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi.

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7. Received June 16, 2016; accepted October 7, 2016.

Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus.

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P < 0.05) inhibition of growth of V. splendidus VS-ABTNL compared with untreated V. splendidus VS-ABTNL with the cocktail being superior to any of the 3 phages used individually. The lytic capability of the 3 phages was subsequently determined with a Spot Assay Technique performed with 4 isolates of V. splendidus, 3 other Vibrio species and 2 environmental isolates. Both PVS-1 and PVS-2 were lytic to all 4 isolates of V. splendidus while PVS-3 only inhibited the growth of 3 of them. V. splendidus VS-ABTNL was more susceptible to phage PVS-2 than the other 2 phages. In an in vivo performance trial, 360 sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 10(6) CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P < 0.05) increased compared with those fed the control diet. However, no significant differences (P > 0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid of the sea cucumbers. The control was injected with 1 ml of sterilized seawater while the untreated group and the test group were injected with the same volume of V. splendidus-ABTNL culture (3 × 10(5) CFU/mL). Then, the test group was injected with 1 ml of the 3 phage cocktail (MOI = 10). After 48 h, the activities of lysozyme, acid phosphatase and superoxide dismutase were elevated in the untreated group while the levels of these enzymes in the test group were similar to the blank control. After 10-day observation, the survival rate of the sea cucumber was 100% for the blank control, 80% for the test group and 20% for the negative control. The overall results of this experiment indicate that phage therapy increased the survival of sea cucumber infected with V. splendidus VS-ABTNL. The above results demonstrate that using phages, especially a combination of different phages, may be a feasible way to control Vibrio infection in the sea cucumber industry.

Complete Genomic Sequence of Bacteriophage H188: A Novel Vibrio kanaloae Phage Isolated from Yellow Sea.

Phage H188, a novel Vibrio kanaloae phage, was isolated from the surface water of Yellow Sea. Morphological analysis by transmission electron microscopy reveals that it belongs to the family Myoviridae. Present result suggests that the phage is stable at pH between 4.0 and 12.0. No significant difference in phage titers is noted at temperature 30-50 °C. A latent period of approximately 96 mins is indicated by the one-step growth curve. And, the burst size is about three virions per cell. Furthermore, genomic analysis of H188 reveals a genome size of 50364 bp with 43.63 % G+C content, and 76 putative open reading frames. There is no obvious similarity between H188 and other known phages by genomic comparison. Moreover, the H188 genome includes modules for phage structure, phage packaging, DNA replication and regulation, and some additional functions.

Cold-active bacteriophages from the Baltic Sea ice have diverse genomes and virus-host interactions.

Heterotrophic bacteria are the major prokaryotic component of the Baltic Sea ice microbiome, and it is postulated that phages are among their major parasites. In this study, we sequenced the complete genomes of six earlier reported phage isolates from the Baltic Sea ice infecting Shewanella sp. and Flavobacterium sp. hosts as well as characterized the phage-host interactions. Based on the genome sequences, the six phages were classified into five new genera. Only two phages, 1/4 and 1/40, both infecting Shewanella sp. strains, showed significant nucleotide sequence similarity to each other and could be grouped into the same genus. These two phages are also related to Vibrio-specific phages sharing approximately 25% of the predicted gene products. Nevertheless, cross-titrations showed that the cold-active phages studied are host specific: none of the seven additionally tested, closely related Shewanella strains served as hosts for the phages. Adsorption experiments of two Shewanella phages, 1/4 and 3/49, conducted at 4 °C and at 15 °C revealed relatively fast adsorption rates that are, for example, comparable with those of phages infective in mesophilic conditions. Despite the small number of Shewanella phages characterized here, we could already find different types of phage-host interactions including a putative abortive infection.

Vibriophages Differentially Influence Biofilm Formation by Vibrio anguillarum Strains.

Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis. Bacteriophages can potentially be used to control bacterial pathogens; however, successful application of phages requires a detailed understanding of phage-host interactions under both free-living and surface-associated growth conditions. In this study, we explored in vitro phage-host interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in microcolonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20 to strain BA35 showed efficient control of biofilm formation and density of free-living cells. The interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage. Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the aggregation of host cells, which protected the cells against phage infection. By the formation of biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together, the results demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.

[THE IDENTIFICATION AND DIFFERENTIATION OF BACTERIOPHAGES OF HUMAN PATHOGENIC VIBRIO].

The issue of identification and differentiation of large group of bacteriophages of human pathogenic vibrio is still unresolved. In research and practical applied purposes it is important to consider characteristics of bacteriophages for establishing similarity and differences between them. The actual study was carried out to analyze specimens of DNA-containing bacteriophages of pathogenic vibrio. The overwhelming majority of them characterized by complicated type of symmetry--phages with double-helical DNA and also phages with mono-helical DNA structure discovered recently in vibrio. For the first time, the general framework of identification and differentiation of bacteriophages of pathogenic vibrio was developed. This achievement increases possibility to establish species assignment of phages and to compare with phages registered in the database. "The collection of bacteriophages and test-strains of human pathogenic vibrio" (No2010620549 of 24.09.210).

A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.

Phylogenomic network and comparative genomics reveal a diverged member of the ΦKZ-related group, marine vibrio phage ΦJM-2012.

Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ΦJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ΦJM-2012 was connected to only five members of the Pseudomonas ΦKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ΦKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ΦJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ΦJM-2012 genome showed that segments of the conserved N-terminal parts of ΦKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ΦKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.