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Isolation and characterization of the new mosaic filamentous phage VFJ ? of Vibrio cholerae.

ABSTRACT: Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJ?, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJ? is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJ? was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCY?. This 700 bp region gave VFJ? several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJ? was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJ? updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.

SUBMITTER: Wang Q

PROVIDER: S-EPMC3731265 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Isolation and characterization of the new mosaic filamentous phage VFJ Φ of Vibrio cholerae.

Wang Qiuya Q Kan Biao B Wang Ruibai R

PloS one 20130801 8

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which ...[more]

PMID: 23936475

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Project description:The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Project description:BackgroundEpidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholera serogroups O1 and O139. In recent years, specific lytic phages of V. cholera have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholera O1 strains.MethodsSixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry.ResultsSixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function.ConclusionsØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations.

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Project description:Filamentous phages are ubiquitously distributed in the global oceans. However, little is known about their biological contribution to their host's genetic and phenotypic diversity. In this study, a filamentous phage, Vaf1, was isolated and characterized from the emerging marine pathogen strain Vibrio alginolyticus AP-1. We explored the effects of the resident phage Vaf1 on the host physiology under diverse conditions by precisely deleting the entire phage Vaf1. Our results demonstrate that the presence of phage Vaf1 significantly increased biofilm formation, swarming motility, and contact-dependent competition. Furthermore, the gene expression profile suggests that several phage genes were upregulated in response to low-nutrient conditions. Unexpectedly, an in vivo study of zebrafish shows that fish infected with strain ΔVaf1 survived longer than those infected with wild-type strain AP-1, indicating that Vaf1 contributes to the virulence of V. alginolyticus. Together, our results provide direct evidence for the effect of Vaf1 phage-mediated phenotypic changes in marine bacteria V. alginolyticus. This further emphasizes the impressive complexity and diversity that filamentous phage-host interactions pose and the challenges associated with bacterial disease control in marine aquaculture. IMPORTANCE Non-lytic filamentous phages can replicate without killing their host, establishing long-term persistence within the bacterial host. In contrast to the well-studied CTXφ phage of the human-pathogenic Vibrio cholerae, little is known about the filamentous phage Vaf1 and its biological role in host fitness. In this study, we constructed a filamentous phage-deleted strain, ΔVaf1, and provided direct evidence on how an intact phage, φVaf1, belonging to the family Inoviridae, helps the bacterial host AP-1 to overcome adverse environmental conditions. Our results likely open new avenues for fundamental studies on how filamentous phage-host interactions regulate different aspects of Vibrio cell behaviors.

Dataset Information

Isolation and characterization of the new mosaic filamentous phage VFJ ? of Vibrio cholerae.

Publications

Isolation and characterization of the new mosaic filamentous phage VFJ Φ of Vibrio cholerae.

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