Regulation of the \u3cem\u3eVibrio anguillarum\u3c/em\u3e Metalloprotease EmpA by Posttranslational Modification

The zinc metalloprotease EmpA is a virulence factor in the fish pathogen Vibrio anguillarum. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. In this study, we use real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a ∼48-kDa proenzyme and that activation occurs extracellularly by the removal of a ∼10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic

Identification and Characterization of Epp, the Secreted Processing Protease for the \u3cem\u3eVibrio anguillarum\u3c/em\u3e EmpA Metalloprotease

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease

Development of methods for the genetic manipulation of Flavobacterium columnare

<p>Abstract</p> <p>Background</p> <p><it>Flavobacterium columnare </it>is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the <it>Bacteroidetes</it>, including members of the genus <it>Flavobacterium</it>, have been described, but these methods were not adapted to work with <it>F. columnare</it>.</p> <p>Results</p> <p>As a first step toward developing a robust set of genetic tools for <it>F. columnare</it>, a protocol was developed to introduce the <it>E. coli </it>– <it>Flavobacterium </it>shuttle vector pCP29 into <it>F. columnare </it>strain C#2 by conjugal mating at an efficiency of 1.5 × 10^-3antibiotic-resistant transconjugants per recipient cell. Eight of eleven <it>F. columnare </it>strains tested were able to receive pCP29 using the protocol. pCP29 contains the <it>cfxA </it>and <it>ermF </it>genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into <it>F. columnare </it>was dependent on <it>cfxA</it>, as <it>ermF </it>was found not to provide strong resistance to erythromycin. This is in contrast to other <it>Flavobacterium </it>species where <it>ermF</it>-based erythromycin resistance is strong. The green fluorescent protein gene (<it>gfp</it>) was introduced into <it>F. columnare </it>strains under the control of two different native <it>Flavobacterium </it>promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn<it>4351 </it>was successfully introduced into <it>F. columnare</it>, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 μg ml^-1) were used, and high levels of background growth occurred. These results demonstrate that Tn<it>4351 </it>functions in <it>F. columnare </it>but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in <it>F. columnare</it>.</p> <p>Conclusion</p> <p>The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of <it>F. columnare</it>. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.</p

Development of Methods for the Genetic Manipulation of \u3cem\u3eFlavobacterium columnare\u3c/em\u3e

Background: Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare. Results: As a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli – Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 × 10-3 antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 μg ml-1) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare. Conclusion: The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen

Regulation of the Vibrio anguillarum Metalloprotease EmpA by Posttranslational Modification

The zinc metalloprotease EmpA is a virulence factor in the fish pathogen Vibrio anguillarum. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. In this study, we use real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a ∼48-kDa proenzyme and that activation occurs extracellularly by the removal of a ∼10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic

Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease ▿

The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease

Novel Features of the Polysaccharide-Digesting Gliding Bacterium \u3cem\u3eFlavobacterium johnsoniae\u3c/em\u3e as Revealed by Genome Sequence Analysis

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae

Novel Features of the Polysaccharide-Digesting Gliding Bacterium Flavobacterium johnsoniae as Revealed by Genome Sequence Analysis▿ †

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae