Distinguishing between the extracellular DNases of <i>Vibrio cholerae</i> and development of a transformation system

Focareta, Tony; Manning, Paul A.

doi:10.1111/j.1365-2958.1991.tb02101.x

Cited by 61 publications

(64 citation statements)

References 37 publications

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“…In fact, prior evidence for a second prepilin peptidase in V. cholerae can be found in the observation that TcpA processing is not completely abolished in a tcpJ mutant (29). Moreover, the precedent for multiple prepilin peptidase genes has already been established in E. coli (15). Although our experiments do not show that the observed modification of the EpsG is required for its function in the GSP, they constitute an indication, making the search for a second prepilin peptidase in V. cholerae interesting and important.…”

Section: Discussionmentioning

confidence: 89%

“…5, lanes 2 to 5) is probably due to endogenous prepilin peptidase, since some strains have been demonstrated to express proteolytic activity analogous to that of prepilin peptidases (33). The K-12 genome has been reported to carry at least two genes which by all criteria encode homologs of the prepilin peptidases associated with the GSP and type IV pilus biogenesis systems (15). The failure to detect this endogenous processing in the T7 expression studies may reflect differences in strain backgrounds.…”

Section: Resultsmentioning

confidence: 99%

“…While CT and protease use the general secretion pathway, the secretion of DNase occurs by a different, as yet unidentified secretion mechanism. V. cholerae is known to secrete two different DNases into the extracellular environment (15,47). In our assay for total DNase activity, we cannot determine whether we are detecting one or the other, or both DNases.…”

Section: Discussionmentioning

confidence: 99%

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General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae

et al. 1997

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The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.In Vibrio cholerae, the general secretion pathway (GSP) is required for extracellular secretion of several proteins including chitinase, cholera toxin (CT), and protease (51, 60). This pathway likely plays a significant role in the survival of Vibrio in different environments as well as in its pathogenicity. For instance, secreted chitinase and protease may assist in the detachment of V. cholerae from chitinous waterborne particles and from host epithelial cells, respectively, thus facilitating dissemination (14, 71). CT is the major component responsible for inducing the diarrheal disease cholera and is therefore also involved in the dissemination process (for a review, see reference 30).Escherichia coli heat-labile enterotoxin (LT) is very similar to CT in its structure, biochemical function, and ability to translocate across the outer membrane of V. cholerae (66). It is a multimeric protein which consists of a single A subunit and a pentamer of identical B-subunit polypeptides (18,64,65). Its secretion from V. cholerae proceeds in two steps, similar to the secretion of CT. The first step is translocation of the individual subunit polypeptides across the cytoplasmic membrane. This is mediated by a pathway which is presumably similar to the sec system of E. coli (63). During this step, the signal peptides of the A and B precursor polypeptides are cleaved off (8,25,52) a...

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Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae

et al. 1997

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“…At the protein level C. jejuni Dns displays considerable sequence similarity with other bacterial nucleases, e.g., Dns of Vibrio cholerae (14), EndA of E. coli (22), Dns of A. hydrophila (5), NucM of Erwinia chrysanthemi (33), and Vvn of Vibrio vulnificus (53). In addition, Dns of C. jejuni is predicted to contain a domain typical of endonuclease I proteins.…”

Section: Discussionmentioning

confidence: 99%

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

et al. 2009

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The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNAhydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

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“…These studies highlighted, among other factors, the importance of enzymic barriers preventing the successful establishment of foreign DNA in the microbial cell. Besides restriction enzymes, non-specific nucleases may be involved too, as in the case of Vibrio cholerae and Vibrio vulnificus, where representatives of these mostly extracellular enzymes having no sequence and sugar specificity (Rangarajan & Shankar, 2001;JarvillTaylor et al, 1999) have been shown to inhibit transformation (Focareta & Manning, 1991;Wu et al, 2001). Besides inhibiting transformation, they have been ascribed various roles in nutrition (Benedik & Strych, 1998), in the processing of DNA during natural transformation, e.g.…”

Section: Introductionmentioning

confidence: 99%

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Accetto

Avguštin

2007

Microbiology

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Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1 Available tools for genetic analysis in the anaerobic rumen bacterium Prevotella bryantii are limited to only two known systems for gene delivery, and no genes, with the exception of plasmid maintenance and selection genes, have been successfully expressed from plasmids in any species of the genus Prevotella until now. It is shown here that nucB, a newly cloned nuclease gene from P. bryantii, can be controllably expressed from shuttle vector pRH3 in P. bryantii strain TC1-1, depending on the tetracycline concentration in the growth medium. nucB expression is also growth-medium dependent and this regulation presumably takes place at the translational level. His-tagged NucB was purified from P. bryantii TC1-1 culture supernatant and was shown to degrade DNA as well as RNA; it is most likely a minor 36 kDa P. bryantii non-specific nuclease. INTRODUCTIONMembers of the bacterial genus Prevotella have been found in the rumen as well as in the oral cavity and large intestine of man and animals (Edwards et al., 2004;Paster et al., 2001;Duncan et al., 2003;Eckburg et al., 2005; Leser et al., 2002). In the rumen they are symbionts contributing to the degradation of plant cell-wall polysaccharides (Miyazaki et al., 2003) and protein metabolism (Walker et al., 2003), whereas oral prevotellas are implicated in dental plaque establishment (Kolenbrander et al., 2002), periodontitis (Paster et al., 2001), advanced caries (Martin et al., 2002), oro-pharyngeal abscesses (Brook, 2004) and noma, a grotesque childhood orofacial gangrene, now confined mostly to sub-Saharan Africa (Enwonwu et al., 2006). The genetic tools that would make possible more thorough studies of prevotellas are still undeveloped despite their ecological and medical importance. This appears to be primarily due to the large phylogenetic distance between prevotellas and other well-characterized micro-organisms, and as a consequence of the rather specific genetic elements, exemplified by promoters, containing distinct 27/233 consensus sequences that are recognized by unusual primary s factor (Vingadassalom et al., 2005). Nevertheless, in two studies (Shoemaker et al., 1991;Accetto et al., 2005) the plasmid replicons and tetQ selectable marker derived from Prevotella and related colonic Bacteroides strains were used successfully for shuttle-vector introduction into the xylan-degrading ruminal species Prevotella bryantii, which forms a distinct phylogenetic lineage apparently somewhat closer to oral prevotellas than the other ruminal Prevotella species (Avguštin et al., 2001). Based on the conjugal transfer protocol developed in the first study, a P. bryantii B 1 4 carboxymethylcellulase gene, fused to a cellulose-binding domain of Thermomonospora fusca cellulase, and driven by a Prevotella ruminicola 23 xylanase promoter, was introduced into P. bryantii B 1 4. The gene was expressed in Bacteroides uniformis 1108, but not in P. bryantii B 1 4, possibly ...

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Distinguishing between the extracellular DNases of Vibrio cholerae and development of a transformation system

Cited by 61 publications

References 37 publications

General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae

General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

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