Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Zhang, Jin; Zou, Hong; Wang, Liangfa; Huang, Bei; Li, Nan; Wang, Guitang; Nie, Pin

doi:10.1007/s00343-012-1077-z

Cited by 5 publications

(7 citation statements)

References 49 publications

(42 reference statements)

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“…The fragment was digested with KpnI and cloned into the corresponding sites of pCP23 to generate pCP23-rpsl. pCP23-rpsl was introduced into streptomycin-resistant F. columnare by conjugation as previously described (12)(13)(14). All primers used in this study are listed in Table 2.…”

Section: Methodsmentioning

confidence: 99%

“…Staroscik et al (12) developed techniques to introduce replicative plasmids into F. columnare by conjugation and also to construct mutants by insertional mutagenesis and transposon-mediated mutagenesis. However, both processes remain inefficient, and few subsequent reports utilizing these techniques have been published (12,13).A technique to construct in-frame deletions was recently developed for the genetically tractable bacterium Flavobacterium johnsoniae (14). This involved the isolation of a streptomycinresistant rpsL mutant of F. johnsoniae and the use of wild-type rpsL as a counterselectable marker (14).…”

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confidence: 99%

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Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Qin

Zhang

et al. 2015

Appl Environ Microbiol

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cFlavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ⌬cslA and ⌬cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (⌬cslA ⌬cslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G 4 and of the chondroitin lyase-deficient ⌬cslA ⌬cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes. C olumnaris disease can affect almost all species of freshwater fish, including cultured and wild fish, often resulting in heavy economic losses in aquaculture worldwide (1, 2). Its causative agent, Flavobacterium columnare, has been the focus of microbiological as well as aquaculture research for decades, with respect to diagnosis, epidemiology, pathology, preventive measures, and possible virulence factors (2-4). The genetic diversity of F. columnare has been recognized by the presence of at least three distinct genomovars (5-7). However, the pathogenesis of this important pathogen is still rather unclear. Although a few molecules, such as chondroitin AC lyase, have been reported as possible virulence factors (8-11), the lack of genetic manipulation systems suitable for F. columnare has been an obstacle in understanding the mechanisms involved in the pathogenesis of this bacterium. Staroscik et al. (12) developed techniques to introduce replicative plasmids into F. columnare by conjugation and also to construct mutants by insertional mutagenesis and transposon-mediated mutagenesis. However, both processes remain inefficient, and few subsequent reports utilizing these techniques have been published (12,13).A technique to construct in-frame deletions was recently developed for the genetically tractable bacterium Flavobacterium j...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Qin

Zhang

et al. 2015

Appl Environ Microbiol

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“…The integrative plasmid pGL006 which was constructed and conserved in our laboratory has ampicillin resistance in E. coli but not in F. columnare, and contains a specific chromosome fragment amplified from F. columnare G 4 , for homogenous recombination between the integrative plasmid and host chromosome (Zhang et al, 2012). It can coexist with pACYC184, as the replicon of pGL006 was derived from pUC19.…”

Section: Bacterial Strains Plasmidsmentioning

confidence: 99%

“…Bernardet et al, 1996;Bader et al, 2003), pathology and epidemiology (e.g. Foott, 2002;recent reports on the establishment of genetic manipulation system for the bacterium (Staroscik et al, 2008;Zhang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare

Zhang

et al. 2012

Veterinary Microbiology

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The Family Flavobacteriaceae

McBride¹

2014

The Prokaryotes

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101

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Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Cited by 5 publications

References 49 publications

Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare

The Family Flavobacteriaceae

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