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Development of methods for the genetic manipulation of Flavobacterium columnare.

Staroscik AM, Hunnicutt DW, Archibald KE, Nelson DR - BMC Microbiol. (2008)

Bottom Line: Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA. amstar@etal.uri.edu

ABSTRACT

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 x 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 microg 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.

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Photomicrograph of F. columnare colonies. Colonies were grown for 2 days at 27°C on Ordal's agar medium. (A) Wild-type F. columnare C#2. (B) gldJ knockout mutant FcAS44. Both panels are drawn to the same scale.
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Figure 3: Photomicrograph of F. columnare colonies. Colonies were grown for 2 days at 27°C on Ordal's agar medium. (A) Wild-type F. columnare C#2. (B) gldJ knockout mutant FcAS44. Both panels are drawn to the same scale.

Mentions: A cefoxitin based F. columnare suicide vector, pAS42, was created by replacing Flavobacterium replicative functions of pCP29 with a truncated gldJ sequence as described in Methods. Using the mating protocol described below, pAS42 was introduced into C#2 resulting in the successful isolation of non-motile, cefoxitin resistant colonies (Figure 3). Mutants were isolated at an efficiency of roughly 1 × 10-6 cefoxitin-resistant mutants per recipient cell. This is 1,000-fold lower than the rate at which the pCP1 based shuttle vector, pCP29, can be introduced to strain C#2. Disruption of gldJ was confirmed by PCR amplification and sequencing of the novel junction formed by the insertion of the mutagenesis vector in to the gldJ gene. PCR was done using primers pr88 and pr93. Sequencing across the novel junction was accomplished from both directions using primers pr88 and pr104 (data not shown).


Development of methods for the genetic manipulation of Flavobacterium columnare.

Staroscik AM, Hunnicutt DW, Archibald KE, Nelson DR - BMC Microbiol. (2008)

Photomicrograph of F. columnare colonies. Colonies were grown for 2 days at 27°C on Ordal's agar medium. (A) Wild-type F. columnare C#2. (B) gldJ knockout mutant FcAS44. Both panels are drawn to the same scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483708&req=5

Figure 3: Photomicrograph of F. columnare colonies. Colonies were grown for 2 days at 27°C on Ordal's agar medium. (A) Wild-type F. columnare C#2. (B) gldJ knockout mutant FcAS44. Both panels are drawn to the same scale.
Mentions: A cefoxitin based F. columnare suicide vector, pAS42, was created by replacing Flavobacterium replicative functions of pCP29 with a truncated gldJ sequence as described in Methods. Using the mating protocol described below, pAS42 was introduced into C#2 resulting in the successful isolation of non-motile, cefoxitin resistant colonies (Figure 3). Mutants were isolated at an efficiency of roughly 1 × 10-6 cefoxitin-resistant mutants per recipient cell. This is 1,000-fold lower than the rate at which the pCP1 based shuttle vector, pCP29, can be introduced to strain C#2. Disruption of gldJ was confirmed by PCR amplification and sequencing of the novel junction formed by the insertion of the mutagenesis vector in to the gldJ gene. PCR was done using primers pr88 and pr93. Sequencing across the novel junction was accomplished from both directions using primers pr88 and pr104 (data not shown).

Bottom Line: Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA. amstar@etal.uri.edu

ABSTRACT

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 x 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 microg 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.

Show MeSH
Related in: MedlinePlus