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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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Demonstration of Gfp expression levels in F. columnare strain C#2 containing plasmids pAS29 (A and B) pAS36 (C and D) and pAS43 (E and F) using epifluorescence (A, C and E) and transillumination/phase contrast (B, D and F) microscopy. The same field is shown for epifluorescence and phase contrast micrographs for each strain. Exposure was varied in the pictures using transillumination to optimize each image, but for comparative purposes the excitation energy and image exposure times were held constant in the three epifluorescence images. All six panels are drawn to the same scale.
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Figure 1: Demonstration of Gfp expression levels in F. columnare strain C#2 containing plasmids pAS29 (A and B) pAS36 (C and D) and pAS43 (E and F) using epifluorescence (A, C and E) and transillumination/phase contrast (B, D and F) microscopy. The same field is shown for epifluorescence and phase contrast micrographs for each strain. Exposure was varied in the pictures using transillumination to optimize each image, but for comparative purposes the excitation energy and image exposure times were held constant in the three epifluorescence images. All six panels are drawn to the same scale.

Mentions: Introduction of the Gfp gene into F. columnare strain C#2 under control of the map promoter on plasmid pAS36 resulted in expression of the gene at levels that could be detected by both a fluorescence plate reader and by epifluorescence microscopy (Figure 1c). This result demonstrates that gfp expression can be used to detect and quantify expression of native F. columnare genes.


Development of methods for the genetic manipulation of Flavobacterium columnare.

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

Demonstration of Gfp expression levels in F. columnare strain C#2 containing plasmids pAS29 (A and B) pAS36 (C and D) and pAS43 (E and F) using epifluorescence (A, C and E) and transillumination/phase contrast (B, D and F) microscopy. The same field is shown for epifluorescence and phase contrast micrographs for each strain. Exposure was varied in the pictures using transillumination to optimize each image, but for comparative purposes the excitation energy and image exposure times were held constant in the three epifluorescence images. All six 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 1: Demonstration of Gfp expression levels in F. columnare strain C#2 containing plasmids pAS29 (A and B) pAS36 (C and D) and pAS43 (E and F) using epifluorescence (A, C and E) and transillumination/phase contrast (B, D and F) microscopy. The same field is shown for epifluorescence and phase contrast micrographs for each strain. Exposure was varied in the pictures using transillumination to optimize each image, but for comparative purposes the excitation energy and image exposure times were held constant in the three epifluorescence images. All six panels are drawn to the same scale.
Mentions: Introduction of the Gfp gene into F. columnare strain C#2 under control of the map promoter on plasmid pAS36 resulted in expression of the gene at levels that could be detected by both a fluorescence plate reader and by epifluorescence microscopy (Figure 1c). This result demonstrates that gfp expression can be used to detect and quantify expression of native F. columnare genes.

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