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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 Tn4351 transposable element integration into the genome of F. columnare strain AL-203-94 following conjugative mating. Primers pr54 and pr56 targeting a 435 bp fragment of the tetX gene contained within the transposon were used to screen for the presence of the transposon in F. columnare genomic DNA. PCR products were run on a 1% agarose gel at 80 V for 45 min and visualized after staining with ethidium bromide. Lane 1: 1 kb ladder, markers range from 250 to 10,000 bp; Lanes 2–11: PCR product from genomic DNA extracted from colonies that grew on an Ordals agar plate augmented with 1 μg ml-1 of erythromycin and Lane 12: Tn4351 containing plasmid pEP4351 (positive control).
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Figure 2: Demonstration of Tn4351 transposable element integration into the genome of F. columnare strain AL-203-94 following conjugative mating. Primers pr54 and pr56 targeting a 435 bp fragment of the tetX gene contained within the transposon were used to screen for the presence of the transposon in F. columnare genomic DNA. PCR products were run on a 1% agarose gel at 80 V for 45 min and visualized after staining with ethidium bromide. Lane 1: 1 kb ladder, markers range from 250 to 10,000 bp; Lanes 2–11: PCR product from genomic DNA extracted from colonies that grew on an Ordals agar plate augmented with 1 μg ml-1 of erythromycin and Lane 12: Tn4351 containing plasmid pEP4351 (positive control).

Mentions: Transposon mediated random mutagenesis was performed using the Bacteroides transposon Tn4351 [22]. Tn4351 contains the erythromycin resistance gene ermF, necessitating the use of erythromycin as the selective marker. As with efforts to use erythromycin to introduce pCP29 into F. columnare, antibiotic concentrations of 1 μg ml-1 or lower were required for any growth to occur. At these low concentrations, a significant amount of background growth was observed. Transposon mutagenesis was attempted in three strains (C#2, AL-203-94 and Fc14-56) and Tn4351 was successfully introduced into F. columnare strain AL-203-94. Only two of ten colonies isolated from plates containing 1 μg ml-1 erythromycin contained the transposon (Figure 2). While the two identified insertions demonstrate that the transposon is capable of integrating into the F. columnare genome, the high number of false positives suggests that this ermF based transposon is not a useful tool for the generation of mutants in this organism.


Development of methods for the genetic manipulation of Flavobacterium columnare.

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

Demonstration of Tn4351 transposable element integration into the genome of F. columnare strain AL-203-94 following conjugative mating. Primers pr54 and pr56 targeting a 435 bp fragment of the tetX gene contained within the transposon were used to screen for the presence of the transposon in F. columnare genomic DNA. PCR products were run on a 1% agarose gel at 80 V for 45 min and visualized after staining with ethidium bromide. Lane 1: 1 kb ladder, markers range from 250 to 10,000 bp; Lanes 2–11: PCR product from genomic DNA extracted from colonies that grew on an Ordals agar plate augmented with 1 μg ml-1 of erythromycin and Lane 12: Tn4351 containing plasmid pEP4351 (positive control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Demonstration of Tn4351 transposable element integration into the genome of F. columnare strain AL-203-94 following conjugative mating. Primers pr54 and pr56 targeting a 435 bp fragment of the tetX gene contained within the transposon were used to screen for the presence of the transposon in F. columnare genomic DNA. PCR products were run on a 1% agarose gel at 80 V for 45 min and visualized after staining with ethidium bromide. Lane 1: 1 kb ladder, markers range from 250 to 10,000 bp; Lanes 2–11: PCR product from genomic DNA extracted from colonies that grew on an Ordals agar plate augmented with 1 μg ml-1 of erythromycin and Lane 12: Tn4351 containing plasmid pEP4351 (positive control).
Mentions: Transposon mediated random mutagenesis was performed using the Bacteroides transposon Tn4351 [22]. Tn4351 contains the erythromycin resistance gene ermF, necessitating the use of erythromycin as the selective marker. As with efforts to use erythromycin to introduce pCP29 into F. columnare, antibiotic concentrations of 1 μg ml-1 or lower were required for any growth to occur. At these low concentrations, a significant amount of background growth was observed. Transposon mutagenesis was attempted in three strains (C#2, AL-203-94 and Fc14-56) and Tn4351 was successfully introduced into F. columnare strain AL-203-94. Only two of ten colonies isolated from plates containing 1 μg ml-1 erythromycin contained the transposon (Figure 2). While the two identified insertions demonstrate that the transposon is capable of integrating into the F. columnare genome, the high number of false positives suggests that this ermF based transposon is not a useful tool for the generation of mutants in this organism.

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