Chinese Journal of Oceanology and Limnology

, Volume 30, Issue 2, pp 269–278 | Cite as

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

  • Jin Zhang (张金)
  • Hong Zou (邹红)
  • Liangfa Wang (王良发)
  • Bei Huang (黄贝)
  • Nan Li (李楠)
  • Guitang Wang (王桂堂)
  • Pin Nie (聂品)


Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers (chloramphenicol and spectinomycin resistance) for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.


Flavobacterium columnare selectable marker integrative/conjugative plasmid gene disruption 



polymerase chain reaction


outer membrane protein


specific chromosome fragment


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  1. Agarwal S, Hunnicutt D W, McBride M J. 1997. Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc. Natl. Acad. Sci. U.S.A., 94: 12 139–12 144.CrossRefGoogle Scholar
  2. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. 1990. Basic local alignment search tool. J. Mol. Biol., 215: 403–410.Google Scholar
  3. Alvarez B, Secades P, McBride M J, Guijarro J A. 2004. Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Appl. Environ. Microbiol., 70: 581–587.CrossRefGoogle Scholar
  4. Alvarez B, Alvarez J, Menéndez A, Guijarro J A. 2008. A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease. Microbiology, 154: 1 144–1 151Google Scholar
  5. Bader J A, Shoemaker C A, Klesius P H. 2003a. Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based primer for Flavobacterium columnare. J. Microbiol. Methods, 52: 209–220.CrossRefGoogle Scholar
  6. Bader J A, Nusbaum K E, Shoemaker C A. 2003b. Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque). J. Fish Dis., 26: 461–467.CrossRefGoogle Scholar
  7. Bayley D P, Rocha E R, Smith C J. 2000. Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure. FEMS Microbiol. Lett., 193: 149–154.CrossRefGoogle Scholar
  8. Berg P. 1956. Acyl adenylates; an enzymatic mechanism of acetate activation. J. Biol. Chem., 222: 991–1 013.Google Scholar
  9. Bernardet J F, Segers P, Vancanneyt M, Berthe F, Kersters K, Vandamme P. 1996. Cutting a Gordian knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. Nov (Basonym, Cytophaga aquatilis Strohl and Tait, 1978). Int. J. Syst. Bacteriol., 46: 128–148.CrossRefGoogle Scholar
  10. Black T A, Cai Y, Wolk C P. 1993. Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol. Microbiol., 9: 77–84.CrossRefGoogle Scholar
  11. Bouhenni R, Gehrke A, Saffarini D. 2005. Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon. Appl. Environ. Microbiol., 71: 4 935–4 937.CrossRefGoogle Scholar
  12. Braun T F, Khubbar M K, Saffarini D A, McBride M J. 2005. Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis. J. Bacteriol., 187: 6 943–6 952.Google Scholar
  13. Braun T F, McBride M J. 2005. Flavobacterium johnsoniae GldJ is a lipoprotein that is required for gliding motility. J. Bacteriol., 187: 2 628–2 637.Google Scholar
  14. Chen S, Bagdasarian M, Bates A K, Kaufman M G, Walker E D. 2007a. Mutational analysis of ompA promoter in Flavobacterium johnsoniae. J. Bacteriol., 189: 5 108–5 118.Google Scholar
  15. Chen S, Bagdasarian M, Kaufman M G, Walker E D. 2007b. Characterization of strong promoters from an environmental Flavobacterium hibernum strain by using a green fluorescent protein-based reporter system. Appl. Environ. Microbiol., 73: 1 089–1 100.Google Scholar
  16. Cozzone A J. 1998. Regulation of acetate metabolism by protein phosphorylation in enteric bacteria. Ann. Rev. Microbiol., 52: 127–164.CrossRefGoogle Scholar
  17. Darwish A M, Ismaiel A A, Newton J C, Tang J. 2004. Identification of Flavobacterium columnare by a speciesspecific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae. Mol. Cell Probes, 18: 421–427.CrossRefGoogle Scholar
  18. Decostere A, Haesebrouck F, Devriese L A. 1997. Shieh medium supplemented with tobramycin for selective isolation of Flavobacterium columnare (Flexibacter columnaris) from diseased fish. J. Clin. Microb., 35: 322–324.Google Scholar
  19. Decostere A, Haesebrouck F, Devriese L A. 1998. Characterization of four Flavobacterium columnare (Flexibacter columnaris) strains isolated from tropical fish. Vet. Microbiol., 62: 35–45.CrossRefGoogle Scholar
  20. Decostere A, Haesebrouck F, Van Driessche E, Charlier G, Ducatelle R. 1999a. Characterization of the adhesion of Flavobacterium columnare (Flexibacter columnaris) to gill tissue. J. Fish Dis., 22: 465–474.CrossRefGoogle Scholar
  21. Decostere A, Haesebrouck F, Charlier G, Ducatelle R. 1999b. The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops). Vet. Microbiol., 67: 287–298.CrossRefGoogle Scholar
  22. Figueiredo H C P, Klesius P H, Arias C R, Evans J, Shoemaker C A, Pereira D J Jr, Peixoto M T D. 2005. Isolation and characterization of strains of Flavobacterium columnare from Brazil. J. Fish Dis., 28: 199–204.CrossRefGoogle Scholar
  23. Griffin B R. 1991. Characteristics of a chondroitin AC lyase produced by Cytophaga columnaris. Trans. Am. Fish. Soc., 120: 391–395.CrossRefGoogle Scholar
  24. Guiney D G, Hasegawa P, Bouic K, Matthews B. 1989. Genetic transfer systems in Bacteroides: cloning and mapping of the transferable tetracycline-resistance locus. Mol. Microbiol., 3: 1 617–1 623.CrossRefGoogle Scholar
  25. Hawley D K, McClure W R. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res., 11: 2 237–2 255.CrossRefGoogle Scholar
  26. Hunnicutt D W, McBride M J. 2000. Cloning and characterization of the Flavobacterium johnsoniae gliding motility genes gldD and gldE. J. Bacteriol., 183: 4 167–4 175.Google Scholar
  27. Kempf M J, McBride M J. 2000. Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division. J. Bacteriol., 182: 1 671–1 679.CrossRefGoogle Scholar
  28. Koski P, Hiervelä-Koski V, Bernardet J F. 1993. Flexibacter columnaris infection in arctic char (Salvelinus alpinus L.): First isolation in Finland. Bull. Eur. Assoc. Fish Pathol., 13: 66–69.Google Scholar
  29. Liu G Y, Nie P, Zhang J, Li N. 2008. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare. J. Fish Dis., 31: 269–276.CrossRefGoogle Scholar
  30. Lu Q Z, Ni D S, Ge R F. 1975. Studies on the gill diseases of the grass carp (Ctenopharyngodon idellus): I. Isolation of a myxobacterial pathogen. Acta Hydrobiol. Sin., 5: 315–334. (in Chinese with English abstract)Google Scholar
  31. MacLean L L, Perry M B, Crump E M, Kay W W. 2003. Structural characterization of the lipopolysaccharide Opolysaccharide antigen produced by Flavobacterium columnare ATCC 43622. Eur. J. Biochem., 270: 3 440–3 446.CrossRefGoogle Scholar
  32. McBride M J, Kempf M J. 1996. Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. J. Bacteriol., 178: 583–590.Google Scholar
  33. McBride M J, Braun T F, Brust J L. 2003. Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization. J. Bacteriol., 185: 6 648–6 657.CrossRefGoogle Scholar
  34. Nematollahi A, Decostere A, Pasmans F, Ducatelle R, Haesebrouc F. 2003a. Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss. Dis. Aquat. Org., 55: 101–107.CrossRefGoogle Scholar
  35. Nematollahi A, Decostere A, Pasmans F, Haesebrouc F. 2003b. Flavobacterium psychrophilum infections in salmonid fish. J. Fish Dis., 26: 563–574.CrossRefGoogle Scholar
  36. Ringquist S, Shinedling S, Barrick D, Green L, Binkley J, Stormo G D, Gold L. 1992. Translation initiation in Escherichia coli: sequences within the ribosome-binding site. Mol. Microbiol., 6: 1 219–1 229.CrossRefGoogle Scholar
  37. Sambook J, Russell D W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY.Google Scholar
  38. Salyers A A, Shoemaker N, Cooper A, D’Elia J, Shipman J A. 1999. Genetic methods for Bacteroides species. In: Smith M C M, Sockett R E eds. Genetic Methods for Diverse Prokaryotes. Academic Press, London, United Kingdom. p.229–249.CrossRefGoogle Scholar
  39. Shieh H S. 1980. Studies on the nutrition of a fish pathogen, Flexibacter columnaris. Microbios. Lett., 13: 129–133.Google Scholar
  40. Shoemaker N B, Getty C, Gardner J F, Salyers A A. 1986. Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome. J. Bacteriol., 165: 929–936.Google Scholar
  41. Shoemaker N B, Anderson K L, Smithson S L, Wang G R, Salyers A A. 1991. Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B14. Appl. Environ. Microbiol., 57: 2 114–2 120.Google Scholar
  42. Shotts E B, Starliper C E. 1999. Flavobacterial diseases: columnaris disease, cold-water disease and bacterial gill disease. In: Woo P T K, Bruno D W eds. Fish Disease and Disorders, Vol. 3: Viral, Bacterial and Fungal Infections. CABI Publishing, Oxon. p.559–576.Google Scholar
  43. Simon R, Priefer U, Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology, 1: 784–791.CrossRefGoogle Scholar
  44. Smith C J, Rogers M B, McKee M L. 1992 Heterologous gene expression in Bacteroides fragilis. Plasmid, 27: 141–154.CrossRefGoogle Scholar
  45. Staroscik A M, Hunnicutt D W, Archibald K E, Nelson D R. 2008. Development of methods for the genetic manipulation of Flavobacterium columnare. BMC Microbiol., 8: 115–125.CrossRefGoogle Scholar
  46. Stringer-Roth K M, Yunghans W, Caslake L F. 2002. Differences in chondroitin AC lyase activity of Flavobacterium columnare isolates. J. Fish Dis., 25: 687–691.CrossRefGoogle Scholar
  47. Suomalainen L R, Tiirola M, Valtonen E T. 2006. Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare. J. Fish Dis., 29: 757–763.CrossRefGoogle Scholar
  48. Teska J D. 1993. Assay to evaluate the reaction kinetics of chondroitin AC lyase produced by Cytophaga columnaris. J. Aquat. Anim. Health, 5: 259–264.CrossRefGoogle Scholar
  49. Wagner B A, Wise D J, Khoo L H, Terhune J S. 2002. The epidemiology of bacterial diseases in food-size channel catfish. J. Aquat. Anim. Health, 14: 263–272.CrossRefGoogle Scholar
  50. Welker T L, Shoemaker C A, Arias C R, Klesius P H. 2005. Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus. Dis. Aquat. Organ., 63: 129–138.CrossRefGoogle Scholar
  51. Xie H X, Nie P, Chang M X, Liu Y, Yao W J. 2005. Gene cloning and functional analysis of glycosaminoglycandegrading enzyme chondroitin AC lyase from Flavobacterium columnare G4. Arch. Microbiol., 18: 49–55.CrossRefGoogle Scholar
  52. Xie H X, Nie P, Sun B J. 2004. Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4. J. Fish Dis., 27: 719–729.CrossRefGoogle Scholar
  53. Zhang J, Zou H, Yao W J, Nie P. 2011. Acetyl-coenzyme A synthetase gene and its upstream promoter in the bacterial pathogen Flavobacterium columnare. J. Fishery Sciences of China, 18: 1 100–1 107. (in Chinese with English abstract)Google Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Jin Zhang (张金)
    • 1
    • 2
  • Hong Zou (邹红)
    • 1
  • Liangfa Wang (王良发)
    • 1
  • Bei Huang (黄贝)
    • 1
  • Nan Li (李楠)
    • 1
  • Guitang Wang (王桂堂)
    • 1
  • Pin Nie (聂品)
    • 1
  1. 1.State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of HydrobiologyChinese Academy of SciencesWuhanChina
  2. 2.Graduate University of Chinese Academy of SciencesBeijingChina

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