Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Abstract

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

FIG. 1.

Maps of the plasmids pCP1, pCP23, and pCP29. Numbers in parentheses indicate map positions in nucleotides. The relative sizes of the maps do not correspond exactly to the sizes of the plasmids. Abbreviations: oriT, origin of transfer for conjugation; ori, ColE1 origin of replication, which functions in E. coli but not in F. psychrophilum; bla, β-lactamase, which confers resistance to ampicillin to E. coli but not to F. psychrophilum. The exact site of the pCP1 origin of replication, which functions in F. psychrophilum, has not been determined but is probably between ORF3 and repA.

FIG. 2.

Expression of lacZ from the ORF1 promoter of pCP1. Transconjugants of F. psychrophilum containing the lacZY genes inserted into the SphI site of pCP23 downstream of the ORF1 promoter were grown on EAOS agar containing X-Gal. 1, F. psychrophilum THC02-90-p; 2, F. psychrophilum THC02-90-β.

FIG. 3.

Southern blot hybridization of F. psychrophilum transconjugants carrying Tn4351. (A) Restriction map of pEP4351 showing the probes used for Southern blot hybridization. cat, a 633-bp fragment containing the structural part of the cat gene; Tn4351, the 6.2-kb SalI fragment containing the Tn4351 transposon. S, SalI; X, XbaI. (B and C) Chromosomal DNA from the transconjugants was digested with XbaI and Southern blot hybridization was carried out with the Tn4351 (B) and cat (C) probes. Lanes 3 to 13, different isolated transconjugants; lanes 2, DNA from the wild-type strain; lanes 14, pEP4351 DNA digested with SalI; lanes 1, λ DNA digested with PstI.

FIG. 4.

Restriction analysis of genomic DNA and of pCP29 isolated from E. coli λ pir and F. psychrophilum THCO2-90. Genomic or plasmid DNA was obtained as described in the text and was digested with different restriction enzymes. Lanes 2 to 5, F. psychrophilum genomic DNA; lanes 6 to 9, pCP29 isolated from F. psychrophilum; lanes 10 to 13, pCP29 isolated from E. coli. DNA was digested with BamHI (lanes 3, 7, and 11), MboI (lanes 4, 8, and 12), or Sau3AI (lanes 5, 9, and 13). No restriction enzymes were added to the samples in lanes 2, 6, and 10. Lane 1 contained λ DNA digested with PstI as a reference.