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.

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

“…Efficiency of electrotransformation (0-5 antibioticresistant clones per electroporation sample) was still much lower compared with conjugation (4.0 × 10 -5 to 4.6×10 -5 transconjugants per recipient cell). Similar phenomena were observed when exogenous DNA was transferred into other Bacteroides strains, including F. psychrophilum, F. hibernum, and P. bryantii B 1 4 (Shoemaker et al, 1991;Alvarez et al, 2004;Chen et al, 2007b), and these authors also suggested that restriction barriers caused the failure of electroporation transfer. Conjugation was commonly used to transfer exogenous genes from E. coli into cells of Bacteroides bacteria, indicating that conjugation can partially overcome the restriction barriers.…”

Complete genome sequence of the fish pathogen Flavobacterium psychrophilum

“…These features, together with its strong adhesive properties, probably contribute to its dissemination in salmonid farms worldwide. The availability of different genetic tools 8 and transposon mutant libraries 24 will allow large-scale functional analysis to assess the precise role of the predicted proteins identified. Thus, the F. psychrophilum genome sequence should facilitate the understanding of flavobacterial virulence mechanisms in fish and provide a basis for the development of better control strategies.…”

“…1 and Table 1) and the pCP1 cryptic plasmid 8 . The origin of replication has been identified ( Supplementary Fig.…”

Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC , sprD , and sprF

“…This suggests that the native promoter for expression of sprC and sprD lies further upstream and that sprC and sprD are expressed from the pCP1 orf1 vector promoter in pSN80. The pCP1 orf1 promoter is in the correct orientation in pSN80 to allow expression of sprC and sprD, and it has been shown to express other genes cloned into pCP23 (3,5,28).…”