Affiliation:
1. Área de Microbiologia, Departamento de Biología Funcional, IUBA, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
2. Department of Biological Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201
Abstract
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
Sau
3AI and
Bam
HI, indicating that a
Sau
3AI-like restriction modification system may constitute part of this barrier. Tn
4351
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
62 articles.
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