Development of a genetic system for the transfer of DNA into Flavobacterium heparinum.
Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments. It synthesizes five enzymes that degrade glycosoaminoglycan molecules. The study of this unique bacterium has been limited by the absence of a genetic manipulation system. In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F. heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described. Both plasmids were introduced into F. heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained. Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA. The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR. The existence of a derivative of the broad-host-range plasmid pBBR1 in F. heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.
Su
Hongsheng
H
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
Shao
Zhongqi
Z
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
Tkalec
Lydia
L
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
Blain
Françoise
F
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
Zimmermann
Joe
J
IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada1.
D015139:Blotting, Southern; D003227:Conjugation, Genetic; D004261:DNA Replication; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial; D014295:Trimethoprim / Q000494:pharmacology
D014295:Trimethoprim