Development of a genetic system for the transfer of DNA into Flavobacterium heparinum

IBEX Pharmaceuticals Inc., 5485 Pare, Montreal, Quebec H4P 1P7, Canada 1 Author for correspondence: Hongsheng Su. Tel: +1 514 336 7800. Fax: +1 514 336 7242. e-mail: hsu{at}theratech.com 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 . Keywords: Flavobacterium heparinum , integration vector, plasmid replication, heterologous DNA transfer Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; Gm, gentamicin; HT, trimethoprim-resistance gene (cassette); Km, kanamycin; Tc, tetracycline; Tp, trimethoprim The GenBank accession number for the sequence reported in this paper is AF221716 . a Present address: Theratechnologies Inc., 2310 Alfred-Nobel Blvd, Saint Laurent, Quebec H4S 2A4, Canada. b Present address: Merck Frosst Canada and Co., 16711 Trans Canada Hwy, Kirkland, Quebec, Canada H9H 3L1. c Present address: The Millennium Three Group, 919 Conestoga Rd, Rosemont, PA 19010, USA.