DEVELOPMENT OF A GENETIC SYSTEM FOR A PURPLE SULFUR BACTERIUM - CONJUGATIVE PLASMID TRANSFER IN CHROMATIUM-VINOSUM

Establishment of a system for manipulative genetics in phototrophic sulfur bacteria of the family Chromatiaceae has mainly been hampered by the lack of reliable methods for growth of these organisms on agar surfaces, techniques for streaking, growth on selective media, screening for antibiotic resistance markers, and most importantly by the lack of a system for DNA transfer. We, therefore, developed minimal and complex agar media for Chromatium vinosum strain D (DSM 180(T)), a representative of the purple sulfur bacteria. Sensitivity of C. vinosum towards a broad range of antibiotics was tested in liquid cultures and solidified media, allowing us to select appropriate antibiotic resistance markers. Furthermore, a system for conjugative transfer of IncP-mobilizable plasmids from Escherichia coli to C. vinosum was established. Broad-host-range IncQ vectors were mobilized to C. vinosum with the aid of plasmid RP4 either present extrachromosomally or integrated in the chromosome of E. coli S17-1. Conjugation efficiencies of up to 1 were observed. Agarose gel electrophoretic analysis showed that transconjugants contained the transferred plasmids in addition to the two detectable plasmids of wild-type C. vinosum. All genetic markers tested (kanamycin, gentamicin, ampicillin, amikacin, tetracycline) were expressed in C, vinosum. Furthermore, high-frequency transfer of plasmid RP4 from C, vinosum to E, coli and to Rhodospirillum rubrum K100 was demonstrated.