Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degreesC on agar plates could be completely inhibited by 100 mug of gentamicin ml(-1), 2 mug of erythromycin ml(-1), 30 mug of chloramphenicol ml(-1), or 1 mug of tetracycline ml(-1) or a combination of 300 mug of streptomycin ml(-1) and 150 mug of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h, Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids, This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.