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

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natu ral transformation and homologous recombination were established. As a mode l, mutants unable to perform nitrogen fixation were constructed by interrup ting nifD with various antibiotic resistance markers. Growth of wild-type C . tepidum at 40 degreesC on agar plates could be completely inhibited by 10 0 mug of gentamicin ml(-1), 2 mug of erythromycin ml(-1), 30 mug of chloram phenicol ml(-1), or 1 mug of tetracycline ml(-1) or a combination of 300 mu g of streptomycin ml(-1) and 150 mug of spectinomycin ml(-1). Transformatio n 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 gentam icin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequ ency of spontaneous mutants resistant to gentamicin, erythromycin, or spect inomycin-streptomycin was undetectable or significantly lower than the tran sformation frequency. Transformation with the gentamicin marker was observe d when the transforming DNA contained 1 or 3 kb of total homologous flankin g sequence but not when the transforming DNA contained only 0.3 kb of homol ogous sequence. Linearized plasmids transformed at least an order of magnit ude better than circular plasmids, This work forms a foundation for the sys tematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.