Functional and Transcriptional Analyses of a Fengycin Synthetase Gene, fenC, from Bacillus subtilis

Abstract

ABSTRACTA 37-kb DNA fragment containing five fengycin synthetase genes, includingfenC,fenD,fenE,fenA, andfenB, was cloned and sequenced. Among these genes,fenCencodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activatel-glutamic acid andl-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon offenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon offenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription offenCis positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis byB. subtilisF29-3.