Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum: a possible regulatory gene.

In the present study, the transcriptional properties of the nitrogen fixation gene cluster of Hbt. chlorum, a strictly anaerobic, gram-positive, phototrophic bacterium, were explored. The cluster consisted of eleven genes in the same orientation in the order nifI ( 1 ) , nifI ( 2 ) , nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB, and nifV as detected previously. An open reading frame (orf1) preceding these genes was revealed by further cloning. The orf1 was co-transcribed with downstream nif genes in a single polycistronic transcript, the transcription start site (TSS) was located upstream of the orf1, and a putative promoter was identified 10 bp preceding the TSS. Unlike most diazotrophs which have a sigma(54)-dependent -24/-12 promoter, the promoter was similar to the -35/-10 E. coli promoter. The orf1 had no nif homolog in DNA databases, and the highest level of identity (27% at amino acid level) was found with hutP, a positive regulatory gene of the histidine utilization (hut) operon in B. subtilis. Analogous to the regulatory mechanism of the hut operon in B. subtilis, it is conceivable that the orf1 product interacts with the terminator-like structure located downstream of the orf1 during N-deficient condition and prevents transcription termination; thus, the transcription continues into the nif structural genes.