SEQUENCE, GENETIC, AND LACZ FUSION ANALYSES OF A NIFR3-NTRB-NTRC OPERON IN RHODOBACTER-CAPSULATUS

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regu latory genes required to sense and relay the nitrogen status of the ce ll are glnB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and n ifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absenc e of both fixed nitrogen and oxygen. In this study we report the seque nce and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protei n with significant homology to an upstream open reading frame cotransc ribed with the Escherichia coli regulatory gene, fis. Analysis of ntrC -lacZ fusions and complementation data indicate that nifR3 ntrBC const itute a single operon. nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription). This gene organiz ation suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen f ixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal ni fR3 gene was constructed. This nifR3 strain is able to fix nitrogen an d activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R. capsulatus.