Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA.

Northern blot analysis of RNA prepared from cells of Rhodobacter capsulatus derepressed for nitrogenase (N2ase) synthesis, using a 6.0-kb DNA probe containing the entire nifHDK operon, revealed the presence of at least six hybridizing species of the estimated sizes, 4.4, 3.5, 2.7, 1.3, 0.9 and 0.38 kb. No hybridization was detected with RNA prepared from cells grown in the presence of an excess of NH4+, which represses N2ase synthesis. Hybridization with gene-specific probes revealed that the 4.4-kb species hybridized with all three probes, and presumably corresponded to the full-length nifHDK transcript, whereas the 3.5-kb species hybridized with nifD and nifK only, and the 2.7-kb transcript hybridized with nifH and nifD. The 1.3 and 0.9-kb species hybridized with all three probes, but appeared to hybridize most strongly with nifH. In contrast, the 0.38-kb species hybridized with none of the gene-specific probes, and was also detected in RNA from cells of strain RcM1, which contains a chromosomal deletion of the nifHDK operon. This species probably corresponds to the transcript of a gene, named fdxD, which was found to be located just upstream from the nifHDK operon. Nucleotide (nt) sequencing of the nifH-D and nifD-K intergenic regions revealed the presence of inverted repeat (IR) sequences potentially capable of forming stable stem-loop structures in mRNA. Primer extension analysis of the nifDK-homologous species showed that the 5' end was located one or two nt downstream from the IR sequence between nifH and nifD, suggesting that the putative stem-loop structure may be a target for intramolecular processing of the nifHDK mRNA.