SEQUENCE AND TRANSCRIPT ANALYSIS OF THE NITROGENASE STRUCTURAL GENE OPERON (NIFHDK) OF RHODOBACTER-CAPSULATUS - EVIDENCE FOR INTRAMOLECULARPROCESSING OF NIFHDK MESSENGER-RNA

Northern blot analysis of RNA prepared from cells of Rhodobacter capsu latus derepressed for nitrogenase (N2ase) synthesis, using a 6.0-kb DN A 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 prepare d from cells grown in the presence of an excess of NH4+, which repress es N2ase synthesis. Hybridization with gene-specific probes revealed t hat the 4.4-kb species hybridized with all three probes, and presumabl y corresponded to the full-length nifHDK transcript, whereas the 3.5-k b species hybridized with nifD and nifK only, and the 2.7-kb transcrip t 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 n ifH. 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 Rc M1, which contains a chromosomal deletion of the nifHDK operon. This s pecies probably corresponds to the transcript of a gene, named fdxD, w hich was found to be located just upstream from the nifHDK operon. Nuc leotide (nt) sequencing of the nifH-D and nifD-K intergenic regions re vealed the presence of inverted repeat (IR) sequences potentially capa ble of forming stable stem-loop structures in mRNA. Primer extension a nalysis of the nifDK-homologous species showed that the 5' end was loc ated one or two nt downstream from the IR sequence between nifH and ni fD, suggesting that the putative stem-loop structure may be a target f or intramolecular processing of the nifHDK mRNA.