Clostridium pasteurianum W5 synthesizes two NifH-related polypeptides under nitrogen-fixing conditions

Abstract

Previous studies identified fivenifH-like genes (nifH2throughnifH6) inClostridium pasteurianum(strain W5), where thenifH1gene encodes the nitrogenase iron protein. Transcripts of thesenifHgenes, with the exception ofnifH3, were detected in molybdenum-sufficient nitrogen-fixing cells. However, the size of the transcripts, the level of transcription and the presence of polypeptides encoded by thenifH-like genes were not reported. ThenifH2andnifH6genes were extremely similar, as they seemed to differ by only two bases in a span of 2481 bp, one in the coding region and another in the upstream region. Re-examination of the DNA sequences revealed that the coding region ofnifH2andnifH6was identical, whereas the difference in the upstream region was confirmed. Results from the authors' ongoing study of thenifgenes of single-colony isolates ofC. pasteurianumsuggest that thenifH6designation should be eliminated. Here the size of mRNA fromnifH2and the detection of the NifH2 polypeptide in nitrogen-fixing cells ofC. pasteurianumare reported. Northern blot analysis of periodically collected nitrogen-fixing cells showed that thenifH1andnifH2mRNAs were present throughout growth. Addition of ammonium acetate repressed the transcription of both these genes similarly. Using an antiserum raised against NifH ofAzotobacter vinelandii, two NifH-related bands were detected by Western blot analysis after electrophoretic separation of proteins in extracts of nitrogen-fixingC. pasteurianumcells. After separation of proteins by preparative SDS-PAGE, the NifH polypeptides were characterized by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and by ES-MS/MS (electrospray tandem mass spectrometry) analyses. The results confirmed the presence of NifH2, in addition to NifH1, in nitrogen-fixingC. pasteurianumcells.