The molecular analysis of the nitrogen fixation (nif) system of Klebsiella pneumoniae is of relevance both for the basic scientific questions it addresses as well as for its implications for the understanding of the more agronomically important rhizobia. The general problems addressed by this proposal are the functions of certain of the proteins encoded by the system and the nature of an unusual post-transcriptional regulatory system involving mRNA stability. In the case of the nifX, Y and U genes, where the function is obscure, strains with characterized mutations in these genes will be analyzed for effects on growth phenotype, protein accumulation, localization, and stability, and mRNA processing. The nifNG gene products are known to be involved in FeMo-co biosynthesis, but the precise biochemical role in unknown. This will be addressed by completing the purivication of these proteins, characterizing the purified proteins and analyzing their function in vivo and in vitro by assays aimed at molybdenum metabolism. The final topic in the proposal addresses the unusual stability and even more unusual regulated destabilization of the nif mRNA's. The precise ends of these mRNA's will be determined and sites and proteins relevant to the stability/instability will be identified. These analyses will largely depend on the use of mutants and protein expression vectors coupled with biochemical analysis of transcript stability. An analysis of this post-trancriptional system will increase our understanding of the range of regulatory mechanisms employed by living systems.
