Transcriptional regulation of Rhizobium meliloti nitrogen fixation genes Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ns064829c

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  • The transcriptional promoter sequences for the Rhizobium meliloti nitrogen fixation genes nifA and nifB were cloned to a β-galactosidase gene fusion plasmid vector and transferred by homologous recombination to a specialized transducing phage. The promoter fusions were then transduced to a recombination deficient strain of Escherichia coli as single-copy lysogens and analysed under defined aerobic and anaerobic conditions. The lysogenic strains contained plasmids encoding either of two transcriptional activator proteins, NifA or FixJ, produced from a constitutive plasmid promoter. The expression of the nifA and the nifB promoters was found to be sensitively regulated by the carbon source used for anaerobic fermentation or anaerobic respiration, the redox potential of the terminal electron acceptor used for anaerobic respiration, and the growth phase of anaerobic cultures. The repression of nit promoter expression by oxygen respiration was specifically compared to anaerobic respiration of alternative electron acceptors. Both nifA and nifB promoter expression decreased exponentially as the reduction potential of the terminal respiration reaction increased. The repressive effect of oxygen appears to be due soley to the exponential relationship between nit promoter expression and the redox potential of oxygen respiration. In addition to separate fusions of the nifA and nifB promoters to β-galactosidase, a single-copy fusion of the entire nifA-nifB region was constructed. In this construct, plasmid-encoded FixJ protein stimulated the expression of a chromosomal nifA gene to produce the NifA protein, which then stimulated the expression of the nitB promoter. This strain produced 20-fold lower activity than a strain in which nifB promoter expression was stimulated by plasmid-encoded NifA protein. Finally, the nifA locus was found to contain a transcriptionally active element, oriented opposite to the nifA promoter.
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