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. / Graduation date: 1992
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36259 |
| Date | 13 June 1990 |
| Creators | Evans, Paul D. |
| Contributors | Brown, Lyle R. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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