Nitrate- and molybdenum-independent signal transduction mutations in narX that alter regulation of anaerobic respiratory genes in Escherichia coli.
Escherichia coli can respire anaerobically by reducing nitrate, trimethylamine-N-oxide, dimethyl sulfoxide, or fumarate. When nitrate is present, expression of the genes for fumarate (frdABCD), trimethylamine-N-oxide, and dimethyl sulfoxide (dmsABC) is repressed while expression of the nitrate reductase (narGHJI) gene is induced. This regulation requires molybdate and is mediated by the narX and narL gene products, which together form a two-component regulatory system. We provide evidence that NarX is a nitrate and molybdenum sensor which activates NarL when nitrate is available to cells. Mutants generated by hydroxylamine mutagenesis were repressed for frdA-lacZ expression even when cells were grown in the absence of nitrate. The mutations responsible for three of these nitrate independence (NarX*) phenotypes were localized to narX and further characterized in vivo for their ability to repress frdA-lacZ expression. Two of the mutants (the narX64 and narX71 mutants) had a greatly reduced requirement for molybdenum to function but still responded to nitrate. In contrast, a third mutant (the narX32 mutant) required molybdenum but did not exhibit full repression of frdA-lacZ expression even when nitrate was present. These narX* alleles also caused the induction of nitrate reductase gene expression and the repression of a dmsA-lacZ fusion in the absence of nitrate. Each narX* mutation was determined to lie in an 11-amino-acid region of the NarX polypeptide that follows a proposed transmembrane domain. We suggest that the conformation of the narX* gene products is altered such that even in the absence of nitrate each of these gene products more closely resembles the wild-type NarX protein when nitrate is present. These data establish a clear role for the narX gene product in gene regulation and strongly suggest its role in sensing nitrate and molybdenum.
Kalman
L V
LV
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Gunsalus
R P
RP
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000502:physiology*; D009154:Mutation; D009566:Nitrates / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics; D015183:Restriction Mapping; D015398:Signal Transduction; D013385:Succinate Dehydrogenase / Q000235:genetics
D007506:Iron-Sulfur Proteins; D009566:Nitrates; D008982:Molybdenum; D010088:Oxidoreductases; D013385:Succinate Dehydrogenase; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C045664:dimethyl sulfoxide reductase