Abstract
One of the most efficient systems of acid resistance in Escherichia coli, the gad system, is based on the coordinated action of two isoforms of glutamate decarboxylase (GadA and GadB) and of a specific glutamate/gamma-aminobutyrate antiporter (GadC). The gadA/BC genes, activated in response to acid stress and in stationary phase cells, are subjected to complex circuits of regulation involving sigma70, sigmaS, cAMP receptor protein, H-NS, EvgAS, TorRS, GadE, GadX, GadW, and YdeO. Herein, we provide evidence that the nucleoid-associated protein H-NS directly functions as repressor of gadA, one of the structural genes, and gadX, a regulatory gene encoding one of the primary activators of the gad system. Band shift and DNase I footprints reveal that H-NS indeed binds to specific sites in the promoter regions of gadA and gadX and represses the transcription of these genes both in an in vitro system and in vivo. Moreover, we show that a maltose-binding protein MalE-GadX fusion is able to stimulate the promoter activity of gadA/BC, thus indicating that GadX is by itself able to up-regulate the gad genes and that a functional competition between H-NS and GadX takes place at the gadA promoter. Altogether, our results indicate that H-NS directly inhibits gadA and gadX transcription and, by controlling the intracellular level of the activator GadX, indirectly affects the expression of the whole gad system.
Highlights
Colonization of the mammalian gastrointestinal tract by enteric bacteria requires the induction of genes, the protein products of which participate, either as regulators or as structural components, in survival mechanisms aimed at counteracting the life-threatening consequences of internal pH acidification [1,2,3]
H-NS Interacts with the Promoter Region of gadA and gadX Genes— many authors have shown that gadA and gadBC genes are derepressed in an hns genetic background during the exponential growth in neutral medium, a condition in which these genes are normally silenced [7, 10, 18], so far, no evidence has been provided that H-NS is directly involved in repressing transcription of gadA, gadBC, and other genes belonging to the gad system
We focused on the molecular mechanism adopted by the nucleoid-associated protein H-NS and the AraC-like activator GadX to cause antagonistic effects, negative and positive, respectively, on the expression of gad genes that play a major role in E. coli acid resistance
Summary
Colonization of the mammalian gastrointestinal tract by enteric bacteria requires the induction of genes, the protein products of which participate, either as regulators or as structural components, in survival mechanisms aimed at counteracting the life-threatening consequences of internal pH acidification [1,2,3]. We found that the fusion protein MalE-GadX is able both to stimulate in vitro the transcription of gadA and gadBC genes and to antagonize in a dose-dependent manner the repressive activity of H-NS at the level of the gadA promoter.
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