Antagonistic role of H-NS and GadX in the regulation of the glutamate decarboxylase-dependent acid resistance system in Escherichia coli.
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.
Giangrossi
Mara
M
Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy.
Zattoni
Stefano
S
Tramonti
Angela
A
De Biase
Daniela
D
Falconi
Maurizio
M
D051777:AraC Transcription Factor / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D017931:DNA Primers; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C446316:GadX protein, E coli; C031908:H-NS protein, bacteria; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; D005968:Glutamate Decarboxylase