Functional characterization and regulation of gadX, a gene encoding an AraC/XylS-like transcriptional activator of the Escherichia coli glutamic acid decarboxylase system.
The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H(+) and producing gamma-aminobutyric acid. This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB. Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family. The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites. Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes. However, P(T5)lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed. Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription. Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities.
Tramonti
Angela
A
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Visca
Paolo
P
De Canio
Michele
M
Falconi
Maurizio
M
De Biase
Daniela
D
D018528:ATP-Binding Cassette Transporters; D051777:AraC Transcription Factor / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000502:physiology; D005809:Genes, Regulator; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000502:physiology; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation
D018528:ATP-Binding Cassette Transporters; D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C446316:GadX protein, E coli; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C461599:maltose transport system, E coli; C069567:sigma factor KatF protein, Bacteria; D004247:DNA