Genes of the GadX-GadW Regulon inEscherichia coli

Abstract

Acid in the stomach is thought to be a barrier to bacterial colonization of the intestine. Escherichia coli, however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. The gadX gene and a second downstream araC-like transcription factor gene, gadW, were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in a gadX mutant and even more so by combined mutation of gadX and gadW. However, there was no defect in colonization of the streptomycin-treated mouse model by the gadX mutant in competition with the wild type, and the gadX gadW mutant was a better colonizer than the wild type. Thus, E. coli colonization of the mouse does not appear to require glutamatedependent acid resistance.

Role of the Multidrug Resistance Regulator MarA in Global Regulation of the hdeAB Acid Resistance Operon in Escherichia coli

“…Regulation of the hdeAB operon. The figure has been produced using data from the literature (8,13,16,20,22,24,26,27,30,33,37,39,42,43,44,46) as well as the results obtained in this study. Thick arrows represent genes.…”

“…The GadE binding site has been located between Ϫ158 and ϩ4 but not identified (20). The consensus binding sequences we used were the following: 5ЈWYAGGMWWWWDYTW YWWW for binding of GadE, GadX, and GadW (gadbox [26,43]); 5ЈYAGHA WATTWTDCTR for Lrp (11); and 5ЈCTGTTCATAT for TorR (2,40); where D is A, T, or G; H is A, C, or T; M is A or C; R is A or G; W is A or T; and Y is C or T. Only sequences with 70% or higher identity to the consensus sequences were selected.…”

“…The hdeAB operon is induced in stationary phase (39,44) and at acid pH (19,43). Transcription of hdeAB depends on complex circuits of regulation involving RpoD ( 70 ), RpoS ( 38 ), H-NS, cyclic AMP receptor protein, GadE, GadX, GadW, GadY, EvgAS, YdeO, Lrp, MarA, SoxRS, TorRS, and TrmE, among other regulators (see Fig.…”

The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His–Asp phosphorelay RcsCDB/AF

“…A 25 % reduction of gadWlacZ fusion expression was observed in the rcsB strain, leaving the possibility that RcsB might activate gadAB expression through gadW. This is however unlikely, as GadW was reported to be a negative regulator of gadAB expression (Ma et al, 2002Tucker et al, 2003). In addition, a gadW mutant was reported to resist acid as well as a wild-type strain .…”

GntP Is the Escherichia coli Fructuronic Acid Transporter and Belongs to the UxuR Regulon

“…Transcript levels were confirmed by quantitative PCR (QPCR). Cells were grown on MOPS minimal medium to mid-log growth phase and total RNA was prepared by using RNAlater (Ambion) and purified using an RNeasy MinElute (QIAGEN) column (31). Taqman probes and primers were designed by using Primer Express software provided with the ABI Prism 7000 sequence detector (Applied Biosystems).…”