Characterization of EvgAS-YdeO-GadE branched regulatory circuit governing glutamate-dependent acid resistance in Escherichia coli

Escherichia coli prefers growth in neutral pH environments but can withstand extremely acidic conditions (pH 2) for long periods. Of the four E. coli systems that contribute to acid resistance, one, the glutamate-dependent system, is remarkable in its efficacy and regulatory complexity. The resistance mechanism involves the intracellular consumption of protons by the glutamate decarboxylase isozymes GadA and GadB. The antiporter GadC then exports the product, gamma-aminobutyric acid, in exchange for fresh glutamate. A microarray study using overexpressed regulators uncovered evgAS and ydeO as potential regulators of gadE, now known to encode the essential activator of the gadA and gadBC genes. Examination of evgA and ydeO under normal expression conditions revealed that their products do activate gadE expression but only under specific conditions. They were important during exponential growth in acidified minimal medium containing glucose but were unnecessary for gadE expression in stationary-phase cells grown in complex medium. The response regulator EvgA activates gadE directly and indirectly via induction of the AraC-like regulator ydeO. Evidence obtained using gadE-lacZ operon fusions also revealed that GadE was autoinduced. Electrophoretic mobility shift assays indicated that EvgA, YdeO, and GadE bind to different regions upstream of gadE, indicating they all act directly at the gadE promoter. Since GadE controls the expression of numerous genes besides gadA and gadBC, the relevance of these regulatory circuits extends beyond acid resistance.     

GadE (YhiE) activates glutamate decarboxylase-dependent acid resistance in Escherichia coli K-12

Commensal and pathogenic strains of Escherichia coli possess three inducible acid resistance systems that collaboratively protect cells against acid stress to pH 2 or below. The most effective system requires glutamate in the acid challenge media and relies on two glutamate decarboxylases (GadA and B) combined with a putative glutamate:gamma-aminobutyric acid antiporter (GadC). A complex network of regulators mediates induction of this system in response to various media, pH and growth phase signals. We report that the LuxR-like regulator GadE (formerly YhiE) is required for expression of gadA and gadBC regardless of media or growth conditions. This protein binds directly to the 20 bp GAD box sequence found in the control regions of both loci. Two previously identified AraC-like regulators, GadX and GadW, are only needed for gadA/BC expression under some circumstances. Overexpression of GadX or GadW will not overcome a need for GadE. However, overexpression of GadE can supplant a requirement for GadX and W. Data provided also indicate that GadX and GadE can simultaneously bind the area around the GAD box region and probably form a complex. The gadA, gadBC and gadE genes are all induced by low pH in exponential phase cells grown in minimal glucose media. The acid induction of gadA/BC results primarily from the acid induction of gadE. Constitutive expression of GadE removes most pH control over the glutamate decarboxylase and antiporter genes. The small amount of remaining pH control is governed by GadX and W. The finding that gadE mutations also diminish the effectiveness of the other two acid resistance systems suggests that GadE influences the expression of additional acid resistance components. The number of regulatory proteins (five), sigma factors (two) and regulatory feedback loops focused on gadA/BC expression make this one of the most intensively regulated systems in E. coli.     

EvgA of the two-component signal transduction system modulates production of the yhiUV multidrug transporter in Escherichia coli

Overexpression of the EvgA regulator of the two-component signal transduction system was previously found to modulate multidrug resistance of Escherichia coli by increasing efflux of drugs (K. Nishino and A. Yamaguchi, J. Bacteriol. 183:1455-1458, 2001). Here we present data showing that EvgA contributes to multidrug resistance through increased expression of the multidrug transporter yhiUV gene.     

Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP

Shigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri. These assays showed that the expression of genes involved in iron acquisition and acid response was induced by low-iron availability and by inactivation of fur. A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB, a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP, which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP. These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA.     

Gene expression profiling of the pH response in Escherichia coli

Escherichia coli MG1655 acid-inducible genes were identified by whole-genome expression profiling. Cultures were grown to the mid-logarithmic phase on acidified glucose minimal medium, conditions that induce glutamate-dependent acid resistance (AR), while the other AR systems are either repressed or not induced. A total of 28 genes were induced in at least two of three experiments in which the gene expression profiles of cells grown in acid (pH 5.5 or 4.5) were compared to those of cells grown at pH 7.4. As expected, the genes encoding glutamate decarboxylase, gadA and gadB, were significantly induced. Interestingly, two acid-inducible genes code for small basic proteins with pIs of >10.5, and six code for small acidic proteins with pIs ranging from 5.7 to 4.0; the roles of these small basic and acidic proteins in acid resistance are unknown. The acid-induced genes represented only five functional grouping categories, including eight genes involved in metabolism, nine associated with cell envelope structures or modifications, two encoding chaperones, six regulatory genes, and six unknown genes. It is unlikely that all of these genes are involved in the glutamate-dependent AR. However, nine acid-inducible genes are clustered in the gadA region, including hdeA, which encodes a putative periplasmic chaperone, and four putative regulatory genes. One of these putative regulators, yhiE, was shown to significantly increase acid resistance when overexpressed in cells that had not been preinduced by growth at pH 5.5, and mutation of yhiE decreased acid resistance; yhiE could therefore encode an activator of AR genes. Thus, the acid-inducible genes clustered in the gadA region appear to be involved in glutatmate-dependent acid resistance, although their specific roles remain to be elucidated.     

Global analysis of genes regulated by EvgA of the two-component regulatory system in Escherichia coli

The response regulator EvgA controls expression of multiple genes conferring antibiotic resistance in Escherichia coli (K. Nishino and A. Yamaguchi, J. Bacteriol. 184:2319-2323, 2002). To understand the whole picture of EvgA regulation, DNA macroarray analysis of the effect of EvgA overproduction was performed. EvgA activated genes related to acid resistance, osmotic adaptation, and drug resistance.     

Regulation of acid resistance by connectors of two-component signal transduction systems in Escherichia coli

Two-component signal transduction systems (TCSs), utilized extensively by bacteria and archaea, are involved in the rapid adaptation of the organisms to fluctuating environments. A typical TCS transduces the signal by a phosphorelay between the sensor histidine kinase and its cognate response regulator. Recently, small-sized proteins that link TCSs have been reported and are called "connectors." Their physiological roles, however, have remained elusive. SafA (sensor associating factor A) (formerly B1500), a small (65-amino-acid [65-aa]) membrane protein, is among such connectors and links Escherichia coli TCSs EvgS/EvgA and PhoQ/PhoP. Since the activation of the EvgS/EvgA system induces acid resistance, we examined whether the SafA-activated PhoQ/PhoP system is also involved in the acid resistance induced by EvgS/EvgA. Using a constitutively active evgS1 mutant for the activation of EvgS/EvgA, we found that SafA, PhoQ, and PhoP all contributed to the acid resistance phenotype. Moreover, EvgS/EvgA activation resulted in the accumulation of cellular RpoS in the exponential-phase cells in a SafA-, PhoQ-, and PhoP-dependent manner. This RpoS accumulation was caused by another connector, IraM, expression of which was induced by the activation of the PhoQ/PhoP system, thus preventing RpoS degradation by trapping response regulator RssB. Acid resistance assays demonstrated that IraM also participated in the EvgS/EvgA-induced acid resistance. Therefore, we propose a model of a signal transduction cascade proceeding from EvgS/EvgA to PhoQ/PhoP and then to RssB (connected by SafA and IraM) and discuss its contribution to the acid resistance phenotype.     

痢疾杆菌酸抗性系统相关基因缺失突变体的构建

依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础.