Escherichia coli gene expression responsive to levels of the response regulator EvgA

Abstract

To investigate the function of the EvgA response regulator, we compared the genome-wide transcription profile of EvgA-overexpressing and EvgA-lacking Escherichia coli strains by oligonucleotide microarrays. The microarray measurements allowed the identification of at least 37 EvgA-activated genes, including acid resistance-related genes gadABC and hdeAB, efflux pump genes yhiUV and emrK, and 21 genes with unknown function. EvgA overexpression conferred acid resistance to exponentially growing cells. This acid resistance was abolished by deletion of ydeP, ydeO, or yhiE, which was induced by EvgA overexpression. These results suggest that ydeP, ydeO, and yhiE are novel genes related to acid resistance and that EvgA regulates several acid resistance genes. Furthermore, the deletion of yhiE completely abolished acid resistance in stationary-phase cells, suggesting that YhiE plays a critical role in stationary-phase acid resistance. The multidrug resistance in an acrB deletion mutant caused by EvgA overexpression was completely abolished by deletion of yhiUV, while the emrKY deletion had no effect on the increase in resistance by EvgA overexpression. In addition, EvgA overexpression did not confer resistance in a tolC-deficient strain. These results suggest that YhiUV induced by EvgA overexpression is functionally associated with TolC and contributes to multidrug resistance.

FIG. 1.

Acid resistance of EvgA overexpression strains. Various strains were grown to mid-log phase in LB broth (pH 7.0) with or without IPTG. Cells were diluted 40-fold into LB broth (pH 2.5) or EG (pH 2.5) and were incubated for 1 h at 37°C. Initial cell densities ranged from 1.2 × 10⁶ to 1.6 × 10⁷ CFU/ml. Error bars represent standard errors of the means.

FIG. 2.

Acid resistance as a function of exposure time. The percentage of survival of exponential-phase MG1655/pQEevgA grown in the absence (○) or presence (•) of IPTG and stationary-phase MG1655 (▵) after acid exposure in LB broth (pH 2.5).

FIG. 3.

Acid survival of exponential-phase cells. Various deletion mutants harboring pQEevgA were grown to mid-log phase in LB broth (pH 7.0) containing IPTG. Cells were diluted 40-fold into LB broth (pH 2.5) and were incubated for 1 h at 37°C. Initial cell densities ranged from 7.4 × 10⁵ to 1.3 × 10⁷ CFU/ml. Error bars represent standard errors of the means. wt, wild-type MG1655; ydeP-O, ydeP-b1500-ydeO; yegR-Z, yegR-b2084-yegZ; yfdX-E, yfdXWUVE.

FIG. 4.

Acid survival of stationary-phase cells. Various deletion mutants were grown overnight in LB broth (pH 7.0). Cells were diluted 1,000-fold into LB broth (pH 2.5) and were incubated for 2 h at 37°C. Initial cell densities ranged from 2.3 × 10⁶ to 9.5 × 10⁶ CFU/ml. Error bars represent standard errors of the means. wt, wild-type MG1655; ydeP-O, ydeP-b1500-ydeO; yegR-Z, yegR-b2084-yegZ; yfdX-E, yfdXWUVE.