Alkali metals in addition to acidic pH activate the EvgS histidine kinase sensor in Escherichia coli

Abstract

Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception.

FIG 1

Activation of theEvgS sensor triggers a transcriptional cascade of the EvgA, YdeO, and GadE regulators which induces expression of AR genes (white arrows). The EvgS/EvgA system further activates the PhoQ/PhoP system, via a small protein, SafA, which increases the cellular RpoS level via IraM and RssB. Other AR genes induced by the EvgS/EvgA and PhoQ/PhoP systems and RpoS are not shown in this diagram.

FIG 2

Alkali metals, in addition to acidic pH, are required for EvgS/EvgA activation. Transcriptional activities of ydeP in strain MG1655 ydeP-lacZ (A and B) and emrKY in strain MG1655 emrKY-lacZ (C) were measured to examine EvgS/EvgA activity. β-Galactosidase activity was measured in cultures grown to the mid-exponential phase. (A) Transcriptional activity of ydeP was induced only in minimal A medium adjusted to pH 5.5. (B) Addition of KCl, NaCl, and LiCl to M9 medium (pH 5.5) at final concentrations of 50 and 100 mM induced the transcriptional activity of ydeP but not MgCl₂. (C) Addition of KCl and NaCl to M9 medium (pH 5.5) at final concentrations of 50 and 100 mM induced the transcriptional activity of emrKY. Columns represent the means of the results of three independent experiments ± standard errors.

FIG 3

EvgS/EvgA is activated under mildly acidic conditions. Strain MG1655 ydeP-lacZ was grown in M9 medium (pH 7.5) until the exponential phase (OD₆₀₀ = 0.1 to 0.2), and cells were collected by centrifugation and resuspended in M9 medium at various pH levels with the addition of KCl at a final concentration of 100 mM. Cells were cultured for 30 min at 37°C, and the transcriptional activity of ydeP was measured. Columns represent the means of the results of three independent experiments ± standard errors.

FIG 4

Activation of the EvgS/EvgA system by acidic pH and alkali metals is EvgS dependent. (A) Strain MG1655 ydeP-lacZ and its evgS-deleted mutant were grown in M9 medium (pH 5.5) with the addition of KCl or NaCl at a final concentration of 100 mM until the exponential phase, and the transcriptional activity of ydeP was measured. (B) Complementation of ΔevgS mutant. Strain MG1655 evgS ydeP-lacZ transformed with pBAD18 or pBADevgS was assayed as described for panel A with the addition of 0.002% arabinose for EvgS induction. Columns represent the means of the results of three independent experiments ± standard errors.

FIG 5

Acid resistance is induced by acidic pH and K⁺ in an EvgS-dependent manner. Strains MG1655 and MG1655 evgS, which were grown to the mid-exponential phase in M9 medium (pH 7.5 and 5.5) with the addition of KCl at final concentrations of 0, 50, and 100 mM, were treated in acidic (pH 2.5) LB medium for 1 h, and their survival rates were determined. Survival rates are shown as the ratios of viable cells remaining after acid treatment. Columns represent the means of the results of three independent experiments ± standard errors.

FIG 6

L152 in the periplasmic region is involved in EvgS activation. (A) Transcriptional activities of emrKY in strains MG1655 emrKY-lacZ and KMY1 were measured in M9 medium (pH 5.5) with and without the addition of KCl at a final concentration of 100 mM. (B) Transcriptional activity of emrKY in strain KMY1 evgS transformed with plasmids pBAD18, pBADevgS, and pBADevgS L152F grown in M9 medium (pH 5.5) with and without the addition of KCl at a final concentration of 100 mM and 0.002% arabinose for induction. The inset shows a Western blot using EvgS-antiserum against the cells used for the β-galactosidase assay. Numbers indicated above the lanes correspond to those shown below the columns. (C) Transcriptional activity of ydeP in strain MG1655 evgS ydeP-lacZ transformed with plasmids pBAD18, pBADevgS, and pBADevgS L152F was measured as described for panel B. The inset shows a Western blot as described for panel B. (D) Activity of EvgS L152-substituted mutants. Transcriptional activity of ydeP in strain MG1655 evgS ydeP-lacZ transformed with plasmids pBADevgS L152A, pBADevgS L152R, pBADevgS L152E, pBADevgS L152I, pBADevgS L152Y, pBAD18, and pBADevgS was measured as described for panel B. The inset shows a Western blot as described for panel B. Expression levels of EvgS and its mutants were examined only in cells grown in the presence of 100 mM KCl. Statistical significance is indicated by asterisks as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Columns represent the means of the results of at least three independent experiments ± standard errors.

FIG 7

The periplasmic region of EvgS is necessary for activation. (A) Diagram of PhoQ-EvgS chimera (EvgP) and EvgSΔSD. Residues 43 to 190 of PhoQ were fused between residues 1 and 23 and residues 531 and 1197 of EvgS in chimera EvgP, and residues 30 to 530 in EvgSΔSD were deleted. (B) Transcriptional activities of ydeP in strain MG1655 evgS ydeP-lacZ transformed with plasmids pBAD18, pBADevgS, pBADevgP, and pBADevgSΔSD were measured in M9 medium (pH 5.5 and 7.5) with or without the addition of KCl at a final concentration of 100 mM and 0.002% arabinose for induction. The insets show Western blots using PhoQ antiserum and EvgS antiserum against the cells used for the β-galactosidase assay. White arrows indicate the EvgS, EvgP, and EvgSΔSD bands. Columns represent the means of the results of three independent experiments ± standard errors.

FIG 8

Signal recognition by EvgS-PhoQ chimera sensors. (A) Diagram of EvgS-PhoQ chimeras, PvgS-A, and PvgS-B. Residues 1 to 558 of EvgS were fused to residues 215 to 486 of PhoQ in chimera PvgS-A, and residues 1 to 710 of EvgS were fused to residues 267 to 486 of PhoQ in chimera PvgS-B. (B and C) Transcriptional activities of mgtA in strain MG1607 transformed with plasmids pBAD18, pBADphoQ, pBADpvgS-B, and pBADpvgS-A were measured in M9 medium (pH 5.5 and 7.5) with and without the addition of KCl at a final concentration of 100 mM. Cells in panel B were induced with 0.002% arabinose and those in panel C with 0.2% arabinose. The insets show Western blots using PhoQ antiserum against the cells used for the β-galactosidase assay. Columns represent the means of the results of at least three independent experiments ± standard errors. Statistical significance is indicated by asterisks as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.