Multistress regulation in Escherichia coli: expression of osmB involves two independent promoters responding either to sigmaS or to the RcsCDB His-Asp phosphorelay

Abstract

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a sigma(S)-dependent manner. The upstream promoter, osmBp1, is independent of sigma(S) and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its -35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a sigma(S)-dependent promoter and a second one independent of sigma(S) but controlled by stress-specific transcription factors.

FIG. 1.

Sequence of the osmB promoter region. Arrows indicate the oligonucleotides used for mRNA 5′-end mapping or to amplify the DNA fragments used to construct transcriptional lac fusions. B and E, BamHI and EcoRI restriction sites. The −35 and −10 elements of osmBp1 and osmBp2 are boxed. Broken arrows indicate the transcription starts. The Shine-Dalgarno sequence (SD) and translation start of osmB are shown in bold letters.

FIG. 2.

osmB transcription is stimulated by RcsB but not by its cofactor, RcsA. Strain SK1755 (Φ[osmBp1-osmBp2-lac]), transformed with plasmid pHRcsB or pHRcsA, as indicated, was grown in LB broth, and the optical density at 600 nm (OD₆₀₀) (open symbols) and β-galactosidase activity (filled symbols) were followed during growth. At time zero, half of each culture was kept in LB broth (circles, dashed lines) and the other half (triangles, full lines) was treated with IPTG (0.5 mM) to induce the production of RcsB or RcsA.

FIG. 3.

osmBp2 is induced upon entry into stationary phase in a σ^S-dependent manner but is not stimulated by overexpression of RcsB. (A) Strains CLG250 (Φ[osmBp2-lac]) (circles) and CLG254 (Φ[osmBp2-lac] rpoS::Tn10) (squares) were grown in LB0N, and OD₆₀₀ (open symbols) and β-galactosidase activity (filled symbols) were monitored during growth. (B) CLG250 transformed with plasmid pHRcsB was grown in LB broth, and the OD₆₀₀ (open symbols) and β-galactosidase activity (filled symbols) were monitored during growth. At time zero, half of the culture was kept in LB broth (circles, dashed lines) and the other half was treated with IPTG (0.5 mM) to induce the production of RcsB (triangles, full lines).

FIG. 4.

σ^S is responsible for osmotic shock induction of osmBp2. Strains CLG250 (Φ[osmBp2-lac]) (dark gray bars) and CLG254 (Φ[osmBp2-lac] rpoS::Tn10) (light gray bars) were grown in LB0 until early exponential phase. At time zero, half of the culture was kept in LB0 (− NaCl) and the other half was treated with 0.4 M NaCl (+ NaCl), and β-galactosidase activity was monitored at 0, 20, 40, and 60 min.

FIG. 5.

osmBp1 is stimulated by overexpression of RcsB but neither upon entry into stationary phase nor by osmotic shock. (A) Strain CLG249 (Φ[osmBp1-lac]) transformed with plasmid pHRcsB was grown in LB broth, and the OD₆₀₀ (open symbols) and β-galactosidase activity (filled symbols) were monitored during growth. At time zero, half of the culture was kept in LB (circles, dashed lines) and the other half was treated with IPTG (0.5 mM) to induce the production of RcsB (triangles, full lines). (B) CLG249 was grown in LB0N, and the OD₆₀₀ (open symbols) and β-galactosidase activity (filled symbols) were monitored during growth. At time zero, half of the culture was kept in LB0N (circles, dashed lines) and the other half (triangles, full lines) was treated with NaCl (0.4 M).

FIG. 6.

Determination of osmBp1 mRNA 5′ end. Total RNAs were extracted from a strain that was (+) or was not (−) overproducing RcsB_D56E, hybridized with ³²P-labeled osmB17 or osmB18 oligonucleotides, and treated with AMV reverse transcriptase before analysis on a denaturing polyacrylamide gel and autoradiography. The sequence ladders (lanes T, A, G, and C) were obtained by sequencing the osmBp region by using the same primers and the CircumVent sequencing kit (Biolabs) as described by the manufacturer.

FIG. 7.

Activation of osmBp1 by RcsB necessitates a site upstream of the −35 element. (A) Arrows indicate the oligonucleotides used for the construction of Φ[osmBp1-lac] transcriptional fusions. osmB11 and osmB20 carry the GA-to-TC double mutations indicated. B and E, BamHI and EcoRI restriction sites. The consensus sequence of RcsB binding sites is shown above the osmB sequence, with the most-conserved positions in bold letters. (B) Strains carrying the indicated transcriptional fusions were transformed with plasmid pHRcsB and grown in LB broth. At time zero (OD₆₀₀ of 0.1), cultures were not treated or were treated with IPTG (0.5 mM) to induce the production of RcsB, and β-galactosidase activity was assayed over time.