Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D

Abstract

The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

FIG. 1

Identification of the rsd promoters. (A) Primer extension analysis was carried out for RNA extracted from the exponentially growing cells (Log lane) and the stationary-phase cells (Stationary lane). (B) Primer extension products were analyzed by electrophoresis on a denatured polyacrylamide gel together with sequence ladders. P1, transcript from rsdP1 promoter; P2, transcript from rsdP2 promoter. (C) Primer extension analysis was carried out for RNA extracted from stationary-phase cells of wild-type (WT) strain ZK126 (rpoS⁺) and mutant strain ZK1000 (rpoS). (D) In vitro transcription of the rsd promoters by Eς⁷⁰ or Eς^S holoenzymes. Transcription products were analyzed by polyacrylamide gel electrophoresis in the presence of 8 M urea. (E) Nucleotide sequence of the upstream region of rsd. (F) Primers used in this study. (G) Sequence comparison of rsdP2 with the known gearbox promoters.

FIG. 2

Growth-dependent expression of the rsd-lacZ fusions. (A) Strain MJ6 [MC4100 λ(rsdI-lacZ); open symbols] and an isogenic rpoS mutant, MJ27 [MC4100 rpoS::Km λ(rsdI-lacZ); solid symbols], both carrying the rsdP1P2-lacZ fusion integrated in the genome (Table 1), were grown in LB medium. Cell growth (circles) was monitored by measuring turbidity, while rsd promoter activity was determined by measuring β-galactosidase activity (triangles). (B) Strain MJ19 [MC4100 λ(rsdIII-lacZ)] carrying the rsdP2-lacZ fusion was grown in LB medium. The cell growth (circles) and the β-galactosidase activity (triangles) were measured at the indicated times. (C) MJ6 [MC4100 λ(rsdI-lacZ)] was grown in LB medium (medium 1), M9–0.4% glucose–0.4% Casamino Acids (medium 2), M9–0.4% glycerol–0.4% Casamino Acids (medium 3), M9–0.4% glycerol (medium 4), and M9–0.4% acetate (medium 5). Exponentially growing cells at A₆₀₀ of 0.4 to 0.5 were used for the assay of β-galactosidase activity. (D) Strain MJ6 [MC4100 λ(rsdI-lacZ); open symbols] and its isogenic spoT mutant, MJ39 [MC4100 rpoT::Cm λ(rsdI-lacZ); solid symbols], were grown in LB medium. The β-galactosidase activity (triangles) and cell growth (circle) were measured at the indicated times.

FIG. 3

Effect of the rsd mutation on bolAP1-directed transcription. Cells were grown in LB medium. Growth (circles) was monitored by measuring turbidity, while β-galactosidase activity (triangles) was determined at the indicated times. Aliquots containing the same cell numbers were subjected to a quantitative Western blot assay for measurement of ς^S levels. (A) Strain MJ31 [MC4100 λ(bolA-lacZ); open symbols] and its isogenic rsd mutant, MJ35 [MC4100 rss::Km λ(bolA-lacZ); solid symbols]. (B) MJ31 [MC4100 λ(bolA-lacZ)] carrying either pACYC184 (open symbols) or pACYCRsd (solid symbols). (C) MJ35 [MC4100 rsd::Km λ(bolA-lacZ)] carrying either pACYC184 (open symbols) or pACYCRsd (solid symbols). (D) MJ35 [MC4100 rsd::Km λ(bolA-lacZ)] carrying either pBAD22A (open symbols) or pBF1 (solid symbols). Arabinose was added at 2 h of culture (time point 1).

FIG. 4

Effect of the rsd mutation on ompF promoter-directed transcription. Cells were grown in LB medium. Growth (circles) was monitored by measuring turbidity, while β-galactosidase activity (triangles) was determined at the indicated times. Expression of ς^S was induced by adding arabinose (0.02%) at the middle of the exponential phase. All E. coli strains carried the ompF-lacZ fusion integrated in the genomes. (A) Strain MH513 [MH20 λ(ompF-lacZ); open symbols], its isogenic rpoS mutant, MJ83 [MH20 rpoS::Km λ(ompF-lacZ); solid symbols], and an rsd mutant, MJ57 [MH20 rsd::Km λ(ompF-lacZ); crosses]. (B) MJ513(pACYC184; open symbols), MJ57(pACYC184; solid symbols), and MJ57(pACYCRsd; crosses). (C) MH513(pACYC184; open symbols) and MH513(pACYCRsd; solid symbols). (D) MH513(pBAD22A; open symbols) and MH513(pBF1; solid symbols).

FIG. 5

Effect of Rsd production on ompF-directed transcription. Cells of MH513 [MH20 λ(ompF-lacZ)] carrying either pBAD22A (open symbols) or pBADRsd (closed symbols) were grown in LB medium. Growth (circles) was monitored by measuring turbidity, while β-galactosidase activity (triangles) was determined at the indicated times. Overproduction of Rsd was induced by adding arabinose (0.02%) at the early exponential phase (A), at the middle of the exponential phase (B), and after entry into the stationary phase (C). Aliquots containing the same cell numbers were subjected to quantitative Western blotting for measurement of the Rsd level.