Regulation of osmC gene expression by the two-component system rcsB-rcsC in Escherichia coli

Abstract

The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp(1) and osmCp(2). Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp(1) and activates osmCp(2), the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor sigma(s) specifically recognizes osmCp(2). This work reports the identification of an additional regulatory element, the two-component system rcsB-rcsC, affecting positively the distal promoter osmCp(1). The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp(2). Deletion analysis located the site necessary for RcsB activation just upstream of osmCp(1). In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp(1).

FIG. 1

Sequence of the osmC promoter region and of its RcsB box. (A) The −10 and −35 regions of the two osmC promoters, osmCp₁ and osmCp₂, are underlined. Bent arrows show the osmCp₁ transcription starts. The A-to-G substitutions in the −10 boxes that abolish either osmCp₁ or osmCp₂ activity (osmCp₁₁ or osmCp₂₁, respectively) are represented by G's in parentheses above the sequence. Lines under the sequence show the extents of the DNA fragments carrying osmCp₁ that were used to locate the RcsB target site. The first three nucleotides at the 5′ ends of these fragments are given. The sequence required for activation of the fts genes by RcsB is shown above the osmC sequence. The bases in that sequence whose mutations have a strong or mild effect on RcsB activity are indicated by asterisks or circles, respectively (5). (B) Alignment of the sequences of the RcsB and RcsAB boxes. The RcsB box is proposed from the comparison between the fts and osmC regulatory regions (W stands for A or T, K stands for T or G, M stands for C or A, R stands for A or G, Y stands for C or T, and S stands for C or G). Bases important for activation of the fts genes by RcsB are underlined. The symmetry of the motifs is highlighted by the vertical broken line. The sequence of the RcsAB box is from reference .

FIG. 2

Induction of osmCp₁ following an osmotic shock does not require RcsB. Bacterial cells of wild-type or rcsB strains were grown at 30°C in LB0, and β-galactosidase specific activity was assayed at the times shown. At the time indicated by the arrow, NaCl was added to a final concentration of 0.5 M (+). The same volume of water was added to unshocked controls (−). (A) Expression of an osmCp₁-lacZ fusion; (B) expression of a cps-lacZ fusion.

FIG. 3

RcsB activates transcription from osmCp₁ in vitro. The transcription reaction was performed with (+) or without (−) 10 μM RcsB. The same downstream primer used to synthesize the templates was also used to generate a sequence ladder. The −10 sequence of the osmCp₁ promoter and the transcription starts are shown. The origin of the transcript marked by a star is unknown. Note that the sequence carries the A-to-G mutation abolishing osmCp₂ activity.

FIG. 4

Stimulation by RcsB of the binding of RNA polymerase to osmCp₁ in a gel shift assay. Binding reactions were performed with or without 5 nM RNA polymerase and with or without 10 μM RcsB. The retarded complex discussed in the text is indicated by an arrow. The signal marked by a star is inconsistently also observed in reactions without RcsB.