Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of sigmas activity and levels

Abstract

Upon environmental changes, bacteria reschedule gene expression by directing alternative sigma factors to core RNA polymerase (RNAP). This sigma factor switch is achieved by regulating relative amounts of alternative sigmas and by decreasing the competitiveness of the dominant housekeeping sigma(70). Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, sigma(S) (RpoS), in its competition with sigma(70) for core RNAP by increasing the formation of sigma(S)-containing RNAP holoenzyme, Esigma(S). Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through sigma(S), that is, on sigma(S)-dependent genes only. Not a specific promoter motif, but sigma(S) availability determines the ability of Crl to exert its function, rendering it of major importance at low sigma(S) levels. By promoting the formation of Esigma(S), Crl also affects partitioning of sigma(S) between RNAP core and the proteolytic sigma(S)-targeting factor RssB, thereby playing a dual role in fine-tuning sigma(S) proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.

Figure 1

Crl does not alter significantly in vitro transcription mediated by Eσ^S alone. Single-round and multi-round in vitro transcription assays using synthetic promoter 9 (synp9, with an rrnB (T₁,T₂) terminator cloned in the place of lacZ; see Materials and methods) were performed at 30°C. RNAP reconstituted with a five-fold excess of either σ^s or σ⁷⁰, and increasing amounts of Crl (0.5-, 1-, 2- and 10-fold more than core RNAP; only 10-fold more for the experiment with σ⁷⁰), were used to transcribe synp9 (upper panel). The RNA I transcript encoded by the vector (lower panel, obtained from the same gel) was used for normalisation in the quantification of the transcripts (data not shown).

Figure 2

Crl shifts sigma factor competition for core RNAP in favour of σ^S. In vitro sigma competition and single-round transcription assays were performed in the presence of 200 mM potassium glutamate at different temperatures: (A) 30°C and (B) 37°C. Purified σ^s and/or σ⁷⁰ were added in equimolar amounts to core RNAP (σ⁷⁰ also in four-fold excess where stated in the figure) for holoenzyme reconstitution, in the presence or absence of excess Crl (10-fold more than σ^s and core RNAP; note that the sigma factors were preincubated with Crl for 10 min at 30°C before addition to core RNAP). The mixture was used to transcribe synp9 as in Figure 1 (upper panel). The RNA I transcript (lower panel), also encoded by the template plasmid, was used for normalising quantification of synp9-derived transcripts (presented below the corresponding gel). For each gel, the amount of Eσ⁷⁰-derived transcript was set to 100%.

Figure 3

In vivo, Crl supports Eσ^S formation in stationary phase at the expense of Eσ⁷⁰. Wild-type MC4100 (A) and its crl⁻ mutant (B; NT190) were grown in LB at 30°C until the onset of stationary phase (OD_{578 nm}=3; cells growing in rich medium have a wide range of time duration during which they do not completely cease growing, but grow considerably slower: we denote this time as the onset/entry into stationary phase). Cells were harvested and lysed in order to obtain whole-cell extracts, which were further fractionated by gel filtration. Fractions were analysed by SDS–PAGE and visualised by immunoblots using monoclonal antibodies against the σ^S, σ⁷⁰ and β′ subunits of RNAP and a polyclonal antibody against Crl. (C) Results of the quantification performed for the two Western blots using the IMAGE GAUGE software. The ratio of free to bound sigma factor was calculated for both σ^S and σ⁷⁰ in the different genetic backgrounds (bound σ^S: in fractions A1–A3; free σ^S: A7–A9; bound σ⁷⁰: A2–A4; and free σ⁷⁰: A6–A8). The experiments were performed twice with reproducible results.

Figure 4

Crl stimulates σ^S degradation in vivo. (A) Increased σ^S levels in the crl⁻ mutant are observed only in the presence of RssB. Immunoblots depict cellular σ^S levels at different stages of growth at 30°C, in the presence or absence of Crl and in rssB⁺ or rssB-deficient backgrounds. (B) Cellular σ^S levels were monitored in the presence or absence of Crl at 30°C by immunoblot analysis after the addition of bacteriostatic amounts of chroramphenicol at an OD₅₇₈ of 3.0 (identical results were obtained also after addition of spectinomycin). The quantification of σ^S degradation is shown in Supplementary Figure S5.

Figure 5

rssB expression is reduced in the crl mutant. Expression of a single-copy rssAB:lacZ operon fusion was determined in wild-type (squares), rpoS⁻ (circles) and crl⁻ (diamonds) backgrounds. Cells were grown in LB medium at 30°C and optical densities and specific β-galactosidase activities were measured along the growth curve.

Figure 6

Uncoupling rssB expression from Crl/σ^S control results in decreased σ^S levels in the absence of Crl. In the rssB mutant background, RssB was expressed ectopically from pMP8 under the control of the p_tac promoter (no inducer present; RssB levels obtained are nevertheless slightly higher than those in the wild-type strain). An immunoblot depicting σ^S levels during different stages of growth at 30°C (o/n stands for overnight), in otherwise isogenic crl⁺ and crl mutant backgrounds, is shown; for reference, σ^S levels at an OD₅₇₈ of 3.0 in the wild-type strain (MC4100) are also shown (last lane).

Figure 7

Crl rescues σ^S from RssB/ClpXP-mediated degradation in vitro, but only in the presence of core RNAP. In vitro degradation of σ^S (A, I and III and B, I–III) was assayed in reaction mixtures containing 2 μM σ^S, 0.2 μM RssB, 0.2 μM reconstituted ClpXP, 5 mM ATP, 10 mM acetyl phosphate and where applicable 4 μM Crl (A, II and B, III), 0,29 μM core RNAP (B, II, III) or 2 μM BSA (B, I). In panel A, II, a control in vitro degradation assay for Crl alone is presented, using the same conditions and reagents as for σ^S (note that Crl was also stable in an in vitro degradation assay in which RssB was omitted; data not shown). For more experimental details, see Materials and methods, and for a more complete picture of the stained SDS–PAGE gels, see Supplementary Figure S8. Below the in vitro degradation assays, densitometric quantifications of the data are depicted. The intensity of bands representing σ^s (or Crl in panel A, II) was calculated relative to the intensity of bands representing a stable protein that was always present in the assay, that is, ClpX. Each experiment was repeated two or three times with highly reproducible results; a representative of those experiments is shown here. The half-life of σ^S is 14.5 min (±1.2) in the absence of Crl, 15 min (±2) in its presence (two-fold excess), 34 min (±3) in the presence of sub-stoichiometric amounts of core RNAP (1:7 molecular ratio) and 57.5 min (±3.5) in the presence of both Crl and core RNAP. Note that the presence of BSA (in amounts similar to those of Crl) in the mixture did not influence the degradation rates of σ^S.