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Keywords:

  • RNA polymerase;
  • RpoS;
  • sigma factor competition;
  • stress;
  • transcription factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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 σ70. Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, σS (RpoS), in its competition with σ70 for core RNAP by increasing the formation of σS-containing RNAP holoenzyme, EσS. Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through σS, that is, on σS-dependent genes only. Not a specific promoter motif, but σS availability determines the ability of Crl to exert its function, rendering it of major importance at low σS levels. By promoting the formation of EσS, Crl also affects partitioning of σS between RNAP core and the proteolytic σS-targeting factor RssB, thereby playing a dual role in fine-tuning σS proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacteria are able to massively reprogram gene expression when confronted with changes in their environment. An efficient way to accomplish this is by competition of promoter-specific sigma subunits for the RNA polymerase (RNAP) core enzyme (for review see Nyström, 2004). Control of expression, stability and/or availability of alternative sigma factors define the conditions under which an alternative sigma factor is able to substantially compete with the vegetative σ70 for limiting amounts of core RNAP. However, as σ70 is abundant throughout the growth cycle and shows the highest affinity for core RNAP in vitro (Jishage et al, 1996; Maeda et al, 2000), the cell obviously uses additional strategies beyond simple competition in order to ensure the switch between σ70 and appropriate alternative sigma factors in the RNAP holoenzyme in response to physiological stresses.

The alarmone ppGpp plays a major role in sigma factor competition for core RNAP upon entry into stationary phase (Jishage et al, 2002; Laurie et al, 2003; Magnusson et al, 2003; Costanzo and Ades, 2006). DksA protein was recently shown to act synergistically with ppGpp (Paul et al, 2004, 2005; Perederina et al, 2004). As rRNA transcription employs 70% of the σ70-containing RNAP holoenzyme (Eσ70) during exponential growth (Raffaelle et al, 2005), factors like DksA and ppGpp, which actively dissociate Eσ70 from rRNA loci upon entry into stationary phase, provide more free core RNAP for alternative sigmas (Bernardo et al, 2006). Furthermore, overexpression of Rsd, a protein with affinity for σ70 and core RNAP (Ilag et al, 2004), whose cellular level increases in stationary phase (Jishage and Ishihama, 1998), has similar effects as ppGpp with respect to ‘holoenzyme switching’ (Jishage et al, 2002; Laurie et al, 2003). Finally, 6S RNA, a conserved small RNA (Barrick et al, 2005; Trotochaud and Wassarman, 2005), is active in stationary phase and structurally mimics an open promoter complex that can ‘fool’ only Eσ70 to recognise it (Wassarman and Storz, 2000). Its presence ensures downregulation of activity of the housekeeping RNAP holoenzyme, thus allowing alternative RNAPs to take over (Wassarman and Storz, 2000; Trotochaud and Wassarman, 2004).

The common characteristic of these factors is that all are active upon entry into stationary phase and that their main target of action is σ70 effectiveness; by decreasing it, they make room for alternative sigma factors to act. However, stationary phase is mainly the territory of the master regulator for stress responses, σS. EσS is actively engaged in the transcription of more genes than any other alternative sigma factor, with the majority of them being also activated in stationary phase (Weber et al, 2005). Despite the strong increase in its protein levels upon entering stationary phase (Hengge-Aronis, 2002), σS only reaches about one-third of the σ70 levels under these conditions (Jishage et al, 1996) and exhibits the lowest affinity for core RNAP of all sigma factors in vitro (Maeda et al, 2000; Colland et al, 2002). Therefore, we reasoned that apart from factors that decrease σ70 effectiveness in stationary phase and thereby give a collective advantage to all alternative sigmas, there should also be mechanisms dedicated to specifically increase the performance of σS and thereby allow EσS to gain its dominant role in stationary phase and several other stress conditions.

Crl protein was initially identified as an activator of genes for curli fimbriae formation (Arnqvist et al, 1992). Later, its role was extended to that of an auxiliary factor for EσS activity at certain genes (Pratt and Silhavy, 1998). Recently, Crl was shown to bind specifically to free σS, and proposed to increase the affinity of EσS for certain promoters at low temperatures (30°C; Bougdour et al, 2004). In this study, we show that Crl positively regulates a large subset of σS-dependent genes that do not share a common promoter motif, and its action strongly depends on σS availability. In in vitro transcription assays, Crl aids σS-dependent transcription, especially when σS is competing with σ70 for limiting amounts of core RNAP. Consistently, during entry into stationary phase, crl mutant cells possess relatively lower levels of EσS, but enhanced amounts of Eσ70. Owing to its role in controlling the partitioning of σS between RNAP core and the proteolytic targeting factor for σS, RssB, Crl also plays a complex role in controlling σS levels. We conclude that the common basis of all these effects is the ability of Crl to specifically aid σS in sigma factor competition for core RNAP during stationary phase.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The Crl regulon

As existing studies had monitored the role of Crl for a limited number of genes known to be controlled by σS, we evaluated its global function in transcriptional regulation in E. coli by genome-wide transcriptional profiling. Using similar conditions as for microarray analyses previously conducted in our laboratory for the σS regulon (Weber et al, 2005) allowed us to directly compare the results. The E. coli K12 strain MC4100 and its isogenic crlcat mutant were grown in rich medium at 30°C, as the stationary phase-induced curli genes are only expressed at such reduced temperatures, which had also previously been suggested to play a significant role in Crl activity (Bougdour et al, 2004). Total RNA was extracted at an OD578 nm of 4.0 (i.e. during entry into stationary phase) and further processed for genome-wide microarray analysis (see Materials and methods for details). Genes with expression ratios in MC4100 and its crl mutant derivative of >2-fold or <0.5-fold (average of three independent experiments) were considered relevant and are presented in Table I. The results indicated that all of these genes were either part of the known σS regulon at 37°C (denoted by an asterisk in Table I; Weber et al, 2005) or were shown to be expressed under the control of σS only at lower temperatures (denoted by an asterisk in parentheses in Table I; H Weber and R Hengge, unpublished microarray results). In addition, all of the genes positively controlled by Crl showed lower ratios of Crl dependency than previously observed ratios of σS dependency, consistent with the assumption that Crl is not essential for but modulates the activity of σS, and the effects of the latter are epistatic to those of the former (Pratt and Silhavy, 1998). It should also be noted that many genes known to be under the control of σS are not listed in Table I, but exhibited ratios just below the cutoff mentioned above (data not shown). Thus, among the genes (approximately 55) with expression ratios between 1.60 and 2, the vast majority (∼90%) was also under the control of σS (data not shown). Furthermore, our microarray analysis identified the csgBA operon as part of the Crl regulon and could also detect the previously reported (Pratt and Silhavy, 1998; Robbe-Saule et al, 2006) relatively modest effects of Crl on the expression of bolA, katE and csgD (data not shown; all three genes exhibited ratios of 1.5–2 at both 30 and 37°C).

Table 1. The Crl regulon (30°C)a
Gene nameIDAverage of ratio of mediansMember of the RpoS regulonFunction
  • a

    Genes are ordered according to their chromosomal position, which is reflected in their b-number. The complete data sets can be found at ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MEXP-720. Genes associated with an asterisk are part of the σS regulon at 37°C (Weber et al, 2005), whereas those associated with an asterisk in parentheses are part of the σS regulon at lower temperatures, that is, 28°C (H Weber and R Hengge , unpublished microarray results).

Crlb0240139.7 Regulatory protein for curli, transcriptional regulator
psiFb03842.2*Pho regulon member, requiring PhoRB system
ybaYb04532.2*Glycoprotein/polysaccharide metabolism
allRb05062.2(*)AllR transcriptional regulator
ybgSb07532.5*Putative homeobox protein
ycaCb08972.4*Putative cysteine hydrolase
ycdFb10053.0*Hypothetical protein
csgBb10412.9(*)Curlin, minor subunit precursor
csgAb10423.5(*)Curlin, major subunit
csgCb10432.1(*)Putative curli production protein
ymdAb10442.0(*)Conserved hypothetical protein
ymfEb11382.4(*)Hypothetical protein
narUb14692.3*MFS nitrite transporter
gadCb14923.1*Putative glutamate:gamma-aminobutyric acid antiporter (APC family)
gadBb14933.6*Glutamate decarboxylase B subunit
yeaHb17842.4*Conserved hypothetical protein
ybeVb18362.4*Stimulates the ATPase activity of Hsc62, possibly component of a new Hsp70 chaperone system
luxSb26872.5*Quorum sensing, autoinducer II synthase
tdcCb31162.3(*)TdcC threonine STP transporter
bfrb33362.2*Bacterioferritin monomer
hdeBb35092.3*10K-L protein, related to acid resistance protein of Shigella flexneri
hdeAb35102.3*Acid-resistance protein, possible chaperone
hdeDb35112.4*Protein involved in acid resistance
gadEb35123.2*Transcriptional regulator, activates glutamate decarhboxylase-dependent acid resistance
gadWb35152.4*Transcriptional regulator (AraC/XylS family)
gadAb35173.6*Glutamate decarboxylase A subunit
yhjRb35352.1*Hypothetical protein
yiaGb35552.8*Putative transcriptional regulator
yjbJb40452.5(*)Highly abundant nonessential protein
     
ydhYb16740.4 Putative oxidoreductase, Fe-S subunit
iadAb43280.5 Subunit ofisoaspartyl dipeptidase
yjiGb43290.5 Putative membrane protein

To further confirm that the role of Crl in global gene expression in stationary phase is mediated exclusively by σS, we extended our genome-wide transcriptional profiling approach to an rpoS background. Here, Crl did not control any significant regulon (it only slightly repressed the expression of the paa operon, responsible for phenylacetic acid degradation; Ferrandez et al, 1998; see Supplementary Figure S1 and Supplementary Table S1).

As some effects of Crl on σS-dependent gene expression were previously observed at 37°C (Pratt and Silhavy, 1998), we performed an analogous microarray analysis at this temperature and found that Crl exerted effects similar to those at 30°C on an overlapping subset of σS-dependent genes (data not shown). Thus, Crl effects seemed to be temperature independent, in contrast to a recent report that proposed a thermosensitive function of Crl in regulating σS activity (Bougdour et al, 2004). Assaying Crl protein levels at different stages of growth revealed only slightly higher expression of Crl at 30°C than at 37°C (∼70% more Crl at 30°C), and weak stationary-phase induction at both temperatures (∼2-fold; Supplementary Figure S2). In a study published during the revision of this manuscript, it was shown that a similar accumulation of Crl takes place in Salmonella typhimurium during growth at 28°C (Robbe-Saule et al, 2006). To summarise, we propose that Crl modulates the activity of σS in stationary phase and thereby plays a global role in the control of σS-dependent genes in a temperature-independent manner.

Crl acts on σS activity by affecting sigma factor competition for core RNAP

The moderate and rather uniform differences in the expression ratios observed for all genes controlled by Crl suggested that Crl might not have specific sequence requirements for exerting its action. Indeed when aligning the known promoters of the positively regulated genes shown in Table I, no sequence pattern specific for this group of σS-dependent genes emerged (data not shown). In addition, assaying synthetic promoters carrying different cis features known to contribute to σS promoter selectivity (Typas et al, 2007) revealed no correlation between those cis elements and Crl dependency of the promoter (Supplementary Figure S3); all the promoters exhibited a similar reduction of expression in the crl mutant strain. The fact that Crl could activate σS-dependent synthetic promoters that do not require any additional transcription factors for their maximal expression also excluded the possibility that Crl facilitates EσS function by optimising its cooperation with trans-acting factors (Bougdour et al, 2004). All these data suggest that Crl does not aid EσS in the specific recognition of promoters (see also Discussion).

When monitoring the role of Crl in the expression of various synthetic and natural promoters in vivo, we noticed an inverse correlation between σS levels and the ability of Crl to activate σS-dependent promoters. When σS levels were kept relatively low by using various combinations of genetic backgrounds and/or growth conditions, Crl had a more pronounced role in the expression of these σS-controlled promoters (Supplementary Figure S4). On the contrary, when the intracellular σS concentration was relatively high, Crl did not stimulate σS-dependent promoter activity. This correlation of low intracellular σS levels with Crl function is also consistent with Crl exerting its role before promoter recognition by EσS.

To clarify this role at the molecular level, we directly monitored the effect of Crl on σS-dependent transcription in vitro. A synthetic promoter with strong σS preference was chosen (synp9) and single-round and multiround transcription assays were performed (Figure 1). Using an excess of sigma-saturated RNAP over the supercoiled DNA template (20:1) enabled us to distinguish between initial recruitment of RNAP to the promoter and later stages of transcription (under such conditions, the multiround transcription assays are more sensitive in detecting effects on initial recruitment). In both assays, preincubation of increasing amounts of Crl with σS before RNAP holoenzyme reconstitution had only a marginal effect in promoter utilisation by EσS alone. Also Eσ70-derived transcription was not influenced by the addition of the maximal amount of Crl (10-fold more than RNAP; note that these ratios of Crl/RNAP are within the physiological range; see also Discussion), which was expected as Crl does not interact with Eσ70 (Bougdour et al, 2004). Thus, Crl does not seem to significantly affect the ability of EσS per se to recognise a promoter sequence and initiate transcription, at least when core RNAP is saturated with sigma (five-fold excess of sigma).

image

Figure 1. Crl does not alter significantly in vitro transcription mediated by EσS 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 σs 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).

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Using the same concept as for the single-round in vitro transcription assays above, we established an in vitro competition assay for the two sigma factors. Different molecular ratios of σ70 and σS (and each sigma factor on its own for control) were preincubated with Crl or buffer alone and then added to limiting amounts of core RNAP for reconstitution. The resulting RNAP holoenzyme mixtures were used to transcribe the synthetic promoter synp9. As shown in Figure 2, Crl shifts the competition balance in favour of σS, that is, the presence of Crl can counteract the reduction in synp9 expression caused by the presence of σ70. The supportive effect of Crl on EσS and its output was especially pronounced when competition for core RNAP was harsher for σS, for example, when σ70 was present in four-fold excess, whereas in the absence of Crl, synp9 expression was as low as with σ70 alone (i.e. no EσS is present at all; Figure 2A and B). In addition, the effect of Crl was at least as pronounced at 37°C as at 30°C (Figure 2B). Again, in the absence of competition, that is, with either σS or σ70 alone, Crl had no or only minor effects (Figure 2A and B). It should be noted that the positive effect of Crl on EσS-derived transcription—upon σ factor competition—might seem relatively small (∼2-fold in most cases), but this reflects the fact that the synp9 promoter retains a basal Eσ70-dependent transcription that can reach up to 20% of that of EσS. In any case, the main difference between the ‘simple’ and the competition in vitro transcription assays was that in the latter case core RNAP was not saturated with σS alone, and therefore the effects of Crl on EσS holoenzyme formation in a sigma factor competition situation could be monitored.

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Figure 2. Crl shifts sigma factor competition for core RNAP in favour of σS. 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 σs 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 σs 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%.

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In order to test whether Crl also has an impact on sigma factor competition in vivo, thereby facilitating the formation of EσS, we measured the relative in vivo amounts of σS and σ70 bound to core RNAP in wild-type and crl mutant strains during the onset of stationary phase. After harvesting the cells, whole-cell extracts were fractionated by gel filtration and the amounts of sigma factors (σS and σ70), Crl and the β′ RNAP subunit in each fraction were quantified by immunoblot analysis. Both sigmas were found to elute in two separate sets of the fractions collected (Figure 3). σS coeluted with the β′ subunit, as part of the RNAP (EσS), in fractions A1–A3, whereas fractions A7–A9 contained σS in its free form. On the other hand, Eσ70 eluted mostly in fractions A2–A4, whereas free σ70 was found in fractions A7–A9. The fractionation pattern was also verified by experiments using purified free σS and σ70 or their reconstituted RNAP forms (data not shown). It is apparent that a significantly larger fraction of σS was bound to RNAP in the wild-type strain compared with the crl mutant strain, whereas the opposite tendency can be observed for σ70 (Figure 3). This result verified that Crl supports σS in its competition with σ70 for core RNAP in vivo, and stimulates the formation of EσS at the expense of formation of the vegetative holoenzyme, Eσ70.

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Figure 3. In vivo, Crl supports EσS 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 (OD578 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 σS, 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 σS and σ70 in the different genetic backgrounds (bound σS: in fractions A1–A3; free σS: A7–A9; bound σ70: A2–A4; and free σ70: A6–A8). The experiments were performed twice with reproducible results.

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In conclusion, both in vitro and in vivo data shown above indicate that Crl plays a role in σS-dependent transcription by affecting sigma factor competition for limiting amounts of core RNAP in favour of σS, and that this role becomes particularly important at low σS levels, that is, before σS reaches its maximal level in stationary phase.

Interplay between Crl, RNAP and RssB: Crl also regulates intracellular σS levels by affecting σS proteolysis

In parallel with enhancing σS activity in stationary phase, Crl also seems to reduce intracellular σS levels (Pratt and Silhavy, 1998). Although at first glance this may seem paradoxical, this means that Crl allows σS to be effective at lower levels, such that high levels of σS are not needed. We observed that this reducing effect of Crl on σS concentration is apparent at all stages of growth both at 30 and 37°C, but it is eliminated in an rssB background (Figure 4A and data not shown). RssB, which is the target of complex signal transduction pathways, shows phosphorylation-dependent affinity for σS and serves as its targeting factor to ClpXP protease (Muffler et al, 1996; Pratt and Silhavy, 1996; Bouché et al, 1998; Becker et al, 1999; Klauck et al, 2001; Zhou et al, 2001; Stüdemann et al, 2003; Mika and Hengge, 2005). The observation that Crl exerts its effect on σS levels via RssB suggested that Crl influences σS degradation. Indeed, σS proteolysis (measured during entry into stationary phase) was slowed down in the crl mutant (Figure 4B). σS half-lives were approximately 4–5 and 10–12 min in crl+ and crl mutant backgrounds, respectively (Supplementary Figure S5).

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Figure 4. Crl stimulates σS degradation in vivo. (A) Increased σS levels in the crl mutant are observed only in the presence of RssB. Immunoblots depict cellular σS 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 σS 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 σS degradation is shown in Supplementary Figure S5.

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In the absence of strong stress signals that interfere with RssB activity, the cellular RssB level is the limiting factor in σS degradation. Consistently, the control of rssB expression by σS provides the system with a homeostatic feedback loop that sets the threshold for titration of RssB and therefore for the stabilisation of σS by certain stress conditions that rapidly and strongly induce σS synthesis (Pruteanu and Hengge-Aronis, 2002). Thus, we reasoned that, Crl could affect, via its effect on σS activity, rssB expression and thereby σS proteolysis (Supplementary Figure S6). Using a transcriptional fusion of the rssAB operon promoter to lacZ, we could verify that rssB behaves like other σS-dependent genes, that is, its expression is reduced in the absence of Crl (Figure 5). We conclude that Crl stimulates the expression of the limiting factor of σS proteolysis, RssB, and thereby increases σS degradation rates. Consequently, cellular σS levels are higher in the crl knockout strain.

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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.

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On the other hand, we wondered what would happen if RssB expression was uncoupled from this σS/Crl control. We suspected that in such a situation, the effect of Crl on σS70 competition for limiting amounts of core RNAP might be revealed: as Crl favours σS in this competition, more σS would be bound to RNAP in the presence of Crl (Figure 3), and thus be protected against proteolysis. In other words, a crl mutant strain would be expected to show increased σS proteolysis and therefore lower σS levels when Crl does not affect the expression of rssB (opposite to what is observed when rssB is expressed from its chromosomal locus with its natural σS/Crl-controlled promoter; see Figure 4)

In order to test this hypothesis, we used a moderate-copy-number plasmid with rssB under ptac promoter control (pMP8; Pruteanu and Hengge-Aronis, 2002). RssB expression from this plasmid is only slightly higher than that from its chromosomal wild-type gene, when no inducer is added, which leads to somewhat higher σS degradation rates (Pruteanu and Hengge-Aronis, 2002) and therefore lower but still detectable σS levels (Figure 6). As hypothesised above, introducing the crl mutation in this background resulted in σS levels that were below the limit of detection (Figure 6). This correlated perfectly with the expression of a synthetic σS-dependent promoter assayed in the same genetic backgrounds, which showed only residual σ70-dependent expression throughout the whole growth curve in the crl mutant (data not shown). To summarise, when the negative feedback link between σS/Crl and rssB expression is eliminated by expressing rssB ectopically from a constitutive promoter, the function of Crl in favour of EσS formation results in increased σS stability, which becomes visible as higher σS levels in the presence of Crl (Figure 6). This increased stability derives from σS being protected within the holoenzyme (Zhou et al, 2001).

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Figure 6. Uncoupling rssB expression from Crl/σS control results in decreased σS levels in the absence of Crl. In the rssB mutant background, RssB was expressed ectopically from pMP8 under the control of the ptac promoter (no inducer present; RssB levels obtained are nevertheless slightly higher than those in the wild-type strain). An immunoblot depicting σS 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, σS levels at an OD578 of 3.0 in the wild-type strain (MC4100) are also shown (last lane).

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Next, we were interested to clarify whether Crl can also directly compete with RssB for binding to σS, or whether, alternatively, all three proteins can form a ternary complex. Phosphorylated RssB (RssB-P) is known to strongly interact with σS (Becker et al, 1999; Zhou et al, 2001). Crl, on the contrary, seems to exhibit rather weak binding to σS (Bougdour et al, 2004). To test if and how Crl influences the interaction of σS with RssB-P, we used an established coelution protocol (Becker et al, 1999; Klauck et al, 2001) and gel filtration analysis (Supplementary Figure S7). In both cases, Crl could not compete with RssB for σS binding, and also no ternary complex formation was apparent.

In addition, the role of Crl in σS proteolysis was also assessed more directly by using in vitro degradation assays (Figure 7, for a more detailed version of this figure, see Supplementary Figure S8). The presence of a two-fold molecular excess of Crl over σS had no effect on the rate of RssB/ClpXP-dependent degradation of σS, that is, σS half-life remained the same (∼15 min) in the absence or presence of Crl (Figure 7A and Supplementary Figure S8A and C). Thus, Crl on its own could not protect σS from being degraded. In addition, Crl itself (with an N-terminal His6 tag) was not a substrate of the ClpXP proteolytic machinery (Figure 7A and Supplementary Figure S8B). However, Crl enhanced the protection provided by core RNAP to σS and further slowed down σS proteolysis about two-fold (Figure 7B and Supplementary Figure S8D). Binding of σS to RNAP polymerase is known to protect σS from degradation (Klauck et al, 2001; Zhou et al, 2001), and even sub-stoichiometric amounts of core RNAP (core RNAP:σS=1:7) were shown here (Figure 7B and Supplementary Figure S8D) to substantially stabilise σS (increasing core RNAP to a ratio of 1:5 slowed down σS proteolysis even more dramatically, leading to a σS half-life of >60 min; data not shown). This stabilisation of σS was further enhanced by the presence of Crl (Figure 7B and Supplementary Figure S8D), in concert with the role of Crl in increasing the formation of EσS, and thereby, protecting σS from degradation. In conclusion, Crl can affect the partitioning of σS between RssB and RNAP in favour of the latter and thus rescue σS from proteolysis, both in vivo and in vitro.

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Figure 7. Crl rescues σS from RssB/ClpXP-mediated degradation in vitro, but only in the presence of core RNAP. In vitro degradation of σS (A, I and III and B, IIII) was assayed in reaction mixtures containing 2 μM σS, 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 σS (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 σs (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 σS 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 σS.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Increased formation of EσS is the basis of the opposing effects of Crl on σS levels and activity

In this study, we demonstrate that Crl is a global regulatory factor in stationary phase, which functions through σS. Crl exerts multiple effects on σS activity and levels, and we present evidence that the common basis of all these effects is the ability of Crl to influence sigma factor competition for core RNAP in favour of σS and thereby facilitate the formation of EσS.

Crl affects σS levels and activity in an opposite manner. This apparently paradoxical behaviour leads to less, but more active σS, when Crl is present. On the one hand, by influencing the partitioning of core RNAP between the competing sigma factors, σ70 and σS, in favour of the latter, Crl positively affects σS-dependent expression of RssB, and thereby stimulates σS proteolysis and reduces σS levels (Figures 4 and 5). On the other hand, by ‘driving’ σS into RNAP, Crl also affects the partitioning of σS between RssB and RNAP in favour of the latter; as a consequence, Crl can protect σS against degradation (as observed both in vivo and in vitro; Figures 6 and 7 and Supplementary Figure S8), thus increasing σS levels. In the wild-type strain, this proteolysis-protective effect is masked by the dominant first effect, that is, Crl stimulating σS proteolysis via RssB. Therefore, in the presence of Crl, overall σS levels are decreased. Taking into consideration that the role of Crl is more profound when σS levels are low (Supplementary Figure S4 and Robbe-Saule et al, 2006), Crl in fact seems to generate the conditions where it gains a significant physiological role. In other words, the opposing effects of Crl on σS levels and activity are both necessary for Crl to be able to fine-tune the σS output. That Crl indeed significantly affects the in vivo output of σS is also supported by the finding that a crl mutation confers a selective advantage in long-term stab cultures of E. coli (Faure et al, 2004).

Crl supports EσS formation and thereby stimulates σS-dependent gene expression in a way that is independent of a specific promoter motif. Why then does it influence only a specific subset of σS-controlled genes in our genome-wide analysis (Table I)? First, many more σS-controlled genes seem to be affected by Crl, but their expression ratios are just below the threshold we have set here (among them also rssB). Second, as Crl functions by directly aiding σS in its competition with σ70 and therefore increasing EσS levels, it would be expected to more strongly influence (i) weak promoters, that is, those with a relatively low affinity for EσS (Grigorova et al, 2006) or (ii) genes the expression of which is σS-controlled at multiple stages, for example, in feedforward loops such as for gadA/BC and csgBA (Weber et al, 2005, 2006). These genes or operons exhibit the strongest regulation by Crl (Table I and Supplementary Figure S4D) and at the same time are extremely sensitive to variations in σS levels and activity (A Typas and R Hengge, unpublished data).

Molecular mechanism of Crl action in competition of σS and σ70 for core RNAP

How exactly does Crl support σS-dependent transcription? Our data indicate that the primary effect of Crl is to significantly bias transcription in favour of EσS under conditions where σS has to compete with the predominant σ70 for binding to limiting amounts of core RNAP (Figure 2). In the presence of σ70, few if any σS-containing RNAP is formed without Crl (Figure 2A and B). In the presence of Crl, however, this disadvantage of σS to compete with σ70 for binding to core RNAP is alleviated, presumably because Crl directly facilitates EσS formation. Other lines of evidence also verify that Crl promotes EσS formation. In the presence of Crl, the EσS holoenzyme is increased at the expense of Eσ70 in stationary phase cells (Figure 3), and Crl can protect σS against proteolysis in vivo and in vitro, but only when it can usher σS to RNAP (Figures 6, 7 and Supplementary Figures S7 and S8).

Consistent with our results, a publication submitted while our study was under review also proposed that Crl facilitates the formation not only of EσS, but also other holoenzymes to a more modest degree (Gaal et al, 2006). Although only preliminary in vitro experiments were presented there (using a system with non-physiologically high amounts of Crl), their data together with our in vitro and in vivo experiments provide strong evidence that the main mechanism of action of Crl on EσS activity is at the initial step of holoenzyme formation. Whether the reported modest effects of Crl on the formation of Eσ32 (Gaal et al, 2006) are physiologically relevant remains to be further tested.

In addition to its major effect on EσS formation under sigma competition conditions, Crl also seems to have a minor positive influence on in vitro transcription mediated by EσS alone (Figure 1), which seems to be identical in the single- and multiround transcription assays (which differ in their ability to sense changes in the initial recruitment of the holoenzyme to the promoter). Thus, these data indicate that Crl does not aid EσS in being recruited by its cognate promoters, but may have some minor effect in steps following EσS recruitment to the promoter (open complex formation, abortive initiation) or in the kinetics of the various steps of transcriptional initiation (which are difficult to detect in in vitro transcription assays). Consistently, during the revision of this paper, Crl was reported to exert a subtle activation in σS-dependent transcription in vitro, mainly due to an increase in open complex formation (Robbe-Saule et al, 2006). The ∼2-fold effects reported in these in vitro transcription assays are comparable to our observations with EσS alone (Figures 1 and 2, about 50% activation in the presence of Crl), if we do not normalise against the RNA I transcript (assuming that Crl enhances the performance of EσS with both our test promoter and the RNA I promoter).

The finding that Crl binds to free σS, but RssB ‘chases’ Crl out of a complex with σS (Supplementary Figure S7B) has interesting implications for the mechanism of action of Crl. First, RssB binding results in a structural change in σS, which exposes an otherwise cryptic binding site for the ClpX hexameric ring (Stüdemann et al, 2003) and in parallel may also reduce affinity for Crl. Alternatively, the binding region on σS for RssB (i.e. region 2.5/3.0) and that for Crl may partially overlap. However, we consider the latter possibility less likely as K173 in region 2.5/3.0 of σS in the RNAP holoenzyme provides an important contact to the promoter (to a C at position –13; Becker and Hengge-Aronis, 2001), which should not be occluded by binding of Crl. In addition, Crl can still aid σS-dependent transcription even with the K173E variant of σS (data not shown), which is defective for RssB binding (Becker et al, 1999). Moreover, taking into consideration that Crl binds only weakly to σS and that it is not completely clear if and how it binds to EσS (see also Figure 3, where most Crl is found to be free and not as part of the EσS complex in vivo), it seems possible that Crl binds only transiently to σS and either imposes a lasting modification or, by acting in a chaperone-like manner, confers a conformational change to σS; both mechanisms could increase the affinity of σS to core RNAP.

The regulation of Crl and its role in cellular physiology

Crl, σS, core RNAP and RssB are components of a complex protein–protein interaction network, whose proper functioning in the control of σS activity and degradation exquisitely depends on the relative affinities and actual cellular levels of all components involved. This requires complex fine-tuning of the regulation of at least Crl, σS and RssB, whose cellular levels have to be adjusted to each other in adequate ratios. For σS and RssB, this is achieved by σS control of the weak rssB transcription, which results in RssB being present at 10- to 20-fold lower levels than σS (Becker et al, 2000). Quantitative immunoblotting indicates that Crl in turn is present in a 5- to 10-fold excess over σS (our unpublished data), and similar to σS, exhibits increased expression during entry into stationary phase and/or at reduced temperature (Supplementary Figure S2). Crl expression, however, does not seem to be σS dependent, as it is not part of the σS regulon under various conditions tested (Weber et al, 2005). Moreover, like σS, Crl was found in the ‘ClpXP-trap’ and therefore may also be regulated by proteolysis (Flynn et al, 2003), although the N-terminally tagged protein used in this study for in vitro experiments was stable (Figure 7A and Supplementary Figure S8B).

Even our current limited knowledge about regulation of Crl raises interesting questions regarding its physiological role. Crl expression patterns (Supplementary Figure S2) and our microarray analysis (Table I) indicate that Crl plays a global role during entry into stationary phase. In addition, its effects are more pronounced when σS levels are relatively low (Supplementary Figure S4). This may result in σS becoming active earlier during entry into stationary phase in the presence of Crl. Does this also mean that Crl could support σS-dependent transcription during exponential phase? Slow but exponential growth on energy-poor carbon sources (e.g. alanine, acetate and proline) causes accumulation of σS and increased σS-dependent gene expression (Liu et al, 2005), suggesting that the role of Crl should be studied under such conditions. Moreover, there may be situations where intracellular σS levels do not significantly change, but increased expression of certain σS-dependent genes may take place owing to the induction of Crl. For example, the presence of external acetate in rich medium (at neutral pH; with low amounts of acetate that do not affect growth) significantly stimulates the σS regulon, but σS levels are not increased in parallel (Kirkpatrick et al, 2001; Polen et al, 2003). In addition, MqsR, a regulator that responds to autoinducer II, strongly and positively regulates crl expression as shown be genome-wide transcription analysis (Gonzalez Barrios et al, 2006), but the impact of this system on σS-dependent gene expression is unknown. Thus, a complete understanding of the physiological role of Crl will also require further studies of its regulation.

The major target process of Crl, that is, the competition between σS and σ70, is the key regulatory process for the transition from exponential to stationary phase. During this transition, the cell reorganises its transcriptional machinery in a way that favours transcription by EσS and other alternative RNAPs, with EσS being the most prominent one. Apart from targeting its own broad regulon, EσS also assumes control of housekeeping functions in stationary phase, for example, basal expression of ribosomal RNAs (Raffaelle et al, 2005). This holoenzyme switch is supported by the strong resemblance of the promoter consensus sequence for the two sigmas (Typas et al, 2007). In addition, numerous genes possess overlapping σS and σ70-specific promoters, in order to secure continuous (but differential) expression during entry into stationary phase. Thus, EσS induces the expression of a plethora of new genes and at the same time takes over the ‘housekeeping’ duties of the cell from Eσ70. Of course, this does not mean that Eσ70 is dispensable or nonfunctional at this stage of growth, as it continues to express many genes important for the cell's nutritional competence. However, its significance is clearly reduced. Proteins like Crl ensure a balanced allocation of duties between the two sigmas in stationary phase and presumably also under other long-term stress conditions. Maintaining this balance is vital to the cell as it allows it to adjust its trade-off between self-preservation and nutritional competence according to the external milieu (King et al, 2004; Ferenci, 2005).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Owing to space constraints and the multitude of methods used in this study, the detailed description of strains and experimental procedures has been moved to Supplementary data.

Supplementary data

Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Janine Kirstein for significant help with the gel filtration experiments, Eberhard Klauck and Nicole Lange from the Hengge group for help with in vitro degradation experiments and B Bukau for the pUHis-ClpX plasmid. Financial support for this study was provided by the Deutsche Forschungsgemeinschaft (He 1556/12-1) and the Fonds der Chemischen Industrie.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supplementary data

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