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 crl∷cat 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the control of σS only at lower temperatures (denoted by an asterisk in parentheses in Table I; H Weber and R Hengge, unpublished microarray results).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 name||ID||Average of ratio of medians||Member of the RpoS regulon||Function|
|Crl||b0240||139.7|| ||Regulatory protein for curli, transcriptional regulator|
|psiF||b0384||2.2||*||Pho regulon member, requiring PhoRB system|
|allR||b0506||2.2||(*)||AllR transcriptional regulator|
|ybgS||b0753||2.5||*||Putative homeobox protein|
|ycaC||b0897||2.4||*||Putative cysteine hydrolase|
|csgB||b1041||2.9||(*)||Curlin, minor subunit precursor|
|csgA||b1042||3.5||(*)||Curlin, major subunit|
|csgC||b1043||2.1||(*)||Putative curli production protein|
|ymdA||b1044||2.0||(*)||Conserved hypothetical protein|
|narU||b1469||2.3||*||MFS nitrite transporter|
|gadC||b1492||3.1||*||Putative glutamate:gamma-aminobutyric acid antiporter (APC family)|
|gadB||b1493||3.6||*||Glutamate decarboxylase B subunit|
|yeaH||b1784||2.4||*||Conserved hypothetical protein|
|ybeV||b1836||2.4||*||Stimulates the ATPase activity of Hsc62, possibly component of a new Hsp70 chaperone system|
|luxS||b2687||2.5||*||Quorum sensing, autoinducer II synthase|
|tdcC||b3116||2.3||(*)||TdcC threonine STP transporter|
|hdeB||b3509||2.3||*||10K-L protein, related to acid resistance protein of Shigella flexneri|
|hdeA||b3510||2.3||*||Acid-resistance protein, possible chaperone|
|hdeD||b3511||2.4||*||Protein involved in acid resistance|
|gadE||b3512||3.2||*||Transcriptional regulator, activates glutamate decarhboxylase-dependent acid resistance|
|gadW||b3515||2.4||*||Transcriptional regulator (AraC/XylS family)|
|gadA||b3517||3.6||*||Glutamate decarboxylase A subunit|
|yiaG||b3555||2.8||*||Putative transcriptional regulator|
|yjbJ||b4045||2.5||(*)||Highly abundant nonessential protein|
| || || || || |
|ydhY||b1674||0.4|| ||Putative oxidoreductase, Fe-S subunit|
|iadA||b4328||0.5|| ||Subunit ofisoaspartyl dipeptidase|
|yjiG||b4329||0.5|| ||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).
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.
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.
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).
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.
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 σS/σ70 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).
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.
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, I–III) 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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