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Originally published In Press as doi:10.1074/jbc.M314145200 on February 21, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19540-19550, May 7, 2004
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Crl, a Low Temperature-induced Protein in Escherichia coli That Binds Directly to the Stationary Phase {sigma} Subunit of RNA Polymerase*

Alexandre Bougdour, Cécile Lelong{ddagger}, and Johannes Geiselmann

From the Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier, CNRS UMR 5163, F-38041 Grenoble Cedex 9, France

Received for publication, December 24, 2003 , and in revised form, February 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The alternative sigma factor {sigma}S (RpoS) of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress-response genes. {sigma}S is also required for the transcription of the cryptic genes csgBA that encode the subunits of the curli proteins. The expression of the csgBA genes is regulated in response to a multitude of physiological signals. In stationary phase, these genes are transcribed by the {sigma}S factor, and expression of the operon is enhanced by the small protein Crl. It has been shown that Crl stimulates the activity of {sigma}S, leading to an increased transcription rate of a subset of genes of the rpoS regulon in stationary phase. However, the underlying molecular mechanism has remained elusive. We show here that Crl interacts directly with {sigma}S and that this interaction promotes binding of the {sigma}S holoenzyme (E{sigma}S) to the csgBA promoter. Expression of Crl is increased during the transition from growing to stationary phase. Crl accumulates in stationary phase cells at low temperature (30 °C) but not at 37 °C. We therefore propose that Crl is a second thermosensor, besides DsrA, controlling {sigma}S activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA polymerase of Escherichia coli is composed of a core enzyme (E) with subunit structure {alpha}2{beta}{beta}' which associates with one of seven different {sigma} subunits to form the holoenzyme (E{sigma}). The core enzyme carries the RNA polymerization function, and the {sigma} subunit is required for promoter recognition and binding (1, 2). Each {sigma} subunit targets RNA polymerase to a different set of promoters, thereby profoundly modulating the gene expression pattern (3, 4). RNA polymerase holoenzyme containing the {sigma}70 subunit is responsible for the transcription of the majority of genes during exponential growth. Upon entry into stationary phase, {sigma}38 also called {sigma}S and encoded by the rpoS gene, begins to accumulate in the cell, associates with the core enzyme, and directs the transcription of genes essential for stationary phase survival (5–7). The synthesis of {sigma}S is also induced in response to many other stress conditions such as high osmolarity, low pH, and high temperature. {sigma}S is therefore considered a general stress-response regulator (8–10).

Regulation of {sigma}S occurs at transcriptional and post-transcriptional levels and involves numerous regulators. In brief, {sigma}S accumulates at the beginning of stationary phase because of many factors acting in concert: increased transcription of the rpoS gene, stabilization and activation of the rpoS mRNA, as well as increased protein stability (8). A number of small molecules, RNAs, and proteins belong to the complex regulatory network that controls {sigma}S expression; ppGpp (the stringent control signal) (11), homoserine lactone (12), inorganic phosphate (13), UDP-glucose (14), and cAMP-CRP (8) have been reported to influence the transcription of rpoS. The nucleoid-associated protein HU, the histone-like protein H-NS, and the small regulatory RNAs DsrA and OxyS in conjugation with the RNA chaperone Hfq control the translation of rpoS by modulating the secondary structure of the rpoS mRNA (15, 16). {sigma}S proteolysis is controlled by the response regulator RssB (the {sigma}S recognition factor) and the ClpXP protease (recently reviewed in Ref. 17).

Despite this abundance of information about the regulation of {sigma}S expression, little is known about the regulation of {sigma}S activity. The total concentration of {sigma}S does not exceed one-third of the concentration of {sigma}70 even at the onset of stationary phase when {sigma}S concentration is at its highest (18, 19). Moreover, among all {sigma} factors, {sigma}S has the lowest affinity for the core enzyme (20). {sigma}S thus appears to be in a difficult position in the competition for binding to core enzyme. Recently, it has been shown that ppGpp modulates this competition in favor of {sigma}S (21). Other factors, such as glutamate, trehalose, and inorganic polyphosphate, modulate the activity of {sigma}S holoenzyme at the steps of holoenzyme formation and/or holoenzyme binding to promoters (22–24). In addition, recent studies have identified the crl gene product as a regulator of {sigma}S activity (25, 26).

Crl is known for stimulating the transcription of csgBA, the operon encoding for the two curli subunits, in a {sigma}S-dependent manner (27–29). In a crl null strain, the transcriptional activity of the csgBA promoter is reduced ~4-fold compared with the wild type strain (25). Curli fibers are thin aggregative surface fimbriae, which are involved in cell-cell attachment (30, 31) and adhesion to extracellular matrices (32–34). Curli are expressed under special environmental conditions, such as low temperature, low osmolarity, and in stationary phase (32, 35). Control of curli production in Escherichia coli and Salmonella typhimurium involves a complex network of regulatory proteins. Global regulators such as H-NS (27, 28), OmpR (30), and IHF (36) control the expression of the curli. Osmolarity sensing occurs through the two-component regulatory systems EnvZ/OmpR and CpxA/CpxR (37, 38), but temperature-sensing of curli expression is not understood.

In the present study we have investigated, at the molecular and the physiological levels, the mechanism by which Crl influences {sigma}S activity. We report that Crl interacts directly with the {sigma}S subunit in vitro. Electrophoretic mobility shift assays (EMSA)1 indicate that Crl associates with the transcriptional complex formed by the E{sigma}S ({sigma}S holoenzyme) and the csgBA promoter (pcsgBA). Our work provides evidence that Crl positively controls E{sigma}S recruitment to pcsgBA. Immunoblot analysis of Crl demonstrates that Crl expression is growth phase- and temperature-dependent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Plasmids—The genotypes of E. coli K12 strains and plasmids used in this study are listed in Table I. Media used were Luria-Bertani broth with 5 g/liter NaCl (LB) and LB containing 12 g/liter agar (LA) (39). Media were supplemented with 15 µg/ml chloramphenicol (Cm), 25 µg/ml kanamycin (Kn), 100 µg/ml ampicillin, 40 µg/ml 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal), or 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside as required. Strains were grown at 37 or 30 °C with shaking (200 rpm).


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  		TABLE I
Bacterial strains and plasmids

 
The crl mutation was transferred from E. coli LP895 (25) to W3110 using P1(vir)-mediated transduction (40). Transduction of the crl::Cmr allele was obtained by selection on Cm plates (41). The rpoS mutation was transferred from the strain ZK1000 (42) to W3110. Transduction of the rpoS::Knr allele was obtained by selection on Kn plates. We verified by Southern blot that the mutations were introduced at the correct locus. All clones were stored at –80 °C in a 20% glycerol solution.

Overproduction and Purification of His6-{sigma}s and His6-Crl Proteins— The coding sequence of the rpoS gene was cloned on a His6 tag PQE-30 vector (Qiagen), yielding the pHis6-{sigma}S plasmid as described (43). To produce the His6-Crl fusion protein, we PCR-amplified the coding region of the crl gene using chromosomal DNA from the W3110 strain as template and the primers C5 (5'-ACGTTACCGAGTGGACAC-3') and C6 (5'-CGCCGTTAACTTCACCGG-3'). The PCR fragment was cloned into pQE-30 UA vector (Qiagen), yielding the pHis6-Crl plasmid. The E. coli strains FI1202 and M15[pREP4], transformed with pHis6-{sigma}S and pHis6-Crl, respectively, were grown in LB medium containing selective antibiotics until an A600 of 0.6. We then added isopropyl-{beta}-D-thiogalactopyranoside to 1 mM. After 5–6 h of induction, cells were harvested and stored at –80 °C until use. Cell lysates were prepared under native conditions. Cell pellets were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) before sonication. The lysates were centrifuged at 10,000 x g for 30 min at 4 °C, and the soluble fractions were applied to a Ni2+-NTA column (Qiagen) equilibrated with lysis buffer. The column was washed with 10x column volumes of buffer A (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), followed by elution with buffer E (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). In control experiments we used a similar protocol except that the sample was incubated with either 600 units of micrococcal nuclease or 300 µg/ml ethidium bromide for 30 min at room temperature before the washes.

When needed, the eluates were purified by gel filtration using an Amersham Biosciences PD-10 desalting column equilibrated with storage buffer (50 mM Tris-HCl, pH 7.5, EDTA 0.1 mM, 150 mM KCl, 5 mM CaCl2, 5 mM MgCl2, 0.1 mM DTT). Glycerol was added to a final concentration of 10% (v/v), and the samples were stored at –20 °C until use. His6-{sigma}70 was a generous gift of A. Kolb. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad) and bovine serum albumin as a standard.

Electrophoresis and Immunoblot Analysis of Proteins—Crude cell extracts under native conditions were prepared as follows. Cells were grown in LB medium at 30 or 37 °C for the time indicated. Cell pellets were resuspended in buffer B (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT, 10% glycerol) before sonication. The lysates were centrifuged at 10,000 x g for 30 min at 4 °C, and the soluble fractions were stored at –80 °C. The protein concentrations were determined using the Bradford protein assay kit (Bio-Rad) and bovine serum albumin as a standard. Protein samples were analyzed by SDS-PAGE (39). Amersham Biosciences prestained protein standards were used for molecular weight estimation.

Antibodies against Crl were produced in rabbits by injecting highly purified His6-Crl protein (Eurogentec). Antibodies against {sigma}S were a gift from R. Hengge-Aronis (8). No cross-reaction was observed for the antibodies used in this study.

The immunoblot analysis of proteins electrotransferred (Bio-Rad system) onto nitrocellulose or polyvinylidene difluoride membranes (Amersham Biosciences) was performed with polyclonal antibodies raised against Crl and {sigma}S as described previously. The blots were developed either with ECL (Amersham Biosciences) or nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma) systems, and staining intensity was quantified with the ImageGauge (Fujifilm) software.

Gel Filtration—Gel filtration was performed using the AKTA system (Amersham Biosciences). Samples of 500 µl containing 167 µg of either His6-Crl (13.5 nmol) or His6-{sigma}S (5 nmol) or both proteins in a 20 mM Tris-HCl, pH 8, buffer (containing EDTA 0.2 mM, 500 mM KCl, 0.5 mM DTT, 10% glycerol, and 0.1% Nonidet P-40) were incubated for 20 min at room temperature. The mixture was loaded onto a Superdex 200 HR 10/30 column (Amersham Biosciences) equilibrated with the same buffer. Filtration was performed at 4 °C at a flow rate of 0.250 ml/min. Fifty fractions of 500 µl each were collected and analyzed by immunoblot, as indicated previously, to determine the protein content of each fraction. The column was calibrated using molecular weight standards from Amersham Biosciences.

EMSA—The E. coli RNA polymerase core enzyme was purchased from Epicenter Technologies. DNA for binding assay was generated by PCR from pTOPO-pcsgBA plasmid and by using the primers C1 (5'-ATACTTTGGTATGAACTAAAAAAGAA-3') and C2 (5'-CTGGTCGTACATTTAAGAAATT-3'). The PCR product (158 bp) from –81 to +77 of csgBA promoter region (named pcsgBA) was purified (Qiagen) and labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Binding assays with purified proteins were conducted in 20-µl reaction mixtures containing 20 mM HEPES, pH 7.4, 50 mM KCl, 1 mM DTT, 10% glycerol, 3 mM MgCl2, 0,1 µg/µl bovine serum albumin, 6 ng/µl herring sperm DNA as competitor, 1 nM labeled DNA, 20 nM RNA polymerase core enzyme, 60 nM His6-{sigma}S protein or 60 nM His6-{sigma}70, and 120 nM His6-Crl protein. The purified proteins were diluted into the reaction buffer (see above) before use. The {sigma} subunits were pre-incubated with His6-Crl for 10 min at room temperature before reconstitution of holoenzymes (see below). The core enzyme and the {sigma} subunits (with or without His6-Crl) were mixed at a ratio of 1:3 in reaction buffer and incubated at 30 °C for 15 min. Binding of E{sigma}S or E{sigma}70 to the csgBA promoter region was allowed to proceed for 20 min at 30 °C in order to allow open complex formation. The reaction mixtures were loaded on a 5% non-denaturing polyacrylamide gel and then run with HEPES buffer (50 mM HEPES, pH 7.4) at 3 watts for 30–45 min at room temperature. Binding assays with crude extracts (1 µg/µl) were conducted in 20-µl reaction mixtures containing 20 mM HEPES, pH 7.4, 50 mM KCl, 1 mM DTT, 10% glycerol, 1 mM EDTA, 10 nM labeled DNA, and 6 ng/µl herring sperm DNA as competitor. Binding was allowed to proceed for 15 min at room temperature. The reaction mixture was loaded on a 4–10% non-denaturing polyacrylamide gradient gel and then run with TBE-1x buffer at 2 watts for 72 min with a cooling system. Gels were dried before being scanned and quantified using a FLA-8000 PhosphorImager (Fujifilm). Intensity profiles of the retarded bands were fitted to a Lorentzian curve shape as shown in Equation 1,

(Eq. 1)
in order to separate and quantify accurately the overlapping peaks.

Footprinting Experiment—A 0.175-kb fragment containing the csgBA promoter was produced by PCR using primers P1(5'-ATCGGTCGACCTTTGGTATGAACTAAAAAAGAA-3') and P2(5'-CGATCTCGAGCTGGTCGTACATTTAAGAAATT-3') containing SalI and XhoI restriction sites, respectively. The 175-bp DNA products were labeled with [{gamma}-32P]ATP, using T4 polynucleotide kinase prior to being digested with either SalI or XhoI in order to produce a DNA fragment with only one labeled end. The reaction mixture (50 µl) containing end-labeled DNA (1 nM) and reconstituted RNA polymerase (20 nM core enzyme and 60 nM His6-{sigma}S), with or without His6-Crl (20–200 nM) in reaction buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 10% glycerol, 3 mM MgCl2, 0,1 µg/µl bovine serum albumin), was incubated for 30 min at room temperature. The {sigma}S subunit was pre-incubated with His6-Crl for 10 min at room temperature before reconstitution of the holoenzyme. DNase I (3 ng) was added, and the mixture was incubated for 5 min at 22 °C. The reaction was stopped by adding 25 µl of stop solution (4 M ammonium acetate and 0,2 µg/ml herring sperm DNA). DNA was precipitated from the reaction mixture with LiCl and ethanol. The reaction products were analyzed by electrophoresis in a 8% polyacrylamide sequencing gel containing 8 M urea. Gels were dried before being autoradiographed and finally quantified using a FLA-8000 PhosphorImager (Fujifilm). Unmodified bands were used for normalization of the results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Crl interacts directly with the {sigma}S subunit—The crl gene product stimulates the expression of curli fimbriae (44), which are involved in cell-cell aggregation and adhesion to extracellular matrices (32). Transcription of csgBA, coding for the two subunits of the curli, is activated by Crl in a {sigma}S-dependent way (27, 44). Furthermore, Crl has a stimulatory effect on the expression of many other {sigma}S-activated genes (25), and it participates in the negative effects of {sigma}S on the expression of ompF (45) and the bgl operon (26). Crl therefore either (i) increases {sigma}S expression or (ii) somehow stimulates {sigma}S activity. We eliminated the first possibility because neither the expression of a transcriptional rpoS::lacZ fusion (data not shown) nor the {sigma}S levels are reduced in a crl mutant strain (Fig. 6, A and B, lanes 1 and 2). These data agree with the results published by Pratt and Silhavy (25), who observed a greater amounts of {sigma}S in a crl null strain than in wild type cells.



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  		FIG. 6.
Expression of {sigma}S is not up-regulated by Crl. Samples were separated by 15% SDS-PAGE and immunoblotted with polyclonal anti-{sigma}S antibodies. Each lane contains 20 µg of total proteins. The W3110 (lane 1), BL001 (lane 2), BL002 (lane 3), BL003 (lane 4), W3110/pTOPO-crl (lane 5), W3110/pDEB2 (lane 6), BL002/pTOPO-crl (lane 7), and BL001/pDEB2 (lane 8) were grown in LB medium for 24 h at 30 °C (A) and 37 °C (B).

 
The most straightforward way to investigate the second possibility would be to test the effect of Crl on {sigma}S-dependent transcription in vitro. Unfortunately, Crl did not significantly modify the activity of {sigma}S as measured by this assay (data not shown). We therefore used a less direct approach to investigate this second hypothesis. We used His6-tagged protein fusions and Ni-NTA-agarose as an affinity matrix for His6-Crl in an in vitro binding assay. This method was chosen in order to get rid of possible aggregation of His6-Crl proteins when purified. Aggregation is prevented by immobilizing the protein on the gel matrix, thus avoiding excessively high local concentrations (46). Considering that the carboxyl-terminal end of Crl is responsible for its ability to stimulate csgBA transcription (44), we decided to add the His6 tag to the amino-terminal end of Crl. For {sigma}S, the His6 derivative has already been constructed by Becker et al. (43). The His6-Crl and His6-{sigma}S fusion proteins were purified, using Ni2+-NTA technology, from crude extracts of E. coli strains M15 and FI1202, respectively. The major His6 protein fusion peaks were recovered by elution of the native proteins with 250 mM imidazole (Fig. 1A). As expected, His6-Crl (Mr, 15,5137) migrates with a relative mobility of ~15 kDa in an SDS-PAGE gel, as does wild type Crl (data not shown). Despite the presence of minor contaminating proteins, His6-Crl and His6-{sigma}S represented about 95% of the eluted protein, as judged by Fig. 1A. The yield of His6 fusion proteins was 0.2 mg from 100 ml of induced culture.



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  		FIG. 1.
In vitro interaction of Crl with {sigma}S. A, Coomassie Blue-stained SDS-PAGE gel. Cells of E. coli K12 containing pHis6-Crl (lane 1) or pHis6-{sigma}S (lane 2) were grown at 30 °C into stationary phase (5–6 h after isopropyl-1-thio-{beta}-D-galactopyranoside was added). Cell lysates were prepared as described under "Materials and Methods." His6 fusion proteins were isolated and separated on a 12% acrylamide gel (His6-Crl and His6-{sigma}S are indicated on the right). B, immunodetection of {sigma}S in the fraction eluted with 250 mM imidazole from the Ni2+-NTA-agarose columns loaded with crude extracts containing His6-Crl and endogenous {sigma}S. Elution with buffer A and 250 mM imidazole (lane 1); wash with buffer A and 20 mM imidazole before elution (lane 2); elution with 250 mM imidazole from the Ni2+-NTA-agarose matrix containing only {sigma}S (lane 3). C, immunodetection of {sigma}S in the fraction eluted with 250 mM imidazole from the Ni2+-NTA-agarose matrix containing {sigma}S and His6-Crl in the presence of either micrococcal nuclease or ethidium bromide. Wash with buffer A and 20 mM imidazole before elution (lanes 1, 3, and 5); elution with buffer A and 250 mM imidazole without treatment (lane 2), after treatment with micrococcal nuclease (lane 4), after treatment with ethidium bromide (lane 6).

 
Immunoblot analysis of the eluted fraction containing His6-Crl with anti-{sigma}S polyclonal antibodies allowed us to demonstrate that {sigma}S coeluted with His6-Crl from the Ni2+ column (Fig. 1B). The interaction specificity was verified using salt gradients (data not shown). As a control, a cleared extract from the host strain that does not express the fusion protein was applied to the Ni2+-NTA-agarose matrix. Immunoblot analysis of the eluted fraction with imidazole did not revealed the presence of proteins, thus suggesting that the interaction between Crl and {sigma}S is specific. To establish that the association of these two proteins was not mediated through DNA, we repeated the purification protocol in the presence of either ethidium bromide (300 µg/ml) or micrococcal nuclease (600 units) as described (47, 48). The association of {sigma}S with His6-Crl was unaffected by either treatment (Fig. 1C).

In order to delineate which part of {sigma}S is responsible for the interaction with Crl, we repeated the His6-Crl purification in strain MNC10, which expresses relatively high amounts of a {sigma}S mutant called {sigma}S({Delta}7–35) (49). The smaller protein {sigma}S({Delta}7–35) still co-purified with His6-Crl, thus indicating that the amino-terminal region of {sigma}S is not essential for Crl binding in vitro (data not shown).

The inverse protocol of using His6-{sigma}S to co-purify Crl was not possible because Crl appeared to bind non-specifically to the column. Therefore, the interaction between Crl and {sigma}S was further assessed using gel filtration. Purified proteins were applied to a Superdex 200 HR 10/30 column (Amersham Biosciences), and column samples were collected and analyzed by immunoblotting. Purified His6-{sigma}S alone, a 38-kDa protein, eluted earlier than expected (mainly in fractions 31–34), at the position predicted for a globular protein of ~48 kDa (Fig. 2). This result is consistent with a previous analysis of this protein using the same method (50). Crl, a 15-kDa protein, eluted at the position expected (mainly in fraction 36). When His6-{sigma}S and His6-Crl were incubated together and analyzed by gel filtration, the two proteins coeluted at a position predicted for a globular protein of ~85 kDa (fractions 28–30). Part of His6-Crl still eluted at the position of the uncomplexed protein. This can be attributed to the fact that an excess of His6-Crl was used in the experiment (His6-Crl/His6-{sigma}S ratio of ~3:1). This result demonstrates a direct interaction between {sigma}S and Crl in solution.



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  		FIG. 2.
Gel filtration analysis of the His6-Crl-His6-{sigma}S interactions. Proteins were preincubated and injected on a Superdex 200 column as described under "Materials and Methods." The composition of the mixture injected on the column is indicated on the left. An ~3-fold molar excess of His6-Crl over His6-{sigma}S was used when the two proteins were loaded together. Five µl of the column input was loaded in the leftmost lane of a 15% acrylamide gel. Eleven µl of the different fractions were loaded in the subsequent lanes. Immunodetection of His6-Crl and His6-{sigma}S is indicated on the right. The elution standards of molecular weights are indicated at the bottom.

 
Crl Forms a Complex with the E{sigma}s Holoenzyme—In order to investigate the mechanism by which Crl stimulates the activity of {sigma}S, we measured the interaction of Crl with a transcriptional initiation complex. We incubated a 158-bp DNA fragment encompassing the csgBA promoter region, extending from positions –81 to +77 with respect to the transcription start site, with purified proteins and analyzed the complex on native polyacrylamide gels (Fig. 3). The extent of binding in each lane was determined by PhosphorImager quantification and normalized to the unbound probe. The lower band (lanes 1 and 2, band c) in the absence of {sigma} subunits represents core polymerase binding non-specifically to the probe. The addition of the {sigma}S subunit leads to the appearance of the upper band corresponding to the binding of E{sigma}S to pcsgBA (lane 3, band b). Addition of an excess of His6-Crl to the reaction mixtures does not influence the binding efficiency of E{sigma}S to the csgBA promoter. Even under these conditions, the intensity of the retarded band remains the same (compare lanes 3 and 4). Nevertheless, the presence of Crl leads to a slight shift of the position of the band (lane 4, band a). It is very likely that Crl is fixed to the transcriptional complex and retards the migration of the complex. No shifted band was observed when His6-Crl was incubated with free His6-{sigma}S (not associated with E) and the radiolabeled DNA fragment (data not shown). As a control, we used the {sigma}70 subunit instead of {sigma}S in the same experiment. No differences in the migration were detected in the presence of Crl, neither with core RNA polymerase (E), nor with E{sigma}70 (bands c and d, respectively). The slight shift observed with E{sigma}S is therefore indicative of a specific interaction between Crl and E{sigma}S on the csgBA promoter region. These data substantiate our hypothesis of a direct interaction between Crl and {sigma}S and show that Crl can be part of the transcriptional complex containing {sigma}S.



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  		FIG. 3.
Crl binds to E{sigma}S at the csgBA promoter. One nM of the radiolabeled csgBA promoter region was run on a 5% polyacrylamide gel alone (lane 0) or after incubation with 120 nM of purified His6-Crl (lane 7); with 20 nM of core RNA polymerase in the absence and presence of His6-Crl (lanes 1 and 2, respectively); with E{sigma}S in the absence and presence of His6-Crl (lanes 3 and 4, respectively). As a control, E{sigma}70 was incubated in the absence and presence of His6-Crl (lanes 5 and 6, respectively). Sixty nM of His6-{sigma}S or His6-{sigma}70 were used for the reconstitution of holoenzymes. Band a corresponds to the His6-Crl-E{sigma}S-csgBA promoter complex; band b corresponds to the E{sigma}S-csgBA promoter complex; band c corresponds to the nonspecific binding of E to the csgBA promoter; band d corresponds to E{sigma}70-csgBA promoter complex; and band e corresponds to the free csgBA promoter probe.

 
Crl Modifies the E{sigma}S DNase I Footprint at the csgBA Promoter—Because Crl can bind to the E{sigma}S-pcsgBA complex, it is tempting to speculate that Crl somehow modifies the interaction of the transcriptional complex with the promoter. To investigate this possibility, we performed DNase I footprinting in the presence of Crl, on both template and non-template strands of the csgBA promoter. As shown in Fig. 4A, when the template strand was labeled, E{sigma}S protected a large region extending from the –70.5 to +42.5. When the non-template strand was labeled, E{sigma}S protected a smaller region extending from –33.5 to +61. Protection of the –10 region was detected in both the upper and the lower strands. Surprisingly, after a 10-min incubation of His6-Crl (20–200 nM) with the {sigma}S subunit prior to the reconstitution of holoenzyme, no additional protected regions could be detected. However, in the presence of higher concentrations of His6-Crl, several sites in both coding and template strands showed enhanced cleavage. His6-Crl modified the footprint at positions –32, –11, and +25 on the template strand and –37, –12, +26, and +36 on the non-template strand (Fig. 4, A and B). Quantification of the bands on the gel confirms that Crl modifies the pattern of the E{sigma}S footprint on the csgBA promoter without enlarging the protected region.



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  		FIG. 4.
DNase I footprinting analysis of E{sigma}S holoenzyme and His6-Crl binding to the csgBA promoter region. A, the reactions were carried out as described under "Materials and Methods" using 20 nM core enzyme, 60 nM His6-{sigma}S, and 1 nM DNA. The promoter fragment was labeled at the 5' end of the template strand (left panel) or at the 5' end of the non template (right panel). With DNA alone (lane 0), with reconstituted E{sigma}S alone (lane 1), and with E{sigma}S in the presence of increasing concentrations of His6-Crl: 20 nM (lane 2), 60 nM (lane 3), 120 nM (lane 4) and 200 nM (lane 5) of purified His6-Crl protein. The sequence of csgBA promoter region is shown in B. Broken arrows indicate the location and orientation of the transcription start site. The –10 and –35 regions are indicated with boxes in A, and are underlined in B. The areas protected from DNase I digestion are indicated by [and] and bases that react more strongly in the presence of His6-Crl are indicated by asterisks.

 
Crl Promotes the Recruitment of E{sigma}S to the Promoter—As described above, purified His6-Crl modulates the conformation of the open complex but does not seem to increase the recruitment of E{sigma}S to the promoter. However, some factor(s) necessary for transcriptional activation by Crl may be missing in the purified system. We therefore repeated the EMSA experiments with crude extracts from E. coli cells (Fig. 5A). Given that curli proteins are expressed only under certain conditions and that their transcription is activated by Crl at growth temperatures below 30 °C (32, 44), the cells were grown to stationary phase in liquid LB medium at 30 °C. A W3110 strain was used because it carries the rpoS gene in its intact form (51). The experiments were conducted at room temperature with 20 µg of total protein in each reaction. In these conditions, only two retarded bands are observed (bands b and c). In order to identify the proteins responsible for the retarded bands, the experiment was done with extracts from strains carrying different mutations. As shown in Fig. 5A, two retarded bands (named b and c) were observed with the wild type strain. Because band b is no longer observed in the absence of {sigma}S (Fig. 5A, lanes 3 and 4), we assign it to the binding of E{sigma}S to the promoter (E{sigma}S-pcsgBA complex). This assignment was confirmed by the immunodetection of {sigma}S in this band b (data not shown). Band c then corresponds to the E{sigma}70-promoter complex (E{sigma}70-pcsgBA), because the difference in migration corresponds exactly to the one observed previously (3).



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  		FIG. 5.
Crl promotes the recruitment of E{sigma}S to the csgBA promoter. 10 nM of the radiolabeled csgBA promoter region was run on 4–10% gradient polyacrylamide gels, alone (lane 0) or after incubation with crude extracts (20 µg of proteins) from the indicated E. coli strains grown for 24 h at 30 °C (A) or 37 °C (B): W3110 (wt; lane 1); BL001 (crl–; lane 2); BL002 (rpoS–; lane 3); BL003 (crl–, rpoS–; lane 4); W3110/pTOPO-crl (crl+++; lane 5); W3110/pDEB2 (rpoS+++; lane 6); BL002/pTOPO-crl (crl+++, rpoS–; lane 7); BL001/pDEB2 (crl–, rpoS+++; lane 8). Profiles of the intensity of the retarded bands b and c from A with the strains wt (C), crl– (D), and crl+++ (E). The total signal (solid line), the E{sigma}70 (dotted line), and the E{sigma}S (dashed line) signals are shown. F, EMSA performed as before with cell crude extracts from the indicated E. coli strains grown for 24 h at 30 °C: W3110 (wt; lane 1); BL001 (crl–; lane 2); BL001 (crl–) with 1.7, 3.5, and 7 µM of His6-Crl (lanes 3–5, respectively); BL003 (crl–, rpoS–) with 7 µM of His6-Crl (lane 4). When His6-Crl was used, the purified protein was incubated for 15 min at 15 °C before 10 nM of a radiolabeled pcsgBA DNA fragment was added to the reaction.

 
The extent of E{sigma}S binding in each lane was determined by PhosphorImager quantification and normalized to E{sigma}70 binding. The data obtained were further quantitated by fitting the lane profile to two Lorentzian peaks in order to deconvolute the overlapping signals (Fig. 5, C–E). With the crl null strain BL001 (Fig. 5A, lane 2), the intensity of band b is decreased ~3-fold in comparison to wild type cells (Fig. 5, C and D). In the same way, with the strain that overexpresses Crl (Fig. 5A, lane 5), the intensity of band b is more than 3-fold higher than with the wild type strain, whereas the signal corresponding to E{sigma}70 (band c) remains constant in all strains used (Fig. 5, A, C, and E). These results indicate that Crl specifically modulates E{sigma}S binding to the promoter but does not affect binding of E{sigma}70. As a control, we verified that neither band b nor band c are affected in the wild type strain carrying the vector without insert (data not shown).

Given that the intracellular concentration of {sigma}S is a critical parameter of its activity (3, 52), we also tested whether an overexpression of {sigma}S can affect the level of E{sigma}S bound to the promoter. With the strain that overexpresses {sigma}S (Fig. 5A, lane 6), the retarded band b is ~3-fold more intense than with the wild type cells, whereas with the strain overexpressing {sigma}S in a crl null background (Fig. 5A, lane 8), the band b signal is diminished by ~2-fold relative to the wild type strain. These results clearly show that in a crude cell extract, Crl enhances the binding of E{sigma}S to the csgBA promoter. In addition, the position of the E{sigma}S-pcsgBA band shifts from position 3.51 to 2.76 mm when Crl is absent or overexpressed, respectively (Fig. 5, D and E). With the wild type strain, the band b is at an intermediate position (3.21 mm), suggesting that the E{sigma}S-pcsgBA molecules were not all complexed with Crl (Fig. 5C). Position and the amplitude of the E{sigma}70-pcsgBA peak remain virtually unchanged in the different strains used, confirming that the effect of Crl is specific for E{sigma}S.

We also tested the ability of the purified protein His6-Crl to modulate the E{sigma}S + pcsgBA {leftrightarrow} E{sigma}S-pcsgBA equilibrium. An increasing amount of His6-Crl was added to the crude extract prepared from the crl null strain grown at 30 °C for 24 h. Fig. 5F shows that the exogenous His6-Crl restored the signal corresponding to E{sigma}S-pcsgBA at least to wild type level, without modifying the E{sigma}70-pcsgBA signal. As a control, a crude extract from the double mutant strain BL003 (rpoS– and crl–) was used with a large amount of His6-Crl (Fig. 5F, lane 6). The E{sigma}S-pcsgBA signal was not observed, showing that the traces of {sigma}S present in the eluted fraction of His6-Crl were not sufficient to allow E{sigma}S binding. Taken together, these results clearly suggest that Crl promotes the recruitment of E{sigma}S to the curli promoter. Fig. 5, A and F, shows that increasing Crl expression or increasing amounts of purified His6-Crl added to the experiments lead to greater retardation of band b (migrating at a higher position) and the formation of larger quantities of E{sigma}S-pcsgBA complexes (see Fig. 5, C—E, for quantification). Because the E{sigma}70-pcsgBA complex is unaffected by Crl, this reveals once more that Crl binds specifically to the E{sigma}S-pcsgBA complex.

It has been reported that Crl down-regulates the level of {sigma}S protein in the E. coli MC4100 strain probably due to a feedback regulation of {sigma}S on its own expression (25). We therefore measured the concentration of {sigma}S by immunoblot analysis. Fig. 6 shows the amount of {sigma}S in the different strains used in the previous experiment. In agreement with published data, the level of {sigma}S is slightly increased in the crl null strain (compare the lanes 1 and 2). The effect of the crl null allele is more dramatic at 37 than at 30 °C (compare Fig. 6 A and B). Furthermore, the crl null strain that overexpresses {sigma}S shows a higher level of this protein than with the wild type allele of crl (compare lanes 6 and 8). In addition, we observed a slight decrease in the amount of {sigma}S in a strain overexpressing Crl compared with the wild type cells (compare lanes 1 and 5). Collectively, these results further strengthen our conclusion: an even greater amount of {sigma}S in crl null strains is less active than the lower amount of {sigma}S present in bacteria expressing crl. Crl clearly enhances the activity of {sigma}S at the csgBA promoter, further strengthening the conclusion of Pratt and Silhavy (25).

Crl Is Induced at Low Temperature and in Stationary Phase—Because the transcription of csgBA is activated by Crl at low temperature but not at 37 °C (44), it has been hypothesized that the crl gene itself might be thermoregulated. To test this, we measured the intracellular concentration of Crl using a quantitative immunoblot. Fig. 7A shows Crl levels in different strains at 30 and at 37 °C. At 37 °C, Crl is hardly detectable, except in the strain with a high copy number of the crl gene, indicating that Crl is actually overexpressed. On the other hand, at 30 °C Crl is detected in the wild type and rpoS null strains, showing that more chromosomal Crl is produced at 30 °C than at 37 °C regardless of the presence of {sigma}S.



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  		FIG. 7.
Temperature and growth phase dependence of Crl expression. A, Crl was detected by immunoblotting. The BL001 (lane 1), W3110 (lanes 2 and 3), BL002 (lane 4), W3110/pTOPO-crl (lanes 5 and 7), and W3110/pDEB2 (lanes 4 and 6) were grown in LB medium for 24 h at the temperature indicated. B, growth phase-dependent expression of Crl. The W3110 and BL001 strains were grown in LB medium at the temperature indicated. At the times indicated, cell lysates were prepared as described under "Materials and Methods." For comparison, the intracellular level of {sigma}S was also quantified. Each samples contains 20 µg of total protein and was subjected to 12 or 15% SDS-PAGE and immunoblot analysis with polyclonal antibodies against Crl or {sigma}S. C, growth curve of the strain wild type grown at 30 °C (filled squares) and 37 °C (open squares), and the strain BL001 (crl–) grown at 30 °C (open circles). D, EMSA demonstrates the binding of E{sigma}70 and E{sigma}S to the csgBA promoter region. 10 nM of a radiolabeled csgBA promoter fragment was run on polyacrylamide gels, after incubation with crude extracts (20 µg of proteins) from the indicated E. coli strains grown at the temperature and harvested at the times indicated.

 
The intracellular level of Crl was further analyzed by quantitative immunoblotting during growth. Crude cell extracts were prepared from the E. coli wild type strain at various stages of growth. As shown in Fig. 7, B and C, the Crl level started to increase when the growth rate began to decrease. The level becomes maximal when cell growth stopped and stayed constant thereafter. At 37 °C, some Crl is detected at 4, 6, and 8 h and then disappears (Fig. 7B, 37 °C). Moreover, after 8 h of growth, the Crl level was ~30-fold higher at 30 than at 37 °C, thus confirming the effect of growth temperature on Crl synthesis. These data show that Crl is expressed in E. coli in a temperature- and growth phase-dependent manner.

In order to assess the functional relevance of the regulation of Crl expression, we repeated the EMSA experiment with crude extracts from strains grown at 37 °C (Fig. 5B). As described before, we observed two retarded bands (named b and c), the lower one corresponding to E{sigma}S bound to pcsgBA. The intensity of band b is at the limit of detection in all lanes containing the {sigma}S subunit, whereas with the strains that overexpress either Crl or {sigma}S (Fig. 5B, lanes 5 and 6, respectively), the E{sigma}S-pcsgBA signal is increased. A reasonable interpretation is that when Crl is poorly expressed (at 37 °C), E{sigma}S is not able to bind the promoter efficiently. This result further corroborates our conclusion that Crl acts as a positive regulator of transcription initiation mediated by the {sigma}S subunit.

Because growth temperature also influences {sigma}S expression, we quantified the intracellular levels of {sigma}S by immunoblotting in all strains used. As published previously (16, 53), we found that in wild type cells, {sigma}S levels are higher at 30 than at 37 °C (Fig. 6, A and B). Nevertheless, in crl null strains grown at either 30 or 37 °C, E{sigma}S binding to the curli promoter was strongly reduced or not allowed even though {sigma}S levels were higher than in wild type cells (Fig. 5B). The E{sigma}S-pcsgBA signal could be detected at 37 °C only in strains overexpressing either Crl or {sigma}S. The intracellular concentrations of both Crl and {sigma}S therefore constitute two independent parameters that control E{sigma}S binding to the promoter. The direct correlation between the amount of Crl and the intensity of the retarded band of E{sigma}S-pcsgBA in our EMSA experiments with crude extracts show that increasing concentrations of Crl lead to better binding of E{sigma}S to the csgBA promoter.

As curli expression is restricted to the stationary phase state, EMSA experiments were conducted with crude cell extracts prepared from strains growing at various growth phases. E{sigma}S binding to pcsgBA takes place after 4 h of growth at 37 °C and after 6 h at 30 °C (Fig. 7D). This difference can be attributed to the difference in growth rate at 30 and 37 °C (Fig. 7C). A strong E{sigma}S-pcsgBA signal was detected only with wild type strain grown at 30 °C but not at 37 °C or with the crl null strain grown at 30 °C. Our experiments thus show that E{sigma}S binding to pcsgBA occurs only in stationary phase (as soon as {sigma}S can be detected), preferentially at 30 °C, and in a Crl-dependent manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Crl Regulates the Transcriptional Activity of Target Promoter through a Direct Interaction with the {sigma}S Subunit—We report here that Crl, a small E. coli regulatory protein, interacts directly with the {sigma}S subunit in vitro and controls E{sigma}S binding to a target promoter. Several lines of evidence support our assertion that Crl controls the activity of the {sigma}S subunit through direct protein-protein interaction. (i) Crl stimulates, in vivo, {sigma}S activity in stationary phase (25). (ii) Crl interacts with the {sigma}S subunit in vitro (Figs. 1 and 2). (iii) Crl is produced at the onset of stationary phase, in parallel with the appearance of {sigma}S in cells, and therefore with the outset of transcription of {sigma}S-dependent genes (Fig. 7, B–D). (iv) Crl does not seem to be a DNA-binding protein. It does not contain any known DNA-binding motif, nor does it bind to DNA-cellulose beads (25) or to the csgBA promoter region (Fig. 3). (v) Crl is associated with the E{sigma}S-pcsgBA open complex but not with E{sigma}70-pcsgBA (Figs. 3 and 5). (vi) Some {sigma}S-dependent genes require additional regulatory factors besides {sigma}S because high cellular levels of {sigma}S do not result in high expression of these target genes (54). Crl may well be this missing factor as suggested by our in vitro studies; even if high amount of {sigma}S subunit is present in crude cell extract, an efficient binding of E{sigma}S to the promoter is not allowed in the absence of Crl. Thus, Crl appears to be required for full {sigma}S activity at the level of transcription initiation of target promoters.

In this paper, we have focused on csgBA transcription because this promoter is well known for being regulated by both Crl and {sigma}S (25, 28, 44). The stimulatory effect of Crl observed in vivo could not be reproduced by in vitro transcription experiments (data not shown), presumably because an as yet unidentified co-factor(s) is missing in the purified system. Our experiments using crude cell extracts confirm this interpretation; enhanced recruitment of E{sigma}S is only observed with these extracts and not in the purified system (discussed below). Furthermore, this missing factor is not essential for the interaction of Crl with the {sigma}S subunit and the transcriptional complex (Figs. 1, 2, 3, 4). We exclude the possibility that the His-tagged fusion proteins Crl and {sigma}S were inactive because the effect of His6-Crl on the formation of the His6-{sigma}S-holoenzyme-pcsgBA complex was observed with crude cell extracts from the double mutant strain (crl– and rpoS–) in EMSA experiments (data not shown). We can, however, not formally exclude the possibility that the His tag on Crl interferes with transcriptional activation in our in vitro transcription experiments.

Crl Regulates {sigma}s Subunit Activity at the Level of Transcriptional Initiation—All available in vivo and in vitro data indicate that Crl functions as a transcriptional regulator. Given that a drastic competition is exerted between the seven {sigma} subunits in E. coli and that {sigma}S is the one with the lowest affinity for the core enzyme (20), an exciting hypothesis is that Crl may favor the association of {sigma}S with RNA polymerase core enzyme to control the amount of E{sigma}S within the cell. However, our experiments show that Crl modulates E{sigma}S binding to pcsgBA without affecting the E{sigma}70-pcsgBA signal (Fig. 5). This indicates that Crl does not influence the equilibrium {sigma} + E {leftrightarrow} E{sigma}. In other words, Crl has no effect on the competition between {sigma}S and {sigma}70 for E. Instead, Crl must therefore strengthen the association of E{sigma}S with pcsgBA (see the proposed model in Fig. 8). An additional set of EMSA experiments, using a mix of purified His6-{sigma}S and His6-{sigma}70 with E, demonstrates that Crl does not favor {sigma}S association with E at the detriment of {sigma}70 (data not shown). Moreover, given that {sigma}S association with E protects {sigma}S from degradation via the RssB/ClpXP pathway, we would expect that Crl contributes to stabilizing {sigma}S (see review by Hengge-Aronis (17)). Quite to the contrary, we observed that intracellular levels of {sigma}S are inversely correlated with Crl concentration (Figs. 6 and 7A). Along the same lines, our in vitro binding assay using Ni-NTA technology showed that His6-Crl binding to {sigma}S is not affected by the {Delta}7–35 deletion in {sigma}S (data not shown). Studemann et al. (49) demonstrated that this amino-terminal deletion corresponds to an element essential for {sigma}S proteolysis. We conclude that the amino-terminal region of {sigma}S is not essential for interaction with Crl and that Crl does not interfere with ClpXP-mediated proteolysis of {sigma}S.



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  		FIG. 8.
Model of the regulatory network controlling curli transcription in E. coli. The curli subunits are transcribed from the csgBA promoter. In the presence of the histone-like protein H-NS, E{sigma}70 poorly recognizes this promoter and provides a low, basal level of transcription. At low temperature (30 °C) and in stationary phase, Crl is expressed and stimulates the activity of E{sigma}S through direct interaction with the {sigma}S subunit. Strong transcription by E{sigma}S-Crl leads to full expression of the operon. The OmpR pathway further activates this {sigma}S-dependent transcription at low osmolarity. At higher osmolarity, the Cpx system inhibits transcription of the csgBA promoter. H-NS inhibits csgBA expression mediated by E{sigma}70. Activation is indicated by an arrow, repression by a line ending in a perpendicular bar.

 
Considering our in vitro results, showing that Crl increases the level E{sigma}S bound to the promoter and modifies the E{sigma}S-pcsgBA footprint (Figs. 5 and 4, respectively), we favor the alternative hypothesis: Crl stimulates E{sigma}S binding to pcsgBA. Crl could thus increase the affinity of E{sigma}S for the promoter and/or stimulate the ability of E{sigma}S to melt promoter DNA and produce an open complex (see proposed model in Fig. 8). Crl may need a co-factor to do so. Brombacher et al. (55) have recently identified an 11-bp sequence (CGGGKGAKNKA) located upstream of the csgBA promoter elements (from –42 to –32) that is necessary for the transcriptional activation by CsgD. This region overlaps some of the bases, whose reactivity was modified by Crl in our DNase I footprinting experiments (Fig. 4). Even though CsgD belongs to the LuxR family and behaves like a transcription factor that up-regulates csgBA expression (29), a direct demonstration of its DNA binding activity is still missing. Given these data, an attractive hypothesis is that CsgD could be the missing factor necessary for transcriptional activation by Crl in the purified systems.

Crl, a Thermometer Regulating the Activity of {sigma}s—Because the crl gene product stimulates transcription of csgBA at low but not high temperature, we hypothesized that the crl gene itself might be thermoregulated. We observed indeed that the synthesis of Crl is temperature-dependent, operating efficiently at 30 °C but not at 37 °C (Fig. 7). Because a previous study (44) has reported that the transcription of the crl gene is not thermoregulated, we suggest that crl expression may be controlled post-transcriptionally. Transcriptional fusions of crl with lacZ allowed us to confirm that the crl promoter is not regulated by temperature (data not shown). However, in an hns and rpoS double mutant strain, transcription of the csgBA gene remains temperature-regulated (27), showing that another means of temperature control must exist. The temperature-dependent expression of Crl thus appears to fine-tune {sigma}S activity, whereas the DsrA-mediated temperature control modulates the amount of {sigma}S in the cell. The two mechanisms act in concert because at low temperature DsrA stimulates {sigma}S expression (16) and Crl enhances the activity of {sigma}S.

In conclusion, our in vitro data are consistent with the observed physiological effect of Crl on {sigma}S activity in vivo: Curli formation and {sigma}S- and Crl-mediated transcription of csgBA genes are observed only at low temperature and in stationary phase (27, 32, 35, 44). Indeed, under these conditions, Crl is present in cells and facilitates {sigma}S-mediated transcription from the curli promoters and probably of other genes that belong to rpoS regulon.

According to the model, Crl would be a prerequisite for an efficient transcription initiation from promoters with a low affinity for E{sigma}S. The production of Crl is concomitant with the accumulation of {sigma}S (Fig. 7B), suggesting that crl transcription may be dependent on the {sigma}S subunit. However, the intracellular level of Crl remained unaffected in a rpoS null strain, thus precluding a simple transcriptional control by {sigma}S. Future studies are needed to elucidate the molecular mechanisms of the control of Crl expression. Our data promote Crl to an important new member of the stress regulon of E. coli, which acts by influencing the activity of the central player of this complex regulatory network, {sigma}S.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier, BP53, F-38041 Grenoble Cedex 9, France. Tel.: 33-4-76-51-40-28; Fax: 33-4-76-63-56-63; E-mail: cecile.lelong{at}ujf-grenoble.fr.

1 The abbreviations used are: EMSA, electrophoretic mobility-shift assays; Cm, chloramphenicol; DTT, dithiothreitol; Ni-NTA, nickel-nitrilotriacetic acid; Kn, kanamycin; wt, wild type. Back


   ACKNOWLEDGMENTS
 
We thank the reviewer(s) for interesting and useful comments on our work. We are very grateful to D. Faure and D. Schneider for providing strains and plasmids. We thank A. Kolb for the gift of purified {sigma}70 protein. We also acknowledge J. Lacoste for advice and technical help with the bandshift experiments and B. Franzetti for help with gel filtration experiments. We thank M. Cashel for helpful discussions and suggestions. Finally, we particularly appreciated the contributions of M. A. Hakimi who always showed an interest in our work, provided helpful advice in biochemistry, and a critical scientific evaluation of our results. Finally, we thank the lab staff for contributions to this work.



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 ABSTRACT
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 MATERIALS AND METHODS
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 DISCUSSION
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