Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli

Mei-Shiue Kuo, Kuei-Peng Chen and Whi Fin Wu

Department of Agricultural Chemistry, Bldg 2, R311, National Taiwan University, Taipei (106), Taiwan, ROC

Correspondence
Whi Fin Wu
whifinwu{at}ccms.ntu.edu.tw


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for {beta}-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBADrcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsARed–lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased {beta}-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.


Abbreviations: HA, haemagglutinin; MMS, methyl methanesulfonate


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In Escherichia coli, the ATP-dependent proteases have been characterized (see the review by Gottesman, 1996), with the Clp proteases (Porankiewicz et al., 1999) and Lon (Goldberg et al., 1994) being the two major groups. The Clp protease complex consists of two components, a proteolytic component ClpP (Maurizi et al., 1990a, b) or ClpQ (Chuang et al., 1993; Yoo et al., 1996), and an ATPase subunit of chaperone-like activities, ClpA (Wickner et al., 1994), ClpX (Wawrzynow et al., 1995) or ClpY (Seong et al., 2000). ClpAP (Gottesman et al., 1998; Katayama-Fujimura et al., 1987), ClpXP (Gottesman et al., 1993, 1998) and ClpYQ (Kanemori et al., 1999; Khattar, 1997; Missiakas et al., 1996; Wu et al., 1999) are recognized for their proteolytic activities against specific short-lived proteins.

The genes encoding ClpYQ (HslUV) are part of a heat-shock operon (Chuang et al., 1993). In the clpQ+clpY+ operon, the first gene encodes ClpQ (19 kDa), a small-subunit peptidase, and the second gene encodes ClpY (49 kDa), a large subunit (Missiakas et al., 1996; Rohrwild et al., 1996; Yoo et al., 1996). Each ClpQ or ClpY self-oligomerizes as a hexamer, and four hexamers constitute a dumb-bell-shaped complex in an Y6Q6Q6Y6 configuration (Kessel et al., 1996; Rohrwild et al., 1997). ClpY transfers substrates from outside the hexameric cylinder into the catalytic core, wherein ClpQ degrades the substrates (Kessel et al., 1996; Rohrwild et al., 1997).

ClpQ has a catalytic amino-terminal threonine residue with sequence similarity to the eukaryotic proteasome {beta} subunit (Seemüller et al., 1995) and can be covalently modified by an inhibitor in the presence of ATP and ClpY (Bogyo et al., 1997). The ClpY molecule is structurally divided into three domains: N, I and C (Bochtler et al., 2000). The N domain, containing an ATP-binding site, is divided into two parts; the I domain recognizes substrates and the C domain is involved in inter-subunit interaction (Bochtler et al., 2000). Both in vitro and in vivo studies have demonstrated that ClpY recognizes specific substrates (Kanemori et al., 1999; Khattar, 1997; Seong et al., 1999; Smith et al., 1999; Song et al., 2000; Wu et al., 1999).

The ClpYQ protease has an overlapping function with Lon, a single-component energy-dependent protease (Kanemori et al., 1999; Khattar, 1997; Wu et al., 1999). E. coli cells containing a lon mutation are sensitive to DNA damage due to an accumulation of a cell division inhibitor, SulA, induced by DNA damage agents, e.g. UV light or methyl methanesulfonate (MMS) (Chung & Goldberg, 1981; Maurizi et al., 1985). In vivo, lon mutants deleted for clpQ or clpY were much more sensitive to MMS than single lon mutants, and ClpY interacts with SulA as detected by a yeast two-hybrid system (Lee et al., 2003; Wu et al., 1999).

Using either in vitro-purified proteins or in vivo-overexpressed clpQ+clpY+ from plasmids, ClpYQ degrades SulA (Kanemori et al., 1999; Khattar, 1997; Wu et al., 1999; Song et al., 2000). In addition, lon mutants stabilize RcsA (Stout et al., 1991; Torres-Cabassa & Gottesman, 1987), a positive activator for transcription of cps (capsule synthesis) genes (Stevenson et al., 1996; Stout et al., 1991; Trisler & Gottesman, 1984). RcsA, in combination with RcsB, binds to the RcsAB box upstream from the cps promoter to activate capsule gene expression (Ebel & Trempy, 1999; Wehland & Bernhard, 2000). However, RcsA, in lon rcsB mutants lacking the co-activator RcsB (Stout & Gottesman, 1990), is short-lived (Ebel & Trempy, 1999). Therefore, it is postulated that another protease is involved in degrading RcsA. Although overproduction of ClpYQ represses cpsB–lacZ expression (Wu et al., 1999), until now there has been no evidence demonstrating that ClpYQ targets RcsA. Here, using cpsB–lacZ (Trisler & Gottesman, 1984), a capsule gene fused with a lacZ reporter, we again analyse this gene fusion expression under different circumstances. We show that the ClpYQ proteases have effects on cpsB–lacZ expression, indicating that ClpYQ is involved in the regulation of RcsA. In addition, we show that an inducible RcsA has an effect on cpsB–lacZ expression and that its influence is significantly altered along with the ClpYQ activities. Moreover, the {beta}-galactosidase levels of a rcsARed–lacZ translational gene fusion are altered by the ClpYQ proteases. Our results indicate that one function of ClpYQ is to regulate RcsA, in an overlapping role with the Lon protease.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and materials.
Bacterial strains, phages and plasmids used in this study are listed in Table 1. Bacteria were grown in Luria–Bertani (LB) broth and supplemented with appropriate antibiotics or sugars as required. Supplements were added at the following concentrations when necessary: 5 µg tetracycline (Tc) ml-1; 100 µg ampicillin (Ap) ml-1; 40 µg X-Gal ml-1. Restriction endonucleases, Taq DNA polymerase and other enzymes were obtained from Takara or New England Biolabs. Chemicals were obtained from Sigma or Waco.


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Table 1. Bacterial strains, plasmids and phages used in this study

 
General methods.
P1vir and {lambda} were prepared as described by Silhavy et al. (1984). Mutant strains were constructed by P1 transduction, using procedures as described by Miller (1972). For plasmid constructions, E. coli XL-1 Blue was used. Plasmid transformations were performed as described by Chung et al. (1989). Plasmid purification was performed according to the manufacturer's instructions (Qiagen or Viogen).

PCR, plasmid construction and DNA sequencing.
Plasmids pEC01, pEC02 and pEC03 were constructed by subcloning the clpQ, clpY and clpQclpY genes into pBAD24 (Guzman et al., 1995) at NheI–EcoRI sites from pVEX11-clpQ (Kessel et al., 1996), pVEX11-clpY (Kessel et al., 1996) and pVEX11-clpQclpY (Kessel et al., 1996), respectively.

PCR was used to amplify the rcsA gene, encompassing the complete open reading frame or/and promoter region and the Shine–Dalgarno (SD) region. To perform these reactions, MG1655 chromosomal DNA was used as template, and three sets of oligonucleotide primers with restriction enzyme sites (underlined) at the ends were designed as follows. Set 1: forward primer 5'-CCGGAATTCTCACTCACATATCGCAACATTTAC-3'; reverse primer 5'-CGCGGATCCCGCATGTTGACAAAAATACCATTAGT-3'. Set 2: forward primer 5'-CCGGAATTCATGTACGACGTACCAGATTACGCTATGTCAACGATTATTATGGAT-3'; reverse primer 5'-CGGGGTACCTTAGCGCATGTTGACAAAAATACCATTAGT-3'. Set 3: forward primer 5'-CCGGAATTCGAGGGTATGCCATGTACGACGTACCAGATTAC-3'; reverse primer 5'-CGGGGTACCTTAGCGCATGTTGACAAAAATACCATTAGT-3'. E. coli MG1655 genomic DNA was extracted according to the methods described by Silhavy et al. (1984) and the PCR conditions were performed according to the manufacturers' instructions. Using set 1 primers, the amplified DNA products, carrying the rcsA promoter and structural gene, were first purified by using the Viogene PCR clean-up kit. The purified PCR products were digested with EcoRI and BamHI followed by gel purification. The isolated fragments were ligated with pRS414 (Simons et al., 1987) digested with the corresponding restriction enzymes to construct pRS414-rcsA, with rcsA fused in-frame to lacZ. The resulting ligated mixtures were transformed into E. coli strain XL-1 Blue and selected on ampicillin-lactose MacConkey agar plates. Plasmids were then isolated from the colonies grown on selective plates for insertion analyses, followed by DNA sequencing of plasmids with the correct insertions.

By analogous procedures, the set 2 primers were used to amplify the full-length rcsA DNA fragment, which was then cloned into pBAD24 at the EcoRI–KpnI sites to construct pBAD24-ha-rcsA, such that the rcsA gene carrying the haemagglutinin (HA) tag at the 5' end (amino-terminus) is under control of the PBAD promoter.

Also, the rcsA DNA fragment, amplified by set 3 primers, was cloned into pBAD18 (Guzman et al., 1995) at EcoRI–KpnI sites to construct pSN181, in which the rcsA gene, carrying its own SD sequences, is under PBAD promoter control (Guzman et al., 1995).

DNA sequencing of the cloned genes was performed using an ABI 377 automated sequencer. All the cloned rcsA genes had the wild-type sequences, except that pRS414-rcsARed carries three point mutations in rcsA.

Construction of the {lambda}RS45rcsARedlacZ translational fusion and tests of a single lysogen.
Plasmid pRS414-rcsARed was transformed into XL-1 Blue cells and selected for ampicillin resistance. The resulting transformants were infected with the lambda derivative {lambda}RS45 and selected for blue plaques on X-Gal plates, indicating that the rcsARed–lacZ translational fusion had been transferred into {lambda}RS45 (Simons et al., 1987). A single lysogen was selected by directly measuring the {beta}-galactosidase levels of five lysogens, and the single lysogens with the lowest value were selected.

Cell growth and {beta}-galactosidase assays.
Cells carrying pBAD plasmids were grown overnight in LB with the appropriate antibiotic and glucose; the cells were then washed twice and subcultured in the same media except with the addition of the specified concentration of arabinose. The cells were collected in exponential phase and subsequently subjected to {beta}-galactosidase assay.

The {beta}-galactosidase activities were assayed as described by Miller (1972). All the {beta}-galactosidase activities reported are the means of two or more assays in which the values were determined in triplicate for each assay.

Immunoblot analysis.
Cultures of AC31133/pBAD24-ha-rcsA (pEC04) were grown overnight in LB medium with ampicillin and glucose, then washed twice, subcultured and grown to early exponential phase in LB medium with ampicillin and 0·002–2 % (w/v) arabinose. The exponential-phase cells grown in different arabinose concentrations were collected, subjected to {beta}-galactosidase assays and extracted to determine the cellular levels of RcsA by separating the extracts on 12·5 % SDS-polyacrylamide gels (Laemmli, 1970) followed by electroblotting to nitrocellulose membranes. The blots were probed with a 1 : 1000 dilution of monoclonal HA antibody (Roche). Immunological detection was performed with mouse monoclonal antibody (Roche). Washes and detection were done with the ECL chemiluminescence system (Amersham).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Overproduction of clpQ+clpY+ suppresses lon mucoidy and cpsB–lacZ expression increases in lon strains with clpQ, clpY or clpQclpY mutations
In E. coli, lon mutants have two phenotypes: UV (or MMS) sensitivity and mucoidy. The UV sensitivity is due to an accumulation of SulA and the mucoidy is due to stable RcsA, which activates capsule synthesis (cps) genes. Independently, SulA and RcsA are both highly unstable and appear to be degraded by a Lon-dependent route. Nevertheless, the MMS sensitivity was suppressed by clpQ+clpY+ plasmids (Wu et al., 1999). To show that the capsule synthesis of lon mutants was also repressed by an overproduction of clpQ+clpY+, we cloned clpQ or/and clpY into pBAD24, resulting in plasmids pEC01 (clpQ+), pEC02 (clpY+) and pEC03 (clpQ+clpY+). These plasmids were then transformed into the lon mutant MS30001, and the phenotypes of the transformed cells were inspected. The mucoid phenotype of lon mutant was repressed by overproduction of clpQ+clpY+ (Fig. 1). MS30001 cells carrying the control or clpQ or clpY plasmids were mucoid (Fig. 1).



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Fig. 1. Phenotypes of lon mutant MS30001 carrying different plasmids: pBAD24, pEC01 (pBAD24+clpQ+), pEC02 (pBAD24+clpY+) or pEC03 (pBAD24+clpQ+clpY+). Ten microlitres of each exponential-phase cell sample was plated out on LB-ampicillin plates plus 0·2 % (w/v) arabinose and incubated for 4 days at 30 °C. The clpQ+, clpY+ and clpQ+clpY+ genes were expressed under PBAD control with the addition of arabinose.

 
To investigate whether a chromosomal copy of clpQ or/and clpY regulates capsular gene expression, clpQ or/and clpY deletions were introduced into the wild-type and lon mutant chromosomes by P1vir transduction. Since all the strains carried cpsB–lacZ, the effects of ClpQ or/and ClpY on cps expression were examined by measuring their {beta}-galactosidase levels. The lon+ control cells, SG22622, had low cps expression (Table 2, line 1). The lon+ clpQ clpY mutant AC3111, however, also showed low cps expression (Table 2, line 3). As expected, the single lon mutant SG22623 had higher {beta}-galactosidase levels (Table 2, line 2). There was an approximately twofold increase in cps expression in AC3112 (lon clpQ clpY), AC3113 (lon clpQ) and AC3114 (lon clpY) as compared to the single lon mutant (Table 2, compare lines 4, 5 and 6 with line 2). These results indicate that cells lacking ClpQ and/or ClpY in the absence of Lon increase cps expression.


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Table 2. cpsBlacZ expression in the wild-type and various protease-deficient mutants

All strains were incubated at 30 °C. The {beta}-galactosidase assays were carried out as described by Miller (1972). Values are means of three independent assays±SD.

 
Effect of ClpQ, ClpY or ClpYQ overproduction on cpsB–lacZ expression
It was previously shown that overproduction of clpQ+clpY+ decreased cpsB–lacZ expression (Wu et al., 1999). However, we used the heterologous promoter PBAD to quantitatively express the ClpQ or/and ClpY and examine their effects on cpsB–lacZ expression. Unexpectedly, we found that overproduced ClpQ protein alone reduced expression of cpsB–lacZ about twofold as compared to the control (Table 3, compare lines 2 and 1, columns 6 to 9: 0·002–2 % arabinose). We also introduced pEC01 (clpQ+) into AC3114, a lon clpY strain, and postulated that cpsB–lacZ expression would decrease further, as would chromosomal ClpQ production. As expected, AC3114, carrying merodiploid clpQ+/clpQ+ in the absence of clpY+, showed about an eightfold decrease in cpsB–lacZ expression with 2 % arabinose (Table 3, compare lines 6 and 5). To examine whether the overproduction of ClpQ resulted in nonspecific effects on {beta}-galactosidase levels in the cell, we measured the level of expression of clpQ–lacZ fusions both in wild-type cells and in cells with overproduced ClpQ. The level of {beta}-galactosidase activity expressed by the clpQ–lacZ fusion in the multi-copy clpQ+ cells was identical to that expressed in the wild-type cells (data not shown), suggesting that the effect of overproducing ClpQ on the expression of cpsB–lacZ demonstrated in this study is specific. However, an overproduction of ClpY alone (pEC02) led to no change in cpsB–lacZ expression (Table 3, compare lines 3 and 1). While both ClpQ and ClpY (pEC03) were increasingly overproduced in the inducing conditions, cpsB–lacZ expression progressively decreased, with an approximately 17-fold reduction under the 2 % arabinose concentration (Table 3, compare lines 4 and 1). These results not only confirm previous findings that ClpQ and ClpY are both required for repression of cpsB–lacZ, but also indicate that there is an inverse correlation between the amount of ClpYQ and the {beta}-galactosidase level of cpsB–lacZ.


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Table 3. Effects on cpsBlacZ expression of overproduction of ClpQ, ClpY or ClpYQ

All strains were grown at 30 °C in LB broth with 2 % glucose or 0·0002–2 % arabinose, or without addition of either sugar. The {beta}-galactosidase assays were carried out as described by Miller (1972). Values are means of three independent assays±SD.

 
Inducible RcsA quantitatively activates cpsBlacZ expression
To show that the expression of cpsB–lacZ reflected the levels of RcsA, we constructed plasmid pBAD24-ha-rcsA (pEC04), in which SD sequences from the plasmids and the N-terminal HA-tagged rcsA gene were under control of the PBAD promoter. pEC04 was transformed into AC31133, a lon rcsA strain. Upon induction of HA-RcsA synthesis with arabinose, the transformants continued to grow at a rate similar to the cultures grown without induction (Fig. 2a). Western blotting with anti-HA antibody was used to detect the appearance of HA-RcsA. As shown in Fig. 2(b), lane 1, when the cells of AC31133(pEC04) were grown in the repressing glucose medium, the {beta}-galactosidase levels of cpsB–lacZ were basal, and no RcsA was detected by Western blot analysis. When arabinose was added to the medium, the cpsB–lacZ expression increased, and RcsA was detected (Fig. 2b). When the arabinose concentration was gradually increased, RcsA accumulated proportionally in the cells, resulting in a progressive increase in cpsB–lacZ expression.



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Fig. 2. cpsB–lacZ and RcsA expression increase after arabinose induction. (a) Growth at different arabinose concentrations of of AC31133 (lon rcsA) carrying the cpsB–lacZ fusion and transformed with pBAD24-ha-rcsA. Cells were grown at 30 °C in LB-ampicillin medium containing 2 % (w/v) glucose as the control (C) and arabinose at the concentrations indicated. (b) cpsB–lacZ expression was assessed by {beta}-galactosidase assays while the RcsA levels were determined by Western blotting. Levels of RcsA proteins and {beta}-galactosidase activity were evaluated from the same sample by splitting the sample equally, using one half for the {beta}-galactosidase assay and the other half for RcsA detection. Specific activity is expressed in Miller units; values are the means of three independent assays. To assess the RcsA levels, equal amounts of protein from each sample were loaded onto a 12·5 % SDS-polyacrylamide gel and analysed by Western blotting with monoclonal anti-HA antibody. The anti-HA antibody reacts with a 23·5 kDa RcsA protein as indicated.

 
ClpYQ regulates cpsBlacZ expression through RcsA
To demonstrate that ClpYQ regulation of cpsB–lacZ expression is through RcsA, we constructed another plasmid carrying an rcsA gene with its own SD sequences under control of the PBAD promoter. The resulting PBAD-rcsA plasmid, pSN181, was introduced into the wild-type and the aforementioned protease-deficient strains. Similarly, all the strains carrying cpsB–lacZ were deleted of the chromosomal rcsA gene by P1vir transduction; RcsA was only expressed from the pSN181 plasmids during arabinose induction. As shown in Table 4, the level of {beta}-galactosidase activity was basal when the cells were grown in medium supplemented with glucose (Table 4). By contrast, when arabinose was added to the medium, the cpsB–lacZ expression increased, due to the activation of RcsA (Table 4). Predictably, all arabinose concentrations resulted in the lon mutants producing higher levels of {beta}-galactosidase activity than the wild-type (Table 4). However, the AC31113 (clpQ clpY lon), AC31130 (clpQ lon) and AC31131 (clpY lon) mutants demonstrated even higher {beta}-galactosidase levels than the single lon mutant (Table 4, compare lines 4, 5 and 6 with line 2). These results suggest that cells lacking ClpQ or/and ClpY in combination with the loss of Lon have increased RcsA levels.


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Table 4. Effects of an inducible RcsA on cpsBlacZ expression in the wild-type and different protease-deficient mutants

All strains were grown at 30 °C in LB broth with 2 % glucose, or 0·2 % or 2 % arabinose. The {beta}-galactosidase assays were carried out as described by Miller (1972). Values are means of three independent assays±SD.

 
We also used the multi-copy plasmid pWF1, in which clpQ+clpY+ were overproduced, to analyse the role of the ClpYQ complex in controlling an inducible RcsA level. At all arabinose concentrations, as compared to the controls, the AC31133 (lon rcsA) mutant carrying pSN181 showed low cpsB–lacZ expression when pWF1 was simultaneously present (Table 5). However, when pWF2 (clpQ : : cat clpY+), pWF3 (clpQ+ clpY : : cat) and pWF4 [{Delta}(clpQ-clpY) : : cat], were separately transformed into AC31133 with pSN181, all of the transformed cells had much higher {beta}-galactosidase levels (Table 5). Thus, cells carrying plasmids overproducing ClpQ and ClpY negatively regulate RcsA, resulting in lower cps expression. When clpQ+ or/and clpY+ were removed from the plasmids, the cpsB–lacZ expression was restored to a higher level due to the accumulation of RcsA.


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Table 5. Overproduction of ClpYQ, ClpY or ClpQ protein, adjusting an inducible RcsA, affects cpsBlacZ expression

Strain AC31133 (lon rcsA) carrying the plasmids shown was grown at 30 °C in LB broth with 2 % glucose or 0·002–2 % arabinose, or without addition of either sugar. The {beta}-galactosidase assays were carried out as described by Miller (1972). Values are means of three independent assays±SD.

 
Construction of an rcsARed–lacZ translational fusion in {lambda}RS45
To determine whether the regulation of RcsA by ClpYQ is a direct reflection of reporter expression, an in-frame rcsA–lacZ translational fusion was constructed by cloning the full-length rcsA gene into plasmid pRS414, generating plasmid pRS414-rcsA. When pRS414-rcsA was transformed into XL-1 Blue E. coli cells and then plated on MacConkey ampicillin plates, two phenotypes emerged: one white and mucoid, and the other red and less mucoid. DNA sequencing was done to verify fusion genes from both phenotypes. The white ones carried the wild-type rcsA whereas the red ones had three point mutations in rcsA: H75R (histidine to arginine, CAT to CGT), I103T (isoleucine to threonine, ATC to ACC) and I176T (isoleucine to threonine, ATT to ACT), and were designated pRS414-rcsARed. Since cells carrying pRS414-rcsARed were red on MacConkey media, we used those to cross the rcsARedlacZ translational fusion gene into {lambda}RS45, and the resultant phage, designated {lambda}RS45rcsARedlacZ, yielded blue plaques on LB X-Gal plates.

Regulation of RcsARed–LacZ fusion protein by the ClpYQ protease
To further analyse whether the targeting of RcsA by ClpYQ is reflected in the RcsARed–LacZ levels, the {lambda}RS45rcsARedlacZ phage were lysogenized into the wild-type and protease-deficient mutants. The wild-type and mutant lysogens were selected and their {beta}-galactosidase levels were measured. As shown in Table 6 (compare lines 1 and 4), the RcsARed–LacZ levels were identical between the wild-type and lon mutants, suggesting that the RcsARed–LacZ translational fusion proteins were insensitive to Lon. However, cells lacking ClpQ or ClpY or both showed a twofold increase in the {beta}-galactosidase activity, independent of the lon+ or lon- background (Table 6, lines 2, 3, 5, 6 and 7). These results suggest that RcsARed–LacZ accumulated in the cells lacking ClpYQ.


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Table 6. rcsARedlacZ expression in the wild-type and various protease-deficient mutants

All strains, which were rcsA+, were grown at 30 °C. The {beta}-galactosidase assays were carried out as described by Miller (1972). Values are means of three independent assays±SD.

 
To identify whether the RcsARed–LacZ was specifically targeted by ClpYQ, a clpA mutation was introduced into the chromosome in the wild-type and the clpQclpY mutant lysogens, by P1vir transduction. The {beta}-galactosidase levels of the two resultant lysogens, MS20009 and MS20010, were then measured, and were found to be similar to those of their isogenic parental strains (Table 6, compare lines 9 and 7, and lines 8 and 1). Thus, the RcsARed–LacZ levels were not affected by ClpA, indicating that RcsARed–LacZ is specifically regulated by ClpYQ.

To examine whether overproduction of ClpY and ClpQ suppresses RcsARed–LacZ levels, mutants MS10008 (rcsA clpQ clpY) and MS20008 (rcsA+ clpQ clpY) were individually transformed with pBAD24 (control) or with pEC03 to overproduce both ClpQ and ClpY. The resulting transformants were subsequently subjected to {beta}-galactosidase assay. Unexpectedly, RcsARed–LacZ expression increased with increasing concentrations of arabinose in the medium (Table 7, lines 1 and 3). However, as shown in Table 7, in both MS10008 and MS20008 overproducing ClpY and ClpQ from pEC03 under arabinose induction, the levels of RcsARed–LacZ decreased compared to the controls (Table 7, compare lines 2 and 1, and lines 4 and 3). Therefore, these results also support the hypothesis that ClpYQ targets RcsARed, leading to the lower levels of RcsARed–LacZ.


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Table 7. Overproduction of ClpYQ protease suppresses expression of rcsARedlacZ

All strains were grown at 30 °C in LB broth with 2 % glucose or 0·0002–2 % arabinose. The {beta}-galactosidase assays were carried out as described by Miller. Values are means of three independent assays±SD.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In E. coli, cells that accumulate RcsA are mucoid in the absence of Lon, which is the major protease for RcsA. However, overproduction of ClpQ and ClpY suppresses the lon mucoidy as well as the cpsB–lacZ expression. Therefore, using lon mutants in combination with clpQ, clpY and clpQ clpY mutations, carrying either cpsB–lacZ or rcsARedlacZ as reporters, we determined that RcsA apparently up-regulates cpsB–lacZ in the absence of the ClpYQ protease. We also demonstrated that an inducible ClpYQ destabilizes the RcsA, reflected in the decrease of both the cps–lacZ expression and the level of RcsARed–LacZ. However, in vivo, the effect of ClpYQ on RcsA was most notable in the absence of Lon, suggesting that ClpYQ acts as a secondary protease in addition to Lon, targeting RcsA. At the highest level of induction, higher amounts of both ClpQ and ClpY were required to decrease cpsB–lacZ expression and RcsARed–LacZ production. These results are consistent with a lower affinity of ClpYQ for RcsA compared to Lon. Additionally, the RcsARed mutant is recognized by ClpYQ but not by Lon, indicating that ClpYQ and Lon are accessible to the RcsA with different specificities.

In the absence of ClpY, overexpressed ClpQ leads to a decrease in cps expression for reasons unknown. Also, from our initial assays, with the introduction of a clpA, clpB or clpX mutant allele into a host carrying the clpQ+ plasmid, cps expression remains low in the transductants overproducing ClpQ despite the lack of the normal ClpA, ClpB or ClpX subunit. These results suggest that an unknown Clp-like molecule associated with ClpQ or a highly concentrated ClpQ alone is likely to regulate RcsA in E. coli. In addition, lon clpQ or lon clpY double mutants increase the RcsA level more than the lon clpQ clpY triple mutant, reflected in the cpsB–lacZ levels (Table 4), indicating that these isogenic strains with similar genotypes have dissimilar influences on RcsA stability. The reason for this is unclear. However, KY2031, the original {Delta}clpQclpY : : tet source, is a recD strain (Kanemori et al., 1999), whose genetic background is different from those of the original clpQ : : cat and clpY : : cat strains (SG12064 and SG12065) (Wu et al., 1999). Hence the observed differences in cpsB–lacZ expression may be due to the strains having subtly different backgrounds.

It was reported recently that RcsA auto-regulates its own expression (Ebel & Trempy, 1999; Wehland & Bernhard, 2000). RcsA, in combination with RcsB, binds to an RcsAB box upstream of the rcsA promoter to activate its own expression. In our studies, the RcsARed–LacZ levels were not affected by RcsA. Since our rcsARedlacZ construct begins with a base-pair far downstream of the RcsAB binding sites, and no RcsAB box was included in its promoter region, it is not surprising that RcsA has no effect on rcsARedlacZ expression. Therefore, the changes in RcsARed–LacZ levels are probably due to the activity of ClpYQ.

Our results also suggest that the mucoidy of transformants overproducing the hybrid fusion protein, RcsA–LacZ, in the presence or absence of a chromosomal rcsA is due to the expression of the wild-type rcsA in the plasmid construct. RcsARed, however, is less active than the wild-type RcsA, as reflected in the decreased colony mucoidy. Additionally, three point mutations were found in RcsARed, generating three amino acid substitutions, H75R, I103T and I176T. Hence, all these residues might be important for an active RcsA.

In this study, we have demonstrated that ClpYQ regulates RcsA by its proteolytic activities. We used pBAD plasmids to manipulate the level of cellular RcsA and employed cpsB–lacZ fusion constructs to reflect its activity. As such we were able to easily modify the RcsA level, to assay cps–lacZ activity, and to observe the influence of ClpYQ. It was previously shown that RcsA was stable in the presence of RcsB (Stout et al., 1991) although in lon rcsB mutants, chromosomal RcsA was not detected in the cells (Ebel & Trempy, 1999). In light of our results, it is likely that ClpYQ targets RcsA in lon rcsB cells as well.


   ACKNOWLEDGEMENTS
 
We thank Dr M. Kanemori, Dr T. Yura, Dr N. Majdalani and Dr Susan Gottesman, for bacterial strains. We also thank Dr Brenda Collins for critical reading of the manuscript. This work was supported by grants from the National Science Council (NSC 88-2313-B-002-029, NSC 89-2313-B-002-035 and NSC 89-2313-B-002-172) of Taiwan, ROC.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 25 April 2003; revised 5 September 2003; accepted 20 October 2003.



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