Department of Agricultural Chemistry, Bldg 2, R311, National Taiwan University, Taipei (106), Taiwan, ROC
Correspondence
Whi Fin Wu
whifinwu{at}ccms.ntu.edu.tw
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ABSTRACT |
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INTRODUCTION |
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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 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 cpsBlacZ expression (Wu et al., 1999
), until now there has been no evidence demonstrating that ClpYQ targets RcsA. Here, using cpsBlacZ (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 cpsBlacZ expression, indicating that ClpYQ is involved in the regulation of RcsA. In addition, we show that an inducible RcsA has an effect on cpsBlacZ expression and that its influence is significantly altered along with the ClpYQ activities. Moreover, the
-galactosidase levels of a rcsARedlacZ 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.
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METHODS |
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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 NheIEcoRI 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 ShineDalgarno (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 EcoRIKpnI 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 EcoRIKpnI 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 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 RS45 and selected for blue plaques on X-Gal plates, indicating that the rcsARedlacZ translational fusion had been transferred into
RS45 (Simons et al., 1987
). A single lysogen was selected by directly measuring the
-galactosidase levels of five lysogens, and the single lysogens with the lowest value were selected.
Cell growth and -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 -galactosidase assay.
The -galactosidase activities were assayed as described by Miller (1972)
. All the
-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 0·0022 % (w/v) arabinose. The exponential-phase cells grown in different arabinose concentrations were collected, subjected to -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).
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RESULTS |
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Regulation of RcsARedLacZ fusion protein by the ClpYQ protease
To further analyse whether the targeting of RcsA by ClpYQ is reflected in the RcsARedLacZ levels, the RS45rcsARedlacZ phage were lysogenized into the wild-type and protease-deficient mutants. The wild-type and mutant lysogens were selected and their
-galactosidase levels were measured. As shown in Table 6
(compare lines 1 and 4), the RcsARedLacZ levels were identical between the wild-type and lon mutants, suggesting that the RcsARedLacZ translational fusion proteins were insensitive to Lon. However, cells lacking ClpQ or ClpY or both showed a twofold increase in the
-galactosidase activity, independent of the lon+ or lon- background (Table 6
, lines 2, 3, 5, 6 and 7). These results suggest that RcsARedLacZ accumulated in the cells lacking ClpYQ.
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To examine whether overproduction of ClpY and ClpQ suppresses RcsARedLacZ 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 -galactosidase assay. Unexpectedly, RcsARedLacZ 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 RcsARedLacZ 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 RcsARedLacZ.
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DISCUSSION |
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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 cpsBlacZ 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
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 cpsBlacZ 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 RcsARedLacZ 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 RcsARedLacZ levels are probably due to the activity of ClpYQ.
Our results also suggest that the mucoidy of transformants overproducing the hybrid fusion protein, RcsALacZ, 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 cpsBlacZ fusion constructs to reflect its activity. As such we were able to easily modify the RcsA level, to assay cpslacZ 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.
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ACKNOWLEDGEMENTS |
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Received 25 April 2003;
revised 5 September 2003;
accepted 20 October 2003.
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