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Unité Postulante de Génétique Bactérienne et Différenciation, CNRS URA 2172, Institut Pasteur, 25 rue Dr Roux, 75724 Paris Cedex 15, France
Correspondence
Philippe Mazodier
mazodier{at}pasteur.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They all contain two distinct domains: an ATPase chaperone domain responsible for substrate recognition and unfolding, and a proteolytic domain. These domains can be either on the same polypeptide chain (Lon and FtsH) or on two different subunits: the ATPase subunits (ClpX, ClpA, ClpC or HslU) and the proteolytic subunits (ClpP or HslV).
Different substrates of Clp proteases in Escherichia coli have been identified. For many of them, Clp ATPases recognize the N- or C-terminal region of the substrate. The MuA transposase and the SsrA-tagged polypeptides, which are degraded by ClpXP, have a substrate recognition motif located at their C-terminal sequence (Gottesman et al., 1997; Levchenko et al., 1995). Recently, analysis of trapped ClpXP substrates revealed five distinct classes of ClpX-recognition motifs; one of these includes the C-terminal motif of SsrA-tagged proteins, ending with two alanine residues (Flynn et al., 2003).
Clp proteases play an important role in biological functions of the Gram-positive soil bacteria of the genus Streptomyces, a model for bacterial differentiation with regard to its complex life cycle. On solid media, spore germination leads to growth of a basal mycelium which then differentiates into an aerial mycelium and finally septates and differentiates into spores. Two classes of mutants have been characterized: bld (bald) mutants which fail to produce aerial hyphae and whi (white) mutants whose aerial hyphae fail to complete the production of normal, grey-pigmented spores (Chater, 2001). The Streptomyces lividans clpP1clpP2 mutant has a bld phenotype and is therefore unable to complete the differentiation cycle (De Crecy-Lagard et al., 1999). This suggests that one or several ClpP1 and/or ClpP2 targets need to be degraded for normal aerial mycelium formation. clpP1 and clpP2 form an operon and thus insertion of an apramycin resistance cassette in clpP1 has a polar effect on clpP2 expression. This was expected as restoration of differentiation in a clp1 bald mutant requires the introduction in trans of both clpP1 and clpP2 (De Crecy-Lagard et al., 1999). Polarity was confirmed by Western blot analysis of clpP expression; indeed, no ClpP1 nor ClpP2 can be detected in the clpP1 mutant (Viala & Mazodier, 2002). Therefore, the clpP1 mutant is referred to as the clpP1P2 mutant to underline the absence of the two proteases.
Only one S. lividans ClpP1/P2 target has been identified to date: PopR, the transcriptional activator of the clpP3P4 operon (Viala et al., 2000). PopR is primarily degraded by ClpP1/P2 and the C-terminal two alanine residues play an essential part in the degradation process (Viala & Mazodier, 2002). Recently, we have shown that ClgR, which is encoded by a gene paralogous to popR, activates expression of clpP1, clpP2, clpC1, lon and clgR (Bellier & Mazodier, 2004). ClgR and PopR DNA-binding domain regions share over 50 % amino acid sequence identity and, like PopR, ClgR has two alanine residues at its C terminus. Therefore, ClgR seemed a good candidate as a substrate for ClpP1/P2.
We investigated ClgR stability in wild-type and clpP1P2 mutant strains. We found that ClpP1/P2 is involved in ClgR degradation and that the two C-terminal alanines are required for degradation. We found that the degradation of Lon is also ClpP1/P2-dependent and that its degradation also involves the two C-terminal alanine residues. Finally, we found that the product of clpC1 accumulates in a clpP1P2 mutant as well. ClgR thus activates clpP1P2 gene expression, and ClpP1/P2 degrades several proteins encoded by genes of the ClgR regulon. Via specific proteolysis, ClpP1/P2 exerts a negative post-translational control on the ClgR regulon.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). YEME medium was used for liquid growth (Hopwood et al., 1985). NE medium (Murakami et al., 1989) and R5 (Hopwood et al., 1985) were used for Streptomyces growth on plates. Antibiotics apramycin, hygromycin and thiostrepton were added to final concentrations of 25, 100 and 25 µg ml–1 to solid medium, and 20, 50 and 10 µg ml–1 to liquid medium, respectively. Stock solutions of antibiotics rifampicin and chloramphenicol were made in 100 % methanol at 20 and 10 mg ml–1, respectively. They were added to final concentrations of 100 and 50 µg ml–1, respectively, in solid medium. E. coli TG1 (Gibson, 1984) was used as the general cloning host and was grown in LB medium. Antibiotics ampicillin and hygromycin were added to final concentrations of 100 µg ml–1.
DNA manipulation and transformation procedures.
Plasmid DNA was extracted from E. coli using a Qiagen kit. DNA fragments were purified from agarose gels with Ultrafree-DA (Amicon-Millipore). Restriction enzymes were used as recommended by the manufacturers. DNA fragments were amplified by PCR (Mullis & Faloona, 1987; Saiki et al., 1988). Standard electroporation procedures were used for E. coli transformation. Streptomyces DNA and protoplasts were prepared and transformed as described (Hopwood et al., 1985).
Plasmids and plasmid constructions.
Primer sequences used in this study are given in Table 1. Plasmids pAB54 and pAB55 for clgR overexpression in Streptomyces have been described previously (Bellier & Mazodier, 2004). To overexpress clpC1 in Streptomyces, the clpC1 gene was amplified with primers AB57 and AB58 and was inserted between the NdeI and HindIII sites of pHM11a. Two M2 Flag epitopes were added by inserting the linker resulting from annealing of primers AB118 and AB119 into the NdeI site, yielding pAB59M2. Correct insertion was checked with M2/clpC1-specific primer AB133. To overexpress lon in Streptomyces, pAB70M2 and pAB71M2 were constructed by inserting the 2350 bp fragment including the coding sequence of the lon gene obtained by PCR amplification with primers AB124 and AB125, and AB124 and AB95, respectively, inserted between the NdeI and HindIII sites of pHM11a. The N-terminal fusion with the tandem M2 flag was constructed by cloning the linker resulting from annealing primers AB118 and AB119 at the NdeI site. Insertion in the appropriate orientation was checked by hybridization with M2/lon-specific primer AB126. The PCR fragment contained in pAB71M2 modified the lon gene in such a way that two aspartic acid residues were encoded instead of two alanines before the stop codon. To express this modified lon gene in Streptomyces under its own promoter, pAB63 was constructed by inserting the 2550 bp fragment including the promoter region and the coding sequence of the lon gene obtained by PCR amplification with primers JU74 and AB96 between the EcoRI and XbaI sites of pSET152. In all the cases, the nucleotide sequences of the cloned DNA fragments were verified by sequencing.
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Real-time quantitative PCR.
Primers were designed with BEACON Designer software. clpP1, clpC1 and hrdB expression were detected with primers MG12 and MG13, MG8 and MG9, and MG14 and MG15, respectively. RNA (10 µg) was treated twice with 30 U RNase-free DNase I (Roche) for 30 min at 37 °C. DNase was removed by applying the sample to an RNeasy mini column (Qiagen) following the RNA clean-up protocol. The RNA sample was eluted in 30 µl deionized water. cDNA synthesis was performed with random hexamers (Roche) using SuperScript II RT (Invitrogen) according to the protocol recommended by the manufacturers. Real-time quantitative PCR was performed in a 25 µl reaction volume containing cDNA, 12·5 µl SYBR PCR master mix (Applied Biosystems) and 1 µl gene-specific primers (10 µM). Amplification and detection of specific products were performed with the iCyclerIQ Multi-Colour real-time PCR detection system (Bio-Rad) with the following cycle profile: one cycle at 95 °C for 3 min followed by 40 cycles at 95 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s. The specificity of the amplified product was verified by generating a melting curve with a final step of 80 cycles of 10 s at an initial temperature of 55 °C, increasing 0·5 °C each cycle up to 95 °C. Loss of fluorescence was observed at the denaturing/melting temperature of the product (Ririe et al., 1997). To check whether contaminating chromosomal DNA was present, each sample was tested in control reactions that did not contain reverse transcriptase. For each condition, quadruple assays were done. The analysis gave a threshold cycle (CT) value for each sample, which is defined as the cycle at which a significant increase in amplification product occurs. The CT value was calculated for each quadruple reaction. A 
CT value was then calculated for each sample by subtracting the mean CT value of the target gene from the mean CT value of the hrdB reference gene (hrdB encodes an essential and constitutively expressed 
factor; Kelemen et al., 1996). The data were transformed from an exponential to a linear scale by using the formula x=2–CT (Livak & Schmittgen, 2001).
Protein extraction and Western blotting experiments.
Cultures of S. lividans 1326 carrying pHM11a (control), pAB54, pAB55, pAB70M2, pAB71M2 or pAB63 were grown on cellophane discs laid down on the surface of solid NE plates. Proteins were prepared from mycelia of the different strains at different stages of growth (basal mycelium, aerial mycelium, sporulation). Mycelium was resuspended in sonication buffer (20 mM Tris, 5 mM EDTA, 1 mM 
-mercaptoethanol, 0·5 mM PMSF) and lysed by sonication. The resulting suspension was centrifuged for 15 min at 4 °C and 20 800 g, and the supernatant was treated with 0·3 % SDS for 5 min at 85 °C. The sample was centrifuged for 15 min at 4 °C and the protein concentration of the supernatant was determined by the method of Bradford (1976). Ten micrograms of protein extract was subjected to SDS-PAGE as described by Laemmli (1970). The proteins were transferred to a nitrocellulose membrane (Hybond C), which was then probed with rabbit polyclonal anti-Streptomyces ClgR (1 : 500) (Agro-Bio), anti-Streptomyces ClpP1 (1 : 10 000), anti-recombinant Synechococcus ClpC (1 : 1000) (Agrisera) (ClpC antibodies were raised against a 13 amino acid epitope of Synechococcus ClpC present in ClpC1 but not in other Streptomyces ClpC), anti-Streptomyces Lon (1 : 5000) or anti-Flag M2 mAbs (Sigma). Signals were detected with the ECL Western Blotting Detection Kit (Amersham Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The level of ClgR-AA is significantly increased in the clpP1P2 mutant compared to the wild-type strain. Moreover, ClgR-DD accumulates to a very high level in the wild-type strain, to a level that is quite similar to that of ClgR-AA in the clpP1P2 mutant.
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clpP1 and clpC1 expression during the cell cycle
In a previous paper (Bellier & Mazodier, 2004), we showed that the ClpP1 level is constant through the differentiation cycle in the wild-type strain, while it is drastically increased in the strain overexpressing clgR-AA and even more in the strain overexpressing clgR-DD. However, we also showed that the ClpC1 protein is only detectable at the beginning of the life cycle in the wild-type strain, but is overproduced in the strains overexpressing clgR-AA and clgR-DD, although the signal decreases over time. To understand if these differences between ClpP1 and ClpC1 levels reflect a different expression profile, clpP1 and clpC1 expression were measured by RT-PCR using RNAs extracted from plate cultures (Fig. 2). In the pAB54 strain (clgR-AA overexpression), expression of clpP1 and clpC1 was higher at the basal mycelium stage and then decreased throughout the developmental cycle. In the pAB55 strain (clgR-DD overexpression), clpP1 and clpC1 were strongly expressed at the basal and aerial mycelium stages and reached a low level of expression during sporulation. Therefore, clpP1 and clpC1 show a relatively similar expression profile, contrary to their protein patterns. The differences detected at the protein level might be due to post-translational controls.
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). While ClpC1 rapidly faded away on rifampicin and chloramphenicol plates, ClpP did not disappear.
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). Only the processed form is stable. ClpP1 is still detected after 2 h in these conditions. Our results indicate that ClpP1 is quite stable, whereas ClpC1 appears to be labile.
ClpP1/P2-dependent ClpC1 degradation
As two components of the ClgR regulon accumulated in the clpP1P2 mutant, we looked at ClpC1 stability in the wild-type and clpP1P2 strains harbouring pAB59M2, where clpC1 is under the control of the erm*p promoter, so that the ClgR stability in a clpP1P2 mutant would not interfere with clpC1 expression. Protein levels in crude extracts from liquid cultures were determined by Western blotting with monoclonal anti-M2 antibodies (Sigma) (Fig. 4). M2-ClpC1 levels were higher in the clpP1P2 mutant than in the wild-type strain, suggesting that ClpC1 might be a substrate of the Clp proteases.
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). In the wild-type strain, Lon is present at a high level in the basal mycelium stage and then disappears. However, in the pAB54 and pAB55 strains, no obvious effects of clgR overexpression on Lon protein levels could be detected.
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Lon: a new target for ClpP1 proteolysis
To distinguish between transcriptional and post-translational ClpP1-dependent control of Lon levels, the lon gene was cloned under the strong erm*p promoter. Moreover, to be able to specifically detect the Lon protein, whose expression is controlled by erm*p, but not the endogenous Lon protein, two M2 Flag epitopes were added to the N terminus of the Lon protein. Finally, to test if the two alanines could be the substrate degradation motif for Lon, both lon-AA (native gene) and lon-DD genes were cloned.
Protein levels from crude extracts of liquid cultures were tested by Western blot experiments with monoclonal anti-M2 antibodies (Fig. 6). The level of M2-Lon-AA is higher in the clpP1P2 mutant than in the wild-type strain. The M2-Lon-DD levels were higher than the M2-Lon-AA levels, but were about the same in the wild-type strain and in the clpP1P2 mutant.
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Effects of ClgR on the stabilized Lon-DD protein
To discriminate the post-translational effects (i.e. degradation of Lon by ClpP1/P2) from the transcriptional effects (i.e. activation of the lon promoter by ClgR) of clgR overexpression on lon expression, the lon-DD gene was cloned under its own promoter in an integrative vector to give pAB63. Lon protein levels were then tested by Western blotting on plate cultures in wild-type or in pAB55 (clgR-DD) strains harbouring pAB63 (Fig. 7). As observed in the wild-type strain, Lon-DD was only present at the beginning of the developmental cycle. However, in the pAB55 strain, the Lon-DD protein was detected throughout the developmental cycle, suggesting that the disappearance of Lon observed previously in the ClgR-DD strain (Fig. 6
) was due to its degradation, and that the absence of Lon persistence in the wild-type strain expressing lon-DD is due to a shut-off of ClgR-dependent transcription after the basal mycelium stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, we identified three new targets of ClpP1/P2-dependent proteolysis: ClgR, ClpC1 and the Lon protease, all encoded by genes that belong to the ClgR regulon. These observations point to the existence of an interactive network within the ClgR regulon members: ClgR activates clpP1 expression and, in response, ClpP1/P2 degrades ClgR, allowing a negative feedback control of ClgR activation.
Three targets of ClpP proteases in Streptomyces, PopR, ClgR and Lon, all end with two alanines preceded by a hydrophobic residue (leucine for PopR and valine for ClgR and Lon) and in each case, the two alanine residues have been shown to be crucial for their degradation by ClpP1. Indeed, replacement of these two C-terminal residues with two aspartates greatly increased the stability of these proteins. Several previously described Clp targets also end with two alanines: the SsrA tag (Gottesman et al., 1998), the LexA autocleavage C-terminal fragment (Neher et al., 2003) and the essential response regulator CtrA in Caulobacter crescentus (Domian et al., 1997). It was therefore tempting to speculate that, in Streptomyces, the two alanine residues at the C terminus could be sufficient to target proteins for ClpP1- and/or ClpP2-dependent degradation. However, we have shown that the protein encoded by the Streptomyces coelicolor SCO3558 gene, homologous to cicA, which encodes a phosphotransferase in Cau. crescentus and which ends with two alanines preceded by a hydrophobic residue (proline) in Streptomyces, does not accumulate in a clpP1P2 mutant (data not shown). Therefore, in Streptomyces, a hydrophobic residue followed by two alanines at the C terminus is not sufficient to target proteins for Clp degradation. We cannot exclude that there may be other features in the sequences, besides these three C-terminal residues, that play an important role for degradation by ClpP1 and ClpP2 in Streptomyces. Moreover, the ClpC1 protein, which also accumulates in a clpP1P2 mutant, does not end with two alanines, confirming that there are probably many different Clp recognition motifs.
Clp-dependent degradation of ClgR might be conserved in all Actinomycetes. Indeed, ClgR was recently shown to be stabilized in a Corynebacterium glutamicum clpP1P2 mutant (Engels et al., 2005). However, the motif responsible for its degradation is probably different since Cor. glutamicum ClgR does not end with two alanines. Moreover, the motif is probably not located at the C terminus since the addition of 10 amino acid residues did not interfere with its degradation (Engels et al., 2004).
We have shown that ClpC1 accumulates in a clpP1P2 mutant. Degradation of Clp subunits by Clp proteases has already been shown. Indeed, in E. coli, the ClpA subunit strongly accumulates in a clpP mutant (Gottesman et al., 1990), and in Bacillus subtilis, ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remain almost stable in a clpP mutant, suggesting ClpP-dependent degradation (Gerth et al., 2004). Moreover, here we show the degradation of Lon by the Clp protease for the first time. Therefore, the levels of ATP-dependent proteases in the cell seem to require fine tuning, combining transcriptional and post-translational regulation. This is in agreement with the control of lon expression by both ClgR and HspR (Sobczyk et al., 2002), probably allowing a flexible response to a variety of signals.
ClgR is not the only example of a regulator that activates expression of proteases responsible for its own degradation. In S. lividans, PopR degradation is primarily dependent on ClpP1 and ClpP2, but can also be achieved by ClpP3/P4, whose expression is activated by PopR (Viala & Mazodier, 2002). In E. coli, the 32 heat-shock transcriptional sigma factor controls ftsH expression and is itself degraded by FtsH (Blaszczak et al., 1999). More generally, degradation of transcriptional activators by ATP-dependent proteases appears to be quite common. In E. coli, the RcsA capsular biosynthesis transcriptional activator is degraded by Lon (Torres-Cabassa & Gottesman, 1987) and the S stress sigma factor is degraded by ClpXP (Schweder et al., 1996), and in Cau. crescentus the CtrA essential regulator is degraded by ClpXP (Jenal & Fuchs, 1998). For regulatory proteins, control by proteolysis allows a rapid reduction of their cellular levels in response to a specific signal, triggering the corresponding response. For example, the 32 and S sigma factor levels rapidly increase after stress, and the CtrA regulator accumulates and then drastically decreases at specific stages of the Cau. crescentus cell cycle. We could assume that ClpP-dependent proteolysis of ClgR is achieved until some stress or specific environmental condition occurs.
ClpP1/P2 may not be the only proteases involved in ClgR and Lon degradation, since in Figs 1 and 6, levels of ClgR-DD and Lon-DD are higher than levels of ClgR-AA and Lon-AA in the clpP1/P2 mutant. Therefore, some proteins ending with -AA are still degraded in the clpP1/P2 mutant. The protease involved in this phenomenon is not known. This protease is not Lon, as ClgR-AA is not stabilized in the lon mutant (data not shown). However, several other proteases, such as FtsH, should be tested for their ability to degrade ClgR and/or Lon.
The stress or input signal has not been identified yet. However, the cinA gene (encoding a homologue of the Streptococcus pneumoniae competence-induced protein) located upstream from clgR, belongs to the disulfide stress R regulon (Paget et al., 2001). The intergenic region is 120 bp long and there is no obvious transcription terminator between the two genes, so it is conceivable that they form an operon. clpP1 expression is not induced by heat shock (Viala et al., 2000), but the ClpP1 complex is likely to be involved in misfolded protein degradation under other stress conditions, which could lead to an increase of ClgR stability, and consequently clgR expression. Analogous mechanisms have been described, such as the HspR regulon, where the DnaK chaperone acts as a transcriptional co-repressor by binding to the HspR repressor. Under heat-stress conditions, DnaK is recruited by misfolded proteins and the HspR regulon is therefore induced (Bucca et al., 2000).
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ACKNOWLEDGEMENTS |
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Received 4 October 2005;
revised 17 January 2006;
accepted 17 January 2006.
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