Unité de Biochimie Microbienne, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France1
VIVALIS SA, CHU de Nantes Hôtel-Dieu, Place A. Ricordeau, 44093 Nantes Cedex 1, France2
Author for correspondence: Philippe Mazodier. Tel: +33 1 45 68 88 42. Fax: +33 1 45 68 89 38. e-mail: mazodier{at}pasteur.fr
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ABSTRACT |
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Keywords: heat shock regulation, Streptomyces, HspR, protease, DnaK
Abbreviations: HSP, heat-shock protein
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INTRODUCTION |
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The ATP-dependent proteases are central enzymes in the regulation of differentiation in several bacteria. Indeed, in E. coli FtsH degrades the specific heat-shock sigma factor, 32 (Herman et al., 1995
), and ClpXP degrades the specific stationary-phase sigma factor,
S (Schweder et al., 1996
). In Bacillus subtilis, ClpP degrades ComK, the central regulator of competence (Turgay et al., 1998
), and at low pHs Lon degrades the sporulation sigma factor,
H (Liu et al., 1999
). The Lon protease also plays an essential role in the formation of asymmetrical flagella in Caulobacter crescentus (Wright et al., 1996
).
Streptomycetes are model bacteria for the study of differentiation mechanisms. In vitro these soil bacteria follow a differentiation cycle that lasts about a week. The germinated spores form basal or vegetative mycelia. A few days later, aerial mycelia are formed from these structures. These new mycelia partition themselves to form chains of spores that are dispersed following maturation (Hopwood, 1999 ). The interest of Streptomyces as a model for studying differentiation is reinforced by the fact that this morphological phenomenon is generally accompanied by the production of secondary metabolites. Currently, 70% of industrially produced antibiotics come from these bacteria.
Regulatory processes associated with spore formation from aerial hyphae and germination take place in cells that do not divide and thus the pre-existing regulatory proteins cannot be diluted. Two types of mechanism can be used to palliate the absence of dilution: the activation/inactivation of central regulators by modification (for example by phosphorylation or methylation), or specific degradation. These considerations led us to study the role of ATP-dependent proteases in the control of the cell cycle (De Crecy-Lagard et al., 1999 ; Viala et al., 2000
).
The ATP-dependent proteases Clp, Lon and FtsH are heat-shock proteins (HSPs) in most bacterial species and their regulation is well documented in E. coli and B. subtilis. Although the induction of HSPs is a universal response, a number of mechanisms control HSP synthesis in different organisms. The transcription of heat-shock genes is regulated by both positive and negative mechanisms. In bacteria, the regulation of the heat-shock response was first studied in E. coli and shown to rely on the level and activity of specific sigma factors, 32 and
24 (for reviews see Bukau, 1993
; Yura et al., 1993
). These sigma factors are required for the recognition of specific heat-shock promoters associated with heat-shock genes by the RNA polymerase. The regulation of expression was shown to depend largely on the stability of the sigma factor. Thus, an increase in temperature leads to a rapid increase in the level of active
32 due to an increase in the synthesis of this molecule and its stabilization. At 30 °C, the DnaK chaperone system destabilizes
32 and sequesters it in an inactive state that can be degraded by the FtsH protease (Herman et al., 1995
). Heat shock causes the denaturation of cellular polypeptides: the DnaK system binds these misfolded polypeptides and releases
32, in a mechanism allowing positive feedback regulation.
This general dependence on sigma factors for heat-shock regulation is not conserved in prokaryotes. Indeed, in most organisms, important hsp genes are controlled exclusively by specific repressors. This is well documented in Bacillus and Streptomyces. In Streptomyces, the synthesis of major HSPs, such as the widespread molecular chaperones DnaK, ClpB, GroEL and HSP18, is negatively controlled at the transcriptional level by at least three different repressors. The control of groE gene expression involves an inverted-repeat element (called CIRCE) that is highly conserved among eubacteria, and the HrcA repressor (Grandvalet et al., 1998 ). The dnaK operon and clpB belong to the HspR/HAIR regulon (Bucca et al., 1997
; Grandvalet et al., 1999
). The HspR repressor-HAIR operator system is used in some bacteria (Spohn & Scarlato, 1999
) but is not widespread. In particular, it is not used in low-G+C Gram-positive bacteria.
In this study, the genome sequence of S. coelicolor (www.sanger.ac.uk/Projects/S_coelicolor/) was searched for the HAIR motif CTTGAGT-N7-ACTCAAG. A HAIR sequence was found upstream of a gene closely related to the lon gene. We demonstrated that lon belongs to the HspR/HAIR regulon.
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METHODS |
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pIJ8600 was used for controlled gene expression in S. lividans using the thiostrepton-inducible promoter pTipA (Sun et al., 1999 ). pGM160
(Muth et al., 1989
) was used to construct the disruption derivatives. Thiostrepton, viomycin and hygromycin were added to plates at final concentrations of 30 µg ml-1, 25 µg ml-1 and 250 µg ml-1, respectively, as appropriate. Cassettes containing resistance genes (Blondelet-Rouault et al., 1997
) were used for gene disruption experiments. E. coli TG1 (Gibson, 1984
) was used as the general cloning host and E. coli strains were grown in Luria-Bertani (LB) broth supplemented with 200 µg hygromycin ml-1, 10 µg viomycin ml-1 or 100 µg ampicillin ml-1 when needed. pUC19, pUC18 (Yanisch-Perron et al., 1985
) and pBluescript-SK were used as cloning vectors in E. coli.
DNA manipulation and plasmid construction.
Standard cloning procedures were used to produce all plasmids (Sambrook et al., 1989 ). Restriction and modification enzymes were used according to the manufacturers recommendations.
Cloning of the lon gene of S. lividans.
A pair of oligonucleotides, JU74 (5'-GAAGAATTCTACGGCGGTGCTGTCCCGAGA-3') and JU76 (5'-AAGAAGCTTCCAACGGCTGACGGCTCCTCC-3'), were designed based on the sequence of the S. coelicolor lon gene (Sanger Centre, Cambridge, UK; http://www.sanger.ac.uk/Projects/S_coelicolor/). These oligonucleotides were used to amplify the lon locus from S. lividans chromosomal DNA. We cloned a 3 kb PCR-amplified fragment containing the S. lividans lon locus and its promoter region. The PCR fragment was digested with EcoRI and HindIII and cloned into EcoRI/HindIII-digested pUC19 to generate pJV300. The 3 kb insert was sequenced and was found to contain one consensus HAIR motif centred 30 bp upstream of the putative lon start codon.
Expression of lon in E. coli.
A 2·4 kb fragment containing the lon coding sequence from S. lividans was amplified by PCR using oligonucleotides AS58 (5'-ATACCATGGCTGCTGAGTCCGCCGCCTTC-3') and AS59 (5'-ATACTCGAGCGCTGCGACCGGAACCTCACG-3'). The PCR fragment was digested with NcoI/XhoI and cloned into NcoI/XhoI-digested pET28a vector to yield pAS45. This plasmid allowed the overexpression of Lon under the control of the T7 promoter. A translational fusion added six carboxy-terminal histidine residues to Lon, which allowed affinity purification of the protein on a nickel column. Purified Lon was used to obtain antibodies in rabbit (carried out by Eurogentec).
Overproduction of HspR in E. coli.
The DNA fragment containing S. lividans hspR was amplified by PCR using oligonucleotides AS7 (5'-ATACATATGGACGGTCGGCGACGCAACCCG-3') and AS11 (5'-ATAAGATCTTCAGTCCGAGGACTGGCCGCG-3'). These primers introduced NdeI and BglII sites into the resulting 450 bp DNA fragment. The NdeI site replaced the original hspR GTG translation start site with an ATG. The NdeIBglII DNA fragment was cloned into the NdeI and BamHI sites of pET28a (Novagen) to yield pAS16, in which the T7 promoter was used to control gene expression. A translational fusion added 20 residues, including six histidines, to the amino terminus of HspR.
Purification of Lon protein to homogeneity and production of antibodies.
E. coli BL21(DE3)(pAS45) cells were grown at room temperature in LB medium containing kanamycin and chloramphenicol. When cultures reached an OD600 of 0·6, the production of Lon-His6 was induced by the addition of 0·1 mM IPTG. After 3 h cells were harvested and washed twice in buffer A (20 mM phosphate buffer pH 7·4, 500 mM NaCl, 20 mM imidazole, 1 mM DTT and 10%, v/v, glycerol). The cell pellet was resuspended in 20 ml buffer A supplemented with one tablet of Complete protease inhibitor cocktail (Boehringer Mannheim). Cells were disrupted in a French press (9000 p.s.i., 62 MPa). The soluble fraction was obtained by centrifugation at 4 °C and 30000 g and was loaded onto a 1 ml Ni-NTA column (Qiagen) that had been equilibrated with buffer A. Lon-His6 was eluted with a linear 20300 mM imidazole gradient. The fractions with the highest protein concentration were pooled, dialysed for 2 h against buffer A and centrifuged at 13000 g for 5 min. The supernatant (5 ml) was loaded onto the Ni-NTA column for a second identical run. The second protein pool was dialysed against storage buffer C (20 mM phosphate buffer pH 7·4, 50 mM NaCl and 10% glycerol) and aliquots were stored at -80 °C. Lon-His6 concentration was determined by the Bradford method. This purified protein was used to raise polyclonal antibodies against Lon.
lon-disrupting plasmid.
The BamHI cassette containing the hygromycin-resistance gene purified from pHP45HygR (Blondelet-Rouault et al., 1997
) was used to disrupt the S. lividans lon gene. The BamHI
HygR cassette was ligated into the lon BamHI site in pAS45, 878 bp downstream from the lon ATG codon. Two plasmids were generated: pAS47 and pAS48. The hygR gene was transcribed in opposite direction to lon in pAS48. pAS48 was further digested with XbaI/NcoI. The sites for these enzymes are located close together in pAS48. Thus, we cloned the whole pAS48 plasmid between the NcoI and XbaI sites of pGM160
. The resulting S. lividans replication-thermosensitive plasmid was called pAS49 and was used to disrupt lon.
hspR-disrupting plasmid.
Inverse-PCR (I-PCR) was used to clone hspR and large DNA fragments of its surrounding sequences from S. lividans. This allowed the region 3' of hspR to be cloned in the absence of sequence data and also created an EcoRI site 100 bp downstream of the hspR translation start site (ATG) into which a resistance gene could be inserted. We used the pHP45VioR (Blondelet-Rouault et al., 1997
) cassette containing the viomycin phosphotransferase gene (vph) from Streptomyces vinaceus for disruption experiments. S. lividans chromosomal DNA (5 µg) was digested with PstI and ligated after dilution to facilitate the intramolecular ligation of the DNA fragments. Two divergent oligonucleotides were designed, AS1 (5'-ATAGAATTCTGGCGCAGCGTCTGCGGGTG-3') and AS2 (5'-ATAGAATTCCGCGACATCGAACTGCTCCG-3'), based on published S. coelicolor (cosmid H35, Sanger Centre sequencing project) and S. albus (Grandvalet et al., 1997
) hspR sequences. PCR was performed on 500 ng ligated S. lividans TK24 chromosomal DNA, 20 pmol AS1 and AS2, 10% DMSO, 200 mM of each dNTP and 1 U Pfu DNA polymerase (Promega) as recommended by the manufacturer. PCR generated a 5·5 kb I-PCR fragment with enough DNA sequence upstream and downstream of hspR to favour double recombination in S. lividans. After purification, the PCR fragment was digested with EcoRI and cloned into pBluescript-SK to generate pAS2. The two hspR DNA fragments obtained after an EcoRI/PstI digestion of pAS2 were purified separately, ligated to the EcoRI viomycin cassette and cloned into the PstI site of pBluescript-SK(-) to give pAS3. pAS3 was selected for its ability to confer both ampicillin and viomycin resistance to E. coli. Inserts from pAS2 and pAS3 were partly sequenced to confirm the constructs. In pAS3, the vph gene is transcribed in the opposite direction to hspR. pAS3 was further digested with XbaI/NcoI to give a 7·6 kb DNA fragment encompassing the following S. lividans sequences: the 3' end of grpE, the dnaJ gene, disrupted hspR and a 3 kb region downstream of hspR. This DNA fragment was cloned into the XbaI/NcoI sites of pGM160
to yield the S. lividans replication-thermosensitive hspR-disrupting plasmid, pAS25.
Transformation procedures and screening of mutants.
The lon gene from S. lividans was disrupted after transformation of protoplasts with pAS49. The TK24 protoplasts were prepared and transformed as described by Hopwood et al. (1985) . After 24 h incubation thiostrepton was added to the plates. TsrR colonies were tested for the presence of hygromycin resistance. Two doubly resistant clones were incubated in 10 ml YEME medium supplemented with hygromycin and grown for 3 days at 30 °C before the crossover selection. To obtain a mutant due to homologous recombination at the lon locus, 10 ml YEME without antibiotics was inoculated with a drop of the pre-culture. The culture was incubated for 3 days with vigorous shaking at 40 °C to prevent pAS49 from replicating. Different dilutions (1/104, 1/105 and 1/106) of cells were plated onto R5 medium supplemented with hygromycin. HygR clones were finally patched on both R5+hygromycin and R5+thiostrepton plates to select HygR and TsrS clones. Insertion of the hygromycin cassette in the lon gene was controlled by a series of PCR amplifications using oligonucleotides specific to lon and to the cassette.
A S. lividans lon hspR double mutant was obtained by transforming protoplasts of the S. lividans lon mutant with the hspR-disrupting plasmid, pAS25. The mutants were selected as above but with viomycin instead of hygromycin.
RNA analysis.
RNA was prepared as described previously (Servant & Mazodier, 1996 ). The transcription start site upstream of lon was located by primer extension using oligonucleotide AB10 (5'-GTCGATGCGCGGGACGAGGAG-3') as previously described (Grandvalet et al., 1999
).
Gel retardation assay.
A 300 bp DNA fragment encompassing 200 bp of lon promoter region and 100 bp of the 5' end of the lon coding sequence was PCR-amplified with oligonucleotides JU74 and JU75 (5'-GCGGGGCTTGCCGGGCTCGGA-3'). The purified fragment was end-labelled with [-32P]ATP by the T4 polynucleotide kinase method. Crude extracts with or without overexpressed HspR in E. coli (IPTG induction) were incubated with the labelled probe for 15 min at 25 °C. These were incubated in 10 µl gel-shift buffer (10 mM Tris/HCl pH 7·5, 10 mM MgCl2, 1 mM DTT, 0·1% Triton X-100, 50 mM NaCl and 10% glycerol) and 1 µg sonicated herring sperm DNA. Samples were subjected to electrophoresis in 6% polyacrylamide gels containing 50 mM Tris/HCl pH 8, 400 mM glycine, 1·7 mM EDTA and 2·5% glycerol. Samples were separated for 1 h at 100 V. Finally, the gels were dried and exposed to film.
Western immunoblot analysis.
Total protein extracts were prepared from S. lividans wild-type strain TK24 or S. lividans mutants after being grown in YEME, supplemented with the appropriate antibiotic if needed. Cells were incubated for 24 h at 30 °C, and then samples were subjected to a 40 min heat shock at 40 °C. Proteins were separated on polyacrylamide denaturing gels (10 % SDS-PAGE) before being electrotransferred to Immobilon membranes (Amersham). Antigens were detected by ECL Western blotting in the presence of rabbit polyclonal antibodies raised against purified S. lividans Lon-His6.
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RESULTS |
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Cloning of the lon gene of S. lividans
A pair of oligonucleotides (JU74 and JU76) were designed based on the sequence of the S. coelicolor lon gene and used to amplify the lon locus from S. lividans chromosomal DNA. The gene was cloned in pUC19, yielding pJV300. Partial DNA sequence determination confirmed the clone and showed more than 99% identity with lon of S. coelicolor.
lon mutant
The chromosomal lon gene was disrupted by a double recombination event using the pAS49 vector containing lon::hygR. Candidate HygR TsrS clones were analysed. The correct integration of hygR in chromosomal lon was checked by PCR using pairs of oligonucleotides annealing to the hyg cassette and to the lon chromosomal locus outside the region cloned in pAS49.
The wild-type strain grew faster than the lon mutant on all the liquid and solid media tested at 30 °C (NE, R5 YEME). However, the lon mutant formed aerial mycelium on plates 30 h after the wild-type and ultimately sporulated. Spores of the lon mutant failed to form colonies on NE plates after 1 week at 40 °C, whereas the wild-type produced colonies within 23 days, suggesting that the germination process is thermosensitive in the mutant (data not shown).
Regulation of expression: lon hspR double mutant
To confirm the role of HspR in the regulation of lon, a lon::hygR hspR::vioR double mutant was constructed. The chromosomal hspR was disrupted by a double recombination event using the pAS25 vector containing hspR:vioR and the procedure described above used to disrupt lon except that candidate VioR TsrS clones were analysed.
Bucca et al. (1997) attempted insertion by a double crossover event of the hygromycin-resistance gene into the 5' end of hspR in S. lividans, but without success. Only the entire mutating plasmid could be integrated, leading to a construction containing the mutated hspR gene and an intact copy of hspR. Our attempts to use pAS25 to integrate a viomycin cassette into the middle of hspR in S. lividans by a double crossover event failed repeatedly in the wild-type strain; however, as, shown here, the hspR::vioR mutant was obtained readily in the lon mutant. These results indicate that a high level of lon expression in Streptomyces may be toxic for the cell (i.e. long-term full induction due to a complete knockout mutation of hspR). Toxicity related to lon overexpression will be investigated in future work.
Western blot of Lon
Proteins extracted from the wild-type strain, the lon mutant and the lon hspR double mutant grown at 30 °C or subjected to heat shock were analysed by Western blotting using anti-Lon antibodies. In the wild-type a 90 kDa heat-inducible protein was detected (Fig. 1, lanes A and B). In the lon mutant the 90 kDa Lon protein was not detected (lanes C and D), but a 30 kDa peptide, corresponding to the predicted (29·5 kDa) truncated Lon peptide, was present. In the lon mutant, this 30 kDa protein was heat induced, whereas in the lon hspR double mutant it was constitutively synthesized at 30 °C and 40 °C (lanes E and F).
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DISCUSSION |
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The co-production of the Lon protease and the DnaK and ClpB chaperones may present some advantages. Indeed, the Lon protease and the DnaK system have been reported to act in synergy in E. coli, since chaperones detect misfolded proteins that will be either refolded or degraded (Tomoyasu et al., 2001 ). The co-chaperone DnaJ has also been reported to be necessary for the Lon-dependent degradation of some abnormal proteins by keeping the substrates soluble (Jubete et al., 1996
).
The HAIR/HspR regulon is not widespread, but it is found in other actinomycetes. HspR controls expression of the hsp70 and clpB genes in Mycobacterium tuberculosis and Mycobacterium leprae, but these bacteria do not contain any gene orthologous to lon. However, lon orthologues have been found in other mycobacteria, such as Mycobacterium smegmatis (Roudiak et al., 1998 ), and genome analysis revealed HAIR motifs upstream of lon in M. smegmatis, suggesting that the HAIR/HspR regulation of lon may be widespread among actinomycetes.
Although Streptomyces spp. consistently display two or more paralogues for many genes, the complete genome sequence surprisingly shows that there is only one copy of lon in S. coelicolor. In contrast, in bacteria that generally have a lower number of paralogues, such as B. subtilis and C. crescentus, there are two copies of lon and they have different roles in the cell (Serrano et al., 2001 ). Likewise, Myxococcus xanthus also has two copies of the lon gene: lonV, which is essential for vegetative growth (Tojo et al., 1993a
), and lonD, which is required for development (Tojo et al., 1993b
).
A lon mutant has previously been constructed in Mycobacterium smegmatis (Knipfer et al., 1999 ). This mutant displayed wild-type growth rates, whereas we observed that the growth rate of Streptomyces was reduced by the lon mutation.
S. lividans has been used industrially as a host for expression of several heterologous proteins (Pozidis et al., 2001 ). Utilization of the lon mutant of S. lividans should be considered when low yield of production points to proteolytic degradation of the protein of interest.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 November 2001;
revised 30 January 2002;
accepted 13 February 2002.