Department of Veterinary Microbiology, Stigboejlen 4, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark1
Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL, USA2
Author for correspondence: H. Ingmer. Tel: +45 3528 2773. Fax: +45 3528 2757. e-mail: ingmer{at}biobase.dk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Salmonella typhimurium, Clp protease, RpoS
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Escherichia coli, several ATP-dependent proteases have been characterized (for reviews see Gottesman, 1996 ; Miller, 1996
; Porankiewicz et al., 1999
). Among these is the Clp protease, which together with Lon accounts for up to 80% of the protein degradation in the cell (Goldberg et al., 1994
; Laskowska et al., 1996
; Porankiewicz et al., 1999
). The Clp protease complex consists of a proteolytic component, ClpP, to which substrate specificity is conferred through association with either of the ATPases, ClpA or ClpX. Beside their functions in proteolysis, both ClpA and ClpX possess chaperone-like activities (Wawrzynow et al., 1995
; Wickner et al., 1994
). The Clp protease complex mediates the turnover of specific short-lived regulatory proteins (Mhammedi-Alaoui et al., 1994
; Schweder et al., 1996
), among them the stationary-phase sigma factor,
S (RpoS). RpoS regulates the expression of more than 50 genes in the response to stress or the entry into stationary phase (Hengge-Aronis, 1996
, 2000
; Loewen & Hengge-Aronis, 1994
; Schweder et al., 1996
). During exponential growth the level of RpoS is low, in part due to degradation by ClpXP (Lange & Hengge-Aronis, 1994
). When cells enter stationary phase or encounter various stress conditions, the concentration of RpoS increases as a result of greater resistance to degradation by ClpXP (Schweder et al., 1996
; Webb et al., 1999
; Zgurskaya et al., 1997
).
Components of the Clp complex are highly conserved in prokaryotic cells (Maurizi et al., 1990a ; Wawrzynow et al., 1996
). In Gram-positive bacteria ClpP is required for survival under various kinds of stress (Frees & Ingmer, 1999
; Msadek et al., 1998
) and it has been shown that ClpP participates in the degradation of misfolded proteins generated under these conditions (Frees & Ingmer, 1999
; Gaillot et al., 2000
; Kruger et al., 2000
). In Gram-negative bacteria the role of ClpP during stress is less clear, as indicated by the lack of obvious phenotypes of an E. coli clpP mutant (Maurizi et al., 1990b
). In recent studies mutants were generated in the clpP gene of Salmonella enterica serovar Typhimurium (referred to as Salmonella typhimurium) (Hensel et al., 1995
; Webb et al., 1999
; Yamamoto et al., 2001
). S. typhimurium is a facultative intracellular pathogen that upon contact with host cells can promote its own entry (Galan, 1996
). In this organism clpP is required for virulence in a mouse assay (Hensel et al., 1995
; Webb et al., 1999
; Yamamoto et al., 2001
) and for growth and survival within peritoneal macrophages (Yamamoto et al., 2001
). ClpP has also been found to be involved in the regulation of flagellum biosynthesis in S. typhimurium, and the lack of ClpP leads to a hyper-flagellate cell (Tomoyasu et al., 2002
).
Since S. typhimurium encounters various hostile conditions during the infection process (Foster & Spector, 1995 ), we were prompted to investigate the importance of ClpP for growth in the presence of stress. We find that clpP mutant cells have a reduced ability to grow compared to wild-type cells when exposed to high temperature, low pH or a high salt concentration. Furthermore, we demonstrate that the clpP mutant degrades puromycyl-containing polypeptides to a lesser extent than the wild-type, indicating that S. typhimurium ClpP is important for the degradation of misfolded proteins generated when exposed to stress.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Construction of a S. typhimurium clpP deletion mutant.
Using a replacement recombination technique, a recombinant strain of S. typhimurium C5 carrying an 80 amino acid in-frame deletion of clpP was constructed. By PCR amplification of chromosomal S. typhimurium DNA a 750 bp fragment carrying part of the upstream region of clpP was obtained using the primers ClpP-B1 (5'-AGTAGATCTCGTCTGCTTACGAAGATCC-3') and ClpP-Ec1 (5'-AGAGAATTCCTGTCCCATACAAATGGTGC-3'), while a 642 bp fragment carrying the downstream part of clpP was obtained using the primers ClpP-Ec2 (5'-CTCGAATTCCCTGAAGCGGTAGAATACG-3') and ClpX-H1 (5'-CCTAAGCTTACGCCATTGCTGGTATCG-3').
The two fragments were digested with EcoRI/BglII and EcoRI/HindIII, respectively, and cloned into the BamHIHindIII sites of the thermosensitive vector pTSA29 (Phillips, 1999 ), resulting in the plasmid pLT11, carrying a 1376 bp insert.
S. typhimurium C5 was transformed with pLT11 by electroporation and integration was promoted by incubation at 42 °C in the presence of ampicillin. To excise the plasmid from the chromosome, the integrants were grown overnight at 30 °C and plated in the presence of ampicillin. The excised plasmid was cured by incubation of the strain in the absence of antibiotics at 42 °C. Forty ampicillin-sensitive colonies were analysed by PCR to identify mutants with an internal deletion. One colony gave a single 1376 bp fragment, corresponding to the clpP gene with a 240 bp deletion; the wild-type strain C5 gave a single 1616 bp fragment (data not shown). The correct construction of the resulting clpP mutant (LT1100) was verified by sequencing the clpP gene.
Growth experiments.
Growth was followed by diluting cultures (grown overnight at 37 °C in LB) 100-fold into LB and incubating either at 37 °C, at 45 °C, at 37 °C with 5% NaCl, or at 37 °C with the pH reduced to pH 4·5. The optical density was measured at 450 nm (OD450).
In plating experiments overnight cultures were diluted 100-fold in LB and allowed to grow to OD450 0·4 at 37 °C. Samples (10 µl) of culture were spotted on plates with or without 5% NaCl. Plates were incubated overnight at either 37 °C or 45 °C.
Immunoblotting.
Western blot analysis using monoclonal anti-S antibodies (obtained from Neoclone) was performed essentially as described by Lee et al. (1995)
. Cells were grown to the mid-exponential growth phase (OD600 0·4) or late stationary phase (15 h growth) in LB. Equal amounts (5 µg) of protein were loaded for each sample.
Two-dimensional protein gel electrophoresis.
Two-dimensional SDS-PAGE analysis was performed as described by Spector et al. (1986) with minor modifications. Strains were grown in M63 supplemented with 0·05% Casamino acids at 37 °C until the OD600 was 0·4. The cultures were then transferred to 45 °C and allowed to grow for 1 h. Samples were labelled with 35S-translabel (40 µCi ml-1; 1·48x106 Bq ml-1; ICN Pharmaceuticals) for 3 min. In the first dimension proteins were separated using ReadyStrip IPG Strips pH 47 (Bio-Rad) and in the second dimension an SDS-11·5% polyacrylamide gel was used.
Measurement of degradation of puromycyl-containing polypeptides.
The experiment was performed essentially as described by Raina & Georgopoulos (1990) . S. typhimurium wild-type and clpP mutant cells were grown at 37 °C in M63 until the OD450 reached 0·4. The cells were subsequently incubated with puromycin (200 µg ml-1, Sigma) for 10 min and then labelled with 30 µCi (1·11x106 Bq ml-1) [35S]methionine per ml for 10 min. The cells were washed and resuspended in M63 containing 500 µg unlabelled methionine ml-1. Samples (300 µl) were collected at 5 min intervals and precipitated with 6% trichloroacetic acid. The radioactivity of the acid-soluble fraction was measured by liquid-scintillation counting.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Recently it was shown that the accumulation of another sigma factor, 28, encoded by fliA, is also affected by a clpP mutation in S. typhimurium leading to increased levels of FliA and a hyper-flagellate phenotype (Tomoyasu et al., 2002
). When we transduced the fliA mutation into C5 and LT1100 and compared growth of the resulting strains under stress we found that fliA did not alter the growth characteristics of the wild-type or the mutant strain, demonstrating that the accumulation of FliA does not account for the observed stress sensitivity of the clpP mutant (data not shown).
During the course of these experiments we observed that LT1100 has a normal colony size. In a recent study, a S. typhimurium clpP mutant displayed a small-colony morphology (Webb et al., 1999 ), suggesting that LT1100 could carry a secondary mutation. We verified this notion by transducing the transposon-disrupted clpP allele, clpP::Tn10dTc (Webb et al., 1999
) into C5, and found that the resulting C5 clpP mutant had a small-colony morphology (data not shown). Interestingly, large-colony revertants arose with high frequency, thus showing that the mutant is unsuitable for growth experiments. When we introduced the clpP::Tn10dTc allele into LT1102, which carries the wild-type clpP allele in the clpP deletion mutant background, the resulting strain, LT1103 (clpP::Tn10dTc), had retained the large-colony morphology, demonstrating that the secondary mutation is unlinked to clpP (data not shown). However, as growth of LT1102 was identical to growth of C5 and, in addition, LT1103 (clpP::Tn10dTc) behaved like LT1100 (clpP) under the various stress conditions tested (Fig. 1
and data not shown), our results show that the impaired growth observed for LT1100 is not a consequence of the secondary mutation but rather it is caused by the lack of ClpP.
The absence of ClpP in E. coli also affects growth during stress
In E. coli the Clp protease degrades intrinsically unfolded protein substrates such as the CRAG protein (Kandror et al., 1999 ) and a non-secreted alkaline phosphatase mutant protein (Huang et al., 2001
), indicating that the proteins formed during stress could be degraded by Clp. The results we obtained with S. typhimurium therefore prompted us to analyse how an E. coli clpP mutant behaved when exposed to stress using the same experimental conditions as for S. typhimurium. In agreement with a previous finding (Maurizi et al., 1990b
), we found that growth of the E. coli clpP mutant was identical to that of the wild-type at 37 °C (Fig. 2
). However, when the mutant was shifted to 5% NaCl, to 45 °C or from neutral pH to pH 4·5, we reproducibly obtained results showing that the growth was impaired compared to the wild-type (Fig. 2
) although not to the same extent as observed for the S. typhimurium clpP mutant. Thus, our results indicate that the S. typhimurium clpP mutant is generally more sensitive to stress than the E. coli clpP mutant.
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
De Mot, R., Nagy, I., Walz, J. & Baumeister, W. (1999). Proteasomes and other self-compartmentalizing proteases in prokaryotes. Trends Microbiol 7, 88-92.[Medline]
Downs, D., Waxman, L., Goldberg, A. L. & Roth, J. (1986). Isolation and characterization of lon mutants in Salmonella typhimurium. J Bacteriol 165, 193-197.[Medline]
Enomoto, M. & Stocker, B. A. (1974). Transduction by phage P1kc in Salmonella typhimurium. Virology 60, 503-514.[Medline]
Foster, J. W. & Spector, M. P. (1995). How Salmonella survive against the odds. Annu Rev Microbiol 49, 145-174.[Medline]
Frees, D. & Ingmer, H. (1999). ClpP participates in the degradation of misfolded protein in Lactococcus lactis. Mol Microbiol 31, 79-87.[Medline]
Gaillot, O., Pellegrini, E., Bregenholt, S., Nair, S. & Berche, P. (2000). The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol Microbiol 35, 1286-1294.[Medline]
Galan, J. E. (1996). Molecular genetic bases of Salmonella entry into host cells. Mol Microbiol 20, 263-271.[Medline]
Goff, S. A. & Goldberg, A. L. (1985). Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41, 587-595.[Medline]
Goldberg, A. L., Moerschell, R. P., Chung, C. H. & Maurizi, M. R. (1994). ATP-dependent protease La (lon) from Escherichia coli. Methods Enzymol 244, 350-375.[Medline]
Gottesman, S. (1996). Proteases and their targets in Escherichia coli. Annu Rev Genet 30, 465-506.[Medline]
Hanahan, D. (1985). Techniques for transformation of Escherichia coli. In DNA Cloning: a Practical Approach , pp. 109-135. Edited by D. M. Glover. Oxford, UK:IRL Press.
Hengge-Aronis, R. (1996). Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli. Mol Microbiol 21, 887-893.[Medline]
Hengge-Aronis, R. (2000). The general stress response in Escherichia coli. In Bacterial Stress Responses , pp. 161-178. Edited by G. Storz & R. Hengge-Aronis. Washington, DC:American Society for Microbiology.
Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E. & Holden, D. W. (1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400-403.[Medline]
Hormaeche, C. E. (1979). Natural resistance to Salmonella typhimurium in different inbred mouse strains. Immunology 37, 311-318.[Medline]
Huang, H. C., Sherman, M. Y., Kandror, O. & Goldberg, A. L. (2001). The molecular chaperone DnaJ is required for the degradation of a soluble abnormal protein in Escherichia coli. J Biol Chem 276, 3920-3928.
Ibanez-Ruiz, M., Robbe-Saule, V., Hermant, D., Labrude, S. & Norel, F. (2000). Identification of RpoS (S)-regulated genes in Salmonella enterica serovar typhimurium. J Bacteriol 182, 5749-5756.
Kandror, O., Sherman, M. & Goldberg, A. (1999). Rapid degradation of an abnormal protein in Escherichia coli proceeds through repeated cycles of association with GroEL. J Biol Chem 274, 37743-37749.
Kessel, M., Maurizi, M. R., Kim, B., Kocsis, E., Trus, B. L., Singh, S. K. & Steven, A. C. (1995). Homology in structural organization between E. coli ClpAP protease and the eukaryotic 26S proteasomes. J Mol Biol 250, 587-594.[Medline]
Kruger, E., Witt, E., Ohlmeier, S., Hanschke, R. & Hecker, M. (2000). The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins. J Bacteriol 182, 3259-3265.
Lange, R. & Hengge-Aronis, R. (1994). The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. Genes Dev 8, 1600-1612.[Abstract]
Laskowska, E., Kuczynska-Wisnik, D., Skorko-Glonek, J. & Taylor, A. (1996). Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro. Mol Microbiol 22, 555-571.[Medline]
Lee, I. S., Lin, J., Hall, H. K., Bearson, B. & Foster, J. W. (1995). The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium. Mol Microbiol 17, 155-167.[Medline]
Loewen, P. C. & Hengge-Aronis, R. (1994). The role of the sigma factor sigma S (KatF) in bacterial global regulation. Annu Rev Microbiol 48, 53-80.[Medline]
Maloy, S. R., Stewart V. J. & Taylor R. K. (1996). Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Maurizi, M. R., Trisler, P. & Gottesman, S. (1985). Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable. J Bacteriol 164, 1124-1135.[Medline]
Maurizi, M. R., Clark, W. P., Kim, S. H. & Gottesman, S. (1990a). ClpP represents a unique family of serine proteases. J Biol Chem 265, 12546-12552.
Maurizi, M. R., Clark, W. P., Katayama, Y., Rudikoff, S., Pumphrey, J., Bowers, B. & Gottesman, S. (1990b). Sequence and structure of ClpP, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J Biol Chem 265, 12536-12545.
Mhammedi-Alaoui, A., Pato, M., Gama, M. J. & Toussaint, A. (1994). A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol Microbiol 11, 1109-1116.[Medline]
Miller, C. G. (1996). Protein degradation and proteolytic modification. In Esherichia coli and Salmonella: Cellular and Molecular Biology, pp. 938954. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Msadek, T., Dartois, V., Kunst, F., Herbaud, M. L., Denizot, F. & Rapoport, G. (1998). ClpP of Bacillus subtilis is required for competence development, motility, degradative enzyme synthesis, growth at high temperature and sporulation. Mol Microbiol 27, 899-914.[Medline]
OCallaghan, D. & Charbit, A. (1990). High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol Gen Genet 223, 156-158.[Medline]
ONeal, C. R., Gabriel, W. M., Turk, A. K., Libby, S. J., Fang, F. C. & Spector, M. P. (1994). RpoS is necessary for both the positive and negative regulation of starvation survival genes during phosphate, carbon, and nitrogen starvation in Salmonella typhimurium. J Bacteriol 176, 4610-4616.[Abstract]
Phillips, G. J. (1999). New cloning vectors with temperature-sensitive replication. Plasmid 41, 78-81.[Medline]
Porankiewicz, J., Wang, J. & Clarke, A. K. (1999). New insights into the ATP-dependent Clp protease: Escherichia coli and beyond. Mol Microbiol 32, 449-458.[Medline]
Raina, S. & Georgopoulos, C. (1990). A new Escherichia coli heat shock gene, htrC, whose product is essential for viability only at high temperatures. J Bacteriol 172, 3417-3426.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schultz, J. E., Latter, G. I. & Matin, A. (1988). Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli. J Bacteriol 170, 3903-3909.[Medline]
Schweder, T., Lee, K. H., Lomovskaya, O. & Matin, A. (1996). Regulation of Escherichia coli starvation sigma factor (s) by ClpXP protease. J Bacteriol 178, 470-476.[Abstract]
Spector, M. P., Aliabadi, Z., Gonzalez, T. & Foster, J. W. (1986). Global control in Salmonella typhimurium: two-dimensional electrophoretic analysis of starvation-, anaerobiosis-, and heat shock-inducible proteins. J Bacteriol 168, 420-424.[Medline]
Tomoyasu, T., Ohkishi, T., Ukyo, Y. & 7 other authors (2002). The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar Typhimurium. J Bacteriol 184, 645653.
Wang, J., Hartling, J. A. & Flanagan, J. M. (1997). The structure of ClpP at 2·3 resolution suggests a model for ATP-dependent proteolysis. Cell 91, 447-456.[Medline]
Wang, L., Elliott, M. & Elliott, T. (1999). Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium. J Bacteriol 181, 1211-1219.
Wawrzynow, A., Wojtkowiak, D., Marszalek, J., Banecki, B., Jonsen, M., Graves, B., Georgopoulos, C. & Zylicz, M. (1995). The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. EMBO J 14, 1867-1877.[Abstract]
Wawrzynow, A., Banecki, B. & Zylicz, M. (1996). The Clp ATPases define a novel class of molecular chaperones. Mol Microbiol 21, 895-899.[Medline]
Webb, C., Moreno, M., Wilmes-Riesenberg, M., Curtiss, R.III & Foster, J. W. (1999). Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium. Mol Microbiol 34, 112-123.[Medline]
Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K. & Maurizi, M. R. (1994). A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci USA 91, 12218-12222.
Yamamoto, T., Sashinami, H., Takaya, A., Tomoyasu, T., Matsui, H., Kikuchi, Y., Hanawa, T., Kamiya, S. & Nakane, A. (2001). Disruption of the genes for ClpXP protease in Salmonella enterica serovar Typhimurium results in persistent infection in mice, and development of persistence requires endogenous gamma interferon and tumor necrosis factor alpha. Infect Immun 69, 3164-3174.
Zgurskaya, H. I., Keyhan, M. & Matin, A. (1997). The sigma S level in starving Escherichia coli cells increases solely as a result of its increased stability, despite decreased synthesis. Mol Microbiol 24, 643-651.[Medline]
Received 31 December 2001;
revised 23 May 2002;
accepted 12 June 2002.