Chaperone-like activities of the CsaA protein of Bacillus subtilis

Jörg P. Müller1, Sierd Bron2, Gerard Venema2 and Jan Maarten van Dijl3

Institute for Molecular Biology, Jena University, Winzerlaer Strasse 10, 07745 Jena, Germany1
Department of Genetics, Center for Biological Sciences, Kerklaan 30, 9751 NN Haren, The Netherlands2
Department of Pharmaceutical Biology, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands3

Author for correspondence: J. P. Müller. Tel: +49 3641 65 7577. Fax: +49 3641 65 7520. e-mail: jmueller{at}imb-jena.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The growth and protein export defects of Escherichia coli secA51(Ts) strains can be suppressed by the CsaA protein of Bacillus subtilis. The present studies indicate that this effect can be attributed to chaperone-like activities of CsaA. First, CsaA stimulated protein export in secB, groES and dnaJ mutant strains of E. coli. Second, CsaA suppressed the growth defects of dnaK, dnaJ and grpE mutants of E. coli. Third, and most importantly, CsaA exhibited chaperone-like properties by stimulating the reactivation of heat-denatured firefly luciferase in groEL, groES, dnaK and grpE mutant strains of E. coli, and by preventing the aggregation of heat-denatured luciferase in vitro. Thus, it seems that CsaA suppresses the growth and secretion defects of E. coli secA(Ts) strains either by improving the translocation competence of exported pre-proteins, thereby making them better substrates for mutant SecA proteins, or by stimulating the translocation activity of mutant SecA proteins.

Keywords: Bacillus subtilis, CsaA, protein export, protein targeting, chaperone

Abbreviations: 6H-CsaA, hexa-histidine-tagged CsaA


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The transport (i.e. export) of proteins across the cytoplasmic membrane of eubacteria depends on properties of the transported protein and the cellular protein export apparatus. Exported proteins are usually synthesized as precursors with an amino-terminal extension, the signal peptide, which directs these proteins into the export pathway (Pugsley, 1993 ; von Heijne, 1990 ). Genetically and biochemically the best characterized bacterial protein export apparatus is that of Escherichia coli. It consists of cytoplasmic and membrane-bound components (Wickner et al., 1991 ; Duong et al., 1997 ; Fekkes & Driessen, 1999 ).

Cytoplasmic proteins, like SecB (Kumamoto & Beckwith, 1985 ), GroEL, GroES (Altman et al., 1991 ; Kusukawa et al., 1989 ), DnaK, DnaJ and GrpE (Altman et al., 1991 ; Wild et al., 1992 , 1993 ), denoted as chaperones, are important for the export competence of pre-proteins, either by stabilizing an unfolded conformation, or by preventing their aggregation (Hendrick & Hartl, 1993 ). In addition, SecB is involved in the targeting of pre-proteins to the membrane-bound pre-protein translocase complex (Fekkes et al., 1997 ; Hartl et al., 1990 ). A second targeting factor, which is homologous to the eukaryotic signal recognition particle (SRP), assists the export of a subset of pre-proteins (De Gier et al., 1997 ; Luirink et al., 1992 ; Phillips & Silhavy, 1992 ; Wolin, 1994 ).

The pre-protein translocase consists of the peripheral membrane protein SecA (Oliver & Beckwith, 1982 ), which acts as a force generator for protein translocation, and a complex of the integral membrane proteins SecD, SecE, SecF, SecG, SecY and YajC, which form the translocation channel (for recent reviews, see Fekkes & Driessen, 1999 ; Duong et al., 1997 ; Pohlschröder et al., 1997 ). SecA plays a crucial role in protein translocation. First, it can function as a receptor for pre-protein–SecB complexes (de Cock & Randall, 1998 ; Fekkes et al., 1997 ; Hartl et al., 1990 ). Second, SecA drives protein transport through cycles of pre-protein binding, membrane insertion, pre-protein release and deinsertion from the membrane (Economou et al., 1995 ; Economou & Wickner, 1994 ; Kim & Oliver, 1994 ). The cycling of SecA is regulated by ATP binding and hydrolysis, which causes major conformational changes in this protein (den Blaauwen & Driessen, 1996 ; van der Does et al., 1998 ). During or shortly after the translocation of the pre-protein across the membrane, the signal peptide is removed by signal peptidase (also known as leader peptidase), which is a prerequisite for the release of the mature protein from the membrane (Dalbey et al., 1997 ).

Compared to E. coli, the protein export apparatus of Bacillus subtilis has been studied in less detail. So far, homologues of GroEL, GroES (Schmidt et al., 1992 ), DnaK, DnaJ and GrpE (Wetzstein et al., 1992 ), SRP (Honda et al., 1993 ; Struck et al., 1988 ), SecA (Overhoff et al., 1991 ; Sadaie et al., 1991 ), SecY (Nakamura et al., 1990 ; Suh et al., 1990 ), SecE (Jeong et al., 1993 ), SecDF and YajC (Bolhuis et al., 1998 ) and several signal peptidases have been identified (Bolhuis et al., 1996 ; Meijer et al., 1995 ; Tjalsma et al., 1997 , 1998 ; van Dijl et al., 1992 ). In an early attempt to clone the B. subtilis secA gene by complementation of the E. coli secA51(Ts) mutation, we identified the B. subtilis csaA gene (Müller et al., 1992 ). The deduced amino acid sequence of CsaA showed no similarities to that of SecA or other known components of the E. coli protein export apparatus. Nevertheless, the expression of the complete csaA gene resulted in a suppression of the growth and protein export defects associated with the secA51 mutation. Unlike other previously identified suppressors of secA51 (Brickman et al., 1984 ; Lee & Beckwith, 1986 ; Oliver, 1985 ; Overhoff-Freundlieb & Freudl, 1991 ), csaA showed no pleiotropic effects on mutations in other sec genes with the exception of the secB::Tn5 mutation (Müller et al., 1992 ). The present studies were aimed at the elucidation of the mechanism by which csaA suppresses secretion defects in E. coli. The results show that the CsaA protein has chaperone-like activities in vivo and in vitro, suggesting that these activities are responsible for the suppression of the secA51 mutation.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Plasmids, bacterial strains and media.
Table 1 lists the plasmids and bacterial strains used. TY medium contained Bacto tryptone (1%), Bacto yeast extract (0·5%) and NaCl (1%). M9 media 1 and 2 used in the labelling of E. coli were prepared as described by van Dijl et al. (1991) . If required, ampicillin (40 µg ml-1), erythromycin (150 µg ml-1), kanamycin (20 µg ml-1) or tetracycline (15 µg ml-1) were added.


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Table 1. Bacterial strains and plasmids used

 
DNA techniques.
Procedures for DNA purification, restriction, ligation, transformation of E. coli, and agarose gel electrophoresis, were carried out as described by Sambrook et al. (1989) . Enzymes were from Roche Molecular Biochemicals.

Purification of hexa-histidine-tagged CsaA.
To provide CsaA with an amino-terminal hexa-histidine tag, the csaA gene was amplified by PCR with the primers csa-5' (5'-GGAGTTATTGGATCCGCAGTTATTGATGAC-3'; containing a BamHI site) and csa-3' (5'-GCCGATCTGCAGGCCTTTACGGCACACACG-3'; containing a PstI site) and cloned into plasmid pQE9, resulting in plasmid pQE9csaA. The sequence of the amplified fragment was verified by DNA sequencing. Hexa-histidine-tagged CsaA (6H-CsaA) was purified from E. coli M15 (pREP4, pQE9csaA) using Ni2+-nitrilotriacetic acid agarose (Qiagen) under non-denaturing conditions, according to the manufacturer’s instructions.

CsaA antiserum.
A CsaA-based peptide with the sequence KAEEFPEARC (residues 19–27 of CsaA: the last C was added for experimental reasons; Müller et al., 1992 ) was cross-linked to KLH (Keyhole Limpet Hemocyanin) from Pierce using the m-maleimidobenzoic acid N-hydroxysuccinimide ester, as described by Närvänen (1990) . Next, the peptide–KLH conjugate was used to immunize rabbits.

Protein labelling, immunoprecipitation, SDS-PAGE and fluorography.
Pulse labelling, or pulse–chase labelling experiments in E. coli and subsequent immunoprecipitations were performed as described by van Dijl et al. (1991) . SDS-PAGE was performed according to Laemmli (1970) . [14C]Methylated molecular mass reference markers were from Amersham. Fluorography was performed as described by Skinner & Griswold (1983) . All pulse–chase labelling experiments were repeated at least twice. Relative amounts of radioactivity in gels were determined with a phosphorimager or by scanning of films with a densitometer. Pre-protein processing was calculated as the percentage of the total labelled protein (precursor + mature) present in the mature form at the time of sampling. Differences of more than 10–15% pre-protein processing were reproducibly detected in parallel labelling experiments. Differences in pre-protein processing that are smaller than 10% are, in general, not reproducible.

Immunoblotting.
Samples for SDS-PAGE were prepared by boiling of cells (5 min) in sample buffer (Laemmli, 1970 ). Separated proteins were transferred to a nitrocellulose membrane (BA85; Schleicher & Schuell) as described by Towbin et al. (1979) . Proteins were detected with specific antisera and 125I-labelled protein A. Relative amounts of radioactivity on blots were determined with a phosphorimager.

ß-Galactosidase activity.
ß-Galactosidase activity assays were carried out as described by Miller (1972) .

Luciferase activity and aggregation.
In vivo luciferase activity assays were performed essentially as described by Schröder et al. (1993) . E. coli cells producing luciferase were grown at 30 °C in TY medium. When the cells reached an absorbance (A600) of 0·5, kanamycin (100 µg ml-1) and chloramphenicol (50 µg ml-1) were added to stop further protein synthesis, and samples of 1 ml were incubated for 10 min at 42 °C to inactivate luciferase. Next, the samples were incubated at 30 °C and the reactivation of luciferase was determined in duplicate experiments. In vitro luciferase aggregation assays were performed as described by Schröder et al. (1993) .


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CsaA affects the expression of secB
Due to the temperature sensitivity of the SecA51 protein, E. coli MM54 is unable to export proteins at 42 °C. Consequently, the strain is unable to grow at this temperature (Oliver & Beckwith, 1981 ). We have previously shown that the CsaA protein from B. subtilis, encoded by plasmid pBNB8, is able to suppress both the protein export defect and the growth defect of E. coli MM54 at 42 °C (Müller et al., 1992 ). Similar effects were observed when pBNB8 was introduced into other E. coli strains with temperature-sensitive SecA proteins (our unpublished results). In addition, the introduction of pBNB8 into E. coli CK1953 (secB::Tn5) which can not grow on complex media, allowed this strain to form colonies on complex media (Müller et al., 1992 ). In contrast, CsaA did not suppress mutations in secDF and secE (our unpublished results), secY and lep (Müller et al., 1992 ). These findings suggested that CsaA, which is a soluble cytoplasmic protein, exerts its effect during the early stages of protein export, involving both SecA and SecB. Thus, CsaA could, in principle, have a function analogous to that of the E. coli SecB protein, which is absent from B. subtilis (Kunst et al., 1997 ). As CsaA lacks amino acid sequence similarity with SecB, immunological approaches were used to investigate whether CsaA was related to SecB. To this purpose plasmid pTZcsa82 was constructed by ligation of an 895 bp SspI–HincII fragment, which contains the complete csaA gene, into the high-copy-number pUC18-derived plasmid pTZ18R (Mead et al., 1986 ). In E. coli MC4100, the expression of the pTZcsa82-located csaA gene resulted in approximately fourfold overproduction of CsaA, as compared to E. coli MC4100(pBNB8) (Fig. 1a). Next, we tried to detect CsaA (12 kDa) with polyclonal antibodies directed against SecB, both by immunoblotting and immunoprecipitation from [35S]methionine-labelled cells of E. coli MC4100(pTZcsa82). The results showed that this was not possible (data not shown), indicating that the antigenic determinants, recognized by the anti-SecB antibodies, are not present in CsaA. Unexpectedly, the amount of SecB immunoprecipitated from E. coli MC4100(pTZcsa82) was approximately 10-fold reduced, as compared to that from E. coli MC4100(pBR322), whereas similar amounts of the outer-membrane protein A (OmpA) and periplasmic ß-lactamase (Bla) were precipitated from both strains (Fig. 1b). Similarly, the presence of pTZcsa82 in E. coli MM54 secA51 resulted in a 10-fold reduction of the synthesis of SecB (data not shown). As cells containing pTZcsa82 or pBR322 incorporated comparable amounts of [35S]methionine (Fig. 1b), the production of CsaA appeared to interfere specifically with the production of SecB.



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Fig. 1. Production of CsaA and SecB in E. coli MC4100. (a) The production of CsaA in E. coli MC4100(pBNB8) (CsaA expression: +) and E. coli MC4100(pTZcsa82) (CsaA expression: +++) was analysed by pulse-labelling at 37 °C for 30 s. E. coli MC4100(pBR322) was labelled as a control (CsaA expression: -). The production of CsaA was monitored by immunoprecipitation with specific antibodies, SDS-PAGE and fluorography. (b) Production of SecB in E. coli MC4100(pBR322) (CsaA expression: -) and E. coli MC4100(pTZcsa82) (CsaA expression: +++) was analysed by pulse-labelling at 42 °C for 30 s. Subsequent immunoprecipitation was carried out with antibodies against SecB, ß-lactamase (Bla) and OmpA. Bands corresponding to SecB, ß-lactamase and OmpA are indicated. The incorporation of 35S-labelled methionine in pulse-labelled cells, indicated as c.p.m., was determined by liquid scintillation counting of 10 µl aliquots of the labelling mixtures. p, precursor; m, mature.

 
To study the effects of CsaA on the production of SecB as a function of growth, E. coli MC4100::pLBL332, which contains a translational secB'–lacZ gene fusion (Müller, 1996 ), was transformed with pBNB8 or pTZcsa82. As a control, this strain was transformed with pBR322. The resulting strains were grown in TY medium and samples, withdrawn at hourly intervals, were assayed for ß-galactosidase activity. Irrespective of the growth phase, the levels of ß-galactosidase activity were approximately two- and fivefold reduced in the pBNB8- and pTZcsa82-containing cells, respectively, compared to the control cells (Fig. 2a). To demonstrate that the effect was specific for secB and to exclude the possibility that CsaA modulates the activity of ß-galactosidase, secA expression was studied in E. coli MM171.3, which contains a chromosomal secA'–lacZ fusion (Schmidt & Oliver, 1989 ). Strain MM171.3 was transformed with plasmids pBR322, pBNB8 or pTZcsa82 and ß-galactosidase activities were determined. As shown in Fig. 2b, the presence of pBNB8 or pTZcsa82 did not significantly affect the expression of secA'–lacZ in strain MM171.3. Taken together, these data show that the production of CsaA in E. coli (pBNB8 or pTZcsa82) results in a strong reduction of the expression of secB.



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Fig. 2. Analysis of the expression of secB and secA. Time courses of the expression of the secB'–lacZ gene fusion of E. coli MC4100::pLBL332 (a) and the secA'–lacZ gene fusion of E. coli MM171.3 (b) in cells growing on TY medium at 37 °C. ß-Galactosidase activities (closed symbols) and optical densities at 600 nm (open symbols) were determined in duplicate for strains containing the plasmids pBR322 (circles), pBNB8 (squares), or pTZcsa82 (triangles).

 
CsaA stimulates protein export in the absence of SecB
The suppression of the growth defect of E. coli MM54 secA51 at 42 °C could either be due to the reduction of the synthesis of SecB, and consequently reduced rates of targeting of SecB-dependent pre-proteins to the SecA51 protein, or to a direct effect of CsaA on protein export. To analyse these possibilities, we investigated the efficiency of processing of pro-OmpA and pre-ß-lactamase by signal peptidase in CsaA-producing cells of E. coli CK1953, which lack SecB. Compared to the wild-type, E. coli CK1953 exports pro-OmpA at a reduced rate (Kumamoto & Beckwith, 1985 ), whilst the rate of pre-ß-lactamase export is not affected (Kumamoto, 1989 ). As shown in Fig. 3, the production of CsaA resulted in increased rates of pro-OmpA and pre-ß-lactamase processing at 42 °C, the highest rates being observed in E. coli CK1953(pTZcsa82). At lower temperatures, no alteration of protein export could be measured in the presence or absence of CsaA (data not shown).



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Fig. 3. CsaA-stimulated precursor processing in the absence of SecB. The processing of pro-OmpA (a) and pre-ß-lactamase (b) in E. coli CK1953 secB::Tn5 was analysed by pulse-labelling for 30 s at 42 °C. The efficiencies of processing of pro-OmpA and pre-ß-lactamase are indicated as the percentage of the mature protein relative to the total amount of protein (precursor+ mature protein). Strains tested were: E. coli CK1953(pBR322) (lanes 1, CsaA expression: -), E. coli CK1953(pBNB8) (lanes 2, CsaA expression: +) and E. coli CK1953(pTZcsa82) (lanes 3, CsaA expression: +++). p, precursor; m, mature.

 
As demonstrated by immunoblotting, the E. coli strains MM54 (secA51), CK1953 (secB::Tn5) and MC4100 (wild-type) produced comparable amounts of the major chaperones DnaK and GroEL, irrespective of the production of CsaA (data not shown). Thus, the effects of CsaA at 42 °C, as described above, were not due to an increased synthesis of DnaK and GroEL, which have been implicated in protein export (Kusukawa et al., 1989 ; Wild et al., 1992 ). Furthermore, suppression of the secA51 and secB::Tn5 mutations required the presence of an intact csaA gene (Müller et al., 1992 ; our unpublished results), ruling out the possibility that the effects of CsaA production are due to protein (i.e. CsaA) overproduction per se, as previously reported for the suppression of cold-sensitive sec mutations through the overproduction of proteins unrelated to the process of protein export (Danese et al., 1995 ).

Taken together, our data imply that CsaA either has a direct effect on protein export in E. coli MM54 secA51 and CK1953 secB::Tn5 at 42 °C, or that CsaA induces the synthesis of as yet unidentified proteins which participate in maintaining pre-proteins in an export-competent conformation, thereby reducing (but not bypassing) the requirements for SecA and/or SecB. In what follows, we show that CsaA can prevent protein aggregation, suggesting that CsaA can have a direct effect on protein export.

CsaA-mediated suppression of the temperature-sensitive growth of dnaK, dnaJ and grpE mutants
Since the observed effects of CsaA on protein export are in accordance with the hypothesis that CsaA itself could have a chaperone-like activity (Müller et al., 1992 ), we tested whether this protein can complement the temperature-sensitive growth phenotype of E. coli strains carrying mutations in various chaperone-encoding genes. To this purpose, strains containing the groEL44, groES619, dnaK756, dnaJ259 or grpE280 mutations were transformed with plasmids pBNB8, pTZcsa82 or pBR322 (control). As essentially the same results were obtained with strains containing pBNB8 or pTZcsa82, only the results obtained with strains containing pBNB8 are documented here. As shown in Fig. 4, at 42 °C, the post-exponential growth in TY medium of strains containing the groEL44 or groES619 mutations was slightly, but reproducibly, impaired due to the expression of CsaA, similar to the wild-type strain MC4100. In contrast, the post-exponential growth of strains containing the dnaK756, dnaJ259 or grpE280 mutations was stimulated by the expression of CsaA, the growth of the dnaJ mutant strain being stimulated most strongly (Fig. 4). To verify these effects of CsaA, the growth of chaperone mutant strains expressing csaA was analysed on plates (Table 2). Whilst the expression of csaA neither exacerbated nor improved the ability of strains containing the groEL44 or groES619 mutations to form single colonies at 42 °C, a stimulatory effect could be observed in strains containing the dnaK756, dnaJ259 or grpE280 mutations (Table 2). Furthermore, the expression of CsaA strongly reduced the formation of long filamentous cells of strains containing the dnaK756, dnaJ259 or grpE280 mutations (data not shown). These findings show that CsaA can suppress growth defects of strains with impaired activity of the DnaK–DnaJ–GrpE chaperone machinery. Nevertheless, CsaA was unable to replace DnaK as the transformation of E. coli BB1553 {Delta}dnaK52, which lacks the dnaK gene (Hesterkamp & Bukau, 1998 ), with pBNB8 or pTZcsa82 did not result in a suppression of the growth defects of this strain at 42 °C (data not shown). Furthermore, at 37 °C and 42 °C, the impaired replication of bacteriophage in the above strains with defective DnaK–DnaJ–GrpE or GroEL–GroES chaperone machineries (see Polissi et al., 1995 ) was not restored by CsaA (data not shown). These observations demonstrate that CsaA can not complement for the malfunction of the major chaperone machineries of E. coli.



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Fig. 4. Effects of CsaA production on the growth of chaperone mutant strains. E. coli strains MC4100 (wild-type), NRK117 (groEL44), NRK223 (groES619), BB1048 (dnaK756), BB1458 (dnaJ259), or BB1752 (grpE280) carrying plasmids pBR322 (circles) or pBNB8 (squares) were grown overnight in TY medium at 30 °C and diluted 100-fold in fresh TY medium. After 2 h growth at 30 °C, the temperature was raised to 42 °C. Growth was monitored by optical density readings at 600 nm.

 

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Table 2. Growth of E. coli chaperone mutant strains containing plasmids pBR322 (control) or pBNB8 as determined by their ability to form single colonies on TY agar plates at 37 °C and 42 °C

 
CsaA-stimulated export of ß-lactamase in groES and dnaJ mutant strains
It was previously shown that mutations in chaperone-encoding genes affect the export of pre-proteins which are exported independently of SecB (Kusukawa et al., 1989 ; Wild et al., 1992 , 1996 ). Therefore, we investigated whether the expression of CsaA in chaperone mutant strains containing pBNB8 affected the export of ß-lactamase, which is one of the proteins not depending on SecB for its export. As shown in Fig. 5, at 42 °C, processing of pre-ß-lactamase to the mature form in the groEL44, dnaK756 or grpE280 mutant strains was not significantly affected by CsaA, like in the wild-type strain MC4100. In contrast, processing of pre-ß-lactamase was stimulated by the expression of CsaA in strains containing the groES619 or dnaJ259 mutations (Fig. 5). These findings show that CsaA can, at least in part, suppress protein export defects of strains with mutations in the GroES and DnaJ chaperones.



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Fig. 5. Effects of CsaA production on the processing of ß-lactamase in chaperone mutant strains. E. coli strains MC4100 (wild-type), NRK117 (groEL44), NRK223 (groES619), BB1048 (dnaK756), BB1458 (dnaJ259), or BB1752 (grpE280) carrying plasmids pBR322 (circles) or pBNB8 (squares) were grown overnight in M9 minimal medium at 30 °C, diluted in fresh M9 medium and incubated at 30 °C until the mid-exponential growth phase. Next, the temperature was raised to 42 °C and growth was continued for 2 h. Cells were pulse-labelled with [35S]methionine for 30 s and chased with non-radioactive methionine at 42 °C. Samples were withdrawn at the times indicated. Processing of labelled pre-ß-lactamase was analysed by immunoprecipitation and SDS-PAGE. The relative amounts of precursor and mature forms of ß-lactamase were determined with a phosphorimager. The kinetics of pre-ß-lactamase processing are plotted as the percentage of the total ß-lactamase (precursor+mature) present in the mature form at the time of sampling.

 
CsaA-stimulated reactivation of heat-denatured luciferase in groEL, groES, dnaK and grpE mutant strains
The reactivation of thermally inactivated firefly luciferase at 30 °C is impaired in certain chaperone mutant strains (Schröder et al., 1993 ). To further characterize the activity of CsaA in E. coli, its effects on the reactivation of luciferase were studied. To this purpose, wild-type and chaperone mutant strains containing plasmids pBNB8 or pBR322 were transformed with the compatible plasmid pSEL16. The latter plasmid contains the luc gene for luciferase under the control of a constitutive promoter (Gräfe et al., 1996 ). As shown in Fig. 6, CsaA restored the reactivation of thermally inactivated luciferase in dnaK756 and grpE280 mutant strains. Interestingly, even though the reactivation of thermally inactivated luciferase was not impaired in strains containing the groEL44 or groES619 mutations, CsaA expression in these strains resulted in significantly increased levels of luciferase activity, suggesting that some luciferase was already in an inactive conformation before the heat treatment and that this luciferase was also reactivated by CsaA (Fig. 6). In contrast, CsaA did not restore the reactivation of luciferase in the dnaJ259 mutant strain, and it interfered with the reactivation of luciferase in the wild-type strain (Fig. 6). As in the dnaJ259 mutant strain, CsaA did not restore the reactivation of luciferase in the {Delta}dnaK52 mutant strain (data not shown). As verified by Western blotting, cells containing pBR322 or pBNB8 produced comparable amounts of luciferase (data not shown), which is important because the reactivation kinetics of luciferase depend on its expression level. Thus, it seems that the expression of CsaA in strains containing different chaperone mutations has different effects on the reactivation of luciferase. In particular, the CsaA-mediated reactivation of thermally inactivated luciferase in dnaK756 and grpE280 mutant strains, and the enhanced reactivation of luciferase in groEL and groES mutant strains indicate that CsaA has a chaperone-like activity.



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Fig. 6. CsaA stimulated reactivation of thermally inactivated luciferase in chaperone mutant strains. E. coli strains MC4100 (wild-type), NRK117 (groEL44), NRK223 (groES619), BB1048 (dnaK756), BB1458 (dnaJ259), or BB1752 (grpE280) carrying plasmids pBR322 (circles) or pBNB8 (squares) were transformed with plasmid pSEL16, which carries the luc gene for firefly luciferase, and grown in TY medium at 30 °C. To inactivate the luciferase present in the cells and to prevent de novo luciferase synthesis, the cells were incubated at 42 °C for 10 min in the presence of kanamycin and chloramphenicol. Next, the cells were reincubated at 30 °C (t=0) and luciferase activities were determined in duplicate as a function of the incubation time. The kinetics of luciferase reactivation were plotted as the percentage of the luciferase activity before thermal inactivation at 42 °C.

 
CsaA prevents the aggregation of luciferase in vitro
To obtain direct evidence for a chaperone-like activity of CsaA, its effects on the aggregation of luciferase at 42 °C and the reactivation of heat-denatured luciferase were determined in vitro, as previously described for DnaK, DnaJ and GrpE (Schröder et al., 1993 ). To this purpose, purified 6H-CsaA was used. As revealed by light-scattering experiments, the presence of 6H-CsaA prevented the aggregation of luciferase at 42 °C. In contrast, luciferase aggregated in the presence of bovine serum albumin, or if no other protein was added to the reaction mixture (Fig. 7). Even though 6H-CsaA neither stimulated the reactivation of heat-denatured luciferase nor prevented its inactivation in vitro (data not shown), the observation that CsaA can prevent luciferase aggregation shows that it has a chaperone-like activity.



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Fig. 7. CsaA prevents the aggregation of luciferase. Luciferase (100 nM) was equilibrated at room temperature in renaturation buffer (Schröder et al., 1993 ) containing 1 mM ATP and 6H-CsaA (750 nM, squares), bovine serum albumin (750 nM, triangles), or no further additions (circles). Next, the mixtures were incubated at 42 °C and light scattering due to luciferase aggregation was monitored at 320 nm in a thermostated cuvette. Aggregation of luciferase in the absence of other proteins was almost complete after 30 min incubation at 42 °C and was set as 100%. The kinetics of luciferase aggregation are plotted as the percentage of this value.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
As evidenced by in vivo and in vitro studies, the CsaA protein of B. subtilis has chaperone-like activities. First, CsaA stimulated protein export in secB, groES and dnaJ mutant strains of E. coli. Second, CsaA suppressed the growth defects of dnaK, dnaJ and grpE mutants of E. coli. Third, CsaA stimulated the reactivation of heat-denatured firefly luciferase in groEL, groES, dnaK and grpE mutant strains of E. coli. Finally, CsaA prevented the aggregation of luciferase in vitro. How can we use these observations to explain the suppression of the growth and protein export defects of secA mutations through the production of CsaA? Other previously identified suppressors of mutations in secA seem to act pleiotropically by reducing the rate of protein synthesis in the cell (Brickman et al., 1984 ; Oliver, 1985 ; Overhoff-Freundlieb & Freudl, 1991 ), or by the overproduction of GroEL (Van Dyk et al., 1989 ). Since the production of CsaA did not affect the rate of protein synthesis, it seems that the suppression of the defects of secA mutant strains containing the complete csaA gene (Müller et al., 1992 ; unpublished observations) must be attributed to the chaperone-like activities of CsaA. At least four mechanisms can be envisaged. First, CsaA could improve the translocation-competence of exported pre-proteins, for example by preventing their aggregation, thereby making them better substrates for the mutant SecA protein. This view would be supported by the observation that CsaA was able to prevent the aggregation of luciferase in vitro. Second, CsaA could stimulate the translocation activity of mutant SecA proteins. In fact, CsaA could have a similar stimulating effect on the DnaK756 mutant protein, as evidenced by the observation that the production of CsaA caused a strong reactivation of heat-inactivated luciferase in the dnaK756 mutant strain, but not in the {Delta}dnaK52 strain, lacking the dnaK gene. Third, CsaA could complement SecA function, which seems to be unlikely considering the pivotal role of SecA in the translocation process. Fourth, CsaA could reduce the detrimental effects of secA mutations through a reduction of SecB synthesis and, consequently, through reduced rates of targeting of SecB-dependent pre-proteins to the translocase complex in the membrane. The latter explanation is, however, in apparent conflict with the observation that CsaA stimulated export of the SecB-dependent protein OmpA in the absence of SecB.

Interestingly, the production of CsaA antagonized the expression of the secB gene specifically, as shown by immunoprecipitation of pulse-labelled SecB and determination of ß-galactosidase activities in a secB'–lacZ strain producing different levels of CsaA. As CsaA has chaperone-like activities, this observation is in accordance with the observation that the expression of secB is regulated in response to the cellular chaperone levels: secB expression was increased in groES, groEL, dnaK, dnaJ and grpE mutant strains (Müller, 1996 ) and reduced in strains overexpressing DnaK and DnaJ, or GroEL and GroES (our unpublished observations). Our present findings indicate that secB expression is not only regulated in response to the levels of homologous chaperones, but also in response to the production of a heterologous protein with chaperone-like activities. Notably, the expression of DnaK and GroEL was not affected by the production of CsaA, indicating that these chaperones are not involved in the CsaA-mediated repression of secB. Recently, it has been demonstrated that secB expression is under the control of catabolic repression by cAMP receptor protein–cAMP complexes at the transcriptional level (Seoh & Tai, 1997 , 1999 ). However, in contrast to catabolic repression, which caused a 1·4-fold reduction of secB expression, the presence of CsaA affected the expression levels of secB about two- to fivefold, depending on the level of CsaA production. This suggests that CsaA does not exert its effects on secB expression via catabolic repression, but we are presently unable to exclude this possibility.

The phenotypes observed upon the production of CsaA in groEL44, groES619, dnaK756, dnaJ259 and grpE280 mutant strains indicate that CsaA can suppress a variety of different defects caused by the malfunction of the corresponding mutant chaperones. Two key observations may, at least in part, provide explanations for the effects of CsaA production in the latter strains. First, 6H-CsaA was shown to prevent the aggregation of luciferase in vitro, but it was unable to reactivate heat-denatured luciferase or to prevent its inactivation. This indicates that CsaA has an aggregation-preventing rather than a folding-promoting chaperone-like function. Second, CsaA was unable to replace DnaK in the {Delta}dnaK52 mutant strain with respect to temperature-sensitive growth and the in vivo reactivation of heat-inactivated luciferase. In contrast, CsaA was able to suppress these defects in the dnaK756 mutant strain. Taken together, the latter findings show that the presence of CsaA supported the activity of the mutant DnaK756 protein. This could be a direct effect of CsaA on the DnaK756 protein (see above). Alternatively, an indirect effect through the prevention of protein aggregation by CsaA would also be a plausible explanation for these findings, the in vivo reactivation of heat-inactivated luciferase in particular. In fact, if CsaA can indeed prevent the in vivo aggregation of denatured luciferase, as it does in vitro, CsaA would keep this denatured protein in a proper condition for refolding/reactivation by the DnaK–DnaJ–GrpE and/or GroEL–GroES chaperone machineries. This would explain why CsaA showed a pleiotropic effect with respect to the reactivation of heat-inactivated luciferase in dnaK756, grpE289, groEL44 and groES619 mutant strains. Notably, the production of CsaA did not induce a general stress response as evidenced by the lack of effect of CsaA on the expression of DnaK and GroEL. In fact, CsaA activity seemed to interfere to some extent with chaperone activity in wild-type cells, as evidenced by the reduced reactivation of inactivated luciferase in cells of E. coli MC4100 producing CsaA.

Interestingly, as reflected by the processing of pre-ß-lactamase to the mature form, CsaA only had a significant stimulating effect on ß-lactamase export in those chaperone mutant strains in which the rate of export was drastically slowed down (i.e. groES619 and dnaJ259). Consistent with the above hypothesis that CsaA keeps unfolded luciferase in a proper condition for in vivo refolding by general chaperones, our observations suggest that CsaA keeps pre-ß-lactamase in a proper condition for translocation, for example by preventing its folding and/or aggregation in the cytoplasm. If so, this implies that CsaA has a pre-protein folding-preventing activity, analogous to that of SecB (Topping & Randall, 1997 ), which would explain the CsaA-mediated suppression of protein export defects in secA51 and secB::Tn5 mutant strains as well (see above). This view is consistent with our unpublished observation that purified SecB prevented the aggregation of denatured luciferase in vitro in a similar manner to CsaA.

Finally, the role of CsaA in B. subtilis remains to be defined. First results indicate that CsaA is required for the efficient secretion of at least a subset of proteins and that it interacts specifically with SecA and pre-proteins, suggesting that it could have protein-secretion-specific chaperone-like activities. This is an intriguing hypothesis in view of the fact that B. subtilis lacks a SecB homologue (Kunst et al., 1997 ).


   ACKNOWLEDGEMENTS
 
We thank Dr A. M. Ledeboer for the synthetic CsaA-specific peptide, Mrs J. C. A. M. Bun and Dr H. G. Seijen for preparing the CsaA antiserum, Professor D. B. Oliver for strain MM171.3, Dr R. Freudl for antibodies against OmpA, Dr J. Tommassen for antibodies against SecB and Mrs S. Gräfe for pSEL16. We thank Dr A. J. M. Driessen and Dr B. Bukau for useful discussions. J.M. was supported by the European Union in the framework of the BRIDGE programme and by the Deutsche Forschungsgemeinschaft. S.B. and J.M.v.D. were supported in part by Biotechnology Grants Bio2-CT93-0254 and Bio4-CT96-0097 from the European Union.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Altman, E., Kumamoto, C. A. & Emr, S. D. (1991). Heat-shock proteins can substitute for SecB function during protein export in Escherichia coli. EMBO J 10, 239-245.[Abstract]

Ang, D., Chandrasekhar, G. N., Zylicz, M. & Georgopoulos, C. (1986). Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature. J Bacteriol 167, 25-29.[Medline]

den Blaauwen, T. & Driessen, A. J. M. (1996). Sec-dependent preprotein translocation in bacteria. Arch Microbiol 165, 1-8.[Medline]

Bolhuis, A., Sorokin, A., Azevedo, V., Ehrlich, S. D., Braun, P. G., de Jong, A., Venema, G., Bron, S. & van Dijl, J. M. (1996). Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I. Mol Microbiol 22, 605-618.[Medline]

Bolhuis, A., Broekhuizen, C. P., Sorokin, A., van Roosmalen, M. L., Venema, G., Bron, S., Quax, W. J. & van Dijl, J. M. (1998). SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins. J Biol Chem 273, 21217-21224.[Abstract/Free Full Text]

Bolivar, F., Rodriguez, R. L., Betlach, M. C. & Boyer, H. W. (1977). Construction and characterization of new cloning vehicles. I Ampicillin-resistant derivatives of the plasmid pMB9. Gene 2, 75-93.[Medline]

Brickman, E. R., Oliver, D. B., Garwin, J. L., Kumamoto, C. A. & Beckwith, J. (1984). The use of extragenic suppressors to define genes involved in protein export in Escherichia coli. Mol Gen Genet 196, 24-27.[Medline]

de Cock, H. & Randall, L. L. (1998). Correlation between requirement for SecA during export and folding properties of precursor polypeptides. Mol Microbiol 27, 469-476.[Medline]

Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. (1997). The chemistry and enzymology of the type I signal peptidases. Protein Science 6, 1129-1138.[Abstract/Free Full Text]

Danese, P. N., Murphy, C. K. & Silhavy, T. J. (1995). Multicopy suppression of cold-sensitive sec mutations in Escherichia coli. J Bacteriol 177, 4969-4973.[Abstract]

De Gier, J. W., Valent, Q. A., von Heijne, G. & Luirink, J. (1997). The E. coli SRP: preferences of a targeting factor. FEBS Lett 408, 1-4.[Medline]

van der Does, C., Manting, E. H., Kaufmann, A., Lutz, M. & Driessen, A. J. M. (1998). Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state. Biochemistry 37, 201-210.[Medline]

van Dijl, J. M., de Jong, A., Smith, H., Bron, S. & Venema, G. (1991). Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli.Mol Gen Genet 227, 40-48.[Medline]

van Dijl, J. M., de Jong, A., Vehmaanperä, J., Venema, G. & Bron, S. (1992). Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J 11, 2819-2828.[Abstract]

Duong, F., Eichler, J., Price, A., Leonard, M. R. & Wickner, W. (1997). Biogenesis of the Gram-negative bacterial envelope. Cell 91, 567-573.[Medline]

Economou, A. & Wickner, W. (1994). SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell 78, 835-843.[Medline]

Economou, A., Pogliano, J. A., Beckwith, J., Oliver, D. B. & Wickner, W. (1995). SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell 83, 1171-1181.[Medline]

Fekkes, P. & Driessen, A. J. M. (1999). Protein targeting to the bacterial cytoplasmic membrane. Microbiol Mol Biol Rev 63, 161-173.[Abstract/Free Full Text]

Fekkes, P., van der Does, C. & Driessen, A. J. M. (1997). The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of the precursor protein translocation. EMBO J 16, 6105-6113.[Abstract/Free Full Text]

Georgopoulos, C. P. (1977). A new bacterial gene (groPC) which affects lambda DNA replication. Mol Gen Genet 151, 35-39.[Medline]

Gräfe, S., Ellinger, T. & Malke, H. (1996). Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A. Med Microbiol Immunol 185, 11-17.[Medline]

Hartl, F.-U., Lecker, S., Schiebel, E., Hendrick, J. P. & Wickner, W. (1990). The binding cascade of SecB to SecA to SecY/E mediates pre-protein targeting to the Escherichia coli plasma membrane. Cell 63, 269-279.[Medline]

von Heijne, G. (1990). The signal peptide. J Membrane Biol 115, 195-201.[Medline]

Hendrick, J. P. & Hartl, F.-U. (1993). Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem 62, 349-384.[Medline]

Hesterkamp, T. & Bukau, B. (1998). Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E. coli. EMBO J 17, 4818-4828.[Abstract/Free Full Text]

Honda, K., Nakamura, K., Nishiguchi, M. & Yamane, K. (1993). Cloning and characterization of a Bacillus subtilis gene encoding a homolog of the 54-kilodalton subunit of mammalian signal recognition particle and Escherichia coli Ffh. J Bacteriol 17, 4885-4894.

Ito, K., Bassford, P.Jr & Beckwith, J. (1981). Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer membrane proteins? Cell 24, 707-714.[Medline]

Jeong, S. M., Yoshikawa, H. & Takahashi, H. (1993). Isolation and characterization of the secE homologue gene of Bacillus subtilis.Mol Microbiol 10, 133-142.[Medline]

Kim, Y. J. & Oliver, D. B. (1994). Escherichia coli SecY and SecE protein appear insufficient to constitute the SecA receptor. FEBS Lett 339, 175-180.[Medline]

Kumamoto, C. A. (1989). Escherichia coli SecB protein associates with exported protein precursors in vivo. Proc Natl Acad Sci USA 86, 5320-5324.[Abstract]

Kumamoto, C. A. & Beckwith, J. (1985). Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol 163, 267-274.[Medline]

Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[Medline]

Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y. & Ito, K. (1989). Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J 8, 3517-3521.[Abstract]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]

Lee, C. A. & Beckwith, J. (1986). Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol 166, 878-883.[Medline]

Luirink, J., High, S., Wood, H., Giner, A., Tollervey, D. & Dobberstein, B. (1992). Signal sequence recognition by an Escherichia coli ribonucleoprotein complex. Nature 359, 741-743.[Medline]

Mead, D. A., Szczesna-Skorupa, E. & Kemper, B. (1986). Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Engng 1, 67-74.[Abstract]

Meijer, W. J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J. M. (1995). The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids. Mol Microbiol 17, 621-631.[Medline]

Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Müller, J. (1996). Influence of impaired chaperone or secretion function on SecB production in Escherichia coli. J Bacteriol 178, 6097-6104.[Abstract]

Müller, J., Walter, F., van Dijl, J. M. & Behnke, D. (1992). Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis. Mol Gen Genet 235, 89-96.[Medline]

Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, H. & Yamane, K. (1990). Cloning and characterization of a Bacillus subtilis gene homologous to Escherichia coli secY. J Biochem 107, 603-607.[Abstract]

Närvänen, A. (1990). Synthetic peptides as probes for protein interactions and as antigenic epitopes. PhD thesis, University of Jyväskylä, Finland.

Oliver, D. B. (1985). Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli. J Bacteriol 161, 285-291.[Medline]

Oliver, D. B. & Beckwith, J. (1981). Escherichia coli mutant pleiotropically defective in the export of secreted proteins. Cell 25, 765-772.[Medline]

Oliver, D. B. & Beckwith, J. (1982). Regulation of a membrane component required for protein secretion in Escherichia coli. Cell 330, 311-319.

Overhoff, B., Klein, M., Spies, M. & Freudl, R. (1991). Identification of a gene fragment which codes for the amino-terminal 364 amino acid residues of a SecA homologue from Bacillus subtilis: further evidence for the conservation of the protein export apparatus in Gram-positive and Gram-negative bacteria. Mol Gen Genet 228, 417-423.[Medline]

Overhoff-Freundlieb, B. & Freudl, R. (1991). Suppression of an Escherichia coli secAts mutant by a gene cloned from Staphylococcus carnosus. FEMS Microbiol Lett 84, 1143-1150.

Phillips, G. J. & Silhavy, T. J. (1992). The Escherichia coli ffh gene is necessary for viability and efficient protein export. Nature 359, 744-746.[Medline]

Pohlschröder, M., Prinz, W. A., Hartmann, E. & Beckwith, J. (1997). Protein translocation in the three domains of life: variations on a theme. Cell 91, 563-566.[Medline]

Polissi, A., Goffin, L. & Georgopoulos, C. (1995). The Escherichia coli heat shock response and bacteriophage lambda development. FEMS Microbiol Rev 17, 159-169.[Medline]

Pugsley, A. P. (1993). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50-108.[Abstract]

Sadaie, Y., Takamatsu, H., Nakamura, K. & Yamane, K. (1991). Sequencing reveals similarity of the wild-type div+ gene of Bacillus subtilis to the Escherichia coli secA gene. Gene 98, 101-105.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schmidt, A. M., Schiesswohl, M., Völker, U., Hecker, M. & Schumann, W. (1992). Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon of Bacillus subtilis. J Bacteriol 174, 3993-3999.[Abstract]

Schmidt, M. G. & Oliver, D. B. (1989). SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli. J Bacteriol 171, 643-649.[Medline]

Schröder, H., Langer, T., Hartl, F.-U. & Bukau, B. (1993). DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J 12, 4137-4144.[Abstract]

Seoh, H. K. & Tai, P. C. (1997). Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across Escherichia coli membranes. J Bacteriol 179, 1077-1081.[Abstract]

Seoh, H. K. & Tai, P. C. (1999). Catabolic repression of secB expression is positively controlled by cyclic AMP (cAMP) receptor protein-cAMP complexes at the transcriptional level. J Bacteriol 181, 1892-1899.[Abstract/Free Full Text]

Skinner, M. K. & Griswold, M. D. (1983). Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem J 209, 281-284.[Medline]

Struck, J. C. R., Vogel, D. W., Ulbrich, N. & Erdmann, V. A. (1988). The Bacillus subtilis scRNA is related to the 4.5S RNA from Escherichia coli.Nucleic Acids Res 16, 2719.[Medline]

Suh, J.-W., Boylan, S. A., Thomas, S. M., Dolan, K. M., Oliver, D. B. & Price, C. W. (1990). Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria. Mol Microbiol 4, 305-314.[Medline]

Sunshine, M., Feiss, M., Stuart, J. & Yochem, J. (1977). A new host gene (groPC) necessary for lambda DNA replication. Mol Gen Genet 151, 27-34.[Medline]

Tjalsma, H., Noback, M. A., Bron, S., Venema, G., Yamane, K. & van Dijl, J. M. (1997). Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes. J Biol Chem 272, 25983-25992.[Abstract/Free Full Text]

Tjalsma, H., Bolhuis, A., van Roosmalen, M. L., Wiegert, T., Schumann, W., Broekhuizen, C. P., Quax, W. J., Venema, G., Bron, S. & van Dijl, J. M. (1998). Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases Genes Dev 12, 2318-2331.[Abstract/Free Full Text]

Topping, T. B. & Randall, L. L. (1997). Chaperone SecB from Escherichia coli mediates kinetic partitioning via a dynamic equilibrium with its ligands. J Biol Chem 272, 19314-19318.[Abstract/Free Full Text]

Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354.[Abstract]

Van Dyk, T. K., Gatenby, A. A. & LaRossa, R. A. (1989). Demonstration by genetic suppression of interaction of GroE products with many proteins. Nature 342, 451-453.[Medline]

Wetzstein, J., Völker, U., Dedio, J., Löbau, S., Zuber, U., Schiesswohl, M., Herget, C., Hecker, M. & Schumann, W. (1992). Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis.J Bacteriol 174, 3300-3310.[Abstract]

Wickner, W., Driessen, A. J. M. & Hartl, F.-U. (1991). The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem 60, 101-124.[Medline]

Wild, J., Altman, E., Yura, T. & Gross, C. A. (1992). DnaK and DnaJ heat shock proteins participate in protein export in Escherichia coli. Genes Dev 6, 1165-1172.[Abstract]

Wild, J., Walter, W. A., Gross, C. A. & Altman, E. (1993). Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response. J Bacteriol 175, 3992-3997.[Abstract]

Wild, J., Rossmeissl, P., Walter, W. A. & Gross, C. A. (1996). Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli. J Bacteriol 178, 3608-3616.[Abstract]

Wolin, S. L. (1994). From elephant to E. coli: SRP-dependent protein targeting. Cell 77, 787-790.[Medline]

Received 17 June 1999; revised 27 September 1999; accepted 12 October 1999.