Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilissecA mutant strains

ELSEVIER

FEMS Microbiology

Letters 131(1995)

271-277

Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains Michael Klein, Jochen Meens, Roland Freud1

*

Institutfir Biotechnologie I, ForschungszentrumJiilich GmbH, D-52425 Jiilich, Germany Received 7 June 1995; accepted

12 July 1995

Abstract The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe. The S. carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B. subtilis secA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other

components of bacterial preprotein translocase are optimized within each organism. Keywords: Staphylococcus carnosus; Protein translocation;

SecA protein; Preprotein

1. Introduction In Escherichia coli, the translocation of proteins across the plasma membrane is mediated by preprotein translocase, a multi-subunit complex consisting of the integral membrane proteins SecY, SecE, SecG and the peripheral subunit SecA [l]. The SecA protein [2] has been shown to initiate the translocation of precursor proteins by undergoing Am-driven cycles of membrane insertion and de-insertion [3], after which further tram&cation might mainly be driven by the protonmotive force 141. Gram-positive bacteria can secrete large amounts of proteins directly into the growth medium and are

* Corresponding author. Tel.: +49 (2461) 613 472; Fax: +49 (2461) 612 710; E-mail: [email protected]. 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(95)00267-7

of European

Microbiological

translocase

considered to be attractive as potential host organisms for the secretory production of homologous and heterologous proteins. In the last few years, substantial progress has been made in the elucidation of the mechanism of protein transport in these microorganisms [5]. Homologues of SecA [6,7], SecY [8,9], and SecE [lo] have been identified in Bacillus subtilis, demonstrating that the components of preprotein translocase and, most likely, the mechanism of protein translocation are highly conserved in eubacteria. In contrast to most Bacillus species, the Grampositive bacterium Staphylococcus carnosus is mainly devoid of extracellular proteases [ll]. Therefore, S. cm-noms seems to be a promising host for the secretory production of protease-labile, heterologous proteins and detailed knowledge of the mechanism of protein translocation in this organism would be desirable. So far, the integral membrane components Societies. AR rights reserved

M. Klein et al. / FEMS Microbiology Letters 131 (1995) 271-277

272

SecY [12] and SecE [13] of the S. carnosus preprotein translocase have been identified. In this communication we describe the cloning and nucleotide sequence of the S. carnosus secA gene. In addition, the corresponding S. car-noms SecA protein was expressed and functionally characterized in E. coli and B. subtilis secA mutant strains.

into pHSG576 [19] and, after transformation into SURETM, by subsequent screening of the resulting transformants by colony hybridization using the B. subtilis secA gene [20] as a probe. Plasmid pMA12 was obtained by cloning a 4-kb HpaI/PstI DNA fragment from pMA10, harboring a promoterless S. carnosus secA gene, into E. coli expression vector pTRC99A [21]. Plasmids pMKL18 (containing the E. coli secA gene) and pMKL40 (containing the B. subtilis secA gene) have been described previously [20]. For the expression of the secA genes in B. subtilis, the corresponding promoterless secA genes were subcloned into B. subtilis expression vector pWH1520 [22]. Plasmid pWAl1 (containing the S. carnosus secA gene) was obtained by cloning a 2.6-kb BstXI/SphI DNA fragment from pMA12 into pWH1520. pWEl0 (containing the E. coli secA gene) was constructed by cloning a 2.7-kb NdeI/XbaI DNA fragment of pMKL18 into pWH1520. Plasmid pWMKL1 (containing the B. subtilis secA gene) has been described previously

2. Materials and methods

2.1. Bacterial strains and growth conditions E. coli SURETM (Stratagene) and MM66 ( geneXom, supF”) [14] were grown in LB medium [15] supplemented with 50 pg ml-’ ampicillin, 30 pg ml-’ chloramphenicol, 0.5% (w/v> glucose, or 1 mM IPTG, as required. B. subtilis NIG11.52 (diu431”) [16] was grown in LB or S7 minimal medium [17] supplemented with 1.5 pg ml-’ chloramphenicol, 15 pg ml-’ tetracycline, 0.5% (w/v) glucose, or 0.5% (w/v) xylose, as required. S. carnosus TM300 [ll] was grown in LB medium.

1231.

2.2. DNA techniques

2.3. Miscellaneous

Isolation of chromosomal DNA, Southern hybridization and other DNA techniques followed standard procedures [181. Plasmid pMA10, containing the S. carnosus secA gene, was obtained by cloning &I-digested chromosomal DNA from S. carnosus

Western blotting and pulse-chase experiments in B. subtilis were done as described previously [17]. Nucleotide and amino acid sequences were analyzed with the PC/Gene software package (IntelliGenetics Inc., Mountain View, CA).

Table 1 Degree of relatedness SC SC EC Bs cc ss Lm PI 01 At

46.6 60.2 42.5 42.4 60.4 38.5 34.1 38.4

techniques

of SecA proteins from various organisms EC

Bs

cc

ss

Lm

Pl

01

At

61.6

73.2 64.5

57.7 64.9 61.2

56.7 58.9 60.8 53.8

73.2 63.7 79.7 57.9 60.2

52.6 51.8 54.1 51.2 62.1 51.9

50.0 51.0 52.2 46.7 58.2 51.4 52.7

53.3 53.7 54.7 52.5 65.4 55.0 59.8 59.2

52.4 51.5 46.2 50.2 38.0 36.4 39.5

47.0 46.1 67.5 39.8 36.0 40.5

42.3 45.5 36.9 31.8 38.6

46.3 47.5 42.6 49.9

38.4 35.4 40.2

39.0 46.9

42.7

Values below the diagonal represent percentage of identical amino acid residues; values above the diagonal represent percentage of identical plus chemically related amino acids. The SecA proteins are from Staphylococcus carnosus (SC; X79725), Escherichia coli (EC; M20790, Bacillus subtilis (Bs; D10279, D90218), Caulobacter crescentus (Cc; UO6928). Synechococcus sp. (Ss; X74592), Listeria monocytogenes (Lm; L.32090), Pauloua futherii (PI; X65961), Olisthodiscus luteus (01; 235718), and Antithamnion sp. (At; X64705). The numbers indicated in parentheses represent the accession numbers of the corresponding nucleotide sequences in the EMBL data library.

M. Klein et al. / FEMS Microbiology Letters 131 (1995) 271-277

1

121

255

392

509

636 763

190

Fig. 1. Nucleotide and predicted amino acid sequences of a 3.7-kb DNA fragment from the S. c(~rnosus secA region. Nucleotides are numbered from the 5’ end. The putative ribosome-binding sites (RBS) and a putative terminator region are underlined. The sequence data reported in this paper appear in the EMBL Nucleotide Sequence Data Library under accession number X79725.

274

M. Klein et al. /FEMS Microbiology Letters 131 (1995) 271-277

12345678

3. Results 3.1. Cloning and nucleotide carnosus secA gene

sequence

of the S.

Chromosomal DNA of S. carnosus was digested with various restriction enzymes and Southern blotting was performed using the B. subtilis secA gene [20] as a probe. In PstI-digested DNA, a single 8.5kb DNA fragment gave rise to a positive hybridization signal. For the cloning of this fragment, chromosomal DNA of S. carnosus was digested with PstI and fragments, 7-10 kb in size, were isolated and cloned into plasmid pHSG576 [19]. Screening of this partial gene library with the B. subtilis secA gene by colony hybridization resulted in the isolation of plasmid pMA10, which contained a PstI insert of 8.5 kb. The nucleotide sequence of a 3.7-kb DNA fragment from pMAl0, encompassing the S. carnosus secA gene is shown in Fig. 1. Two open reading frames (ORFsl were identified on this DNA fragment. The second ORF (ORF2) starts with an ATG codon at position 1012 and encodes a putative protein of 844 amino acid residues. Upstream of ORF2, a putative ribosome binding site (AGGAG) at positions 999-1003 can be identified. Downstream of ORF2, sequences are found between positions 3558-3646 which might constitute a rhoindependent terminator. Computer analysis of the deduced amino acid sequence revealed that ORF2 is highly homologous to other known SecA proteins (Table I), strongly suggesting that we indeed have cloned the S. carnosus secA homologue. The ORF which is located upstream of the S. carnosus secA gene (ORFl; positions 56-619) was found to be homologous to a B. subtilis gene (ORF1891 which, also in this case, is located upstream of the secA gene [24] and which might be involved in the regulation of u54 activity [25]. 3.2. The S. carnosus SecA protein does not complement the growth defect of a conditional-lethal E. coli secA mutant For the functional analysis of the S. carnosus SecA in the temperature-sensitive E. coli secA mutant MM66 (geneXam, supF’“) [14], the S. carnosus sec4 gene was subcloned into the expression vector

6

Fig. 2. Western blot showing expression of SecA proteins in E. coli and B. subtilis. Total cell extracts were subjected to SDSPAGE and immunoblotting using a mixture of anti-E. coli SecA and anti-B. subtilis SecA antibodies. Open circle, E. coli SecA protein; arrowhead, E. subtilis and S. cnrnosus SecA protein. (A) Ceil extracts of E. coli MM66 containing pTRC99A (lanes 1, 2), pMIU18 (lanes 3, 4), pMA12 (lanes 5, 6) or pMKL40 (lanes 7, 8) grown in the presence of 0.5% glucose (odd-numbered lanes) or 1 mM IPTG (even-numbered lanes). (B) Cell extracts of B. subtilis NIG1152 containing pWH1520 (lanes 1, 2), pWEl0 (lanes 3, 4). pWAl1 (lanes 5, 6) or pWMKL1 (lanes 7, 8) grown in the presence of 0.5% glucose (odd-numbered lanes) or 0.5% xylose (even-numbered lanes).

pTRC99A [21] under the regulatory control of the trc-promoter/lac-operator, resulting in plasmid pMA12. Plasmids pMKL18 (containing the E. coli secA gene) and pMKL40 (containing the B. subtilis secA gene) have been described previously 1201. pTRC99A, pMA12, pMKL18, and pMKL40 were transformed into MM66 and expression of the secA genes was analysed under repressed (i.e. in the presence of 0.5% glucose) and induced (i.e. in the presence of 1 mM IPTG) conditions by Western blotting using a mixture of anti-E. coli SecA and anti-B. subtilis SecA antibodies (Fig. 2A). Whereas under inducing conditions high-level synthesis of the corresponding SecA polypeptides was observed, a basal level of expression was found under repressed conditions which, most likely, is due to a leakiness of the strong trc promoter. The apparent molecular masses of the respective SecA proteins were in good agreement with the molecular masses calculated from the corresponding amino acid sequences. Since we previously found that the growth defect of MM66 could be complemented by the B. subtilis SecA protein only under conditions of low-level synthesis [20] and since we noticed that high-level synthesis of the B. subtilis or the S. carnosus SecA protein in MM66 frequently resulted in lysis and cell

M. Klein et al. / FEMS Microbiology Table 2 Growth complementation Plasmid

pTRC99A pMKL18 PMI’=~O pMA12

of E. coli secA mutant MM66

se& allele

None E. coli B. subtilis S. carnosus

Cells of E. coli MM66 carrying plated on LB plates, supplemented 42°C. + , growth; - , no growth.

Letters 131 (1995) 271-277 Table 3 Growth complementation

42°C

+ + + +

+ + -

the respective plasmids were with 0.5% glucose, at 30°C or

death, complementation of the growth defect of MM66 by the various SecA proteins was tested under repressed (i.e. in the presence of glucose) conditions (Table 2). Plating of pTRC99A, pMA12, pMKL18 or pMKL40-containing cells of MM66 at 42°C revealed that, in contrast to the E. coli and B. subtilis secA genes, the presence of the S. carnosus secA gene did not restore growth of MM66 at the non-permissive temperature, suggesting that the S. carnosus SecA could not functionally replace the E. coli SecA protein. 3.3. Complementation of a temperature-sensitive B. subtilis sed mutant by the S. carnosus SecA protein To allow the functional analysis of the SecA proteins in the temperature-sensitive B. subtilis secA mutant NIG1152 (dio431’“) 1161, the secA genes of E. coli (pWElO), B. subtilis (pWMKLl), and S. carnosus (pWA11) were cloned into the B. subtilis expression vector pWH1520 [22] under the regulatory control of the xylose-inducible xylpromoter/operator. As shown in Fig. 2B, induction with xylose resulted in high-level synthesis of the corresponding SecA proteins in B. subtilis NIG1152. In the presence of glucose, hardly any synthesis above the level of chromosomally encoded B. subtilis SecA is detected. The ability of the various SecA proteins to complement the growth defect of NIG1152 was tested under repressed (i.e. in the presence of glucose) and induced (i.e. in the presence of xylose) conditions (Table 31. In contrast to the E. coli secA gene, which did not restore growth of NIG1152 at the non-per-

of B. subtilis sec.4 mutant NlG1152

secA allele

Plasmid

MM66 30°C

215

NIG1152 30°C

None E. coli B. subtilis S. carrwsus

pWH1520 pWEl0 pWMKL1 pWAl1 Cells of B. were plated 0.5% xylose + , growth;

42°C

G

X

+ + + +

+ + + +

G

X

_

_

+ ?-

+ +

subtilis NIG1152 containing the respective plasmids on LB plates in the presence of 0.5% glucose (G) or (Xl at 30°C or 42”C, respectively. - , no growth.

missive temperature under either condition, the B. subtilis and the S. carnosus secA genes fully complemented the growth defect of NIG1152 at 42°C regardless of the state of induction. This finding strongly suggests that even very low levels of the S. carnosus SecA protein are sufficient to substitute for the B. subtilis SecA polypeptide in growth complementation. To test whether the S. carnosus SecA protein can in fact promote the translocation of precursor proteins in B. subtilis, pulse-chase experiments were performed monitoring the processing of a model protein (Staphylococcus hyicus pre-pro-lipase; [ 111) in NIG1152 at the non-permissive temperature (Fig. 3). We have shown previously that in B. subtilis the 1

2

3

4

5

6

7

8

3

10

II

12 0 .

Fig. 3. Processing of pre-pro-lipase in B. subtilis secA mutant NIG1152 at 42°C. Cells of NIG1152 containing pLipPS1 [ll] in addition to pWH1520 (lanes l-41, pWMKL1 (lanes 5-8) or pWAl1 &-mes 9-12) were grown in 8.7 medium at 42°C for 3 h, labelled with [35S]methionine for 1 min and subsequently chased with an excess of non-radioactive methionine. The samples represent chase times of 30 s (lanes 1, 5, 91, 2 min (lanes 2, 6, lo), 5 min (lanes 3, 7, 11) and 10 min (lanes 4, 8, 12). Open circle, pre-pro-lipase; arrowhead, pro-lipase; square, mature lipase-sized degradation product.

276

M. Klein et al. / FEMS Microbiology

S. hyicus pre-pro-lipase

is processed by signal peptidase and, after translocation across the plasma membrane, the resulting pro-lipase is further degraded to, among others, mature lipase-sized polypeptides [17]. Compared to the vector control (lanes l-41, the presence of the S. carnosus SecA protein significantly restored processing of the pre-pro-lipase in NIG1152 at 42°C (lanes 9-121, although with lower efficiency than the B. subtilis SecA protein (lanes 5-8).

4. Discussion In this communication, we describe the cloning and characterization of the secA gene from the Gram-positive bacterium S. carnosus. S. CUT~OSUS secA encodes a polypeptide of 844 amino acid residues which harbors significant homologies to the other known SecA proteins. Regarding the size and amino acid sequence identity, S. carnosus SecA is closely related to the SecA proteins of the Grampositive bacteria B. subtilis (841 amino acid residues; 60.2% identity) and Listeria monocytogenes (836 amino acid residues; 60.4% identity). This is also supported by the fact that the S. carnosus SecA protein strongly cross-reacted with anti-B. subtilis SecA antibodies. Expression of the S. carnosus secA gene in the E. coli secA mutant MM66 did not relieve the growth defect of MM66 at the non-permissive temperature under all conditions tested. In contrast and as has been reported previously [20], the B. subtilis SecA protein allowed growth of MM66 at 42°C provided it was not overproduced. These results could reflect the fact that the B. subtilis SecA is somewhat more closely related (52.4% identity) to the E. coli SecA protein than is the S. carnosus SecA polypeptide (46.6% identity). In contrast to its behavior in the E. cofi secA mutant strain, S. carnosus SecA was fully able to complement the growth defect of the temperaturesensitive B. subtilis secA mutant NIG1152. Furthermore, the presence of S. curnosus SecA in NIG1152 resulted in substantial restoration of the secretion of a model protein (S. hyicus pre-pro-lipase) at 42°C. Although processing of pre-pro-lipase in the presence of S. carnosus SecA occurred with lower effi-

Letters 131 (1995) 271-277

ciency than in the presence of B. subtilis SecA, our results clearly demonstrate that S. curnosus SecA can (at least to a certain extent) functionally replace B. subtilis SecA in protein translocation. In contrast, E. coli SecA did not complement the growth defect of NIG1152 at 42°C a finding which is consistent with the results obtained by Takamatsu et al. [16]. In summary, our results indicate that, although SecA seems to be a highly conserved component of the bacterial protein translocation apparatus, it cannot always be exchanged between different bacteria. This finding most likely reflects an optimization of the interactions between SecA and the integral components SecY/E/G of preprotein translocase and/or SecA and the corresponding precursor/ chaperone complexes within each organism.

Acknowledgements We are very grateful to F. Gotz, Y. Sadaie and W. Hillen for their kind gifts of bacterial strains and plasmids. We thank H. Sahm for his continuous support, A. Bida for excellent technical assistance, J. Carter-Sigglow for critically reading the manuscript and the members of the European Bacillus Secretion Group for valuable discussions. This work was supported by grants from the Bundesministerium hir Bildung, Wissenschaft, Forschung und Technologie and the CEC (Biotech Grant B102 CT 930254).

References ItI Wickner, W., Driessen, A.J.M. and Hart], F.-U. (1991) The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu. Rev. Biocbem. 60, 101-124. PI Oliver, D.B., Cabelli, R.J. and Jarosik, G.P. (1990) SecA protein: autoregulated initiator of secretory precursor protein translocation across the E. coli plasma membrane. J. Bioenerg. Biomembr. 22, 311-336. [31 Economou, A. and Wickner, W. (1994) SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell 78, 835-843. [41 Driessen, A.J.M. (1992) Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force. Trends Biochem. Sci. 17, 219-223. [51 Simonen, M. and Palva, I. (1993) Protein secretion in Bacillus species. Microbial. Rev. 57, 109-137.

M. Klein et al. / FEMS Microbiology Letters 131 (1995) 271-277 [6] Sadaie, Y., Takamatsu, H., Nakamura, K. and Yamane, K. (19911 Sequencing reveals similarity of the wild-type diu + gene of EaciNus subtilis to the Escherichia coli secA gene. Gene 98, 101-105. [7] Overhoff, B., Klein, M., Spies, M. and Freudl, R. (1991) Identification of a gene fragment which codes for the 364 amino-terminal amino acid residues of a SecA homologue from Bacillus subtilis: further evidence for the conservation of the protein export apparatus in Gram-positive and Gramnegative bacteria. Mol. Gen. Genet. 228, 417-423. [8] Suh, J.-W., Boylan, S.A., Thomas, S.M., Dolan, K.M., Oliver, D.B. and Price, C.W. (1990) lsolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria. Mol. Microbial. 4, 305-314. [9] Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, H. and Yamane, K. (19901 Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secy. J. B&hem. 107, 603-607. [lo] Jeong, SM., Yoshikawa, H. and Takahashi, H. (1993) Isolation and characterization of the secE homologue of Bacillus subtihs. Mol. Microbial. 10, 133-142. [ill Wenzig, E., Lottspeich, F., Verheij, B., de Haas, G.H. and Gdtz, F. (1990) Extracellular processing of the Staphylococcus hyicus lipase. Biochemistry (Life Sci. Adv.) 9, 47-56. [12] Tschauder, S., Driessen, A.J.M. and Freudl, R. (1992) Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family. Mol. Gen. Genet. 235, 147-152. [131 Meens, J., Klose, M. and Freudl, R. (19941 The Staphylococcus carnosus secE gene: cloning, nucleotide sequence, and functional characterization in Escherichia coli secE mutant strains. FEMS Microbial. Lett. 117, 113-120. [14] Oliver, D.B. and Beckwith, I. (1982) Regulation of a membrane component required for protein secretion in Escherichia coli. Cell 30, 311-319. [151 Miller, J.H. (19721 Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [16] Takamatsu, H., Fuma, S.-I., Nakamura, K., Sadaie, Y.,

277

Shinkai, A., Matsuyama, S.-I., Mizushima, S. and Yamane, K. (1992) In vivo and in vitro characterization of the secA gene product of Bacillus subtilis. J. Bacterial. 174, 43084316. [17] Meens, J., Frings, E., Klose, M. and Freudl, R. (19931 An outer membrane protein (OmpAl of Escherichia cob can be translocated across the cytoplasmic membrane of Bacillus subtilis. Mol. Microbial. 9, 847-855. [18] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1191 Takeshita, S., Sato, M., Toba, M., Masahashi, W. and Hashimoto-Gotoh, T. (1987) High-copy-number and lowcopy-number plasmid vectors for La& a-complementation and chloramphenicolor kanamycin-resistance selection. Gene 61, 63-74. [20] Klose, M., Schimz, K.-L., van der Wolk, J., Driessen, A.J.M. and Freudl, R. (1993) Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coh secA mutants in vivo. J. Biol. Chem. 268, 4504-4510. [21] Amann, E., Ochs, B. and Abel, K.-J. (1988) Tightly regulated promoter vectors useful for the expression of unfused and fused proteins in Escherichia coh. Gene 69, 301-315. [22] Rygus, T. and Hillen, W. (1991) Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl. Microbial. Biotechnol. 35, 594-599. [231 Klein, M., Hofmann, B., Klose, M. and Freudl, R. (1994) Isolation and characterization of a Bacillus subtihs secA mutant allele conferring resistance to sodium azide. FEMS Microbial. Lett. 124, 393-398. [24] Chen, L. and Helman, J.D. (1994) The Bacillus subtihs aD-dependent operon encoding the flagellar proteins FliD, FliS, and FliT. J. Bacterial. 176, 3093-3101, [25] Merrick, M.J. and Coppard, J.R. (1989) Mutation in genes downstream of the rpoN gene (encoding os4) of Klebsiella pneumoniae affect expression from os4-dependent promoters. Mol. Microbial. 3, 1765-1775.