The twin-arginine signal peptide of PhoD and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system.

The bacterial twin-arginine translocation (Tat) pathway has been recently described for PhoD of Bacillus subtilis, a phosphodiesterase containing a twin-arginine signal peptide. The expression of phoD is co-regulated with the expression of tatA(d) and tatC(d) genes localized downstream of phoD. To characterize the specificity of PhoD transport further, translocation of PhoD was investigated in Escherichia coli. By using gene fusions, we analyzed the particular role of the signal peptide and the mature region of PhoD in canalizing the transport route. A hybrid protein consisting of the signal peptide of beta-lactamase and mature PhoD was transported in a Sec-dependent manner indicating that the mature part of PhoD does not contain information canalizing the selected translocation route. Pre-PhoD, as well as a fusion protein consisting of the signal peptide of PhoD (SP(PhoD)) and beta-galactosidase (LacZ), remained cytosolic in the E. coli. Thus, SP(PhoD) is not recognized by E. coli transport systems. Co-expression of B. subtilis tatA(d)/C(d) genes resulted in the processing of SP(PhoD)-LacZ and periplasmic localization of LacZ illustrating a close substrate specificity of the TatA(d)/C(d) transport system. While blockage of the Sec-dependent transport did not affect the localization of SP(PhoD)-LacZ, translocation and processing was dependent on the pH gradient of the cytosolic membrane. Thus, the minimal requirement of a functional Tat-dependent protein translocation system consists of a twin-arginine signal peptide-containing Tat substrate, its specific TatA/C proteins, and the pH gradient across the cytosolic membrane.

The existence of a protein export pathway structurally and mechanistically similar to the ⌬pH-dependent pathway used for importing chloroplast proteins into the thylakoid has been shown for a variety of bacteria (1)(2)(3). Despite the fact that the mechanism of targeting and the transport of folded proteins via the ⌬pH/Tat 1 route is not yet understood, some common features characterize these translocation systems (for reviews, see Refs. 4 and 5). It has been shown that the Escherichia coli Tat system involves four proteins with calculated membrane-spanning domains (6 -8). TatA/TatE and TatB are sequence-related proteins that are homologous to Tha4 and Hcf106 of the ⌬pH-dependent thylakoid import pathway (7)(8)(9)(10)(11). Chloroplast cp-TatC has been described recently as the ortholog of E. coli TatC (12). While TatB and TatC appear to play a pivotal role in the Tat-dependent protein translocation in E. coli, TatA and TatE seem to fulfill complementary functions as the deletion of TatA or TatE does not block export, while the TatA/TatE double deletion drastically inhibits export (7). Expression studies suggested that tatE may be a cryptic gene duplication of tatA (13). An in vitro reconstituted translocation system demonstrated the necessity of TatA, TatB, and TatC for a functional E. coli Tat-dependent translocation system (14). The information about the structure of the Tat translocase is contradictory. While Bolhuis et al. (15) suggested that TatB and TatC proteins form a functional and structural unit of the E. coli Tat translocase, a recent report from Sargent et al. (16) demonstrated a double-layered ring structure with a central cavity of a complex consisting of TatA and TatB.
The presence of genes encoding TatA-and TatC-like proteins as well as the synthesis of exported proteins containing twinarginine signal peptides are strong indications for the existence of the Tat pathway in eubacteria (17). While most of the bacteria contain one copy of tatA and one copy of tatC (5) sequencing of bacilli genomes (i.e. Bacillus subtilis, Bacillus halodurans, and Bacillus stearothermophilus) indicated the presence of multiple TatA and TatC proteins. In particular, B. subtilis contains two tatC-and three tatA-like genes (18). Both tatC genes are localized directly downstream from a tatA gene (19). A TatB-like protein appears to be absent from bacilli.
The recently described transport of PhoD of B. subtilis revealed that TatC could act as a specificity determinant for this process. While the inactivation of the tatC d completely inhibited the secretion of PhoD, the inactivation of the second tatC gene (tatC y ) had no effect on the secretion of PhoD (19). This observation was the first indication for the existence of multiple Tat pathways in a single bacterial cell with separate substrate specificity. PhoD is a secretory protein with a twinarginine signal peptide. We have shown previously that it is efficiently transported across the cytosolic membrane but only inefficiently processed. Slow processing of the enzymatically active precursor was shown to keep the protein at the outer surface of the cell envelope (20). The tatA/tatC gene pair (designated tatA d /C d ), localized downstream from phoD, is co-regulated with the expression of phoD (19).
To investigate the specificity of the PhoD transport further, we analyzed its transport in E. coli. By using gene fusion technology the particular role of the signal peptide and the mature region of PhoD in canalizing the transport was investigated. A fusion protein consisting of the signal peptide of ␤-lactamase (Bla) and mature PhoD was transported in a Secdependent manner. PhoD, as well as the fusion protein consisting of the signal peptide of PhoD (SP PhoD ) and LacZ, was shown to be export-incompetent in E. coli. The co-expression of the phoD-associated B. subtilis gene pair tatA d /C d resulted in the processing and the translocation of SP PhoD -LacZ. This transport was shown to be ⌬pH-dependent. Since the transport of SP PhoD -LacZ was independent of E. coli tat genes, the minimal requirement of a Tat transport system consists of a twin-arginine signal peptide-containing substrate, an adopted TatA/C pair, and the pH gradient across the bacterial cytosolic membrane. Table I lists the plasmids and bacterial strains used. TY medium (tryptone/yeast extract) contained Bacto tryptone (1%), Bacto yeast extract (0.5%), and NaCl (1%). For pulse-chase labeling experiment M9 minimal medium was prepared as described previously (21). When required, media were supplemented with ampicillin (100 g/ml), kanamycin (40 g/ml), chloramphenicol (20 g/ml), tetracycline (12.5 g/ml), arabinose (0.2%), isopropyl-␤-D-thiogalactopyranoside (IPTG, 1 mM), nigericin (1 M), and/or sodium azide (3 mM). [ 35 S]Methionine was provided by Hartman Analytic (Braunschweig, Germany), and the 14 C-Labeled molecular weight marker was from Amersham Biosciences, Inc.. DNA Techniques-Procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of E. coli were carried out as described in Sambrook et al. (22). Restriction enzymes were from MBI Fermentas. PCR was carried out with the VENT DNA polymerase (New England Biolabs).

Plasmids, Bacterial Strains, and Media-
To construct pAR3phoD, the phoD gene including its ribosome binding site was amplified from the chromosome of B. subtilis strain 168 by PCR using the primers P1 (5Ј-GAG GAT CCA TGA GGA GAG AGG GGA TCT TGA ATG GCA TAC GAC-3Ј) containing a BamHI site and P2 (5Ј-CGA TCC TGC AGG ACC TCA TCG GAT TGC-3Ј) containing a PstI site. The amplified fragment was cleaved with BamHI and PstI and cloned in the corresponding sites of pAR3. The resulting plasmid pAR3phoD allowed the arabinose-inducible expression of wild type phoD in E. coli.
To construct a gene fusion between bla and phoD genes, phoD deleted for its signal sequence was amplified using primer P3 (5Ј-GTA GGA TCC GCG CCT AAC TTC TCA AGC-3Ј) containing a BamHI site and primer P2 containing a PstI site. The amplified fragment was cleaved with BamHI and PstI and cloned in the corresponding sites of pUC19, resulting in plasmid pUC19ЈphoD. Next the 5Ј-region of ␤-lactamase encoding its signal sequence was amplified from plasmid pBR322 by PCR with primer B1 (5Ј-ATA GAA TTC AAA AAG GAA GAG TAT G-3Ј) containing an EcoRI site and primer B2 (5Ј-CTG GGG ATC CAA AAA CAG GAA GGC-3Ј) containing a BamHI site. The amplified PCR fragment was cleaved with BamHI and EcoRI and inserted into pUC19ЈphoD, cleaved with the same restriction enzymes, which resulted in plasmid pUC19bla-phoD. For easy selection of recom-binant clones, plasmid pORI24, containing a tetracycline resistance gene, was inserted 3Ј of the bla-phoD gene fusion using an unique PstI site. From the resulting plasmid pUC19bla-phoD-Tc an EcoRI-BglII fragment containing bla-phoD and the tetracycline resistance gene of pORI24 was isolated and inserted into pMUTIN2 cleaved with EcoRI and BamHI. In the plasmid pMutin2bla-phoD the bla-phoD gene fusion is under control of the IPTG-inducible P SPAC promoter.
To construct a gene fusion consisting of the signal sequence of phoD and lacZ, a DNA fragment encoding the signal peptide of PhoD and the translational start site of phoD was amplified by PCR with primer P1 containing a BamHI site and primer P4 (5Ј-GAG AAG GTC GAC GCA GCA TTT ACT TCA AAG GCC CC-3Ј) containing a SalI site and inserted into the corresponding sites of pORI24 resulting in plasmid pORI24phoDЈ. Next, the lacZ gene lacking nine 5Ј-terminal codons was amplified using primer L1 (5Ј-ACC GGG TCG ACC GTC GTT TTA CAA CG-3Ј) containing a SalI site and primer L2 (5Ј-GGG AAT TCA TGG CCT GCC CGG TT-3Ј) containing an EcoRI site and subsequently inserted into the corresponding sites of pORI24phoDЈ. The resulting plasmid pORI24phoDЈ-lacZ was linearized with BamHI and inserted into pAR3 cleaved with BglII. The resulting plasmid pAR3phoDЈ-lacZ allows the arabinose-inducible expression of the phoDЈ-lacZ gene fusion.
To obtain a plasmid that mediates an inducible overexpression of tatA d /tatC d of B. subtilis, the DNA region containing these genes including their ribosome binding sites was amplified by PCR with the primer T1 (5Ј-CAA GGA TCC CGA ATT AAG GAG TGG-3Ј) containing a BamHI site and primer T2 (5Ј-GGT CTG CAG CTG CAC TAA GCG GCC GCC-3Ј) containing a PstI site. The amplified fragment was cleaved with BamHI and PstI and cloned into the corresponding sites of pQE9 (Qiagen) resulting in pQE9tatA d /C d .
To obtain TG1 ⌬tatABCDE, plasmids pFAT44 and subsequently pFAT126 covering in-frame deletions of E. coli tatE and tatABCD genes, respectively, were transferred to the chromosome of TG1 as described (7). Mutant strain TG1 ⌬tatABCDE was verified phenotypically by mutant cell septation phenotype, hypersensitivity to SDS, and resistance to P1 phages as described previously (23).
SDS-PAGE and Western Blot Analysis-SDS-PAGE was carried out as described by Laemmli (24). After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Schleicher and Schü ll) as described by Towbin et al. (25). Proteins were detected using specific antibodies against PhoD (20), ␤-galactosidase (5 Prime 3 3 Prime, Inc., Boulder, CO), pro-OmpA (provided by R. Freudl), SecB (laboratory collection), and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma) according to the instructions of the manufacturer.
Protein Chase Experiments, Immunoprecipitation, and Quantification of Protein-Pulse-labeling experiments of E. coli strains were performed as described earlier (21). Cultures were pulse-labeled with 100 Ci of [ 35 S]methionine and chased with unlabeled methionine, and then samples were taken at the times indicated immediately followed by precipitation with trichloroacetic acid (0°C). After cell lysis proteins were precipitated with specific antibodies against PhoD (20), OmpA, ␤-galactosidase, or ␤-lactamase (5 Prime 3 3 Prime, Inc.). Relative amounts of radioactivity were estimated by using a phosphorimaging system (Fuji) and the associated image analytical software PC-BAS.
In Vivo Protease Mapping-In vivo protease mapping was carried out according to Kiefer et al. (26). For spheroplast formation, cells were grown in TY medium to exponential growth. To induce the plasmidencoded genes the medium was supplemented with arabinose (0.2%) and/or IPTG (1 mM) for 60 min. After spheroplast formation cells were treated with proteinase K (Sigma) or with proteinase K and Triton X-100 or remained untreated. PhoD or SP PhoD -LacZ were detected by Western blotting. Detection of cytosolic SecB revealed the proteinase K resistance of Triton X-100-untreated spheroplasts.

RESULTS
PhoD Is Not Transported in E. coli-The initial aim was to test whether PhoD could be exported by the Tat pathway in E. coli. For this purpose we placed the gene encoding this peptide under the control of the P BAD promoter of Salmonella typhimurium localized at plasmid pAR3 (27). The resulting plasmid allowed the arabinose-inducible enzymatically active production of PhoD in E. coli TG1 (data not shown). Since phosphodiesterase is highly toxic to E. coli after induction of PhoD synthesis cell growth immediately ceased. To assay transport of PhoD in E. coli TG1(pARphoD) pulse-chase experiments were performed. As shown in Fig. 1A no processing of the wild type pre-PhoD was observed even 60 min after chase, indicating that pre-PhoD was not translocated by the E. coli Tat machinery. Localization of PhoD was further analyzed by in vivo protease mapping. As shown in Fig. 1B pre-PhoD was not accessible to proteinase K at the outer side of the cytosolic membrane, demonstrating that PhoD remained in a cytosolic localization.
PhoD Can Be Transported via the Sec-dependent Protein Translocation Pathway-Absence of pre-PhoD processing in E. coli could be due to inefficient recognition of the signal peptide of PhoD by the E. coli Tat machinery or due to the nature of the mature part of the PhoD peptide. This B. subtilis protein could have unexpected folding characteristics or the necessity of cofactors not present in E. coli. To address this question, the DNA encoding the mature peptide of PhoD was fused to the region encoding the signal peptide of ␤-lactamase (SP Bla ). The resulting gene fusion was cloned into the pMUTIN2 vector containing an IPTG-inducible P SPAC promoter allowing the synthesis of the SP Bla -PhoD peptide. The transport and processing of this fusion protein was analyzed by immunoblotting of whole cell extracts of E. coli strain TG1(pMUTIN2bla-phoD). As shown in Fig. 2A, lane 2, SP Bla -PhoD was completely converted to a protein with a molecular weight of mature PhoD indicating the efficient transport of the protein. To elucidate the export path used for SP Bla -PhoD translocation, Sec-dependent transport was selectively inhibited by addition of sodium azide. While the presence of sodium azide abolished conversion of SP Bla -PhoD to PhoD, addition of nigericin did not retard processing of SP Bla -PhoD ( Fig. 2A, lanes 3 and 4). To analyze the Sec dependence of SP Bla -PhoD transport in a more detailed manner, expression of bla-phoD in E. coli TG1(pMUTIN2bla-phoD) was induced in the presence or absence of sodium azide, pulse-labeling with [ 35 S]-methionine was carried out, and PhoD was subsequently immunoprecipitated. Fig. 2B demonstrates the kinetics of conversion of SP Bla -PhoD to mature PhoD. The presence of sodium azide significantly retarded maturation of SP Bla -PhoD (Fig.  2C). To demonstrate that azide was effective in inhibiting Secdependent translocation, the processing of pro-OmpA was monitored from the same cultures. While in untreated culture pro-OmpA was quickly converted into its mature form, in the azide-treated culture processing of pro-OmpA was efficiently retarded (Fig. 2, D and E). These data indicate that PhoD can be transported in E. coli in a Sec-dependent manner. Thus, it can be concluded that the mature PhoD peptide is not canalizing the export route and does not prevent efficient transport or processing.
The Signal Peptide of PhoD Cannot Mediate Transport of LacZ in E. coli Wild Type Cells-It has been shown that signal peptides containing a twin-arginine motif can canalize transport of heterologous proteins via the Tat-dependent translocation route (for a review, see Ref. 5). The signal peptide of the E. coli trimethylamine-N-oxide reductase (TorA) has been successfully used to mediate Tat-dependent transport of the thylakoidal protein 23K, the glucose-fructose oxidoreductase of Zymomonas mobilis and green fluorescent protein (3,7,28,29). To test whether the signal peptide of PhoD is recognized by the E. coli Tat machinery and could canalize the transport of a protein in E. coli, we constructed a gene fusion consisting of the DNA region encoding the signal peptide of PhoD (SP PhoD ) and the lacZ gene encoding ␤-galactosidase as a reporter protein.
The gene hybrid was inserted into plasmid pAR3 resulting in plasmid pAR3phoDЈ-lacZ. Induction of production of the SP PhoD -LacZ fusion protein in E. coli TG1 resulted in LacZ ϩ colonies (data not shown). Hence, correct folding and tetramerization of the peptide as a prerequisite for its activity does occur in E. coli.
To analyze whether the signal peptide of PhoD could mediate translocation of LacZ into a extracytosolic localization, we studied localization of LacZ by using in vivo protease mapping. As shown in Fig. 3A no processing of SP PhoD -LacZ could be observed. The SP PhoD -LacZ fusion protein was not susceptible to

FIG. 1. Processing and localization of pre-PhoD in E. coli TG1.
A, E. coli TG1 carrying plasmid pARphoD encoding wild type PhoD was grown in M9 minimal medium to early logarithmic phase. 1 h prior to labeling expression of phoD was induced with IPTG (1 mM). Cells were labeled for 1 min with [ 35 S]methionine after which nonradioactive methionine was added. Samples were withdrawn at chase times 10, 20, 40, and 60 min and subjected to immunoprecipitation with monospecific antibodies against PhoD followed by SDS-PAGE using a 10% polyacrylamide gel and fluorography. M, molecular mass marker; Glu, uninduced control. B, in vivo protease mapping of PhoD in E. coli TG1(pAR3phoD). Cells were converted to spheroplasts and treated with proteinase K or with proteinase K and Triton X-100 or remained untreated as indicated. Localization of pre-PhoD is indicated. Accessibility of proteinase K to the cytosol was analyzed by monitoring SecB in a 15% polyacrylamide gel. PhoD and SecB were detected by monospecific antibodies.
protease digestion in spheroplasts. When spheroplasts were destroyed by addition of Triton X-100, the unprocessed SP PhoD -LacZ protein became protease-sensitive. The reliability of the method was verified by using the cytosolic protein SecB as internal control. In spheroplasts SecB was resistant to proteinase K but was digested after solubilizing the spheroplasts with Triton X-100.

Export of SP PhoD -LacZ Fusion Protein in E. coli Needs the Presence of the B. subtilis TatA d and TatC d Transport Components-
The data demonstrated above indicate that the Tat system of E. coli does not mediate transport of pre-PhoD or of the SP PhoD -LacZ fusion protein. Absence of translocation could be due to the necessity of additional components for the translocation of PhoD present only in B. subtilis or due to the specificity of recognition of pre-PhoD as a Tat-dependent substrate. To test the latter hypothesis, the B. subtilis tatA d /C d gene pair was coexpressed in E. coli strains TG1(pARphoD) and TG1(pARphoDЈ-lacZ).
To study the effect of TatA d /C d proteins on localization of PhoD, strain TG1(pARphoD, pREP4, pQE9tatA d /C d ) expression of phoD as well as tatA d /C d was induced with arabinose and IPTG. Unexpectedly, no PhoD could be detected in strain TG1(pARphoD, pREP4, pQE9tatA d /C d ) using Western blotting (data not shown). Induction of TatA d /C d proteins in strain TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ) resulted in stable co-production of TatA d /C d and SP PhoD -LacZ (data not shown). SP PhoD -LacZ processing was analyzed in the presence and absence of TatA d /C d using pulse-chase labeling and subsequent immunoprecipitation with specific antibodies against LacZ. While in TG1(pARphoDЈ-lacZ) no processing of SP PhoD -LacZ could be observed (Fig. 4A), in strain TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ) the peptide was at least partially processed (Fig. 4B).
Since processing of the translocation product is an indication of membrane translocation but does not necessarily prove that export of the protein has occurred, we examined whether LacZ was localized in the periplasmic space in TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ). To monitor localization of the LacZ peptide, cells of strain TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ) were converted to spheroplasts and treated with proteinase K. As shown in Fig. 3B after co-expression of tatA d /C d , SP PhoD -LacZ was completely susceptible to protease digestion in spheroplasts. Unexpectedly, both the processed form and the precursor of the fusion protein were accessible to the protease treatment. The presence of SecB after proteinase K treatment demonstrated the stability of spheroplasts. These results clearly show that the SP PhoD -LacZ fusion protein is exported into the periplasmic space of E. coli when the B. subtilis tatA d /C d genes are co-expressed.
TatA d /C d -mediated Transport of SP PhoD -LacZ Needs ⌬pHdependent Gradient at the Cytosolic Membrane and Is Secindependent-To directly prove that the membrane translocation of the system is dependent on the pH gradient across the cytosolic membrane, Sec-or Tat-dependent protein translocation pathways were selectively blocked. Localization of SP PhoD -LacZ in TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ) was de- . Samples were taken 20 min after induction of SP Bla -PhoD and lysed, and cell extracts were analyzed by SDS-PAGE using a 10% polyacrylamide gel. B-E, TG1(pMUTIN2bla-phoD) was grown in M9 minimal medium to early logarithmic phase. 1 h prior to labeling, expression of phoD was induced with IPTG (1 mM). While one culture remained untreated (B and D), the other was treated with sodium azide (3 mM) upon induction (C and E). Cells were labeled for 1 min with [ 35 S]methionine after which nonradioactive methionine was added. Samples were withdrawn at times after chase as indicated in the figures and subjected to immunoprecipitation with antibodies against PhoD (B and C) or against OmpA (D and E) followed by SDS-PAGE using a 12.5% polyacrylamide gel and fluorography. Localization of SP Bla -PhoD and mature PhoD is indicated. *, unspecific cross-reacting band. tected by Western blotting after in vivo protease mapping. Nigericin, an ionophore inhibiting the Tat-dependent protein translocation as a result of dissipating the ⌬pH (30), did efficiently block both processing and translocation of SP PhoD -LacZ (Fig. 5A). The addition of an equal volume of methanol used to disperse nigericin had no effect on localization of SP PhoD -LacZ (data not shown). Treatment of the culture with sodium azide, which inhibits Sec-dependent protein export by interfering with the translocation-ATPase activity of the SecA protein (31), did result in accumulation of pro-OmpA but did not affect the localization and the processing of the SP PhoD -LacZ fusion protein (Fig. 5B).
Tat d /C d -mediated Transport of SP PhoD -LacZ Is Not Assisted by E. coli Tat Components-To exclude co-operative action of B. subtilis and E. coli Tat proteins, E. coli strain TG1 was deleted for tatABCDE genes and subsequently transformed with plasmids pARphoDЈ-lacZ, pREP4, and pQE9tatA/C d . Processing and localization of the SP PhoD -LacZ fusion protein was analyzed under identical conditions as described for the E. coli tat ϩ strain. As shown in Fig. 6 in the absence of the E. coli tatAB-CDE genes most of SP PhoD -LacZ was protease-accessible demonstrating the extracytosolic localization of the fusion protein.
The resistance of SecB to the proteolytic digestion demonstrated the stability of the spheroplasts (Fig. 6). Surprisingly, no processing of the SP PhoD -LacZ fusion protein could be observed in the absence of tatABCDE.

DISCUSSION
In the present report we have shown that the export signals of PhoD, a Tat-dependent transported phosphodiesterase of B. subtilis, was incompatible with the Tat machinery of E. coli. While the mature part of PhoD could be exported efficiently with the help of the export signals of Sec-dependent ␤-lactamase, wild type PhoD or a fusion protein consisting of the signal peptide of PhoD and LacZ remained cytosolic. The coexpression of the phoD-associated genes tatA d and tatC d mediated the Tat-dependent translocation of the SP PhoD -LacZ fusion protein. Since transport of SP PhoD -LacZ was blocked in the presence of nigericin but not in the presence of sodium azide, it can be concluded that SP PhoD -LacZ is transported in a Secindependent manner. Transport of SP PhoD -LacZ in an E. coli tatABCDE strain revealed that transport was independent of the E. coli Tat components. These data show that the minimal requirement of a specific Tat-dependent protein translocation system is consisting of a pair of TatA and TatC proteins, a signal peptide specifically recognized by these Tat components, and the existence of the ⌬pH gradient across the cytosolic membrane.
PhoD as well as SP PhoD -LacZ is not recognized by the E. coli Tat system. Our previous results obtained in B. subtilis revealed that transport of PhoD is mediated by TatC d but is independent of the TatC y protein (19). These observations implied that B. subtilis contains at least two specific routes for Tat translocation. Further, E. coli tat strains could not be complemented by its B. subtilis Tat proteins (19). Finally, absence of E. coli tat genes did not prevent TatA d /C d -mediated transport of SP PhoD -LacZ in E. coli. These data strongly implicated that transport of hybrid peptides consisting of the signal peptide of PhoD, the reporter protein, and the TatA d /C d protein pair form an autonomous Tat translocation system, and the recognition of Tat substrates is a selective process determined by multiple special protein-protein interactions between a given Tat substrate and its specific Tat proteins.
Most of the twin-arginine signal peptide-containing E. coli precursors associate in the cytosol with cofactors (17). As shown for a mutant glucose-fructose oxidoreductase, association with its cofactor NADP was a prerequisite for the efficient export of the protein (30). We currently cannot exclude that PhoD needs the association of cofactors prior to translocation. Since the protein could be exported efficiently by using the Sec-dependent signal peptide of ␤-lactamase, necessity of a B. subtilis-specific cofactor association prior to transport appears to be unlikely.
The efficiency of SP Bla -PhoD processing in E. coli far exceeded transport kinetics observed for wild type PhoD observed in the gene donor strain. While the half-life of pre-PhoD in B. subtilis far exceeded 40 min (20), the half-life of the SP Bla -PhoD precursor in E. coli was about 10 min. Slow processing kinetics of pre-PhoD could be due to slower transport kinetics of the Tat pathway compared with the efficient Sec-dependent pathway. Overexpression of phoD in E. coli was highly toxic for the cell. Immediately after induction of PhoD synthesis cell growth was blocked. Most likely cytosolic phosphodiesterase activity was highly detrimental for E. coli. After co-expression of tatA d /C d no PhoD could be detected by using Western blotting. Coexpression of TatA d /C d proteins obviously resulted in additional impairment of cell viability preventing further protein synthesis of PhoD or raising protease degradation of the heterologous peptide. The induction of the SP PhoD -LacZ fusion protein was not lethal for the E. coli cell. Several signal peptide-LacZ fusion proteins were previously used for studies of the Sec-dependent protein transport in E. coli. These fusion proteins were usually not transported through the cytosolic membrane. High level induction of these proteins was frequently detrimental for E. coli due to jamming of the Sec machinery (32)(33)(34)(35)(36). Induction of production of SP PhoD -LacZ was not toxic for E. coli either in the absence or in the presence of B. subtilis TatA d /C d proteins. Since SP PhoD is not recognized by the E. coli Sec or Tat machinery the fusion protein remained cytosolic. Co-induction of B. subtilis TatA d /C d proteins transported SP PhoD -LacZ independent of E. coli-specific transport paths. Therefore, its translocation did not interfere with essential export functions of the cell. Tat-mediated export of LacZ is consistent with the capacity of the Tat translocation system to transport proteins that are probably folded prior to translocation.
Despite the fact that SP PhoD -LacZ was partially processed only in E. coli TG1(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ), the protein was entirely proteinase K-accessible. This observation indicates that the TatA d /C d components transport the protein efficiently through the cytosolic membrane, but cleavage of the signal peptide by E. coli LepB was inefficient. In E. coli TG1 ⌬tatABCE(pARphoDЈ-lacZ, pREP4, pQE9tatA d /C d ) no processing of SP PhoD -LacZ could be observed. At the moment there is no experimental knowledge about whether, when, and how the E. coli leader peptidase LepB cleaves signal peptides of Tat substrates. The presence of E. coli Tat components could be a prerequisite for cleavage of twin-arginine leader peptides by LepB.