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Applied and Environmental Microbiology, November 2006, p. 7183-7192, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01528-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869{triangledown}

Yoshimi Kikuchi,1* Masayo Date,1 Hiroshi Itaya,1 Kazuhiko Matsui,1 and Long-Fei Wu2

Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki 210-8681, Japan,1 Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, F-13402 Marseille, France2

Received 3 July 2006/ Accepted 13 September 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In prokaryotes, most secreted proteins are translocated across the cytoplasmic membrane by the Sec pathway, involving the membrane-bound SecYEG and SecDF complexes, which act on unfolded proteins using the energy provided by SecA-dependent ATP hydrolysis (8, 42). Recently, a second general pathway, designated the Tat pathway, has been discovered (3, 6), which differs from the Sec pathway in that it translocates folded proteins, many of which are complexed with cofactors. Most exported proteins contain an N-terminal signal peptide that is cleaved by a membrane-bound signal peptidase. The signal peptide of preproteins translocated via the Sec pathway comprises three distinct regions: an N-terminal positively charged region (n-region), a hydrophobic {alpha}-helical region (h-region), and a C-terminal domain that contains a signal peptidase cleavage site (53). The structure of the Tat pathway-dependent signal peptide is similar to that of the Sec pathway signal peptide, but it possesses a longer n-region, a weakly hydrophobic h-region, and, most notably, a conserved twin-arginine motif around the n- and h-region boundary (2, 3).

The Tat pathway is conserved in bacteria, archaea, and chloroplast thylakoids. The extensively studied bacterial model systems include those from the gram-negative bacterium Escherichia coli and from the gram-positive bacterium Bacillus subtilis. The Tat system of E. coli seems to operate with a mechanism similar to that of the Tat machinery of chloroplast thylakoids; both require three functional distinct membrane-bound components, TatA, TatB, and TatC (37, 40). The current prevalent working model is that TatB and TatC form a complex and are required for the recognition and binding of the twin-arginine signal peptide (1, 17). TatA constitutes a homo-oligomer complex, which is recruited by the TatB-TatC complex and probably fulfills a channel function in the protein export process (7, 19, 20). TatE, a TatA paralogue, exists only in enterobacteria (55) and is functionally overlapped with TatA in E. coli (45). The TatA and TatB proteins show a limited amino acid sequence similarity, mainly in the region of the transmembrane segment and the adjacent amphipathic helix. In this region, a conserved Phe-Gly motif is found in TatA and a conserved Gly-Pro motif is detected in TatB (28). Compared to TatA, TatB proteins contain a C-terminal extension. The B. subtilis genome carries the tatA, tatAyCy, and tatAdCd gene clusters, and one copy each of TatA and TatC is sufficient to sustain a functional Tat system (27). Generally, almost all gram-positive bacteria contain a tatAC cluster, with an additional tatA or tatB gene located at a separated locus (55). In addition to that of B. subtilis, a functional gram-positive bacterial Tat system has been demonstrated for Mycobacterium smegmatis (36, 41) and Streptomycetes lividans (12, 35, 47, 48).

Corynebacterium is a gram-positive, aerobic or facultatively anaerobic, generally nonmotile rod-shaped bacterium. Genomes of four species from this genus have been sequenced, i.e., the pathogen Corynebacterium diphtheriae (5), the nosocomial pathogen Corynebacterium jeikeium (50), the nondiphtheria corynebacterium Corynebacterium glutamicum (22, 29), and Corynebacterium efficiens (38). Like other gram-positive bacteria, C. diphtheriae and C. jeikeium possess tatA, tatB, and tatC genes organized in tatAC and tatB clusters. Remarkably, the genetic structures of the C. glutamicum ATCC 13032 and C. efficiens YS-314 Tat systems are unique in that they contain the tatE gene in addition to the tatA, tatB, and tatC genes (22, 29, 38). The function has not yet been demonstrated for any of the Corynebacterium Tat systems. The C. glutamicum species is widely used for the industrial production of amino acids, such as glutamate and lysine, that have been applied to human food, animal feed, and pharmaceutical products for several decades (33). We recently demonstrated that human epidermal growth factor and Streptomyces mobaraensis transglutaminase (MTG), an enzyme that is useful in the food industry, can be efficiently secreted in active forms by C. glutamicum isolate ATCC 13869 using a signal peptide derived from a corynebacterial cell surface protein (9, 10, 11, 32). For this report, we cloned the tatAC, tatB, and tatE genes from the isolate C. glutamicum ATCC 13869, constructed tat mutants, and assessed the function of the Tat system from Corynebacterium species and the potential of using the Tat system of C. glutamicum for industrial-scale protein production.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria strains, plasmids, and culture medium.
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli JM109 was grown in Luria broth and used as an intermediate host for various plasmid constructions. Arthrobacter globiformis and C. glutamicum were grown at 30°C in CM2G medium (5 g glucose, 10 g tryptone, 10 g yeast extract, 5 g NaCl, 0.2 g DL-methionine per liter distilled water, adjusted to pH 7.2) (32). Protein secretion was examined for C. glutamicum cultures grown in MMTG medium (32) at 30°C for 40 h. C. glutamicum was transformed by electroporation using a Gene Pulser (Bio-Rad) according to the manufacturer's protocol. Kanamycin (Km) (25 mg/liter) or chloramphenicol (Cm) (5 mg/ml) was added to the medium when required. A sodium dodecyl sulfate (SDS) sensitivity test was performed by inoculating cell culture on plates containing SDS at appropriate concentrations. Growth was monitored by absorbance at 660 nm.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
DNA manipulations.
DNA manipulations were carried out using the methods described by Sambrook et al. (43). PCRs with Pyrobest DNA polymerase (Takara Bio, Kyoto, Japan) were performed with 50-µl reaction mixtures for 5 min at 94°C and 25 cycles of 10 s at 98°C, 30 s at 55°C, and 3 min at 72°C, as specified by the manufacturer. Primers used in this study are shown in Table 2. Nucleotide sequences were determined using a BigDye terminator cycle sequencing FS ready reaction kit (Applied Biosystems) and a DNA sequencer (model 377; Applied Biosystems).


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  		TABLE 2. PCR primers used for plasmid constructions

 
Construction of tat gene deletion mutants of C. glutamicum.
All tat gene deletions were constructed by crossover PCR (Fig. 1A). First-round PCRs amplified chromosomal DNA of C. glutamicum ATCC 13869 using primer A (forward primer; Table 2) with primer B (reverse primer) and using primer C (forward primer) with primer D (as reverse primer). Second-round PCRs were performed on the first-round PCR products using primer A and primer D. Amplified fragments were inserted into the SmaI site of pBS4 to obtain pBS{Delta}A, pBS{Delta}B, pBS{Delta}C, and pBS{Delta}E, with a deletion of tatA, tatB, tatC, or tatE, respectively.


Figure 1
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  		FIG. 1. Construction of a deleted or fused gene by crossover PCR. (A) Schematic representation of construction of deletions of tatA, tatB, tatC, and tatE by crossover PCR using primers A and B and primers C and D for first-round PCRs and primers A and D for second-round PCR. (B) Schematic representation of construction of plasmids for secretion of IMD, pro-MTG, and GFP by crossover PCR using primers A and B and primers C and D for first-round PCRs and primers A and D for second-round PCR.

 
YDK010 was transformed with pBS{Delta}A, pBS{Delta}B, pBS{Delta}C, or pBS{Delta}E at 25°C, and obtained transformants were grown at 25°C for 20 h. They were further incubated overnight at 34°C on CM2G plates containing kanamycin. Since pBS{Delta}A, pBS{Delta}B, pBS{Delta}C, and pBS{Delta}E are temperature-sensitive replicons in C. glutamicum, transformants should form kanamycin-resistant colonies at 34°C (nonpermissive temperature) only if they have integrated the plasmid into the chromosome by homologous recombination. The intact tat genes on the chromosomes of the integrants were replaced with deleted tat genes through the second homologous recombination, and candidate tat gene deletion mutants were selected as kanamycin-sensitive colonies. The lengths of the amplified fragments in the kanamycin-sensitive colonies harboring the tatA, tatB, tatC, and tatE deletion mutants were determined by PCR using primers A and D (Table 2), complementary to the upstream and downstream regions of tatA, tatB, tatC, or tatE, respectively. The tatA, tatB, tatC, and tatE deletion mutants were designated YDK010A with nucleotides 911 to 1264 of the C. glutamicum tatA sequence deleted, YDK010B with nucleotides 411 to 490 of the C. glutamicum tatB sequence deleted, YDK010C with nucleotides 1458 to 2402 of the C. glutamicum tatC sequence deleted, and YDK010E with nucleotides 1037 to 1259 of the C. glutamicum tatE sequence deleted, respectively. We also obtained a tatA tatE double mutant using YDK010A and pBS{Delta}E, and this strain was designated YDK010AE.

Construction of a plasmid for IMD secretion.
A plasmid for isomaltodextranase (IMD) secretion containing the promoter of C. glutamicum cspB was constructed by crossover PCR (Fig. 1B). First-round PCR amplified the cspB promoter using primer A (Csp5K, forward primer; Table 2) and primer B (Csp-Imd, reverse primer) and pPSPTG11 DNA as a template; the IMD gene with its signal sequence was also amplified using primer C (Imd5, forward primer) and primer D (Imd3, reverse primer) and A. globiformis IMA12103 chromosomal DNA as a template. Second-round PCR was performed on the first-round PCR products using primer A (Csp5K, forward primer) and primer D (Imd3, reverse primer). The amplified fragment was then digested with KpnI and BamHI, inserted into the KpnI-BamHI site of pPK4 to obtain pPIMD, and confirmed by sequencing.

Construction of plasmids for GFP secretion using TorA, IMD, and CspA signal peptides.
Plasmids for green fluorescent protein (GFP) secretion containing the promoter of cspB from C. glutamicum and the signal sequence of TorA (TorAss) from E. coli, IMD (IMDss) from A. globiformis, or CspA (CspAss) from Corynebacterium ammoniagenes were constructed by crossover PCR (Fig. 1B). First-round PCR amplified the promoter of cspB and the signal sequence of TorA, IMD, or CspA from pPSPTG11, pPIMD, or pPSPTG11, respectively, using primer A (Csp5K, forward primer; Table 2) and primer B (Csp-TorA, Imd-Gfp, or Csp-Gfp, reverse primers); the GFP gene was also amplified from p8760 using primer C (TorA-Gfp5 or Gfp5, forward primers) and primer D (Gfp3, reverse primer). Second-round PCR was performed on the first-round PCR products using primer A (Csp5K, forward primer) and primer D (Gfp3, reverse primer). Amplified fragments were then digested with KpnI and BamHI and inserted into the KpnI-BamHI site of pPK4 to obtain pPTGFP, pPIGFP, and pPSGFP. All cloned fragments were sequenced to confirm the absence of PCR-induced errors.

Construction of a plasmid for pro-MTG secretion using the IMD signal sequence.
The plasmid for pro-MTG secretion containing the promoter of cspB from C. glutamicum and the signal sequence of IMD from A. globiformis was constructed by crossover PCR in a similar fashion (Fig. 1B). First-round PCR amplified the promoter of cspB and the signal sequence of IMD from pPIMD DNA using primer A (Csp5K, forward primer; Table 2) and primer B (Imd-PTg, reverse primer); the pro-MTG gene was also amplified from pPSPTG11 using primer C (PTg5, forward primer) and Primer D (PTg3, reverse primer). Second-round PCR was performed on the first-round PCR products using primer A (Csp5K, forward primer) and primer D (PTg3, reverse primer). The amplified fragment was then digested with KpnI and BamHI and inserted into the KpnI-BamHI site of pPK4 to obtain pPIPTG. The cloned fragment was sequenced to confirm the absence of PCR-induced errors.

Construction of plasmids for expression of tat genes.
Plasmids for expression of each tat gene were constructed by PCR. Amplification of tatA, tatB, or tatE was performed with chromosomal DNA of C. glutamicum ATCC 13869 using primer A (TatA5, TatB5, or TatE5, forward primers) and primer D (TatA3, TatB3, or TatE3, reverse primers) (Table 2). The amplified fragments were inserted into the SmaI site of pVC7 to obtain pVtatA, pVtatB, and pVtatE, respectively.

To obtain a plasmid for tatC expression, we cloned a DNA fragment of the tatAC operon lacking a tatA open reading frame (ORF). First-round PCRs were performed on chromosomal DNA of C. glutamicum ATCC 13869 using primer A (TatA5, forward primer) with primer B (TatAm3, reverse primer) and using primer C (TatAm5, forward primer) with primer D (TatC3, reverse primer) (Table 2). Second-round PCR was performed on the first-round PCR products using primer A (TatA5, forward primer) and primer D (TatC3, reverse primer). The amplified fragment was inserted into the SmaI site of pVC7 to obtain pVtatC.

PCR amplifications of four tatA genes, the C termini of whose products are truncated, were performed on pVtatA DNA using primers TatA5 (forward primer) and TatA3-39, TatA3-42, TatA3-54, and TatA3-87 (reverse primers) (Table 2). These reverse primers each contain the stop codon TAA. The amplified fragments of tatA39, tatA42, tatA54, and tatA87 were inserted into the SmaI site of pVC7 to obtain pVtatA39, pVtatA42, pVtatA54, and pVtatA87, respectively. All cloned fragments were sequenced to confirm the absence of PCR-induced errors.

Protein analysis.
SDS-polyacrylamide gel electrophoresis (PAGE) with 15- to 25%-gradient polyacrylamide gels was carried out as described by Laemmli (34), and gels were stained with SYPRO Orange (Bio-Rad). GFP fluorescence analysis was carried out by mixing cell supernatants with equal volumes of sample loading buffer heated at 37°C for 20 min, followed by SDS-PAGE analysis as described previously (34). GFP fluorescence was detected in the gels at an excitation wavelength of 473 nm with an interference filter for emission at 520 nm using a FLA-3000 fluorescence image analyzer (Fujifilm, Tokyo, Japan). The fluorescence intensity of GFP in the gel was measured using MultiGauge software (Fujifilm, Tokyo, Japan), and we have confirmed that the intensities linearly increased according to the secretion amounts of GFP in our experiments. N-terminal amino acid sequences were determined as described previously (31), using a gas phase protein sequencer (model PSQ; Shimadzu, Kyoto, Japan) equipped with an on-line amino acid analyzer (model RF-550; Shimadzu). Accumulation of IMD and pro-MTG was measured by high-pressure liquid chromatography on a column in a 24 to 40% linear gradient of CH3CN containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min with purified MTG as the standard (56).

Nucleotide sequence accession numbers.
The nucleotide sequences of the tatAC operon, tatB, and tatE of C. glutamicum ATCC 13869 are deposited in the GenBank/EMBL/DDBJ database under accession no. AB247375, AB247376, and AB247377, respectively.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the tat genes of the C. glutamicum isolate ATCC 13869.
The complete genome sequence of C. glutamicum ATCC 13032 has been determined (GenBank accession no. BA000036) (22). Compared to those of other gram-positive bacteria, the tat genetic structure of this genome is unique in that it contains the tatE gene in addition to tatAC and tatB. Based on this genome sequence, we amplified and cloned the corresponding tat genes from C. glutamicum isolate ATCC 13869. Sequencing showed that the predicted ORFs for tatA, tatB, tatC, and tatE encoded proteins of 117, 156, 314, and 60 amino acid residues, respectively. TatB, TatC, and TatE from the two isolates had the same lengths and possessed 99%, 100%, or 100% amino acid sequence identity, respectively. ATCC 13869 TatA was 12 residues longer than the corresponding TatA protein from the isolate ATCC 13032 and was 88% identical in the overlapped region (Fig. 2), which is the greatest difference that has been observed for the Tat protein from the same species. The amino acid sequence comparison of TatA and TatE revealed approximately 67% identity, whereas TatB was approximately 10 to 15% identical to TatA and TatE. Amino acid sequence alignment of the TatA/B/E family of proteins from C. glutamicum ATCC 13869, C. glutamicum ATCC 13032, C. efficiens, C. diphtheriae, C. jeikeium, M. smegmatis, S. lividans, B. subtilis, and E. coli is shown in Fig. 2. In contrast to the case with B. subtilis but similar to that with E. coli, TatA and TatE of C. glutamicum contained only the FG motif, whereas TatB carried the GP motif. The C terminus of C. glutamicum TatA is extremely rich in glutamine (Fig. 2). Their implication for the Tat function was analyzed (see below).


Figure 2
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  		FIG. 2. Amino acid sequence alignment using Vector NTI software. Identical or similar residues are shown as white characters on a black or gray background, respectively. C. glutamicum ATCC 13869 TatA (CG13869tatA), TatB (CG13869tatB), and TatE (CG13869tatE), C. glutamicum ATCC 13032 TatA (CG13032tatA), TatB (CG13032tatB), and TatE (CG13032tatE), C. efficiens TatA (CEtatA), TatB (CEtatB), and TatE (CEtatE), C. diphtheriae TatA (CDtatA) and TatB(CDtatB), C. jeikeium TatA (CJtatA) and TatB (CJtatB), Mycobacterium smegmatis TatA (MStatA) and TatB (MStatB), Streptomyces lividans TatA (SLtatA) and TatB (SLtatB), E. coli TatA (ECtatA), TatB (ECtatB), and TatE (ECtatE), and B. subtilis TatAd (BStatAd) and TatAy (BStatAy) are aligned. The C-terminal end points of the tatA deletion mutants are indicated by arrows.

 
Phenotype analysis of tat deletion mutants.
Using a crossover PCR approach, we constructed tat mutants and compared their growth to that of the wild-type strain (YDK010). As shown in Fig. 3A, the deletion of the tatA or tatC gene severely impaired bacterial growth in CM2G medium, while the growth of the {Delta}tatB strain was slightly slower than that of the wild-type strain. The {Delta}tatE mutation had no influence on growth under these conditions. Meanwhile, the numbers of CFU per absorbance at 660 nm for each tat deletion mutant were the same as that for the wild-type strain (data not shown). In contrast to the case with E. coli tat mutants, no morphological change was observed for the C. glutamicum tat deletion mutants (data not shown).


Figure 3
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  		FIG. 3. Growth defects for C. glutamicum {Delta}tatA, {Delta}tatB, and {Delta}tatC mutants. (A) Representative growth curves for the wild type and {Delta}tatA, {Delta}tatB, and {Delta}tatC, and {Delta}tatE mutants in CM2G liquid medium at 30°C are shown as absorbances at 660 nm. (B) Strains were grown in CM2G liquid medium, and approximately 104 CFU was spotted onto CM2G plates with or without 0.005% or 0.01% SDS.

 
It has been demonstrated that tat deletion mutants of E. coli and M. smegmatis are hypersensitive to the detergent SDS (25, 36). To assess whether the C. glutamicum tat deletion mutants are similarly hypersensitive to SDS, we compared the abilities of the mutant strains to grow on plates containing SDS. The same amounts of cells, adjusted from the optical densities of the precultures, were spotted on the plates. The {Delta}tatC mutant failed to grow in the presence of 0.005% SDS in CM2G agar medium, while the {Delta}tatA and {Delta}tatB mutants failed to grow in the presence of 0.01% SDS, in contrast to the normal growth observed for the wild-type strain and the {Delta}tatE mutant (Fig. 3B).

The Tat pathway of C. glutamicum is functional.
In order to determine whether C. glutamicum had a functional Tat system, we analyzed the secretion of GFP fused with the twin-arginine signal peptide of the E. coli TorA protein. The C. glutamicum ATCC 13869 wild-type strain (YDK010) and its {Delta}tatC derivative (YDK010C) were transformed with pPTGFP (TorAss-GFP). As shown in Fig. 4, GFP was secreted by the wild-type strain but not by the {Delta}tatC mutant. GFP was synthesized and accumulated in the cytoplasm of the {Delta}tatC mutant (data not shown). Therefore, the Tat system of C. glutamicum ATCC 13869 is functional.


Figure 4
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  		FIG. 4. Visualization of SDS-PAGE gels of GFP secreted by C. glutamicum with a fluorescence image analyzer. Ten microliters of supernatant (after adjustment to the equivalent optical density) and equal volumes of sample loading buffer were mixed and heated at 37°C for 20 min, applied to the gels, and analyzed by SDS-PAGE. Fluorescence of GFP in the gels was detected with a fluorescence image analyzer. Lane 1, YDK010/pPIGFP (wild type/IMDss-GFP); lane 2, YDK010C/pPIGFP ({Delta}tatC/IMDss-GFP); lane 3, YDK010/pPTGFP (wild type/TorAss-GFP); lane 4, YDK010C/pPTGFP ({Delta}tatC/TorAss-GFP); lane 5, YDK010/pPSGFP (wild type/CspAss-GFP); lane 6, YDK010C/pPSGFP ({Delta}tatC/CspAss-GFP).

 
Next, the signal peptide of A. globiformis IMD containing the twin-arginine motif was fused with GFP, and the corresponding plasmid pPIGFP (IMDss-GFP) was transformed into the wild type, C. glutamicum ATCC 13869, and its {Delta}tatC derivative. The signal peptide of the cell surface protein CspA from C. ammoniagenes does not contain the twin-arginine motif (52). We fused this signal peptide to GFP to be used as a negative control for the Tat function. As shown in Fig. 4, only the signal peptide of IMD but not that of CspA was capable of secreting GFP in the wild-type strain. Moreover, neither of the two fusions was secreted in the {Delta}tatC mutant. This result showed that the signal peptide of IMD is a bona fide Tat pathway targeting signal and is consistent with the previous report that the Sec-dependent signal peptide is unable to export folded GFP in E. coli (15).

Minimal composition of a functional Tat system in C. glutamicum.
Unlike most other gram-positive bacteria, C. glutamicum possesses a tatE gene in addition to the tatA, tatB, and tatC genes. To evaluate the importance of the tat genes for Tat-dependent protein secretion in C. glutamicum, we investigated the effect of individual deletion of tat genes on TorAss-GFP secretion. pPTGFP (TorAss-GFP) was introduced into the tat deletion mutants, and supernatants of the transformants were analyzed. No GFP could be detected in the supernatants of YDK010A ({Delta}tatA) or YDK010C ({Delta}tatC) (Fig. 5A, lanes 3 and 4, and B, lanes 3 to 5); a small amount of GFP (about 9% of the wild-type strain level) was secreted by YDK010B ({Delta}tatB) (Fig. 5C, lanes 3 and 4), and GFP was secreted by YDK010E ({Delta}tatE) (Fig. 5D, lanes 3 and 4) at close to the wild-type strain level (Fig. 5D, lanes 1 and 2).


Figure 5
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  		FIG. 5. GFP secretion via the TorA signal sequence in various tat deletion mutants. The photographs are fluorescent images of secreted GFP in SDS-PAGE gels. Other details are as described in the legend to Fig. 4. (A) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3 and 4, YDK010C/pPTGFP ({Delta}tatC/TorAss-GFP); lanes 5 and 6, YDK010C/pPTGFP+pVtatC ({Delta}tatC/TorAss-GFP plus tatC). (B) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3, 4, and 5, YDK010A/pPTGFP ({Delta}tatA/TorAss-GFP); lanes 6 and 7, YDK010A/pPTGFP+pVtatA ({Delta}tatA/TorAss-GFP plus tatA). (C) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3 and 4, YDK010B/pPTGFP ({Delta}tatB/TorAss-GFP); lane 5, YDK010B/pPTGFP+pVtatB ({Delta}tatB/TorAss-GFP plus tatB). (D) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3 and 4, YDK010E/pPTGFP ({Delta}tatE/TorAss-GFP). (E) Lane 1, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 2 and 3, YDK010A/pPTGFP ({Delta}tatA/TorAss-GFP); lanes 4 and 5, YDK010A/pPTGFP+pVtatE ({Delta}tatA/TorAss-GFP plus tatE). (F) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3 and 4, YDK010AE/pPTGFP ({Delta}tatA {Delta}tatE/TorAss-GFP); lanes 5 and 6, YDK010AE/pPTGFP+pVtatA ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA); lanes 7 and 8, YDK010AE/pPTGFP+pVtatE ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatE). (G) Lanes 1 and 2, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 3 and 4, YDK010AE/pPTGFP ({Delta}tatA {Delta}tatE/TorAss-GFP); lanes 5 and 6, YDK010AE/pPTGFP+pVtatB ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatB).

 
Next, we performed complementation experiments to restore GFP secretion in each tat gene mutant. GFP secretion in the {Delta}tatA, {Delta}tatC, and {Delta}tatB mutants was partially restored by the tatA, tatC, and tatBgenes on pVtatA, pVtatC, and pVtatB, respectively (Fig. 5A, lanes 5 and 6, B, lanes 6 and 7, and C, lane 5). Like the tatA gene, tatE in pVtatE could also partially complement the defect in GFP secretion in the {Delta}tatA mutant (Fig. 5E, lanes 4 and 5). We constructed a double ({Delta}tatA {Delta}tatE) deletion mutant, YDK010AE, to investigate whether tatE would complement the tatA deletion. The introduction of both tatA and tatE on pVtatA and pVtatE restored GFP secretion by the {Delta}tatA {Delta}tatE mutant (Fig. 5F, lanes 5 to 8). Therefore, TatA and TatE provide overlapping function. However, tatB introduced on pVtatB did not restore GFP secretion in the {Delta}tatA {Delta}tatE mutant (Fig. 5G, lanes 5 and 6).

Deletion analysis of TatA.
The tatA ORF of C. glutamicum ATCC 13869 encodes a protein of 117 amino acids. The unique feature of this TatA protein is that it contains two penta-Gln and four twin-Gln motifs and seven discrete Gln residues between amino acids 55 and 117. To evaluate the importance of these clusters, we constructed four plasmids expressing truncated forms of TatA. pVtatA39, pVtatA42, pVtatA54, and pVtatA87, encoding the first 39, 42, 54, and 87 N-terminal amino acid residues, respectively (Fig. 2), were introduced into YDK010AE, the {Delta}tatA {Delta}tatE mutant carrying pPTGFP encoding a GFP secreted by the Tat pathway. GFP was absent from the supernatants of YDK010AE(pPTGFP) (Fig. 6, lanes 2 and 3). The plasmids pVtatA, pVtat87, and pVtat54 each complemented the defect in the {Delta}tatA {Delta}tatE mutant (Fig. 6, lanes 4 to 9), whereas pVtatA42 and pVtatA39 could not (Fig. 6, lanes 10 to 13). TatA42 or TatA39 partially loses the conserved amphipathic helix which has been shown to be essential for E. coli TatA function. Consistently, our results here show that the first 54 residues of C. glutamicum TatA are critical for Tat-dependent secretion, whereas the C-terminal Gln-rich clusters are not required.


Figure 6
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  		FIG. 6. Effect of truncated TatA on the secretion of GFP via the TorA signal sequence. Other details are as described in the legend to Fig. 5. Lane 1, YDK010/pPTGFP (wild type/TorAss-GFP); lanes 2 and 3, YDK010AE/pPTGFP ({Delta}tatA {Delta}tatE/TorAss-GFP); lanes 4 and 5, YDK010AE/pPTGFP+pVtatA ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA); lanes 6 and 7, YDK010AE/pPTGFP+pVtatA87 ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA87); lanes 8 and 9, YDK010AE/pPTGFP+pVtatA54 ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA54); lanes 10 and 11, YDK010AE/pPTGFP+pVtatA42 ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA42); lanes 12 and 13, YDK010AE/pPTGFP+pVtatA39 ({Delta}tatA {Delta}tatE/TorAss-GFP plus tatA39).

 
Tat-dependent secretion of A. globiformis IMD and S. mobaraensis pro-MTG.
To assess the secretion pathway used for A. globiformis IMD, the plasmid pPIMD (IMDss-IMD), directing the synthesis of the IMD precursor from the cspB promoter of C. glutamicum, was introduced into wild-type C. glutamicum YDK010 and its {Delta}tatC derivative, YDK010C. After incubation in MMTG medium at 30°C for 40 h, the supernatants were analyzed for the presence of IMD. A band with the expected molecular weight of IMD was detected in the culture supernatants only of the wild-type strain; it was absent from the supernatants of the {Delta}tatC mutant (Fig. 7A, lane 6 versus lane 2). N-terminal amino acid sequence analysis determined a sequence of Ala-Thr-Ala-Val-Thr-Ala-Arg-Pro-Gly, which corresponded to the residues from 31 to 39 of the IMD precursor. Therefore, A. globiformis IMD was successfully secreted via the C. glutamicum Tat pathway and was processed between residues 30 and 31 after the canonical signal peptidase recognition motif AQA (Fig. 7B).


Figure 7
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  		FIG. 7. Secretion of IMD and pro-MTG using IMD signal peptide. (A) SDS-PAGE analysis of IMD and pro-MTG secreted by C. glutamicum. Ten-microliter aliquots of supernatant were mixed with equal volumes of sample buffer and analyzed by SDS-PAGE. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. Lane 1, YDK010C ({Delta}tatC); lane 2, YDK010C/pPIMD ({Delta}tatC/IMDss-IMD); lane 3, YDK010C/pPIPTG ({Delta}tatC/IMDss-pro-MTG); lane 4, YDK010C/pPSPTG11 ({Delta}tatC/CspAss-pro-MTG); lane 5, YDK010 (wild type); lane 6, YDK010/pPIMD (wild type/IMDss-IMD); lane 7, YDK010/pPIPTG (wild type/IMDss-pro-MTG); lane 8, YDK010/pPSPTG11 (wild type/CspAss-IMD); lane 9, molecular mass markers. (B) N-terminal amino acid sequence of IMD from Arthrobacter globiformis. Cleavage sites of the signal peptides of IMD secreted in C. glutamicum, E. coli, and A. globiformis are indicated by arrows.

 
Next, we assessed the capacity of the C. glutamicum Tat system for the secretion of MTG from S. mobaraensis. MTG is synthesized as a prepropolypeptide. After being secreted into medium, it requires removal of the prodomain to achieve the active enzyme. Recently, we have reported that C. glutamicum secretes the pro-MTG efficiently when it is coupled to signal peptides derived from the cell surface proteins of corynebacteria. Moreover, the prodomain is processed by a subtilisin-like protease from Streptomyces albogriseolus (32). In this study, we analyzed the secretion of pro-MTG fused with either IMD or CspA signal peptide. The IMDss-pro-MTG fusion was secreted by the wild-type strain but not by the {Delta}tatC mutant (Fig. 7A, lanes 7 versus 3). These observations confirmed that the IMD signal peptide targets the substrate exclusively to the Tat pathway. In contrast, the pro-MTG fused with the CspA signal peptide was secreted in both the wild-type strain and the {Delta}tatC mutant (Fig. 5A, lanes 4 and 8). As secreted by the Sec pathway, the first N-terminal amino acid of the pro-MTG secreted by the Tat pathway was Asp, as in the native pro-MTG. The retention times of the pro-MTGs secreted by the Sec and Tat pathways were identified by reverse-phase high-pressure liquid chromatography analysis (32). Furthermore, these culture supernatants were incubated with purified SAM-P45, a subtilisin-like protease from S. albogriseolus, for 2 h at a 100:1 ratio of pro-MTG to SAM-P45 for processing the prodomain (32). After this, the specific activities of these active-form MTGs were determined by the calorimetric hydroxamate procedure as described by Yokoyama et al. (56). The specific activities of the MTGs secreted by the Sec and Tat pathways were identified at 26 U/mg, as was that of native MTG. These results clearly showed that pro-MTGs were successfully routed to either the Sec or Tat pathway, depending only on the signal peptide, and were secreted by the Sec and Tat pathways in C. glutamicum.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Only the Tat pathway is capable of exporting folded GFP (15, 44); hence, GFP has been widely used as a substrate to assess the Tat function in various bacteria and chloroplasts (13, 14, 30, 41, 49, 51). We used GFP (39) as a substrates to assess the Tat function in C. glutamicum. GFP was efficiently secreted by the Tat pathway with either the IMD or TorA signal peptide in C. glutamicum (Fig. 4, lanes 1 and 3). The amount of GFP released with the TorA signal peptide was approximately 4.4 times greater than with the IMD signal peptide, indicating that the E. coli TorA signal peptide is an efficient signal peptide for the Tat pathway of C. glutamicum, as it is for those of E. coli and S. lividans.

We found that the wild type, C. glutamicum ATCC 13869, was capable of secreting GFP fused with the twin-arginine signal peptide of the E. coli TorA protein. In contrast, the secretion was completely abolished in the {Delta}tatA or {Delta}tatC mutant, and a small amount of GFP (about 9% of the wild-type strain level) was secreted in the {Delta}tatB mutant. These results indicated that the functional C. glutamicum Tat system requires TatA and TatC. In remarked contrast to the Tat systems from gram-negative bacteria and chloroplasts, the C. glutamicum TatB protein seems to be dispensable for the secretion of GFP, although it is important for maximal efficiency. E. coli tatA, tatB, and tatC are essential for Tat pathway function (46), whereas in S. lividans only tatC is essential, while tatA and tatB are important but not essential; moreover, tatA and tatB can complement each other (12). In B. subtilis, a functional Tat pathway requires a minimum of one TatA and one TatC homolog (27). Here, we demonstrated that tatA and tatC are essential for Tat-dependent protein translocation in C. glutamicum and that tatB is important but not essential for the export of GFP mediated by the TorA signal peptide. Expression studies with E. coli have suggested that tatE is a cryptic gene duplication of tatA (26). Interestingly, tatE introduced into C. glutamicum on a plasmid complemented the defect of Tat-dependent GFP secretion in the {Delta}tatA mutant, whereas tatE on the chromosome did not. It is therefore reasonable to propose that tatE is a cryptic gene in C. glutamicum as it is in E. coli. Although it was believed that E. coli TatA and TatB perform distinct functions and cannot substitute each other, this view has been challenged by the use of hybrid twin-arginine signal peptide-colicin V reporter proteins, which are still exported in a TatC-dependent manner by strains lacking either TatA or TatB (24). In addition, Blaudeck et al. have demonstrated with E. coli that TatA and TatC can form an active minimal translocase (4). In S. lividans, TatB complements the defect in Tat-dependent protein secretion in a {Delta}tatA mutant (12). In contrast, tatB could not restore GFP secretion to the {Delta}tatA {Delta}tatE mutant of C. glutamicum (Fig. 5G, lanes 5 and 6); as with other gram-positive bacteria, the minimal functional C. glutamicum Tat translocase is likely to be composed of TatA and TatC.

The unique feature of the C. glutamicum TatA protein is that it contains two penta-Gln and four twin-Gln motifs and seven discrete Gln residues between amino acids 55 and 117. These Gln-rich clusters are located in the cytoplasmic regions of the protein, and such a high level of Gln has not been observed for any other TatA proteins. Interestingly, pVtatA54 and pVtatA87, which direct the synthesis of TatA lacking the whole or part of the Gln-rich C-terminal extension, restored GFP secretion in the {Delta}tatA {Delta}tatE double mutant more efficiently than intact TatA. It has been reported that TatA might undergo topology change with its C terminus exposed to the periplasm during the export of the Tat substrate in E. coli (20). The C-terminal Gln-rich extension in the intact TatA might interfere with the process of topological change and reduce the efficiency of GFP secretion.

The secreted IMD (EC 3.2.1.94; 1,6-{alpha}-D-glucan isomaltohydrolase) of A. globiformis hydrolyzes dextran to release isomaltose units successively from the nonreducing ends of dextran chains. The precursor is synthesized with a 39-residue signal peptide containing a twin-arginine motif (23). It is exported into the periplasm when expressed in E. coli (23). Since this enzyme is widely used in manufacture of the oligosaccharide on an industrial scale (21), we analyzed the pathway used for its secretion and chose it as a substrate to assess the Tat pathway potential for large-scale protein production. A. globiformis IMD was successfully secreted via the C. glutamicum Tat pathway but not via the Sec pathway. When IMD is expressed in E. coli, periplasmic IMD is processed at the same site, whereas the N terminus of IMD secreted by the natural host, A. globiformis, starts from residue 40 of the precursor (23) (Fig. 7B). Since the PGV residues from position 37 to 39 do not correspond to an optimal signal peptidase recognition site (30), the residues from 32 to 39 of the IMD precursor might be removed after being secreted into the medium.

We demonstrated that pro-MTG was successfully routed to either the Sec or Tat pathway, depending only on the signal peptide, and was secreted by the Sec and Tat pathways in C. glutamicum. The fundamental difference between the two systems is that the Sec pathway is capable of secreting only unfolded proteins, whereas the Tat machinery exports folded proteins. Although the quantity of MTG secreted via the Tat pathway seemed to be lower than that via the Sec pathway, the Tat system does provide an interesting alternative for secretion of protein when correct folding could occur only in the cytoplasm or folding before secretion could increase the protein stability.

Although some recent reports have described the use of the Tat pathway for protein production (18, 35, 48), the efficiency seems to be too low for industrial exploitation. Here we assessed the secretion of A. globiformis IMD and S. mobaraensis pro-MTG via the C. glutamicum Tat pathway and observed that the yield can reach approximately 100 mg/liter in flask cultures. This achievement implies a great potential for the industrial-scale production of proteins that are not efficiently secreted via other systems.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Phone: 81-44-244-7123. Fax: 81-44-222-0129. E-mail: yoshimi_kikuchi{at}ajinomoto.com. Back

{triangledown} Published ahead of print on 22 September 2006. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, November 2006, p. 7183-7192, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01528-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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