The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis

Kristien Schaerlaekens, Lieve Van Mellaert, Elke Lammertyn, Nick Geukens and Jozef Anné

Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

Correspondence
Jozef Anné
jozef.anne{at}rega.kuleuven.ac.be


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Streptomyces are Gram-positive soil bacteria that are used industrially, not only as a source of medically important natural compounds, but also as a host for the secretory production of a number of heterologous proteins. A good understanding of the different secretion processes in this organism is therefore of major importance. The functionality of the recently discovered bacterial twin-arginine translocation (Tat) pathway has already been shown in Streptomyces lividans. Here, the aberrant phenotype of S. lividans {Delta}tatB and {Delta}tatC single mutants is described. Both mutants are characterized by a dispersed growth in liquid medium, an impaired morphological differentiation on solid medium and growth retardation. To reveal the extent to which the Tat pathway is used in Streptomyces, putative Tat-dependent precursor proteins of Streptomyces coelicolor, a very close relative of S. lividans, and of Streptomyces avermitilis, of which the genomes have been completely sequenced, were identified by a modified version of the TATFIND computer program designed by Rose and colleagues [Rose, R. W., Brüser, T., Kissinger, J. C. & Pohlschröder, M. (2002). Mol Microbiol 45, 943–950]. A list of 230 precursor proteins was obtained; this is the highest number of putative Tat substrates found in any genome so far. In addition to the Streptomyces antibioticus tyrosinase, it was also demonstrated that the secretion of the S. lividans xylanase C is Tat-dependent. The predicted Tat substrates belong to a variety of protein classes, with a high number of proteins functioning in degradation of macromolecules, in binding and transport, and in secondary metabolism. Only a minor fraction of the proteins seem to bind a cofactor. The aberrant phenotype of the {Delta}tatB and {Delta}tatC mutants together with the high number of putative Tat-dependent substrates suggests that the Streptomyces Tat pathway has a distinct and more important role in protein secretion than in most other bacteria.


Abbreviations: Tat, twin-arginine translocation


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Protein secretion is a universal process that occurs in all organisms. In bacteria, the major routes for secretion through the cytoplasmic membrane are the Sec and SRP secretion pathways, which have already been known for a long time and have been investigated in great detail in several organisms. Proteins to be secreted via these two pathways are tagged by a signal peptide that directs them to the secretion apparatus. Signal peptides consist of three domains, i.e. an N-terminal domain having one or more positive charges, a hydrophobic core region and a C-terminal end containing the recognition site for cleavage by a signal peptidase (von Heijne, 1984).

A different secretion route for protein transport across the cytoplasmic membrane has been described, the twin-arginine translocation (Tat) pathway (Berks, 1996; Santini et al., 1998). This pathway seems to be fundamentally different from the other two pathways for several reasons. In contrast to the Sec and SRP pathways, the energy to drive protein translocation is provided by the proton motive force and not by nucleoside triphosphates (Santini et al., 1998). Furthermore, although a signal peptide that routes a protein to the Tat pathway has the same overall structure as the signal peptides for Sec- and SRP-dependent transport, a characteristic twin-arginine motif is located at the border of the N-terminal domain and the hydrophobic region. Moreover, in contrast to the Sec and SRP pathways for which proteins need to be unfolded to cross the membrane, the Tat pathway allows the secretion of fully folded proteins and binding of a cofactor does not inhibit translocation (Halbig et al., 1999b; Santini et al., 1998). In fact, the secretion of cofactor-containing, folded proteins is regarded as a specific characteristic of the Tat pathway. However, recent data suggest that the role of the Tat pathway in protein secretion might be broader than originally thought from Escherichia coli studies (Bolhuis, 2002; Ding & Christie, 2003; Ochsner et al., 2002; Rose et al., 2002; Voulhoux et al., 2001).

In this report, we discuss the possible role of the Tat pathway in Streptomyces, a soil-dwelling filamentous bacterium responsible for the production of the majority of natural antibiotics used in medicine. Mainly thanks to its high secretion capacity, it is an interesting host for the secretory production of heterologous proteins (Van Mellaert et al., 1994; Binnie et al., 1997; Lammertyn et al., 1997). In this respect, the Sec-dependent protein secretion pathway in Streptomyces lividans has already been investigated to some extent, including the characterization of the signal peptidases (Geukens et al., 2001) and the importance of the signal peptide and its charge in heterologous protein secretion (Lammertyn et al., 1997, 1998). Recently, the genes encoding TatA, TatB and TatC, the three components constituting the Tat machinery, were identified and the functionality of the Tat pathway has been illustrated by comparing the secretion efficiency of the chimeric preTorA23K and the Streptomyces antibioticus tyrosinase in the wild-type and a {Delta}tatC mutant (Schaerlaekens et al., 2001). In this work, a {Delta}tatB mutant is constructed and the aberrant phenotype of both tat deletion mutants is discussed. Because of the availability of the genome sequences of Streptomyces coelicolor and of Streptomyces avermitilis (Bentley et al., 2002; Ikeda et al., 2003), a prediction of putative Tat substrates could be made. For this computer analysis, a modified version of the program TATFIND 1.2, designed by Rose et al. (2002), was used. The result of this analysis will be discussed and related to the aberrant phenotype of the S. lividans tat deletion mutants.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Bacterial strains and growth conditions.
E. coli strain TG1 was used as host for cloning purposes. Cultures were grown at 37 °C (300 r.p.m.) in Luria–Bertani medium in the presence of ampicillin (50 µg ml-1) or apramycin (50 µg ml-1) when applicable. S. lividans TK24 and derivatives thereof were routinely precultured at 27 °C with continuous shaking at 300 r.p.m. in phage medium (Korn et al., 1978). Subsequently, the strains were inoculated in NM medium (Van Mellaert et al., 1994) or minimal medium (Kieser et al., 2000). Regeneration of S. lividans protoplasts and selection of transformants occurred on MRYE medium (Anné et al., 1990). Where appropriate, apramycin (50 µg ml-1), kanamycin (50 µg ml-1) or thiostrepton (10 µg ml-1) were added. Protoplast formation and subsequent transformation of S. lividans were carried out as described by Kieser et al. (2000).

DNA techniques and vector constructions.
DNA manipulations were performed using standard techniques (Sambrook et al., 1989). All PCR fragments were checked by DNA sequence analysis according to the dideoxy chain termination method with the Thermo Sequenase Primer Cycle Sequencing Kit 7-Deaza-dGTP on an ALFexpress apparatus (Amersham Biosciences).

For the construction of a {Delta}tatB mutant, the E. coliStreptomyces shuttle vector pGM160 (Muth et al., 1989), containing a temperature-sensitive ori, was used. The neo gene of plasmid pBSKAN (Schaerlaekens et al., 2001) was removed by digestion with PstI/HindIII and a fragment containing the aac(3)IV gene, encoding resistance to apramycin, was inserted in the same sites. Then, the surrounding regions of the tatB gene were cloned on both sides of aac(3)IV. An upstream fragment of 399 bp was amplified with primers TatB1 (5'-ATCTCGAGTGCCAAGGGCGGCGACGGCG-3') and TatB2 (5'-ATAAGCTTGCCTATGTCATTGAACACCT-3') with an XhoI and HindIII restriction site, respectively (underlined). A downstream fragment of 500 bp was amplified using primers TatB3 (5'-ATGGATCCGCCCTGAAGGCGACGCCCGC-3') and TatB4 (5'-TATCTAGAGGCCTCCAGCAGGCCGGTGC-3'), with a BamHI and XbaI restriction site, respectively (underlined). All PCR reactions were performed with SuperTaq polymerase (HTBiotechnology) in the presence of 10 % DMSO. The obtained fragments were cloned in pGEM-T Easy (Promega) and subsequently digested with the appropriate restriction enzymes. The aac(3)IV-containing derivative of pBSKAN was digested first with XhoI and HindIII to allow insertion of the 5' fragment, and then with BamHI and XbaI for the insertion of the 3' region. To obtain the complete cassette, the resulting vector was partially digested with XbaI and XhoI. The obtained fragment was blunted and ligated into a HindIII-digested and blunt-ended pGM160, giving rise to plasmid pGM{Delta}tatB. After PEG6000-mediated transformation of S. lividans protoplasts with pGM{Delta}tatB, a temperature shift to 39 °C promoted the integration of the temperature-sensitive replicon into the chromosomal DNA. The tatB deletion resulting from double homologous recombination was confirmed by PCR and Southern blot analysis.

For {Delta}tatB complementation tests in S. lividans, a derivative of the integrative plasmid pSET152 (Bierman et al., 1992) containing the tatB gene was constructed. To achieve this, the aac(3)IV gene was removed by SacI restriction and replaced by the neo gene of pBSKAN (Schaerlaekens et al., 2001). Then, the tatB gene with its putative promoter region was amplified by PCR using primers TatB1 and TatBNNID (5'-ATCCATGGCCGCGCGGGCGTCGCCTTCA-3'). After cloning of the fragment in the EcoRI site of pBluescript KS(+) (Stratagene), restriction with EcoRV and XbaI allowed subsequent ligation of the fragment in the modified pSET152, resulting in the complementation plasmid pSETtatB.

To construct a Streptomyces vector overexpressing xylanase C, the S. lividans xylanase C gene was amplified by PCR with chromosomal DNA as template. The reaction was carried out in the presence of primer Xyl1 (5'-ATCTGCAGAGAAAGGAGAACGCATGCAGCAGG-3'), including an optimized Shine–Dalgarno sequence (bold) and a PstI restriction site (underlined), primer Xyl2 (5'-ATAAGCTTAGAGGTCAACCGCTGACCG-3'), with a HindIII restriction site (underlined), Pfu polymerase (Promega) and 10 % DMSO. After cloning in pGEM-T Easy, the PstI–HindIII fragment was ligated in pBS-CBSS, a pBluescript KS(+) derivative containing the vsi promoter (Lammertyn et al., 1997), such that the xlnC sequence was preceded by the vsi promoter. The resulting pBSvsixyl plasmid was digested with BamHI and HindIII to obtain a 1·1 kb fragment with the vsi promoter and the xlnC coding sequence that was subsequently ligated in BamHI/HindIII-digested pIJ486 (Ward et al., 1986), giving rise to pIJvsixyl.

Activity assays.
Xylanase activity was measured using the dinitrosalicylic acid assay (Miller, 1959). We used 48 h precultures grown in phage medium to inoculate 50 ml NM medium and cultures were subsequently incubated for 24–48 h. After centrifugation, extracellular fractions were diluted in assay buffer and the amount of reducing sugar was quantified. The intracellular amount of xylanase was determined on cell lysates obtained by sonication (2 min, 20 000 Hz, 0 °C) of the mycelium suspended in assay buffer. One unit of xylanase was defined as the amount of enzyme that produces 1 mg reducing sugar in 10 min at 60 °C from a saturated xylan solution.

The inhibitory activity of subtilisin inhibitor was determined in the presence of the substrate N-succinyl-L-ala-L-ala-L-pro-L-Phe-p-nitroanilide as described by Kojima et al. (1990). Precultures grown in phage medium (48 h) were used to inoculate 50 ml NM medium and cultures were subsequently cultivated for 24 h. After centrifugation, extracellular fractions were diluted in assay buffer and the percentage of subtilisin inhibition was measured. The intracellular amount of subtilisin inhibitor was measured on cell lysates obtained by sonication (2 min, 20 000 Hz, 0 °C) of the mycelium suspended in assay buffer. One unit was defined as the amount of enzyme that inhibited 1 µg subtilisin during 10 min incubation at 25 °C.

Immunoblot analysis.
Western blot analysis was performed to check the translocation of xylanase C in S. lividans. Extracellular fractions of 24 or 30 h recombinant S. lividans cultures in NM medium after inoculation with a 48 h preculture in phage medium were obtained by centrifugation (10 min, 4200 g). Proteins in the growth medium were precipitated with trichloroacetic acid (20 % final concn) and separated by SDS-PAGE (Laemmli, 1970). Transfer of proteins onto a nitrocellulose Porablot membrane (Macherey–Nagel) was performed using a Bio-Rad Transblot semidry transfer cell (Bio-Rad), according to the manufacturers' recommendations. Xylanase C was detected using rabbit anti-XlnC antibodies (kindly provided by J. Dusart, University of Liege, Belgium), followed by alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Phenotype of S. lividans {Delta}tatB and {Delta}tatC single mutants
S. lividans {Delta}tatB and {Delta}tatC single mutants clearly showed phenotypic differences in comparison with wild-type S. lividans TK24. In liquid medium, the mycelium grew very dispersed, while wild-type S. lividans formed mycelial aggregates. In addition, growth of {Delta}tatB and {Delta}tatC was much slower and mycelial mass remained lower than that of the wild-type in rich medium (Fig. 1a) as well as in minimal medium (data not shown). Also on solid MRYE medium, growth of the mutant strains was hampered in comparison with that of the wild-type (Fig. 1b). Morphological differentiation of the mutant strains from vegetative to aerial mycelium, although delayed, could be observed, but the mutant strains never sporulated. Also the red pigment undecylprodigiosin could not be produced in the mutant strains (Fig. 1c). On solid MS medium, on the contrary, both mutant strains became greyish, indicating the formation of spores (Fig. 1d). Interestingly, growth and differentiation were more affected in {Delta}tatC than in {Delta}tatB, suggesting a more important role for TatC in the translocation process.



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Fig. 1. Growth of S. lividans {Delta}tatB and {Delta}tatC compared to wild-type S. lividans TK24. (a) NM medium (50 ml) was inoculated with 1 ml of a 48 h preculture in phage medium and cultured for another 48 h. Every 6 h, samples were taken to determine the dry weight. Filled squares, S. lividans TK24; grey circles, S. lividans {Delta}tatB; open triangles, S. lividans {Delta}tatC. (b) Top of an MRYE plate inoculated with S. lividans TK24, {Delta}tatB and {Delta}tatC after 7 days growth. (c) Bottom of an MRYE plate inoculated with S. lividans TK24, {Delta}tatB and {Delta}tatC after 7 days growth. (d) Top of an MS plate inoculated with S. lividans TK24, {Delta}tatB and {Delta}tatC after 7 days growth.

 
The S. coelicolor and S. avermitilis translated genomes contain an extremely high number of putative Tat substrates
To find an explanation for the differential phenotype of the S. lividans tat deletion mutants, it was interesting to predict the number and nature of the proteins secreted in a Tat-dependent way and therefore likely to be mislocalized in these mutants. The availability of the genome sequences of S. coelicolor and S. avermitilis allowed us to make a list of all putative Tat-dependent precursor proteins for these organisms. Because S. coelicolor has been shown to belong to the same taxon at the species level as S. lividans (Kawamoto & Ochi, 1998), its genome analysis is regarded as representative for S. lividans.

In the first instance, TATFIND version 1.2 (Rose et al., 2002) was used. For both S. coelicolor and S. avermitilis, 145 Tat-dependent candidates were found. This is the highest number of putative Tat substrates found in any genome so far. However, because the number of predicted ORFs in S. coelicolor is the highest so far recorded among bacteria (Bentley et al., 2002), we reasoned that a comparison of the number of Tat-dependent substrates between different bacteria should be expressed as a percentage of the total number of predicted ORFs. These numbers for Streptomyces and some other micro-organisms are shown in Table 1. The archaeon Halobacterium sp. NRC-1 leads with 2·7 % putative Tat-dependent proteins. Moreover, the vast majority of haloarchaeal preproteins seem to be predicted substrates of the Tat pathway (Rose et al., 2002). S. avermitilis and S. coelicolor have the second highest percentage of putative Tat-dependent precursor proteins. Mycobacterium tuberculosis and Corynebacterium glutamicum, two other bacteria belonging to the Actinomycetales group, do not seem to have the same high number of Tat substrates, an indication that a high number of putative Tat-dependent proteins is not a general feature of this group of bacteria. A more extensive list of bacteria with their number of putative Tat substrates is given by Dilks et al. (2003).


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Table 1. Number of ORFs and putative Tat substrates of some micro-organisms as identified by TATFIND version 1.2 (Rose et al., 2002)

 
The ratio between the estimated number of secreted Tat substrates and the total number of secreted proteins can give an idea about the contribution of the Tat pathway to the total secretome. Of the 145 predicted Tat substrates, 127 proteins are putative secreted proteins. The other proteins identified by TATFIND are putative membrane proteins. Indeed, it is generally believed that the Tat pathway also serves for the targeting and translocation of certain membrane proteins. In E. coli, for example, sequence analysis suggests that one-quarter of all traffic on the Tat pathway is inner-membrane proteins (Sargent et al., 2002). The annotated genome database of S. coelicolor contains 819 potentially secreted proteins; this is 10·5 % of all predicted proteins (Bentley et al., 2002). If 127 of these 819 proteins are predicted to be secreted via the Tat pathway, then 15·5 % of all signal-peptide-dependent transport occurs via the Tat pathway. For comparison, the seven predicted Tat substrates in Bacillus subtilis represent only 3·9 % of its predicted 180 secretory proteins (Tjalsma et al., 2000).

A modified version of the TATFIND program
TATFIND version 1.2 searches for the following pattern between residues 2 and 35 of the predicted proteins: (X-1)R0R+1(X+2)(X+3)(X+4), where the amino acid at position X-1 has a hydrophobicity score <=0·26; X+2 and X+3 have a hydrophobicity score <=0·02 and >=-0·77 (positively charged residues were excluded from this position), respectively, and X+4 is one of the residues ILVMF. This pattern was taken from literature in combination with a list of putative secreted proteins from Halobacterium sp. NRC-1, and then refined with residues found in putative Tat substrates of other Halobacteriaceae. Proteins corresponding to the above pattern and containing a hydrophobic region following the twin-arginine motif, were designated as Tat-dependent (for further details, see Rose et al., 2002).

So far, the only Streptomyces protein experimentally proven to be secreted in a Tat-dependent way is S. antibioticus MelC1, the transactivator protein of tyrosinase (Schaerlaekens et al., 2001). The twin-arginine motif in its signal peptide is TRRQVM. Although this motif fulfils the criteria of TATFIND version 1.2, we reasoned that this program was not ideally adapted for recognition of putative Tat substrates of Streptomyces. Because little is known about the Streptomyces Tat pathway, we preferred to change the program to search for the more theoretically based motif RRx[FLIVAM][FLIVAM], because only the twin arginines and the hydrophobic residues at the +3 and +4 positions have been shown experimentally to be important for Tat-dependent transport (Brink et al., 1998; Halbig et al., 1999a; Gross et al., 1999; Stanley et al., 2000). The additional criteria for the identification of an uncharged region following RR were not changed. When the modified TATFIND program was used to analyse the S. coelicolor genome, a list of 230 putative Tat substrates was obtained. This modified TATFIND program is designed to recognize every possible Tat substrate and might therefore give an overestimation of the number of Tat substrates. Therefore, Tat dependence should be experimentally confirmed for every protein. In B. subtilis for example, it was shown that the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent (Jongbloed et al., 2002). In this bacterium, however, there was no obvious phenotypic difference between the wild-type strain and the tatCd-tatCy double mutant (Jongbloed et al., 2000).

Characteristics of the putative twin-arginine signal peptides of S. coelicolor
The list of 230 putative Tat signal peptides was analysed by the SIGNALP-HMM program which can predict the position and length of the three signal peptide domains and the position of the signal peptidase cleavage site (Nielsen et al., 1997). This analysis revealed a mean Tat signal peptide length of 39 aa and a mean N region length of 13 aa. The mean positive charge of these N regions was 4·2. The H region had a mean length of 15 aa and a mean hydrophobicity of 1·95 as determined by the algorithms of Kyte & Doolittle (1982) using the PROTPARAM tool (http://us.expasy.org/tools/protparam). In addition, 33 % of the Tat signal peptides contained within the C region a positive charged amino acid that might serve as a Sec avoidance signal (Cristóbal et al., 1999; Blaudeck et al., 2003).

Following analysis of the whole S. coelicolor genome with SIGNALP (Nielsen et al., 1997), a mean signal peptide length of 42 aa was found. Since the mean length of the Tat signal peptides was calculated to be 39, it seems that in the case of Streptomyces, the Tat signal peptides are not longer than the Sec signal peptides. Tat signal peptides from Gram-negative bacteria on the contrary, are on average 14 aa longer than Sec signal peptides, with most of this additional length being caused by an extended N-region (Cristóbal et al., 1999).

The only other Gram-positive bacterium for which these values are available is B. subtilis. The mean length of its predicted Tat signal peptides is 36, which is 8 aa longer than the mean length of its Sec signal peptides (van Dijl et al., 2002). This extra length of the Tat signal peptides could be attributed to an N region with a mean length of 13–14 aa. Concerning length (19·2 on average) and hydrophobicity (1·9 on average) of the H region, however, no significant difference with the Sec signal peptides was observed (van Dijl et al., 2002). This contrasts to the situation in E. coli where the H region of Tat signal peptides is significantly less hydrophobic than the H region of Sec signal peptides (Cristóbal et al., 1999).

Putative Streptomyces Tat substrates belong to a variety of protein classes
The 230 proteins identified by the modified version of the TATFIND program were classified according to the protein classification scheme on the S. coelicolor genome project website (http://www.sanger.ac.uk/Projects/S_coelicolor). From this classification (Table 2), it is clear that the Tat substrates do not belong to a few specified protein classes, but instead are members of a variety of classes. However, a number of groups are overrepresented while other groups are completely absent. Besides the expected groups of membrane proteins (II.C.1), lipoproteins (II.C.2) and putative secreted proteins (II.C.2), a high percentage of the putative Tat substrates function in the degradation of macromolecules (II.B) and in transport and binding (III.A). Ten proteins functioning in secondary metabolism (III.C) form a third important group. Although an important fraction of the proteins in this list have only putatively assigned functions, it is clear that only a minority are cofactor-binding proteins. Examples are a putative cytochrome c oxidase subunit II (I.B.1), a putative copper oxidase (III.B) and the tyrosinase cofactor MelC1 (III.C). This contrasts to the situation in E. coli where the presence of a twin-arginine motif in the signal peptide is strongly correlated to the binding of a redox cofactor (Berks, 1996), and where it is believed that the transportation of cofactor-containing folded proteins is a fundamental feature of the Tat apparatus (Berks et al., 2000). The same overall features apply to the list of putative Tat-dependent substrates of Streptomyces avermitilis predicted by the modified TATFIND program. In the case of Halobacterium, it has been suggested that the extensive use of the Tat pathway could be an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion (Bolhuis, 2002; Rose et al., 2002). The reason why the secretion of many precursor proteins in Streptomyces would occur via the Tat pathway is not yet clear. However, it can be postulated that the Tat pathway has a distinct role in Streptomyces compared to E. coli or B. subtilis.


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Table 2. Functional classification of putative S. coelicolor Tat substrates as identified by a modified version of TATFIND 1.2

 
Tat-dependent secretion of tyrosinase and xylanase C
So far, the only Streptomyces protein experimentally proven to be secreted in a Tat-dependent way is S. antibioticus MelC1, the transactivator protein of tyrosinase, which directs the secretion of the MelC1/apotyrosinase complex to the Tat translocon (Schaerlaekens et al., 2001). The modified TATFIND program identified its S. coelicolor homologue as a putative Tat-dependent substrate (Table 2, III.C). Because of the similarity between S. antibioticus MelC1 and S. coelicolor MelC1 (25 % identity), it can be assumed that secretion of S. coelicolor MelC1 is also Tat-dependent.

For experimental confirmation of the Tat dependency of a second predicted protein, xylanase C (XlnC, II.B.2), containing the motif RRGFL, was chosen. Only small amounts of xylanases were detectable in the extracellular fraction of the S. lividans wild-type strain. Therefore, xylanase C was overexpressed in S. lividans. This was achieved by cloning the coding sequence of XlnC downstream from the strong constitutive promoter of the S. venezuelae subtilisin inhibitor gene vsi (Lammertyn et al., 1997) on the multicopy plasmid pIJ486 (Ward et al., 1986). Selected transformants were grown for 24 h in NM medium and the presence of XlnC in the culture medium was checked by Western blot analysis (Fig. 2a). A strong immunoreactive band of 20 kDa could be detected in the wild-type strain containing the XlnC expression vector (lane 1). This band represents the mature XlnC protein of 25·7 kDa because purified recombinant XlnC migrates at the same distance (lane 8). In the extracellular fraction of the {Delta}tatB mutant containing the XlnC expression vector, only a faint band was detected, while no band was visible in the {Delta}tatC mutant containing the same vector. These results indicated that secretion of XlnC was partially blocked in {Delta}tatB and completely blocked in {Delta}tatC. After complementation of the deleted tatB with a chromosomally integrated tatB, a strong immunoreactive band reappeared (lane 6), indicating that the secretion defect in this mutant was a specific consequence of the tatB deletion. Next, the xylanase activity in the different strains was measured using the dinitrosalicylic acid assay (Miller, 1959). Activities measured in 24 h culture filtrates are shown in Fig. 2(b). Values of about 8 units xylanase activity (mg dry wt)-1 could be measured for the wild-type strain, while hardly any activity could be measured in the {Delta}tatB strain and no activity in the {Delta}tatC strain. Because the {Delta}tat mutants showed hampered growth, activities were also measured in 30, 36 and 48 h cultures. Even at these later stages of growth, extracellular XlnC activity did not increase in {Delta}tatB and {Delta}tatC (data not shown). Upon measurement of intracellular activities, secretion efficiencies could be calculated. While 98 % XlnC was secreted in the wild-type strain, this amount was reduced to about 20 % in {Delta}tatB and 0 % in {Delta}tatC (Fig. 2c) mutants. The remaining secretion efficiency in the {Delta}tatB mutant is due to the relatively low intracellular accumulation of XlnC. Others have also observed that depletion of tat genes results in inactivation or degradation of Tat substrate proteins in the cytoplasm (Angelini et al., 2001; Santini et al., 2001; DeLisa et al., 2003). In the complemented {Delta}tatB mutant, XlnC secretion was completely restored to 8 units (mg dry wt)-1. Taken together, these results are evidence that secretion of S. lividans XlnC is dependent on intact TatB and TatC proteins and, therefore, occurs via the Tat pathway. Because of the very high homology between S. lividans XlnC and S. coelicolor XlnC (98 % identity), it can be postulated also that the secretion of S. coelicolor XlnC is Tat-dependent.



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Fig. 2. Secretion of xylanase C is blocked in {Delta}tatB and {Delta}tatC single mutants. (a) Western blot analysis with anti-XlnC antibodies to detect XlnC in extracellular fractions of 24 h cultures. Lanes: 1, S. lividans TK24(pIJvsixyl); 2 and 3, S. lividans {Delta}tatB(pIJvsixyl); 4 and 5, S. lividans {Delta}tatC(pIJvsixyl); 6, S. lividans {Delta}tatB(pIJvsixyl)(pSETtatB); 7, molecular mass marker; 8, 0·5 ng purified recombinant XlnC. (b) Xylanase activities in extracellular fractions of 24 h cultures measured using the dinitrosalicylic acid assay (Miller, 1959). Given values are the mean of values obtained from three different transformants. wt, S. lividans TK24(pIJvsixyl); dB, S. lividans {Delta}tatB(pIJvsixyl); dC, S. lividans {Delta}tatC(pIJvsixyl). (c) Secretion efficiencies of xylanase (extracellular activity as a percentage of total activity) in 24 h cultures. wt, S. lividans TK24(pIJvsixyl); dB, S. lividans {Delta}tatB(pIJvsixyl); dC, S. lividans {Delta}tatC(pIJvsixyl).

 
Tat-independent secretion of subtilisin inhibitor
As already demonstrated, the translocation of the highly secreted S. lividans trypsin/subtilisin inhibitor (Sti1; Strickler et al., 1992) is not affected in the {Delta}tatC mutant (Schaerlaekens et al., 2001). To investigate the effect of the {Delta}tatB mutation on Sti1 secretion, extracellular Sti1 activity was measured according to Kojima et al. (1990) in 24 h cultures. While wild-type S. lividans and the {Delta}tatC mutant gave equal values of about 19 units (mg dry wt)-1, the value in the {Delta}tatB mutant reached 29 units (mg dry wt)-1. Secretion efficiencies of the subtilisin inhibitor could be calculated after measurement of intracellular activity. In S. lividans TK24, about 97 % of this protein was secreted. In the {Delta}tatB and {Delta}tatC mutants the secretion efficiency reached values of 98·5 %. Although the reason for the observed positive effect of {Delta}tatB on Sti1 secretion is not clear yet, these results show that the secretion of subtilisin inhibitor is not dependent on either intact TatB or TatC proteins and, therefore, occurs in a Tat-independent way.

Relationship between aberrant phenotype and predicted Tat substrates
Because a list of putative Tat-dependent substrates is now available, hypotheses to explain the aberrant phenotype of the {Delta}tatB and {Delta}tatC single mutants can be made. The absence of undecylprodigiosin production in the tat mutants, for example, might be explained by the Tat dependency of six proteins involved in ABC transport (III.A). The absence of spore formation on MRYE could be the consequence of the Tat dependency of two polyketide {beta}-ketoacyl synthases encoded by the whiE locus (III.C). Also the impaired growth rate might be explained by the absence of a number of solute-binding and sugar-transporting proteins in the culture medium (III.A). On the other hand, although two amidases (III.B and IV.B) are predicted substrates of the Tat pathway, the {Delta}tatB and {Delta}tatC mutants did not show hypersensitivity to SDS (data not shown). This contrasts with the situation in E. coli, where the Tat dependency of two amidases has been shown to cause the highly defective cell envelope phenotype of the tat mutant strains (Ize et al., 2003).

Future experiments will need to focus on testing the Tat-dependent secretion of the proteins putatively assigned to be Tat-dependent by the modified TATFIND program, on construction of a {Delta}tatA mutant and on analysing possible differential secretion in the different tat mutants. This will lead to a better understanding of the importance and mechanism of the Streptomyces Tat pathway.


   ACKNOWLEDGEMENTS
 
We thank Mechthild Pohlschröder for kindly providing the TATFIND program. Also thanks to Gert Sclep for aid with computer analysis. Kristien Schaerlaekens is a research fellow of IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie). N. G. and E. L. are postdoctoral fellows of Katholieke Universiteit Leuven (PDM/02/200, PDM/00/168 and PDM/01/163). This study was further supported by grants G40271.98 and 1. 5. 107. 01 from Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (FWO), OT/00/37 from Onderzoeksfonds Katholieke Universiteit Leuven and QLK3-2000-00122 from the European Commission.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 4 August 2003; revised 2 October 2003; accepted 8 October 2003.



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