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Journal of Bacteriology, December 2001, p. 6727-6732, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6727-6732.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 11 April 2001/Accepted 10 September 2001
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In bacteria, three different types
of signal peptide-dependent translocation have been described: the
Sec-dependent pathway, the signal recognition particle-dependent
pathway, and the recently discovered twin-arginine-dependent pathway or
Tat pathway, which is structurally and mechanistically related to the
pH-dependent pathway in chloroplast thylakoid membranes (11,
32).
The bacterial Tat pathway has been extensively studied in
Escherichia coli. At least four proteins
TatA, TatB, TatC,
and TatE (7, 36, 43)are involved in Tat-dependent
translocation. The genes coding for the first three components are part
of a polycistronic operon, while tatE is located separately
on the chromosome. TatA, -B, and -E are sequence-related proteins
encoded by homologs of hcf106, a structural gene of the
thylakoid pH-dependent pathway (38). They possess a
single N-terminal transmembrane helix, followed by a cytoplasmically
located amphipatic helix. The TatC protein contains six transmembrane
helices. In E. coli, TatB and TatC are essential for
Tat-dependent translocation, while TatA and TatE are complements since
protein transport is only fully blocked in a tatAE double
mutant (7, 36, 37, 43). Very recently, it was shown that
the Tat complex contains only TatA, TatB, and TatC and that TatB and
TatC act as a unit in both structural and functional terms
(8). The precise function of the different components in
the translocation of precursor proteins is still to be explained.
Bacterial precursor proteins translocated via the Tat pathway have an
unusually long signal peptide with a conserved twin-arginine sequence
S/T-R-R-x-
- that is essential for transport (1, 29).
The main energy source that drives the translocation process is the
proton motive force instead of nucleoside triphosphates, as in the case
of Sec-dependent translocation (35). A striking feature of
this newly discovered translocation system is its ability to transport
folded proteins eventually bound to a cofactor before export (33,
35). In addition to E. coli, the functionality of the
Tat pathway has been demonstrated for Zymomonas mobilis (16), Ralstonia eutropha (3), and
Bacillus subtilis (17).
We describe here the analysis of the Tat pathway in Streptomyces lividans. This species has a naturally high secretion capacity and is used as an efficient host for heterologous protein production (5, 23, 40). First, the tatA, -B, and -C genes of S. lividans were identified. To demonstrate the functionality of the Tat pathway, we constructed a tatC deletion mutant and tested the translocation of two putative Tat-dependent precursor proteins, the Streptomyces antibioticus tyrosinase and the chimeric pre-TorA-23K, shown to be translocated via the Tat pathway in E. coli (7).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and growth conditions. S. lividans TK24 and derivatives were grown at 27°C with continuous shaking at 300 rpm in phage medium (21) or NM medium (41). For solid medium, R2 medium was used (29). Where necessary, thiostrepton (at 50 µg/ml in solid medium or at 10 µg/ml in liquid medium), apramycin (50 µg/ml), or kanamycin (50 µg/ml) was added. Protoplast formation and transformation of S. lividans was carried out as described by Kieser et al. (19).
E. coli TG1 was used as host for cloning purposes. Cultures were grown in Luria-Bertani medium at 37°C (300 rpm) supplemented with ampicillin (50 µg/ml), tetracycline (15 µg/ml), apramycin (50 µg/ml), kanamycin (50 µg/ml), or chloramphenicol (25 µg/ml), if necessary. Plasmids used in this work are listed in Table 1.
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DNA techniques and vector constructions. All DNA manipulations were performed by using standard techniques (34), and the restriction enzymes were from Life Technologies (Rockville, Md.). DNA sequence analysis was carried out according to the dideoxy chain termination method with the Thermo Sequenase Primer Cycle Sequencing Kit 7-Deaza-dGTP on an ALFexpress apparatus (Amersham Pharmacia Biotech, Rainham, United Kingdom).
PCR for isolation of tatA, tatB, and tatC was performed with Pfu polymerase (Stratagene, La Jolla, Calif.) in the presence of 10% dimethyl sulfoxide with the primers tat1 (5'-GAACGGCTGAAACCCGCCAC) and tat2 (5'-CCACGTTCCCATCTCGTGCG) for the amplification of tatA, tat3 (5'-GGGGCGGATGCCGCTCGC) and tat4 (5'-CCTCAGGTCACGTCGTCG) for the amplification of tatC, and tat5 (5'-GTGTTCAATGACATAGGCGC) and tat6 (5'-CCGCGCGGGCGTCGCCTTCA) for the amplification of tatB. A DNA fragment containing both tatA and tatC was isolated by using the primers tat1 and tat4. The PCR products were cloned in pGEM-T Easy (Promega, Madison, Wis.), and the DNA sequence of the inserts was determined. For the construction of a tatC deletion mutant of S. lividans, we used the E. coli-Streptomyces shuttle vector pGM160 (28). First, we amplified the kanamycin resistance gene (neo) from pFD666 (13) with primers KAN1 (5'-CAGGGGGGCGGAGCCTATGG) and KAN2 (5'-TACTGCGGCCGCGATCCAAGC). After cloning of the PCR fragment in pGEM®-T Easy, it was digested with EcoRI and cloned into pBluescript KS(+) (Stratagene), resulting in pBSKAN. Then, via a PCR reaction with primers tat7 (5'-CGCAGGGCCGGTGATCTCC) and tat8 (5'-CGGAGTTCACGCTGTTGT), a DNA fragment containing tatC with neighboring regions was isolated. After cloning of this PCR fragment in pGEM-T Easy, two resulting pGEMtatC vectors with different orientations were digested with SacI and XhoI-SalI. The 0.3-kb SacI fragment consisting of the upstream region of tatC was cloned into the SacI site of pBSKAN, while the 0.6-kb blunted XhoI/SalI fragment containing the downstream region of tatC was cloned into the blunted XhoI site of pBSKAN. The resulting plasmid contains a tatC derivative in which a 730-bp internal fragment was replaced by neo. The complete fragment was amplified by PCR by using the M13 primers, and it was cloned into pGEM-T Easy. Finally, from this vector the 2.2-kb ApaI fragment was cloned into the blunted HindIII site of pGM160, resulting in plasmid pGMtatC. After polyethylene glycol-mediated transformation of
S. lividans protoplasts with pGMtatC, a temperature shift
to 39°C promoted the integration of the temperature-sensitive
replicon into the chromosomal DNA. The tatC deletion
resulting from double homologous recombination was confirmed by PCR analysis.
In order to complement the tatC deletion, a DNA fragment
containing the tatC gene was amplified with primers tat9
(5'-CGCAGGGCCGGTGATCTCC) and tat8
(5'-CGGAGTTCACGCTGTTGT) and cloned in pGEM-T Easy. After treatment of the vector with EcoRI, this fragment was cloned
into the EcoRI site of the integrative vector pSET152
(4), resulting in pSETtatC.
For the expression of pre-TorA-23K in S. lividans, the gene
was placed under control of the Streptomyces venezuelae
subtilisin inhibitor (vsi) promoter. Therefore, a
PstI/XhoI fragment containing pre-TorA-23K was
isolated from pMW18 (7) and cloned into pBS-CBSS (23). From this construct, a
BamHI/XhoI fragment containing the vsi
promoter and the downstream pre-TorA-23K coding region was cloned into
the streptomycete multicopy plasmid pIJ486 (42) to give pVsiTor23K.
Immunoblot analysis. Western blot analysis was performed to check the translocation of pre-TorA-23K in S. lividans. Extracellular fractions of 30-h S. lividans cultures were obtained by centrifugation (10 min, 4,200 × g, 0°C). Proteins in the growth medium were precipitated with trichloroacetic acid (final concentration of 20%) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (22). Transfer of proteins onto a nitrocellulose Porablot membrane (Macherey-Nagel, Düren, Germany) was performed by using a Bio-Rad Transblot semidry transfer cell (Bio-Rad, Hercules, Calif.) according to the manufacturer's recommendations. The 23K derivatives were visualized with rabbit anti-23K antibodies and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma, St. Louis, Mo.).
Activity assays. The tyrosinase activity was measured using the dopachrome assay procedure with L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate (18). We used 48-h precultures in phage medium (21) to inoculate 5-ml cultures of tyrosinase production medium (NM medium supplemented with 0.0375% L-tyrosine, 0.75% tiger milk [19], and 2 µM CuSO4) that were subsequently cultivated for 6 h. Supernatants were diluted in the assay buffer (0.1 M sodium phosphate buffer; pH 6.2), and the tyrosinase activity was measured immediately. The intracellular amount of tyrosinase was determined on cell lysates obtained by sonication (2 min, 20,000 Hz, 0°C) of the mycelia suspended in assay buffer. One unit of tyrosinase was defined as the amount of enzyme that converts 1 µmol of L-DOPA into dopachrome/min.
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. (20). Cultures were grown in tyrosinase production medium, and supernatants and intracellular fractions were obtained in the same way as described for the sampling of the tyrosinase test. One unit of subtilisin inhibitor activity was defined as the percentage of inhibition of 5 µg of subtilisin/ml during 10 min of incubation at 25°C.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cloning and sequencing of the S. lividans tatA, -B, and -C genes. In the S. coelicolor genome bank (http://www.sanger.ac.uk), we found a tatA, tatB, and tatC homolog. The tatA and tatC gene are clustered (cosmid SCI41), while tatB is located elsewhere on the chromosome (cosmid SCP8). With primers based on the S. coelicolor sequence, we were able to amplify the corresponding genes from S. lividans TK24. tatA and tatC were isolated on a 1,617-bp fragment and are separated by 92 bp. tatB, on the other hand, was isolated as a 580-bp fragment. While the S. lividans and S. coelicolor tatA genes are identical, the tatC and tatB genes of both strains show 99.8 and 99.6% identities at the nucleotide level and 100 and 98.1% identities at the amino acid level, respectively. CLUSTALW similarity scores with analogous proteins of E. coli and B. subtilis are rather low (19 to 24% for E. coli and 22 to 27% for B. subtilis). All DNA sequences were submitted to the EMBL bank (accession numbers AJ251128, AJ292256, and AJ251149 for tatA, -B, and -C, respectively).
Construction of a tatC deletion mutant.
TatC is an
essential component of the E. coli Tat pathway
(7). To prove the functionality of the twin-arginine
translocation pathway in S. lividans, we therefore decided
to knock out tatC. To do this, a 730-bp internal fragment of
tatC comprising the six transmembrane domains was replaced
with the neo gene in the S. lividans chromosome
by using the integrative E. coli-Streptomyces shuttle vector
pGMtatC. tatC mutants differed phenotypically from the
wild type. Colonies grown on R2 medium (30) lacked red
pigmentation, and the mycelium grew very dispersed in liquid medium in
contrast to the mycelial aggregates of the wild-type strain.
Translocation of pre-TorA-23K is tatC dependent.
Pre-TorA-23K is a chimeric construct that consists of the signal
peptide and the first six amino acids of the mature E. coli trimethylamine N-oxide reductase (TMAO) fused to the mature
part of the 23-kDa subunit of the plant photosystem II oxygen-evolving complex (7). It has clearly been proven that the
pre-TorA-23K protein is translocated via the Tat pathway in E. coli (7) and via the pH pathway in thylakoid
membranes (44).
tatC
mutant. Western blot analysis of extracellular protein fractions from
the wild-type strain revealed an immunoreactive band of 26 kDa (Fig.
1), somewhat larger than expected for the
fusion protein. On the other hand, no 23K-specific band was detected in
the supernatant of the tatC mutant containing pVsiTor23K
(Fig. 1). These results showed that pre-TorA-23K could not be
translocated in the tatC mutant. Intracellular accumulation of pre-TorA-23K in the tatC mutant could not
be detected. To prove that S. lividans
tatC(pVsiTor23K) could express but not secrete
pre-TorA-23K, a plasmid was constructed to complement the disrupted
tatC gene. To do this, wild-type tatC transcribed from its own promoter was inserted into the integrative plasmid pSET152, resulting in pSETtatC. tatC is transcribed from its
own promoter and does not constitute an operon with tatA, as
revealed by transcription analysis experiments (data to be published). Western blot analysis of supernatants of 30-h cultures of S. lividans tatC(pVsiTor23K) transformed with pSETtatC
showed that the 26-kDa band appeared again (Fig. 1). The result
indicated that the integrated copy of tatC restored the
translocation of pre-TorA-23K in tatC, and
tatC was thus essential for translocation of pre-TorA-23K. The 23K-specific immunoreactive band observed in the supernatant of the
tatC complemented strain was less intense than in the
wild-type strain. Therefore, it is obvious that the integrated
tatC copy complemented the deleted tatC, but
secretion was not at the same level as in the original wild-type
strain.
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Translocation of S. antibioticus tyrosinase is tatC dependent. In addition to the heterologous pre-TorA-23K, we also tested the tatC dependence of the S. antibioticus tyrosinase secretion. Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase that catalyzes the formation of melanin pigment from tyrosine (25). The S. antibioticus tyrosinase is encoded by melC2, the second open reading frame of the melanin operon (melC). The upstream gene melC1 encodes a transactivator protein with a twin-arginine signal peptide that was demonstrated to be essential for both tyrosinase activation and secretion (10, 26).
To test the Tat dependence of tyrosinase secretion, S. lividans TK24 and itstatC mutant were transformed
with plasmid pIJ702 (18) encoding the S. antibioticus melC operon, and the tyrosinase activity was assayed.
Figure 2A shows the tyrosinase activities in cell lysates and supernatants of cultures grown for 6 h in tyrosinase production medium. At this growth stage, the activities reached a maximum value. Thereafter, decreasing values were measured, probably as a consequence of a lack of copper ions in the medium, since
after the addition of extra Cu2+ the tyrosinase activity
increased again (data not shown). The activity of tyrosinase in the
supernatant of the wild-type strain was ca. 112 U/mg (dry weight) (Fig.
2A). In contrast, only trace amounts of tyrosinase activity could be
detected in tatC supernatant, indicating that tyrosinase
was not translocated in this mutant. The rather low intracellular
tyrosinase activity detected in the mutant and as a consequence, a much
lower total amount of activity (ca. 11% of the wild type), indicated
that there was no intracellular accumulation of tyrosinase in the
tatC strain (Fig. 2A), a similar phenomenon as that
observed for pre-TorA-23K. In the wild-type strain, 92.7% of the
tyrosinase was secreted in contrast to almost 0% in the mutant (Fig.
2B).
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tatC(pIJ702) with pSETtatC, the
tyrosinase secretion was restored, although only partially. As
mentioned above, this phenomenon was also observed for pre-TorA-23K
translocation when tatC was complemented with the intact
tatC. The tyrosinase activity in the supernatant was only
ca. 15 U/mg (dry weight) (Fig. 2A, set 3), but the efficiency of
secretion was restored to 61.9% (Fig. 2B). The lack of tyrosinase
secretion in the S. lividans tatC mutant and
the complementation experiments confirmed the tatC
dependence of the tyrosinase secretion. Therefore, it could be
concluded that S. antibioticus tyrosinase is a substrate of
the Tat pathway in S. lividans.
Because it has already been shown that the two components of
tyrosinase, MelC1 and MelC2, form a complex before export (10, 26), our results show that, similar to E. coli and
some other species (3, 15, 16, 35, 40), the Tat pathway of
Streptomyces can translocate folded proteins.
tatC is not involved in Sec-dependent secretion.
As a supplementary proof that S. lividans tatC
is a specific mutant that only affects Tat-dependent translocation and
not the Sec-dependent secretion process, we investigated the secretion of the S. lividans subtilisin inhibitor, a Sec
pathway-dependent substrate (unpublished data). Therefore, S. lividans TK24 and its tatC strain were cultured in
tyrosinase production medium. After 15 and 24 h of growth,
subtilisin inhibition activity in the culture medium was measured.
Values of ca. 26 and 45 U/mg (dry weight) were obtained after 15 and
24 h, respectively, both for wild-type S. lividans and
the tatC mutant (Fig. 3).
These results showed that a disrupted TatC does not affect the
Sec-dependent secretion of subtilisin inhibitor.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The experiments described in this report demonstrate that the translocation of two tested precursor proteins containing a twin-arginine signal peptide, i.e., the heterologous chimeric pre-TorA-23K and the S. antibioticus tyrosinase, is blocked in a tatC deletion mutant of S. lividans TK24. As a consequence, these results prove the existence of a functional twin-arginine-dependent translocation pathway in S. lividans.
The chimeric pre-TorA-23K, known to be translocated via the Tat pathway
in E. coli (7), is also translocated through
this pathway in S. lividans. Moreover, although the
homologous components of the Tat complexes of E. coli and
S. lividans have rather low similarity scores (ranging from
19 to 24%), the signal peptide of the E. coli
TMAO-reductase can direct Tat-dependent transport in S. lividans. This is an indication of the conserved nature of the Tat
mechanism. Also, the signal peptides of the Desulfovibrio vulgaris [NiFe]-hydrogenase (29) and of the
high potential iron-sulfur protein of Chromatium vinosum
(9) can direct Tat-dependent transport in E. coli. Moreover, signal peptides are functionally interchangeable
between the bacterial Tat pathway and the pH pathway in thylakoid
membranes of chloroplasts (15, 27). An example of
species-specific recognition, however, is observed in Zymomonas
mobilis (6). It was shown for this species that the
Tat-dependent glucose-fructose oxidoreductase (GFOR) could only be
exported to the periplasm in E. coli when its own signal peptide was replaced with an E. coli Tat signal peptide. It
was suggested that specific recognition events should exist between Tat
signal peptides and one or more components of the Tat translocase. As a
consequence, signal peptides of Tat-dependent precursors would be
optimally adapted specifically to their cognate export apparatus.
On the other hand, Cristóbal et al. (12) showed that not only the signal peptide but also the mature part of the precursor can be a determinant for Tat-dependent transport. The fusion between the TorA signal peptide and the Sec-dependent leader peptidase, for instance, did not reroute the translocation to the Tat pathway, indicating that Sec-targeting signals in Lep could override the Tat-targeting information in the TorA signal peptide.
The secretion of S. antibioticus tyrosinase has previously been investigated but was not fully understood (26). The melanin operon (melC) consists of two genes coding for MelC1, a transactivator protein, and the apotyrosinase MelC2. Only MelC1 has a signal peptide and can mediate MelC2 activation by a binary complex formation followed by copper insertion (10). A functional MelC1 is also essential for tyrosinase secretion (26). How the MelC1/MelC2 complex could cross the membrane was not evident, because Sec and SRP pathways cannot translocate folded proteins. As experimentally proven in this study and already suggested by Berks et al. (2), tyrosinase is translocated via the Tat pathway. No secretion was detectable in the tatC mutant, while in the wild-type S. lividans secretion of tyrosinase was very efficient and probably occurred cotranslationally or immediately after translation, since only very low amounts of tyrosinase could be detected intracellularly.
The translocation of folded proteins in Streptomyces can now be explained by the functionality of the Tat pathway. E. coli hydrogenase 2 is also encoded by an operon with two genes, of which only one is coding for a protein containing a signal peptide. This type of "hitchhiker" mechanism is also used by S. lividans to cotranslocate the two subunits of tyrosinase.
The complementation of tatC with a wild-type copy was not
complete. The reason for this effect is not yet understood. Gene dose
effect cannot be the reason because the disrupted tatC gene was replaced by one intact copy. However, positional effects of integration or a lack of regulatory sequences present in the
neighborhood of the wild-type tatC gene might result in a
different transcription efficiency.
For both pre-TorA-23K and tyrosinase, an intracellular accumulation of
the preprotein was not observed when the secretion process was blocked.
As a consequence, the total amount of pre-TorA-23K or tyrosinase in the
tatC cultures was much lower than in those of the wild
type. This effect was also noticed by Jongbloed et al.
(17), who investigated the secretion of PhoD in a
tatCd mutant of B. subtilis. These authors
postulated that the intracellular amount of preprotein is not folded
and therefore not stable. In the case of tyrosinase, it has been
described that the two subunits of tyrosinase form an intracellular
complex (10). The proteolytic breakdown of this complex is
unlikely. However, if the total amount of the respective components
should be unequal in the S. lividans tatC, as
already observed with some signal peptide mutants of MelC1
(26), it might be that the component in excess becomes sensitive to proteases. When Leu et al. (26) demonstrated
low intracellular accumulation of tyrosinase, they agreed with the hypothesis suggested by Oliver (31) and Gennity et al.
(14) that a coupling between secretion and translation
exists. A blocked secretion apparatus would have a negative feedback on
translation of the protein. The results obtained in this study and in
our previous work with Sec-dependent translocated proteins (23, 24) are in agreement with a negative feedback on the
translation, or even transcription, of the precursor as a consequence
of inhibiting secretion. The mechanism of this negative feedback has to
be further investigated.
The TatC component of the Tat complex in S. lividans is
essential for the translocation of the two precursor proteins under investigation, i.e., S. antibioticus tyrosinase and chimeric
pre-TorA-23K. In E. coli, the unique tatC copy is
essential for the translocation of all Tat pathway precursors studied
this far (7). However, in B. subtilis, two
tatC copies were detected but only one of these is essential
for the Tat-dependent translocation of PhoD, indicating that
tatC is a specificity determinant for protein secretion via
the Tat pathway (17). In the fully sequenced genomes of
Mycobacterium tuberculosis and Mycobacterium
leprae, two species classified to be in the same order of the
Actinomycetales as Streptomyces, only one
tatC copy was identified. The S. coelicolor
genome project is now finished, but no second tatC homolog
was detected; neither was one observed by hybridization experiments
(data not shown). Further studies are required to characterize the
components of the S. lividans Tat system at the molecular
level and to investigate the interactions between the Tat translocon
components and between these proteins and the Tat-dependent precursors.
In addition, the underlying reason for the altered phenotype observed
for the tatC mutant, indicating that the mutation has a
pleiotropic effect, as also noticed for E. coli, where it
resulted in an impaired cell separation and a defective cell envelope
(39), is not understood.
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ACKNOWLEDGMENTS |
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We thank Tracy Palmer for kindly providing the pMW18 plasmid and Colin Robinson for the 23K antibodies.
Kristien Schaerlaekens and Nick Geukens are research fellows of IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie). Michaela Schierová is a Marie Curie Fellow of the European Commission (MCF1-2000-01142). This study was further supported by grants G.0271.98 and 1.5.1.7.01 from FWO (Fonds Wetenschappelijk Onderzoek-Vlaanderen), grant OT/00/37 from K. U. Leuven, and grant QLK3-2000-00122 from the EC.
K.S. and M.S. contributed equally to this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. Phone: 32-16-33-73-71. Fax: 32-16-33-73-40. E-mail: jozef.anne{at}rega.kuleuven.ac.be.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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