Unité de Génétique Mycobactérienne, Institut Pasteur, 25, Rue du Dr Roux, 75724 Paris cedex 15, France1
Author for correspondence: Jean-Marc Reyrat. Tel: +33 1 40 61 32 74. Fax: +33 1 45 68 88 43. e-mail: jmreyrat{at}pasteur.fr
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ABSTRACT |
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Keywords: mycobacteria, leaderless secretion
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INTRODUCTION |
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Biochemical, bioinformatic and genetic approaches have been used to identify M. tuberculosis secreted proteins. Two-dimensional gel electrophoresis and microsequencing have recently identified novel secreted proteins in culture filtrates of M. tuberculosis (Weldingh et al., 1998 ; Rosenkrands et al., 2000
). The recently published M. tuberculosis genome (Cole et al., 1998
) and the development of new software have allowed the M. tuberculosis genome to be extensively analysed and the identification of proteins which had not been previously predicted to be secreted (Gomez et al., 2000
). Conversely, the fusion of genes to reporter proteins whose activities are easily detected on Petri dishes, such as Escherichia coli ß-lactamase (Bla) (Chubb et al., 1998
) and alkaline phosphatase (PhoA) (Lim et al., 1995
), has identified the coding sequences which trigger the export of the reporter proteins. phoA has been used to create fusion libraries (Lim et al., 1995
; Wiker et al., 2000
; Carroll et al., 2000
) and as a reporter in an in vitro transposition system (Braunstein et al., 2000
). In both cases, the methods have proven to be successful, although they are limited to fast-growing mycobacteria.
Recently, a new reporter system based on the Staphylococcus aureus nuclease (Nuc) was designed to identify exported proteins. This small secreted endonuclease has proved to be an ideal reporter system in Lactococcus lactis, in which the truncated gene, deprived of its signal sequence, has been successfully used to screen a fusion library for exported proteins (Poquet et al., 1998 ) and also to develop an in vitro transposition system (Ravn et al., 2000
). Nuc has several advantages over PhoA: it is a stable monomeric protein and its detection assay is non-toxic for cells. This system has recently been adapted for use in mycobacteria and has led to the characterization of three secreted proteins (Downing et al., 1999
).
To produce an exhaustive list of M. tuberculosis secreted products we sought to use the nuclease as a reporter system but to detect nuclease activity by a more discriminative method (Lachica et al., 1971 ). Contrary to our expectations, we found that the staphylococcal nuclease is exported in Mycobacterium smegmatis irrespective of whether it was made with or without a signal sequence. Biochemical analysis confirmed that the staphylococcal nuclease is secreted in the absence of bacterial lysis. Although Nuc can be fused to 40 amino acids in the N terminus without affecting secretion, some other fusions are not compatible with secretion. Despite extensive mutational analysis, we were not able to characterize components of the secretion apparatus which are responsible for this non-canonical export.
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METHODS |
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Construction of pPRB4 and derivatives.
Oligonucleotides are listed in Table 1.
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BlaF* derivatives were constructed as follows. pIPJ47* (Timm et al., 1994 ) PCR products were amplified with primers BlaPf and BlaPr for pBla1, primers BlaPf and BlaSr for pBla2, and primers BlaSf and BlaSr for pBla3. The pIPJ47* PCR products were digested with BamHI and PstI and inserted into the dephosphorylated BamHI/PstI-digested pPRB4 vector.
SigE derivatives were obtained by cloning the following PCR products into pPRB4. pSigE1 was obtained by amplifying pIPX70 with primers SigEPf and SigErev, and pSigE2 was obtained by amplifying M. tuberculosis genomic DNA with primers SigEPf and SigESr. The UreA derivative, pUreA, was constructed by cloning the PCR product amplified with primers UreAfw and UreArev into pPRB4. The annealing temperature was calculated independently for each primer pair and 30 cycles were carried out. PCR products and plasmids were purified with the Qiagen PCR purification kit.
pNuc2 was constructed as follows. The blaF* promoter was inserted into pPV24 that had been digested with PstI and BamHI. The resulting plasmid was digested with BamHI and then with Mung Bean Nuclease (BioLabs) to remove the 5' protruding ends, and then digested with KpnI. NucA was amplified with primers Nuc2 and Rev using pPRB4 as a template. The PCR product was digested with PstI, blunt-ended with T4 DNA polymerase and digested with KpnI. It was then cloned into the plasmid to give pNuc2. All the plasmids were first amplified in E. coli and sequenced.
Construction and screening of the fusion library.
M. tuberculosis genomic DNA was partially digested with Sau3AI. Fragments of between 2 and 0·5 kbp were excised from the gel and ligated into dephosphorylated pPRB4 which had been linearized with BamHI. Epicurian Coli XL2 BlueMRF' ultracompetent cells (Stratagene) were transformed with the ligation mixture and 24000 clones were obtained. Twelve clones were randomly picked and shown to be distinct by amplifying the inserts by PCR. The 24000 clones were pooled and plasmid DNA was extracted with a Qiagen Kit and used to transform M. smegmatis. We screened 32000 clones of transformed M. smegmatis for nuclease activity with the toluidine blue overlay assay. Inserts in the positive clones were amplified by PCR, using primers F and Nucr, and sequenced.
Detection of nuclease activity.
To identify Nuc+ clones on Petri dishes, a nuclease plate assay was used. Plates were overlaid with 12 ml warm Toluidine Blue-DNA agar (TB-DNA agar) (0·05 M Tris, pH 9, 10 g agar l-1, 10 g NaCl l-1, 0·1 mM CaCl2, 0·03%, w/v, salmon sperm DNA, 130 mg toluidine blue O l-1). Colonies which secreted active Nuc developed an easily detectable pink halo after 1 or 2 h. A sterile net was placed over the colonies to ensure that they did not detach when the TB-DNA agar was poured. Alternatively, transformed and wild-type M. smegmatis were plated on methyl-green-containing plates (Downing et al., 1999 ). Colonies that secreted an active nuclease developed a yellow halo after 60 h.
Zymograms were performed as follows. SDS gels were renaturated as described by Liebl et al. (1992) , placed on a Petri dish containing TB-DNA agar and incubated at 37 °C. After 16 h pink bands indicated the presence of nuclease activity.
Cellular fractionation.
Bacterial cells were grown in LB, supplemented with 0·05% Tween 80, to an OD600 of 1·31·8 (mid-exponential phase) or 6·5 (stationary phase). The optical density was measured in 1 cm cuvettes in a Hitachi U-1100 spectrophotometer after a 1:10 dilution in LB. The bacteria were harvested by centrifuging 20 ml culture at 4000 g. The supernatant was filtered through a Millex-GV filter with 0·22 µm pores and precipitated with TCA (17% final volume). The pellet was washed, resuspended in 500 µl PBS and the cells were mechanically broken with 0·1 mm glass beads. The beads were removed by centrifugation and the suspension was precipitated with TCA. The precipitated samples were washed with cold acetone containing 1% triethanolamine. Finally, all the samples were resuspended in the appropriate volume of loading buffer and boiled for 5 min.
Western blotting.
SDS-PAGE and electroblotting were carried out by standard methods (Sambrook et al., 1989 ). The membrane was incubated in 5% milk PBST (PBS, 0·05% Tween 20) for 1 h, washed briefly and incubated with anti-KatG (diluted 1:1250 in 5% milk PBST) or anti-Nuc (diluted 1:1000 in 5% milk PBST) antisera for 1 h. After extensive washing in PBST the membrane was incubated for 1 h with a horseradish-peroxidase-conjugated anti-rabbit antibody (Bio-Rad) (diluted 1:10000 in 5% milk PBST). After extensive washing the immunodetection was performed by using an ECL kit (Amersham).
Preparation of anti-Nuc antibodies.
New Zealand rabbits were immunized with 250 µg commercial micrococcal nuclease (Sigma). Boosters (200 µg) were given three times at 2-week intervals. Blood was collected, incubated for 1 h at 37 °C and overnight at 4 °C. Sera were recuperated by centrifuging the blood at 3000 g for 20 min and tested by Western blotting.
Integration of 155nuc into M. smegmatis.
pBla1* was constructed as follows. The PstI/KpnI fragment of pBla1 was blunt-ended and ligated into blunt-ended integrative pNIP40b that had been digested with XbaI. The resulting plasmid was selected for the correct insert orientation and sequenced. Sequencing revealed that a guanine residue 54 bp upstream of the ATG site had been deleted. M. smegmatis was transformed with pBla1* and transformed clones were selected on hygromycin plates.
Transposon mutagenesis.
M. smegmatis::pBla1* was transformed with pCG79 and transposon mutagenesis was carried out by the method of Guilhot et al. (1994) , except that the colonies were plated out at 41 °C to select clones in which transposition had occurred. After 3 d the colonies were tested for nuclease activity by the toluidine blue overlay assay. Nuc- mutants were selected and analysed.
DNA sequencing.
A model 373-B DNA analysis system (Applied Biosystems) was used to sequence the double-stranded plasmid DNA or PCR-generated fragments by the dideoxy chain-termination method.
Computer methods.
Signal sequences were searched using the signal IP server at http://www.cbs.dtu.dk/services/SignalIP. Sequences were analysed by the BLAST algorithm at http://www.sanger.ac.uk/Projects/M_tuberculosis/blast_server.shtml
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RESULTS |
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An equivalent of the truncated nuclease used by Poquet et al. (1998) , in which the signal sequence and the first 13 aa of the propeptide are deleted, was named 155Nuc. The 155nuc gene, deprived of its own promoter, was cloned into pPV24, a shuttle plasmid that replicates in both E. coli and mycobacteria (Triccas et al., 1999
). The resulting vector was named pPRB4. M. smegmatis(pPRB4) was tested for nuclease activity by the toluidine blue overlay assay, a test which can detect just 5 ng active nuclease ml-1 (Lachica et al., 1971
) and which is non-toxic for the cells. Positive colonies can be readily detected after 1 or 2 h by their pink halo, which derives from the degradation of the DNA contained in the overlay. A Nuc- phenotype was observed for M. smegmatis(pPRB4), demonstrating the absence of a cryptic promoter and signal sequence in the vector and of endogenous nuclease. pPRB4 was subsequently used to construct an M. tuberculosis genomic library in fusion with the 5' end of the 155nuc gene. The library was amplified in E. coli and then used to transform M. smegmatis. Over 32000 M. smegmatis colonies were tested for nuclease activity by the toluidine blue overlay assay. Approximately 0·15% of clones were positive after 1 h. Some of the inserts contained in these clones were amplified and sequenced.
In 50% of the cases, (approx. 10 clones) 155nuc was in-frame with the beginning of a gene encoding a putative protein. However, a canonical signal sequence was predicted using the signal IP server in only 20% of the cases. These were Rv0347 and Rv2588c, both of which are of unknown function. Most other fusions were either with genes encoding cytosolic proteins (Rv0187, Rv1608c) or with a fragment that was too short to contain a signal sequence (Rv0270, Rv1837c) (Table 2). In the other 50% of the cases, the inserts were in intergenic or intragenic regions. In every case, however, a codon for methionine or valine, which may act as a start codon, was found in-frame with 155nuc. This may be because M. tuberculosis genes are expressed in M. smegmatis and thus in a relatively heterologous context. Some sequences in M. tuberculosis may be artefactually recognized as promoters, ribosome-binding sites and start codons by M. smegmatis. Alternatively, the genome may contain sequences that were not recognized for a particular annotation with the existing software. These results suggest that a promoter and some amino acids in-frame with the nuclease are sufficient to result in a Nuc+ phenotype on the Petri dishes.
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To determine whether a signal sequence was required for secretion a series of pPRB4 derivatives was constructed. In these derivatives 155nuc was fused to (i) a promoter and signal sequence, (ii) a promoter and start codon, or (iii) a signal sequence alone. We aimed to verify whether the nuclease is secreted in the absence of a signal sequence as the data presented above suggested. We used the blaF* promoter and gene from Mycobacterium fortuitum, which encodes an exported ß-lactamase with a 32 aa N-terminal signal sequence (Timm et al., 1994 ). The blaF* gene was chosen because of its mycobacterial origin. In-frame fusions with 155nuc were constructed by cloning the blaF* promoter and start codon (pBla1), the blaF* promoter and the 57 aa N-terminal sequence, which includes the signal sequence (pBla2), or the BlaF* N-terminal region without promoter (pBla3) into pPRB4 (Fig. 1
). M. smegmatis was transformed with each of the constructs and was screened for Nuc activity by the toluidine blue overlay assay. As expected, M. smegmatis(pBla2) was Nuc+ and M. smegmatis(pBla3) was Nuc-, thus confirming the absence of a cryptic promoter in pPRB4. Interestingly, a clear pink halo appeared in M. smegmatis(pBla1) at the same time and with the same intensity as in M. smegmatis(pBla2). This suggests that if an active promoter controls its expression, 155Nuc is efficiently secreted in M. smegmatis, even in the absence of a signal sequence.
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To demonstrate that the Nuc+ phenotype observed was due to protein secretion and not due to cell lysis we assayed the bacterial cell and supernatant fractions of mid-exponential-phase cultures for the presence of nuclease by Western blotting and for nuclease activity by zymogram analysis (Fig. 2). Cell lysis was monitored by Western blotting with an antibody against KatG, a catalase peroxidase (Zhang et al., 1992
) exclusively cytoplasmic in M. smegmatis (Raynaud et al., 1998
). This confirmed the previous observations and revealed that over 40% of the nuclease hybrid proteins was in the supernatant fraction, whereas KatG was exclusively found in the cell fraction. Furthermore, zymograms showed that all the hybrids had a distinct nuclease activity, demonstrating that the fused polypeptides did not prevent nuclease activity of 155Nuc. The activity of the UreA-155Nuc fusion demonstrated that the absence of the halo was not due to a lack of activity, but that the hybrid protein was not secreted. One may argue that the presence of the nuclease in the supernatant could be due to the fact that it is a small protein (15 kDa) and it can easily leak, contrary to KatG which is a protein of high molecular mass (80 kDa) and is retained in the cells. The absence of UreA-Nuc (17 kDa) in the supernatant demonstrates that the low molecular mass cannot explain the secretion per se.
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M. smegmatis(pBla2) contained different bands, some of which may result from the cleavage of its signal sequence. The abnormal migration and the presence of degradation products that could represent the NucA form have been described (Poquet et al., 1998 ).
155Nuc secretion is due neither to overexpression nor to the propeptide
Previous experiments used a derivative of the pAL5000 replicon, which is a replicative plasmid present at about three copies per bacterium (Ranes et al., 1990 ). To exclude the possibility that the secretion of the nuclease was due to its overexpression, we used pNIP40b, an integrative vector present in a single copy. The blaF* promoter and 155nuc of pBla1 were cloned into pNIP40b and the resulting pBla1* was integrated into M. smegmatis. M. smegmatis::pBla1* colonies remained Nuc+ on Petri dishes, even though a longer incubation time was required. As in M. smegmatis(pBla1), fractionation showed that 155Nuc was in the supernatant in the absence of cell lysis (Fig. 3
). Although the Nuc level was greatly diminished in single-copy expression, 50% of nuclease activity was still found in the culture supernatant fraction. This demonstrates that 155Nuc secretion was not due to overexpression.
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Search for alternative secretion pathways
Our results clearly demonstrate that 155Nuc is secreted independently of overexpression, the presence of the propeptide and of the signal sequence. 155Nuc is thus probably secreted independently of the Sec translocating pathway. To characterize this alternative pathway we used M. smegmatis::pBla1* to create a library of Tn611 insertional mutants using the thermosensitive replicon pCG79 as a delivery system (Guilhot et al., 1994 ). The rationale was that we could distinguish Nuc- mutants on plate, mutants that could arise from mutations of the nuc gene itself or more interestingly in genes involved in 155Nuc secretion. Such mutants would display cytosolic nuclease activity. Four independent libraries were constructed and 25000 clones were screened by a plate overlay test. Thirteen Nuc- mutants were found and analysed for the presence of 155nuc by PCR with different pairs of primers and for nuclease activity by the zymogram assay. None of the mutants showed any nuclease activity in the zymogram assay when culture supernatants and cell fractions were tested (data not shown). This suggests that the nuclease gene was either not transcribed or not translated, or the protein was enzymically inactive. Indeed it was not possible to amplify either the promoter region or the promoter and the 155nuc region in mutants 111 (Table 3
). This was probably due to deletions or to the insertion of the transposon in the 155nuc gene or in the blaF* promoter. Two mutants (12 and 13) had point mutations in the C terminus: in mutant 12 a substitution had occurred in the second helix, responsible for Ca2+ binding (Hynes & Fox, 1991
), whereas in mutant 13 a deletion created a stop codon 6 bp downstream, thus eliminating the C-terminal region of the protein (Table 3
). No mutant with a Nuc- phenotype was found outside the nuc gene itself that could provide evidence for a new secretion pathway. Indeed, no mutant that retained a nuclease activity which was exclusively cytosolic could be identified, suggesting that either the nuclease is secreted by a non-specific or redundant system, or that components of the secretion apparatus may be essential for bacterial survival.
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DISCUSSION |
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We used controlled conditions and a cytoplasmic marker to demonstrate that the staphylococcal nuclease is exported by M. smegmatis independently of a signal sequence. We showed that this protein is secreted by M. smegmatis whether it contains a signal sequence or not and even when fused to a short polypeptide. However, another fusion product was not secreted, probably because the polypeptide prevented the proper folding required for secretion or correct interactions with the secretion machinery.
Interestingly, a similar reporter system, also based on the staphylococcal nuclease, was used to characterize three exported products in M. tuberculosis (Downing et al., 1999 ). Our results clearly show that, on the contrary, the nuclease should not be used as a reporter system for the identification of exported products in mycobacteria because it is secreted independently of a signal sequence. In any case, we do not have any obvious explanation for such a discrepancy between the two results.
The nuclease protein sequence does not contain any classical signal sequences or long hydrophobic stretches that might explain its secretion. It is possible that the nuclease uses an endogenous alternative Sec pathway that could be an ABC transporter. For instance, Gey van Pittius et al. (2000) have suggested that Esat6, a small secreted antigen of M. tuberculosis which lacks a signal sequence (Sorensen et al., 1995
), might use an ABC transporter for secretion. Also other species of Gram-positive bacteria secrete small peptides without Sec-dependent signals via ABC transporters (Sahl & Bierbaum, 1998
). Porins and efflux pumps have also been described in mycobacteria (Trias et al., 1992
), but, with a molecular mass of
15 kDa, it is unlikely that the nuclease is transported by such a system. As for the TAT pathway, the nuclease is devoid of signal peptide and consecutive arginines, both of which are required for efficient export and thus, the nuclease probably does not use this translocation pathway.
The fact that UreA-Nuc is not secreted demonstrates that secretion depends on both the sequence and the proper folding of the protein, as already suggested for other mycobacterial proteins (Harth & Horwitz, 1997 , 1999
). The system which allows the secretion of this leaderless protein is present in M. smegmatis as for glutamine synthetase and superoxide dismutase (Harth & Horwitz, 1997
, 1999
). Whether the secretion of all these leaderless proteins in M. smegmatis is mediated by the same translocation pathway or by different systems remains to be clarified.
The system that mediates the secretion of the nuclease could not be identified by transposition mutagenesis, despite the fact that more than 25000 clones, sufficient to cover the entire genome, were analysed. Nuc- mutants resulted exclusively from mutations of the nuc gene itself, which strongly suggests that the genes encoding components of the alternative system may be essential. Alternatively, 155Nuc might be secreted through different systems that may be redundant and thus a single mutation cannot prevent secretion. Exported products play a key role in M. tuberculosis virulence and the identification of the pathways involved in their export is the next challenging step.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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Received 30 July 2001;
accepted 8 October 2001.
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