Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins^{▿ †}

Bacterial strains and culture methods.

Bacterial strains used during this work are listed in Table 1. Luria-Bertani (LB) medium (Fisher) was used for culturing of Escherichia coli. Middlebrook 7H9 or 7H10 medium (Difco; BD Biosciences) was used for the culturing of M. smegmatis and M. tuberculosis. For M. smegmatis, Middlebrook medium was supplemented with 0.5% glycerol and 0.2% dextrose. For M. tuberculosis, Middlebrook medium was supplemented with 0.5% glycerol and 1× ADS (0.5% bovine serum albumin, fraction V [Roche]; 0.2% dextrose; and 0.85% NaCl). When necessary, medium was supplemented with 0.05 to 0.1% Tween 80 (Fisher). As required, antibiotics were added to Middlebrook media at the indicated concentrations: hygromycin B (Roche Applied Science), 50 μg/ml; carbenicillin (Sigma), 50 μg/ml; and kanamycin (Acros Chemicals), 20 μg/ml. l-Lysine at 40 and 80 μg/ml was added to agar and liquid media, respectively, for growth of M. smegmatis strains PM759 and JM578.

Molecular biology procedures.

Standard molecular biology techniques were employed (48). The Expand High-Fidelity PCR system (Roche) was used in all PCRs, and 5.0% dimethyl sulfoxide was included in select reactions. DNA sequencing was performed either by the UNC-CH Automated DNA Sequencing Facility (Chapel Hill, NC) or by Eton Bioscience Inc. (San Diego, CA).

Construction of ′BlaC reporter libraries.

All plasmids used in this study are listed in Table 2. Two library plasmids were constructed with a truncated version of M. tuberculosis blaC (referred to as ′blaC), amplified from pJM106 by PCR using the primers Lib′blaCfor and Lib′blaCrev (see Table S1 in the supplemental material).

(i) Library 1.

The resulting ′blaC amplicon was ligated into the multicopy mycobacterial shuttle vector pMV206.hyg, which had been digested with ClaI and NcoI. The final plasmid was pJES113 (Fig. 1A). Genomic DNA was isolated from M. tuberculosis strain H37Rv as previously described (6) and partially digested with AciI and HpaII, and digests with DNA fragments between 0.5 and 5.0 kbp were selected. The genomic digest was cloned into the unique ClaI site immediately upstream of ′blaC in pJES113. The resulting ligation reaction mixture was transformed into E. coli XL1-Blue (Stratagene). Approximately 1 × 10⁶ hygromycin-resistant E. coli transformants were pooled for plasmid DNA isolation (Qiagen).

(ii) Library 2.

The second library vector, pJM157, was constructed to carry a mycobacterial promoter upstream of the unique ClaI site. A fragment carrying the promoter and the +1 transcriptional start site from M. tuberculosis hsp60 was amplified by PCR from pMV261.hyg using the primers Hsp60for-BstBI and Hsp60rev2-ClaI (see Table S1 in the supplemental material). The resulting PCR product was ligated into pJES113, which had been linearized with ClaI, to produce pJM157 (Fig. 1B). For construction of library 2, genomic DNA was isolated from M. tuberculosis strain PM638 (ΔblaC). Digestion of genomic DNA and ligation into the single ClaI site upstream of ′blaC in pJM157 were conducted as described above. After electroporation into E. coli DH5α (Invitrogen), approximately 8 × 10⁵ CFU were pooled and used to isolate plasmid DNA (Qiagen).

Selection of exported ′BlaC fusions.

Library DNA was electroporated into M. smegmatis ΔblaS strain PM759 (6, 18). The resulting transformants were plated onto 7H10 agar medium without Tween, containing 40 μg/ml lysine (Fischer Scientific), 50 μg/ml hygromycin, and carbenicillin at concentrations that ranged from 35 to 75 μg/ml, and incubated at 37°C for a minimum of 4 days. The drug resistance of colonies that grew up on 7H10 agar with hygromycin and carbenicillin was confirmed by spot test analysis on 7H10 agar plates containing (i) both 50 μg/ml hygromycin and 45 μg/ml carbenicillin and (ii) 50 μg/ml hygromycin only. Strains were further analyzed if spots revealed confluent growth on plates containing hygromycin and carbenicillin in comparison to the negative-control strain (ΔblaS M. smegmatis with plasmid pJM113, which carries a promoterless ′blaC gene) (34).

Recovery of ′blaC fusion plasmids.

Plasmid DNA was transferred from carbenicillin- and hygromycin-resistant ΔblaS M. smegmatis to E. coli DH5α by electroduction (2). A small amount of M. smegmatis was transferred from a colony into 20 μl of ice-cold 10% glycerol. The suspension was mixed by vortexing, incubated on ice for 10 min, and added to 40 μl of electrocompetent E. coli DH5α. The mixture was transferred to a chilled 0.2-cm-gap cuvette and pulsed using conditions typical for E. coli electroporation (25 μF, 200 Ω, and 2.5 kV). Immediately, 1 ml of LB broth was added to the cuvette, incubated at 37°C for 1 h, and then plated on LB with hygromycin. Plasmid DNA was purified from hygromycin-resistant E. coli (Qiagen). The M. tuberculosis genomic DNA insert upstream of ′blaC was identified by sequencing with the primer TnBlaCout (see Table S1 in the supplemental material).

Anti-BlaC antibody.

A six-histidine-tagged copy of M. tuberculosis BlaC was expressed from Y49 pTrcHisB-BlaC in E. coli DH5α (provided by Doug Kernodle) and purified by nickel affinity chromatography using HIS-Select nickel affinity gel (Sigma) as described previously (59). Purified BlaC was eluted from the nickel column with 300 mM imidazole at a concentration of 1.3 mg/ml and used to immunize rabbits together with TiterMax Gold adjuvant (Sigma). Rabbit immunizations and polyclonal antiserum collection were carried out by the BioSource Custom Immunology Department (Hopkinton, MA).

Immunoblot analysis.

Whole-cell lysates of M. smegmatis and whole-cell lysates of formalin-killed M. tuberculosis were prepared as described previously (7, 19, 33) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Polyclonal BlaC antiserum was used in immunoblot analysis at a dilution of 1:10,000, and polyclonal 19-kDa antiserum (provided by Douglas Young) was used at a dilution of 1:40,000. Anti-rabbit peroxidase-conjugated antibodies (Bio-Rad) were used as secondary antibodies for both anti-BlaC and anti-19-kDa antiserum. Both monoclonal hemagglutinin (HA) antiserum (Covance) and monoclonal GroEL HAT5/IT-64 antiserum (World Health Organization collection) were used at a dilution of 1:20,000, and anti-mouse peroxidase-conjugated antibodies were used as secondary antibodies (Bio-Rad).

Construction of specific M. tuberculosis-′BlaC fusions.

The nucleotides encoding the signal sequences of the Rv2041c and Rv2525c genes were PCR amplified using the primers rv2041ssF and 2041ssR or 2525F and 2525R, respectively (see Table S1 in the supplemental material). Both the forward and reverse primers encoded a BstBI site, and the forward primers also included the Shine-Dalgarno sequence from M. tuberculosis hsp60. The amplified fragments were ligated into pCR2.1 (Invitrogen), digested with BstBI, and then ligated into ClaI-digested pJM157, upstream of ′blaC.

Construction of expression constructs for HA-tagged M. tuberculosis proteins. (i) hsp60 promoter-driven HA fusions: Rv0774c-HA, ΔssRv0774c-HA, Rv2843-HA, and ΔssPlcB-HA.

Oligonucleotide primers were designed to amplify full-length or truncated genes from M. tuberculosis genomic DNA. Forward and reverse primers were designed each with 5′ extension sequences carrying MscI and HindIII restriction sites, respectively (see Table S1 in the supplemental material). The resulting PCR product was first cloned into pCR2.1 (Invitrogen) and sequenced. The cloned gene was then digested with MscI and HindIII, and the appropriate fragment was isolated and ligated into the mycobacterial shuttle vector pJSC77 (21), which was digested with MscI and HindIII and which carries the hsp60 promoter and multiple cloning site upstream of a C-terminal HA tag (Table 2). Due to an MscI site within the Rv2843 gene, the strategy was revised in this instance and an NruI site was included instead of MscI on the forward primer (see Table S1 in the supplemental material).

(ii) Native promoter-driven HA fusions: PlcB-HA and Rv0315-HA.

Oligonucleotide primers were designed to amplify a fragment of M. tuberculosis genomic DNA which included the full-length gene of interest and upstream sequence containing the putative native promoter. Forward primers were designed each with 5′ extension sequences carrying XbaI and HindIII restriction sites, respectively (see Table S1 in the supplemental material). The resulting PCR product was first cloned into pCR2.1 (Invitrogen) and sequenced. The cloned gene and promoter were then digested with XbaI and HindIII, and the appropriate fragment was isolated and ligated into the mycobacterial shuttle vector pJSC77, which had been digested with XbaI and HindIII, which removes the hsp60 promoter (Table 2).

(iii) PlcB(KK)-HA.

The construct in which the codons for the twin-arginine pair of PlcB were mutated to encode lysines (KK) was generated as follows. The PCR primers plcBKKfor2 and plcBKKrev3 overlapped at the site of mutation and were used in inverse PCR to generate a product from pJM199. The resulting PCR product was then end repaired using the EndIt kit (Epicentre) following the manufacturer's instructions and self-ligated to create pJM216.

Subcellular fractionation.

Cell wall, membrane, and soluble fractions were prepared by differential ultracentrifugation as described previously (19, 44). Briefly, 100-ml cultures of M. tuberculosis were harvested by centrifugation at 3,000 × g. Cell pellets were sterilized by gamma irradiation in a JL Shephard Mark I 137Cs irradiator (Department of Radiobiology, University of North Carolina at Chapel Hill) with a dose of 2.4 megarads. All subsequent steps were performed at 4°C. Pellets were resuspended in 4 ml of breaking buffer (phosphate-buffered saline, 1 mM phenylmethylsulfonyl fluoride, 0.6 μg/ml each of DNase and RNase, and a cocktail of protease inhibitors [2 μg/ml each of aprotinin, E-64, leupeptin, and pepstatin A and 100 μg/ml Pefabloc SC]) and then lysed in a French pressure cell. Unbroken cells were pelleted at 3,000 × g for 20 min to generate a clarified whole-cell lysate, which was centrifuged at 27,000 × g for 30 min to pellet the cell wall. The supernatant was centrifuged at 100,000 × g for 2 h to separate the membrane fraction from the soluble fraction. The cell wall and membrane fractions were washed once and then resuspended in phosphate-buffered saline.

Bioinformatic identification of putative twin-arginine signal sequences.

Protein sequences corresponding to the 4,056 predicted open reading frames (ORFs) of M. tuberculosis H37Rv were obtained from TubercuList (Institut Pasteur [http://genolist.pasteur.fr/TubercuList/]) and were entered as query sequences in the TATFIND v.1.4 (45) (http://signalfind.org/tatfind.html) and TatP v.1.0 (4) (http://www.cbs.dtu.dk/services/TatP/) search algorithms. The output for TATFIND v.1.4 is either “true” or “false” for a given peptide. TatP v.1.0 has multiple outputs. We used the default search criteria RRx[FGAVML][LITMVF] and selected only those proteins that had a predicted tripartite signal peptide with a Tat motif according to the prediction program. We also reviewed the list of predicted Tat-exported proteins of M. tuberculosis defined by the TigrFAM motif (TIGR01409) (52). This list was obtained directly from the TIGR website (http://cmr.tigr.org/tigr-scripts/CMR/GenomePage.cgi?database=ntmt02).

Selection of exported M. tuberculosis ORF-′BlaC fusions.

A truncated M. tuberculosis β-lactamase ′BlaC, lacking its native signal sequence, can be used in mycobacteria as a reporter of export (34). A special feature of the ′BlaC reporter is that it works only when exported by the Tat pathway. In a β-lactam-sensitive background, such as the M. smegmatis ΔblaS strain, exported ′BlaC fusion proteins can be detected by their ability to promote growth in the presence of the β-lactam antibiotic carbenicillin.

To experimentally identify M. tuberculosis ORFs with functional Tat signal sequences, we constructed genomic M. tuberculosis libraries upstream of the truncated ′blaC reporter. Two libraries were constructed in multicopy vectors, pJES113 and pJM157, each of which carries truncated ′blaC immediately downstream of a unique ClaI cloning site (Fig. 1). The difference between the vectors is that pJM157 contains the mycobacterial hsp60 promoter upstream of the ClaI site to drive expression from genomic fragments that lack a promoter. The hsp60 sequence in pJM157, however, does not include a Shine-Dalgarno site or start codon; these elements must be provided by the genomic insert. For library 1, M. tuberculosis genomic DNA was prepared from wild-type strain H37Rv, and for library 2, the genomic DNA was prepared from the ΔblaC mutant of M. tuberculosis. In both cases the genomic DNA was cut with ClaI-compatible endonucleases for ligation into the vectors.

The libraries were electroporated into the ΔblaS mutant of M. smegmatis and directly plated on 7H10 medium containing carbenicillin. Plasmids expressing exported fusion proteins were selected by their ability to promote growth in the presence of carbenicillin. For library 1, 101 carbenicillin-resistant colonies were obtained from an estimated 1 × 10⁶ M. smegmatis transformants. For library 2, 29 carbenicillin-resistant colonies were obtained from an estimated 9 × 10⁵ transformants. Following confirmation of carbenicillin resistance of individual transformants, plasmid DNA was isolated and sequenced to determine the identity of the genomic DNA insert. With one notable exception, all plasmids sequenced revealed an in-frame fusion with ′blaC. The exception was plasmids in which the full-length M. tuberculosis blaC gene was cloned. In these cases, the blaC insert did not need to be cloned in frame with the reporter sequence on the plasmid. BlaC was identified in 50% of the carbenicillin-resistant clones from library 1. In library 2 this problem was avoided by using genomic DNA from ΔblaC M. tuberculosis.

From the two libraries, we identified amino-terminal sequences of 10 unique M. tuberculosis proteins that promote export of the ′BlaC reporter (Table 3). To confirm that the fusion proteins identified were exported in a Tat-dependent manner, we rescued the fusion plasmids and electroporated them into a double ΔblaS ΔtatA M. smegmatis mutant. This allowed us to test for export in the absence of a functional Tat pathway. All 10 fusions that conferred carbenicillin resistance in a ΔblaS mutant background failed to confer carbenicillin resistance in the ΔblaS ΔtatA strain. This indicated that all the fusion proteins identified require the Tat pathway to export functional ′BlaC.

Direct testing of candidate M. tuberculosis Tat signal sequences.

The M. tuberculosis sequences fused to ′BlaC in the 10 active fusions were all predicted to contain signal sequences, according to the Signal P 3.0 prediction algorithm (Fig. 2A) (3). Evaluation of the exported fusions revealed that the junction with ′BlaC always occurred close to the predicted signal sequence cleavage site of the M. tuberculosis protein. The greatest distance that we observed between a predicted cleavage site and the ′BlaC fusion junction was 34 amino acids. This revealed a requirement for an appropriately positioned restriction enzyme site near the cleavage site in order to identify a Tat signal sequence in our libraries. It also suggested that some proteins may have been missed for this reason. For example, in previous work we showed that the PlcA signal sequence promotes Tat export of the ′BlaC reporter (34), but PlcA was not identified in the libraries. From genome gazing and bioinformatic predictions (discussed below), we selected Rv2041c and Rv2525c as candidate Tat substrates that may have been missed. Construction and direct testing of ssRv2041c-′BlaC and ssRv2525c-′BlaC fusion proteins revealed that both confer resistance to β-lactam in a Tat-dependent manner. This provides experimental validation of these additional M. tuberculosis Tat signal sequences (Fig. 2B).

Alignment of functional Tat signal sequences identified with the ′BlaC reporter.

From studies largely conducted with E. coli, the generally accepted twin-arginine consensus motif is S/T-R-R-x-F-L-K (x is any polar amino acid). The twin arginines are nearly always invariant, and the frequency of occurrence of the other amino acids is reported to exceed 50% (5, 31, 39). Amino acid alignment of the M. tuberculosis signal sequences that we identified revealed that all but one contained the twin-arginine dipeptide (Fig. 2). The exception was the Rv0063 signal sequence, which has a glutamine in the position where the second arginine would be (R-Q-T-F-L). In terms of the other amino acids in the consensus, all were present in ≥40% of the sequences except for the final lysine (K). The alignment also revealed conservation of a hydrophobic residue just prior to the S/T amino acid in the consensus.

Comparison to in silico-predicted Tat signal sequences.

Multiple Tat signal prediction programs exist, but their ability to accurately and comprehensively identify Tat substrates within mycobacteria is unresolved. We applied two of these web-based programs, TatP v.1.0 (4) and TATFIND v1.4 (45), to the M. tuberculosis H37Rv genome sequence and compared the output to Tat signal sequences predicted by a third prediction program devised by The Institute for Genomic Research (TIGR) (TIGR01409) (52). Of the 4,056 ORFs in the M. tuberculosis H37Rv genome (11), 95 were predicted to encode proteins with Tat signal sequences by at least one of the prediction programs (see Table S2 in the supplemental material). There is surprisingly limited overlap between the algorithms, with only 11 proteins being predicted by all three programs (Fig. 3A).

We next compared the signal sequences that we identified experimentally (Table 3; Fig. 2) to the in silico predictions of the annotated M. tuberculosis genome. Eight of the proteins that we identified were predicted by all three programs, and three were predicted by two programs (Fig. 3B). Of the remaining two signal sequences, one was Rv0063, which lacks the twin arginine and was identified only by the TigrFAM prediction program, and the other was LprQ, which was not predicted by any program.

Assessment of Tat dependence for full-length proteins with functional Tat signal sequences.

In addition to there being a requirement for a Tat signal sequence, the mature domain also plays a role in whether a protein is a Tat substrate. For this reason, we tested full-length versions of a subset of the proteins that we identified to see if they were Tat dependent.

Signal sequence cleavage is commonly used as an indicator of protein export (37). To establish the utility of this approach for monitoring export of M. tuberculosis Tat substrates expressed in M. smegmatis, we first assayed signal sequence cleavage of full-length BlaC. Polyclonal antibodies were raised against BlaC and used to detect BlaC expressed in whole-cell lysates of ΔblaS M. smegmatis by immunoblotting ( Fig. 4). In the presence of a functional Tat apparatus, BlaC is observed as a predominant band running at 30 kDa, which is the predicted size of the exported and processed mature species. Expression of BlaC(KK), which has a KK substitution for the RR dipeptide, produced a slower-migrating species, which is consistent with a lack of export and accumulation of full-length unprocessed BlaC precursor (Fig. 4). In the absence of a functional Tat pathway (in a ΔblaS ΔtatA mutant), a larger presumptive precursor species was observed, although some smaller mature protein was detected as well. These experiments indicated that the protein species observed for each of these strains was a good indicator of Tat- and twin-arginine-dependent export.

We then tested full-length versions of four additional proteins identified with the ′BlaC reporter in wild-type and ΔtatC M. smegmatis. Proteins were expressed as a C-terminal fusion to the HA epitope tag, and immunoblot analysis was performed on whole-cell lysates. For Rv0315 and Rv2843, Tat-dependent processing was observed in M. smegmatis, which is indicative of the full-length proteins being Tat exported. A lower-molecular-weight and presumably processed form of the protein was seen when expressed in the wild type, but this species was significantly reduced in abundance in the ΔtatC mutant (Fig. 5A). Three bands were observed for Rv0315-HA expressed in wild-type M. smegmatis. The highest-molecular-mass band is similar in size (∼34 kDa) to that of the predicted Rv0315 precursor and is present in both wild-type and ΔtatC strains. The two smaller protein species are absent or greatly reduced in the ΔtatC mutant background. This suggests that Rv0315 is subject to multiple processing events.

For the other two full-length proteins tested, no obvious Tat dependence was observed in immunoblots of M. smegmatis whole-cell lysates (Fig. 5B). One of these proteins was the virulence factor PlcB-HA. The protein species seen in the wild-type and ΔtatC M. smegmatis strains migrated at the predicted precursor size of 57 kDa. Moreover, compared to a truncated form of PlcB-HA lacking the predicted signal sequence (ΔssPlcB-HA), the full-length product that we expressed migrated slower than the expected mature product did (Fig. 5B). Similar results were obtained with Rv0774c-HA and a truncated ΔssRv0774c-HA protein expressed in M. smegmatis. This suggested that these two predicted Tat substrates were not being processed or exported when expressed in M. smegmatis.

Full-length PlcB is exported by the Tat pathway when expressed in its native host, M. tuberculosis.

Since M. smegmatis does not have phospholipase C homologs, we considered the possibility that PlcB-HA can be exported only by its native host, M. tuberculosis. To test this idea, we expressed full-length PlcB-HA in M. tuberculosis H37Rv and assayed whole-cell lysates by immunoblot analysis. Unlike what was observed in M. smegmatis, immunoblot assays for PlcB-HA in M. tuberculosis whole-cell lysates yielded two products: a larger species that migrated like the full-length precursor seen in M. smegmatis and a smaller species that migrated like the expected mature ΔssPlcB-HA product (Fig. 6A). This suggested that in M. tuberculosis, PlcB-HA is exported and processed. Subcellular fractions prepared from the M. tuberculosis strain were used to show that the observed faster-migrating product was exported. Soluble, membrane, and cell wall fractions were prepared from whole-cell lysates of H37Rv expressing PlcB-HA and analyzed by immunoblotting (Fig. 6B). Of the two protein species seen in the whole-cell lysate, the larger product, the presumptive nonexported precursor, was in the soluble cytosolic fraction and the smaller product was primarily in the cell wall. Thus, when expressed in M. tuberculosis the PlcB-HA protein was processed and exported to the cell wall. As controls for the fractionation, we showed that the GroEL protein and the 19-kDa lipoprotein were enriched in the soluble and cell envelope (cell wall and membrane) fractions, respectively.

The essential nature of the Tat pathway in M. tuberculosis precludes testing full-length PlcB-HA in an M. tuberculosis Δtat mutant. To address whether full-length M. tuberculosis PlcB is a Tat substrate, the RR pair in the PlcB signal sequence was replaced with KK. When the PlcB(KK)-HA protein was expressed in M. tuberculosis, a single precursor-sized species was observed (Fig. 6C). Together, these experiments demonstrated that PlcB is a Tat substrate exported to the cell wall in M. tuberculosis but not in M. smegmatis.