Title: Genetic Toggling of Alkaline Phosphatase Folding Reveals Signal Peptides for All Major Modes of Transport across the Inner Membrane of Bacteria
Abstract: Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen. Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen. Despite recent advances in bioinformatic analysis of N-terminal protein export signals (1.Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5640) Google Scholar, 2.Bendtsen J.D. Nielsen H. Widdick D. Palmer T. Brunak S. BMC Bioinformatics. 2005; 6: 167-175Crossref PubMed Scopus (402) Google Scholar, 3.Bronstein P.A. Marrichi M. Cartinhour S. Schneider D.J. DeLisa M.P. J. 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A. 1985; 82: 8129-8133Crossref PubMed Scopus (659) Google Scholar) has been an extraordinarily useful experimental tool for verifying and discovering signal peptides in numerous bacterial species on a genome-wide scale (8.Bailey J. Manoil C. Nat. Biotechnol. 2002; 20: 839-842Crossref PubMed Scopus (31) Google Scholar, 9.Gutierrez C. Barondess J. Manoil C. Beckwith J. J. Mol. Biol. 1987; 195: 289-297Crossref PubMed Scopus (110) Google Scholar). TnphoA is a derivative of the Tn5 transposon that enables the generation of protein fusions to Escherichia coli PhoA (alkaline phosphatase; EC3.1.3.1) devoid of its native amino-terminal export signal. The resulting fusions only confer phosphate hydrolase activity to cells if they are capable of export out of the cytoplasm. Consequently, TnphoA can be used to detect proteins localized to the periplasm, inner and outer membranes, or extracellularly. Living cells can be assayed for PhoA activity using the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) 3The abbreviations used are: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; SRP, signal recognition particle; HMM, hidden Markov model; MBP, maltose-binding protein. or by selective growth on medium containing a sole carbon or phosphate source that requires PhoA activity to be metabolized (10.Sarthy A. Michaelis S. Beckwith J. J. Bacteriol. 1981; 145: 288-292Crossref PubMed Google Scholar). The general utility of this genetic construct is exemplified by the large number of applications reporting its use, including, for example, identification of cell surface and secreted virulence factors (11.Finlay B.B. Starnbach M.N. Francis C.L. Stocker B.A. Chatfield S. Dougan G. Falkow S. Mol. Microbiol. 1988; 2: 757-766Crossref PubMed Scopus (101) Google Scholar, 12.Huang H.C. Schuurink R. Denny T.P. Atkinson M.M. Baker C.J. Yucel I. Hutcheson S.W. Collmer A. J. 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Biochem. 2008; 77: 643-667Crossref PubMed Scopus (474) Google Scholar, 23.Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). The Sec pathway consists of the SecYEG translocase formed by the SecYEG integral membrane proteins and a molecular motor, SecA, which drives translocation of unfolded substrates in an ATP-dependent manner (22.Driessen A.J. Nouwen N. Annu. Rev. Biochem. 2008; 77: 643-667Crossref PubMed Scopus (474) Google Scholar, 23.Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar, 24.Driessen A.J. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Crossref PubMed Scopus (178) Google Scholar). The bulk of proteins targeted to SecYEG are routed in a post-translational manner with assistance from the dedicated molecular chaperone SecB (25.Collier D.N. Bankaitis V.A. Weiss J.B. Bassford Jr., P.J. Cell. 1988; 53: 273-283Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 26.Weiss J.B. Ray P.H. Bassford Jr., P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8978-8982Crossref PubMed Scopus (186) Google Scholar), although for some substrates, including PhoA, post-translational export can proceed without assistance from SecB (27.Kumamoto C.A. Beckwith J. J. Bacteriol. 1985; 163: 267-274Crossref PubMed Google Scholar). Alternatively, a subset of exported proteins are routed co-translationally by the signal recognition particle (SRP) (28.Valent Q.A. de Gier J.W. von Heijne G. Kendall D.A. ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar, 29.Valent Q.A. Kendall D.A. High S. Kusters R. Oudega B. Luirink J. EMBO J. 1995; 14: 5494-5505Crossref PubMed Scopus (238) Google Scholar) that directs substrates to the FtsY receptor (30.Luirink J. ten Hagen-Jongman C.M. van der Weijden C.C. Oudega B. High S. Dobberstein B. Kusters R. EMBO J. 1994; 13: 2289-2296Crossref PubMed Scopus (211) Google Scholar) and ultimately the SecYEG translocase. This mechanism is responsible for the export of both soluble periplasmic proteins (31.Huber D. Boyd D. Xia Y. Olma M.H. Gerstein M. Beckwith J. J. Bacteriol. 2005; 187: 2983-2991Crossref PubMed Scopus (106) Google Scholar, 32.Schierle C.F. Berkmen M. Huber D. Kumamoto C. Boyd D. Beckwith J. J. Bacteriol. 2003; 185: 5706-5713Crossref PubMed Scopus (166) Google Scholar) and, with assistance from YidC, integral membrane proteins (29.Valent Q.A. Kendall D.A. High S. Kusters R. Oudega B. Luirink J. EMBO J. 1995; 14: 5494-5505Crossref PubMed Scopus (238) Google Scholar, 33.Froderberg L. Houben E. Samuelson J.C. Chen M. Park S.K. Phillips G.J. Dalbey R. Luirink J. De Gier J.W. Mol. Microbiol. 2003; 47: 1015-1027Crossref PubMed Scopus (70) Google Scholar). Importantly, although the use of TnphoA in bacteria is capable of detecting proteins exported via the Sec and SRP pathways, it does not provide sufficient resolution to distinguish between these different modes of SecYEG targeting. To address this limitation, previous studies used TnphoA in combination with Escherichia coli lpp-5508 mutants that have a leaky outer membrane. Free periplasmic PhoA diffused away from these cells and hydrolyzed BCIP in the surrounding medium to yield a "blue halo" around the colony, whereas membrane-bound PhoA could only hydrolyze intracellular BCIP, and the resulting blue color was localized within the colony (34.Giladi M. Champion C.I. Haake D.A. Blanco D.R. Miller J.F. Miller J.N. Lovett M.A. J. Bacteriol. 1993; 175: 4129-4136Crossref PubMed Google Scholar, 35.Strauch K.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1576-1580Crossref PubMed Scopus (296) Google Scholar). 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For instance, in a recent genome-wide screen for N-terminal signal peptides in Psuedomonas aeruginosa, 310 PhoA fusions were identified, of which only one (RnfG, PA3493) was predicted to be a Tat substrate (39.Lewenza S. Gardy J.L. Brinkman F.S. Hancock R.E. Genome Res. 2005; 15: 321-329Crossref PubMed Scopus (100) Google Scholar) despite the fact that as many as 57 Tat-dependent substrates have been predicted for this organism (4.Dilks K. Rose R.W. Hartmann E. Pohlschroder M. J. Bacteriol. 2003; 185: 1478-1483Crossref PubMed Scopus (203) Google Scholar). The reason for this is that the bacterial Tat transporter accepts only those proteins that have attained a native or nearly native structure in the cytoplasm (40.Bruser T. Yano T. Brune D.C. Daldal F. Eur. J. Biochem. 2003; 270: 1211-1221Crossref PubMed Scopus (61) Google Scholar, 41.DeLisa M.P. Tullman D. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6115-6120Crossref PubMed Scopus (259) Google Scholar, 42.Fisher A.C. Kim W. DeLisa M.P. Protein Sci. 2006; 15: 449-458Crossref PubMed Scopus (95) Google Scholar). Since PhoA folding is dependent upon disulfide bonds that can only form in the periplasm (43.Sone M. Kishigami S. Yoshihisa T. Ito K. J. Biol. Chem. 1997; 272: 6174-6178Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), PhoA fusions misfold in the cytoplasm of wild type bacteria and thus are incapable of transiting the Tat pathway (41.DeLisa M.P. Tullman D. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6115-6120Crossref PubMed Scopus (259) Google Scholar). However, PhoA can be exported by the Tat pathway under specific conditions, such as when the cytoplasm is rendered more oxidizing by deletion of the trxB gor genes that encode the two major cytoplasmic reductases in E. coli (41.DeLisa M.P. Tullman D. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6115-6120Crossref PubMed Scopus (259) Google Scholar). In this study, we sought to expand the utility of TnphoA screening for (i) resolving the targeting specificity (e.g. SecB-independent versus SecB-dependent versus SRP-dependent) of exported proteins and (ii) detecting export via the Tat pathway. Using mini-Tn5 phoA-generated libraries in combination with a limited collection of isogenic E. coli strains derived from strain DR473 that allow toggling of PhoA folding, we have isolated PhoA fusions for all major modes of inner membrane transport in E. coli. Additionally, we have employed this strategy to experimentally verify that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signal peptides are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen. Bacterial Strains and Plasmids—The strains and plasmids used in this study are listed in Table 1. E. coli strains were routinely grown in LB medium (Difco) broth at 30 or 37 °C with antibiotics added at the following concentrations: 100 μg/ml ampicillin, 20 μg/ml chloramphenicol, 10 μg/ml gentamycin, 50 μg/ml hygromycin, 50 μg/ml kanamycin, and 50 μg/ml spectinomycin. Mycobacterium smegmatis strains were grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80; l-lysine was added at a concentration of 80 μg/ml for the strains MB692, PM759, and JM578, and all cells were grown at 37 °C with antibiotics added at the same concentrations as listed above. M. smegmatis cells were also grown on solid Middlebrook 7H10 medium (Difco) supplemented as indicated above but with 40 μg/ml l-lysine when necessary.TABLE 1Strains and plasmids used in this studyStrain or plasmidRelevant genotype/phenotypeReference or sourceE. coli strainsMC4100F ΔlacU169 araD139 rpsL150 relA1 ptsF rbs flbB5301Laboratory stockSM10 λ-pirthi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Kmr pirRef. 77.Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1711) Google ScholarDHB4MC1000 phoR Δ(phoA) PvuII (malF)3 F′[lacIqZYA pro]Laboratory stockJW3584secB::kanRef. 78.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2: 1-11Crossref Scopus (5413) Google ScholarJW5580tatB::kanRef. 78.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2: 1-11Crossref Scopus (5413) Google ScholarJW3815tatC::kanRef. 78.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2: 1-11Crossref Scopus (5413) Google ScholarJW3813tatA:::kanRef. 78.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2: 1-11Crossref Scopus (5413) Google ScholarJW0622tatE::kanRef. 78.Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2: 1-11Crossref Scopus (5413) Google ScholarDHAEDHB4 tatA tatEThis workDHBDHB4 tatBThis workDHCDHB4 tatCThis workDR473DHB4 ΔtrxB gor552 Tn10Tet ahpC* Tn10Cm (araC Para-trxB)Ref. 41DRADR473 dsbA::kanRef. 41DRAEDR473 tatA tatEThis workDRBDR473 tatB::kanRef. 41DRCDR473 tatC::specRef. 41DRSDR473 secBThis workTOP10F− mcrA Δ(mrr-hsdRMS-mcrBC) f80lacZDM15 DlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupGLaboratory stockM. smegmatis strainsmc2155ept-1Laboratory stockPM759mc2155 ΔblaS1 ΔlysA4 rpsL6Ref. 79.Flores A.R. Parsons L.M. Pavelka Jr., M.S. Microbiology. 2005; 151: 521-532Crossref PubMed Scopus (172) Google ScholarMB692mc2155 ΔtatARef. 68JM578PM759 ΔtatARef. 68PlasmidspCP20Apr, CmrRef. 44pBR322Apr, TcrLaboratory stockpBR322-GmApr, GmrThis studypBR-BamHI-GmBamHI site cloned into pBR322-GmThis studypBRGC.1Gateway cassette RfC.1 in pBR322-GmThis studypUTphoAApr, Kmr, mini-Tn5 phoA cassette in pUT-based plasmidRef. 55pPhoAE. coli phoA gene cloned in pTrc99ALaboratory stockpTorA-PhoAE. coli ssTorA fused to Δ(1-22)PhoA in pTrc99ARef. 41pMCS-ΔssPhoAΔ(1-22)PhoA cloned in pBR322-GmThis studypssMBP-PhoAE. coli ssMBP fused to Δ(1-22)PhoA in pMCS-DssAPThis studypCueO-PhoAE. coli CueO fused to Δ(1-22)PhoA in pMCS-DssAPThis studypSufI-PhoAE. coli SufI fused to Δ(1-22)PhoA in pMCS-DssAPThis studypΔssPhoAΔ(1-22)PhoA in pBRGC.1This studypssCueO-PhoAE. coli ssCueO fused to Δ(1-22)PhoA in pBRGC.1This studypssSufI-PhoAE.coli ssSufI fused to Δ(1-22)PhoA in pBRGC.1This studypCueOE. coli cueO gene in pBRGC.1This studypSufIE. coli sufI gene in pBRGC.1This studypMalEE. coli malE gene in pBRGC.1This studypDsbAE. coli dsbA gene in pBRGC.1This studypAg85AM. tuberculosis Rv3804c in pBRGC.1This studypAg85CM. tuberculosis Rv0129c in pBRGC.1This studypModDM. tuberculosis Rv1860 in pBRGC.1This studypPepAM. tuberculosis Rv0125 in pBRGC.1This studypVV16Hmr, KmrRef. 45pVV-GC.1-FHGateway cassette RfC.1 in pVV16; introduces C-terminal FLAG and His6 epitope tagsThis studypVV-Ag85A-FHM. tuberculosis Rv3804c in pVV-GC.1-FHThis studypVV-Ag85C-FHM. tuberculosis Rv0129c in pVV-GC.1-FHThis studypVV-ModD-FHM. tuberculosis Rv1860 in pVV-GC.1-FHThis studypVV-PepA-FHM. tuberculosis Rv0125 in pVV-GC.1-FHThis studypVV-BlaC-FHM. tuberculosis Rv2068c in pVV-GC.1-FHThis studypSALectApr, CmrRef. 80.Lutz S. Fast W. Benkovic S.J. Protein Eng. 2002; 15: 1025-1030Crossref PubMed Scopus (29) Google ScholarpMCS-ΔssBlaCΔ(1-31)BlaC cloned in pSALectThis studypVV-ssAg85A-BlaC-FHssAg85A fused to Δ(1-31)BlaC in pVV16This studypVV-ssAg85C-BlaC-FHssAg85C fused to Δ(1-31)BlaC in pVV16This studypVV-ssModD-BlaC-FHssModD fused to Δ(1-31)BlaC in pVV16This studypVV-ssPepA-BlaC-FHssPepA fused to Δ(1-31)BlaC in pVV16This study Open table in a new tab Genetic disruption of DHB4 and DR473 cells was achieved using P1vir transducing phage. Briefly, donor cells containing the gene of interest with a selectable marker were grown in LB broth at 37 °C overnight and subcultured into fresh medium containing 5 mm CaCl2, 10 mm MgSO4, and 0.2% glucose at a 100-fold dilution and allowed to grow for 1.5 h at 37 °C. Lytic phage was then added, and the culture was grown until "clearing" occurred, after which the phage was harvested through centrifugation and stored at 4 °C with chloroform. Recipient DHB4 or DR473 cells were grown in appropriate antibiotics at 37 °C overnight and centrifuged at 5,000 × g for 3 min. Cells were then resuspended in fresh medium containing 5 mm CaCl2, 10 mm MgSO4, and 0.2% glucose, and lytic phage carrying the marked gene of interest was added at a 10-fold dilution. Cells and phage were grown at 37 °C for 30 min, subsequently supplemented with 100 mm sodium citrate, grown for an additional 1 h, and plated on LB agar containing the appropriate antibiotics and 100 mm sodium citrate overnight. Cells containing the genetic disruption were recovered, and the resistance marker was removed using the pCP20 helper plasmid, as previously described (44.Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11197) Google Scholar). Construction of the plasmid pBR322-Gm was carried out by replacement of the Tcr cassette in pBR322 with the gentamycin resistance gene. pBR322-Gm was then converted into a destination vector with an insertion of the Gateway RfC.1 cassette (Invitrogen) into the SspI/ScaI sites, creating the vector pBRGC.1. Plasmid pVV16 (45.Stover C.K. de la Cruz V.F. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. Bansal G.P. Young J.F. Lee M.H. Hatfull G.F. Snapper S.B. Barletta R.G. Jacobs Jr., W.R. Bloom B.R. Nature. 1991; 351: 456-460Crossref PubMed Scopus (1208) Google Scholar) was converted into a destination vector named pVV-GC.1-FH by insertion of a modified version of the Gateway RfC.1 cassette containing a FLAG affinity epitope at the 3′-end into the NdeI/HindIII sites. All derivatives of pVV-GC.1-FH and pBRGC.1 were created using Gateway cloning technology according to the manufacturer's protocols (Invitrogen). The plasmids pCueO-AP and pSufI-AP were constructed by first inserting the 1,412-bp E. coli phoA gene with a modified 5′-end to include a mini multicloning site (NheI, PsiI, and XhoI sites) into the HindIII/SalI sites of pBR322, yielding vector pMCS-ΔssPhoA. Then DNA encoding E. coli cueO or E. coli sufI was PCR-amplified and inserted in the NheI/PsiI sites pMCS-ΔssPhoA. Plasmid pssMBP-PhoA was constructed by PCR amplification of the first 143 bp of malE from pMalE and insertion of the resulting product into the HindIII/XhoI sites of pMCS-ΔssPhoA. Plasmid pMCS-ΔssBlaC was constructed by inserting an 864-bp PCR product corresponding to M. tuberculosis blaC lacking the first 93 bp but with a 33-bp FLAG epitope tag added to the 3′-end into the SpeI/EcoRI sites of pSALect. To construct plasmid pVV-ssAg85A-BlaC-FH, a 120-bp PCR product encoding the M. tuberculosis Ag85A signal peptide was first cloned into the SalI/SpeI sites of pMCS-ΔssBlaC. Then a 14-bp ribosome binding site was appended to the 5′-end of the DNA encoding Ag85A-BlaC via PCR, and the entire 1010-bp construct was cloned into the NdeI/HindIII sites of pVV16. This same procedure was repeated to create pVV-ssAg85C-BlaC-FH, pVV-ssModD-BlaC-FH, and pVV-ssPepA-BlaC-FH by inserting DNA encoding the signal peptides (72, 117, and 96 bp, respectively) inserted at the 5′-end of ΔssBlaC. Plasmid pBR-BamHI-Gm was constructed by inserting the BamHI restriction site into the SspI/PstI sites of pBR322-Gm. Subcellular Fractionation—Cytoplasmic and periplasmic fractions were prepared from cells that had been induced for protein expression at 30 °C for 8 h in the presence of either arabinose or glucose, as indicated. Following protein induction, cells were pelleted by centrifugation at 3,000 × g for 15 min at 4 °C and then subjected to the ice-cold osmotic shock procedure as previously described (46.Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar). The quality of all fractionations was determined by immunodetection of the cytoplasmic GroEL protein (41.DeLisa M.P. Tullman D. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6115-6120Crossref PubMed Scopus (259) Google Scholar). Western Blot Analysis—Proteins were separated by SDS-PAGE, and Western blotting was performed as described previously (47.Chen G. Hayhurst A. Thomas J.G. Harvey B.R. Iverson B.L. Georgiou G. Nat. Biotechnol. 2001; 19: 537-542Crossref PubMed Scopus (112) Google Scholar). Briefly, all lanes of SDS-12% polyacrylamide gels (Bio-Rad) were loaded with samples prepared from an equivalent number of cells harvested for each experiment. The following primary antibodies were used: monoclonal mouse anti-PhoA (Sigma) diluted 1:20,000; monoclonal mouse anti-FLAG (Stratagene) diluted 1:3,000, and polyclonal anti-GroEL (Sigma) diluted 1:10,000. Secondary antibodies were either goat anti-mouse (Promega) or goat anti-rabbit (Promega) diluted 1:2,500. Following development of blots using the Immun-Star horseradish peroxidase substrate kit (Bio-Rad) and visualized using x-ray film (Eastman Kodak Co.), membranes were stripped in a solution consisting of 2.0% SDS, 7.0% β-mercaptoethanol, 0.03% NaCl, and 0.0025% Tris, reblocked, and probed with anti-GroEL antibody. Monitoring of PhoA Activity in Intact Cells—E. coli cells were grown in LB supplemented with appropriate antibiotics at 37 °C overnight and streaked onto LB agar supplemented with appropriate antibiotics, 50 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma), and 0.2% arabinose or 0.2% glucose and grown at 30 °C for 2 days. Streaks that attained a blue phenotype were classified as export competent, whereas cells that appeared white/colorless were classified as incapable of PhoA export from the cytoplasm. Isolation of Mini-Tn5 phoA Insertions—SM10 λ-pir cells carrying pUTphoA and MC4100 cells carrying the expression vector of interest were grown overnight at 37 °C in LB supplemented with appropriate antibiotics. Cells were then pelleted, washed three times with fresh LB, mixed, and spotted in 40-μl aliquots onto a nitrocellulose membrane on LB agar and grown at 30 °C for 16 h. The membrane was then resuspended in 10 ml of fresh LB and used to inoculate 200 ml of LB supplemented with 300 μg/ml kanamycin and 10 μg/ml gentamycin, followed by growth at 37 °C for 16 h. Upon conjugation, ∼104 recipient cells containing both the marked transposon and the expression vector of interest were selected and pooled together. Plasmid DNA was then extracted from these cells and used to transform competent DR473 cells that were grown on LB agar supplemented with appropriate antibiotics, 50 μg/ml BCIP, and either 0.2% glucose or 0.2% arabinose at 30 °C for 2 days. For each library, 12 colonies that exhibited a strong blue phenotype were restreaked onto the same medium and grown at 30 °C for 2 days to confirm the phenotype. Plasmid DNA was then prepared from all positive clones and sequenced using a primer specific for the antisense strand of the 5′-end of the phoA gene. Construction and Screening of Genomic Library—Genomic DNA was isolated from MC4100 cells and partially digested using Sau3AI, as previously described (39.Lewenza S. Gardy J.L. Brinkman F.S. Hancock R.E. Genome Res. 2005; 15: 321-329Crossref PubMed Scopus (100) Google Scholar). Fragments between 0.75 and 3.0 kb were excised from a 1% agarose-Tris borate-EDTA gel and gel-purified (Qiagen) and then ligated into the BamHI site of pBR-BamHI-Gm. This library of genomic DNA was then electroporated into competent MC4100 cells and conjugated with SM10 λ-pir cells carrying pUTphoA as described above. The resulting library, composed of transposon insertions into genomic DNA, was isolated and electroporated into competent DR473 cells. Screening of the library was performed on BCIP indicator plates supplemented with either glucose or arabinose as described above. Cells displaying a bright blue phenotype were reconfirmed, after which their plasmid DNA was isolated and sequenced using a primer specific for the antisense strand of the 5′-end of the phoA gene. Bioinformatic Prediction of Tat Substrates—Analysis was carried out as previously described (3.Bronstein P.A. Marrichi M. Cartinhour S. Schneider D.J. DeLisa M.P. J. Bacteriol. 2005; 187: 8450-8461Crossref PubMed Scopus (63) Google Scholar). Briefly, a hidden Markov model (HMM) was developed using a training set constructed from experimentally confirmed Tat substrates present in E. coli and P. aeruginosa. The previously developed HMM for Tat motifs using hmmbuild (available on the World Wide Web) was calibrated with hmmcalibrate and used to search the annotated proteins from the chromosome of M. tuberculosis H37Rv (GenBank™ accession number NC_000962.2) with hmmsearch. Si