{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T02:54:26Z","timestamp":1792205666006,"version":"4.4.0"},"reference-count":37,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5317","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1997,5,30]]},"abstract":"<jats:p>\n            The dominant exported proteins and protective antigens of\n            <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n            are a triad of related gene products called the antigen 85 (Ag85) complex. Each has also been implicated in disease pathogenesis through its fibronectin-binding capacities. A carboxylesterase domain was found within the amino acid sequences of Ag85A, B, and C, and each protein acted as a mycolyltransferase involved in the final stages of mycobacterial cell wall assembly, as shown by direct enzyme assay and site-directed mutagenesis. Furthermore, the use of an antagonist (6-azido-6-deoxy-\u03b1,\u03b1\u2032-trehalose) of this activity demonstrates that these proteins are essential and potential targets for new antimycobacterial drugs.\n          <\/jats:p>","DOI":"10.1126\/science.276.5317.1420","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:39:52Z","timestamp":1027762792000},"page":"1420-1422","source":"Crossref","is-referenced-by-count":680,"title":["Role of the Major Antigen of\n            <i>Mycobacterium tuberculosis<\/i>\n            in Cell Wall Biogenesis"],"prefix":"10.1126","volume":"276","author":[{"given":"John T.","family":"Belisle","sequence":"first","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Varalakshmi D.","family":"Vissa","sequence":"additional","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Todd","family":"Sievert","sequence":"additional","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kuni","family":"Takayama","sequence":"additional","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick J.","family":"Brennan","sequence":"additional","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gurdyal S.","family":"Besra","sequence":"additional","affiliation":[{"name":"J. T. Belisle, V. D. Vissa, P. J. Brennan, G. S. Besra, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA."},{"name":"T. Sievert and K. Takayama, Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, University of Wisconsin, Madison, WI 53705, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1146\/annurev.bi.64.070195.000333","volume":"64","author":"Brennan P. J.","year":"1995","unstructured":"Brennan P. J., Nikaido H., Annu. Rev. Biochem. 64, 63 (1995).","journal-title":"Annu. Rev. Biochem."},{"key":"e_1_3_1_3_2","first-page":"215","volume":"65","author":"Blanchard J. S.","year":"1996","unstructured":"Blanchard J. S., ibid. 65, 215 (1996).","journal-title":"ibid."},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"12735","DOI":"10.1073\/pnas.91.26.12735","volume":"91","author":"Besra G. S.","year":"1994","unstructured":"Besra G. S., et al., Proc. Natl. Acad. Sci. U.S.A. 91, 12735 (1994).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"23328","DOI":"10.1016\/S0021-9258(17)31657-5","volume":"269","author":"Wolucka B. A.","year":"1994","unstructured":"Wolucka B. A., McNeil M. R., de Hoffmann E., Chojnacki T., Brennan P. J., J. Biol. Chem. 269, 23328 (1994);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_5_3","first-page":"7820","volume":"271","author":"Mikusova K.","year":"1996","unstructured":"Mikusova K., Mikus M., Besra G. S., Hancock I., Brennan P. J., ibid. 271, 7820 (1996);","journal-title":"ibid."},{"key":"e_1_3_1_5_4","doi-asserted-by":"crossref","first-page":"11829","DOI":"10.1021\/ja00153a002","volume":"117","author":"Lee R. E.","year":"1995","unstructured":"Lee R. E., Miku\u0161ov\u00e1 K., Brennan P. J., Besra G. S., J. Am. Chem. Soc. 117, 11829 (1995).","journal-title":"J. Am. Chem. Soc."},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1126\/science.7886450","volume":"267","author":"Dessen A.","year":"1995","unstructured":"Dessen A., Quemard A., Blanchard J. S., Jacobs W. R., Sacchettini J. C., Science 267, 1638 (1995);","journal-title":"Science"},{"key":"e_1_3_1_6_3","doi-asserted-by":"crossref","first-page":"8235","DOI":"10.1021\/bi00026a004","volume":"34","author":"Quemard A.","year":"1995","unstructured":"Quemard A., et al., Biochemistry 34, 8235 (1995).","journal-title":"Biochemistry"},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"13417","DOI":"10.1016\/S0021-9258(19)76442-4","volume":"262","author":"Sathyamoorthy N.","year":"1987","unstructured":"Sathyamoorthy N., Takayama K., J. Biol. Chem. 262, 13417 (1987).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"6630","DOI":"10.1073\/pnas.92.14.6630","volume":"92","author":"Yuan Y.","year":"1995","unstructured":"Yuan Y., Lee R. E., Besra G. S., Belisle J. T., Barry C. E., Proc. Natl. Acad. Sci. U.S.A. 92, 6630 (1995);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_8_3","doi-asserted-by":"crossref","first-page":"27292","DOI":"10.1074\/jbc.270.45.27292","volume":"270","author":"George K. M.","year":"1995","unstructured":"George K. M., Yuan Y., Sherman D. R., Barry C. E., J. Biol. Chem. 270, 27292 (1995).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_9_2","unstructured":"Mycolyltransferase assays were performed by suspending 250 \u03bcg of TMM in 450 \u03bcl of 0.1 M potassium phosphate (pH 7.5) 0.1 mM dithiothreitol (DTT). To this was added 10 to 50 \u03bcg of enzyme and the mixture was sonicated for 30 s. [ 14 C]Trehalose (20 nmol 30.4 mCi\/mmol) was then added and the mixture was incubated at 37\u00b0C for 30 min. The reaction was stopped by adding 5 ml of CHCl 3 :CH 3 OH (2:1) and 1.4 ml of water. This mixture was vortexed and centrifuged and the lower (organic) phase was collected and dried. This organic extractable material containing the products of the mycolyltransferase reaction was suspended in 100 \u03bcl of CHCl 3 :CH 3 OH (2:1) and 50 \u03bcl was added to 5 ml of scintillation fluid and counted."},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/0076-6879(90)82036-2","volume":"182","author":"Dunbar B. S.","year":"1990","unstructured":"Dunbar B. S., Kimura H., Timmons T. M., Methods Enzymol. 182, 441 (1990);","journal-title":"Methods Enzymol."},{"key":"e_1_3_1_10_3","unstructured":". 10. N. C. Price Ed. Protein Labfax (Academic Press San Diego CA 1996) p. 270."},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1128\/iai.58.1.272-274.1990","volume":"58","author":"Wiker H. G.","year":"1990","unstructured":"Wiker H. G., Sletten K., Nagai S., Harboe M., Infect. Immun. 58, 272 (1990).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_12_2","first-page":"3925","volume":"60","author":"Khanolkar-Young S.","year":"1992","unstructured":"Khanolkar-Young S., et al., ibid. 60, 3925 (1992).","journal-title":"ibid."},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1128\/mr.56.4.648-661.1992","volume":"56","author":"Wiker H. G.","year":"1992","unstructured":"Wiker H. G., Harboe M., Microbiol. Rev. 56, 66 (1992);","journal-title":"Microbiol. Rev."},{"key":"e_1_3_1_13_3","doi-asserted-by":"crossref","first-page":"1530","DOI":"10.1073\/pnas.92.5.1530","volume":"92","author":"Horwitz M. A.","year":"1995","unstructured":"Horwitz M. A., Lee B. W., Dillon B. J., Harth G., Proc. Natl. Acad. Sci. U.S.A. 92, 1530 (1995).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_14_2","first-page":"4834","volume":"61","author":"Peake P.","year":"1993","unstructured":"Peake P., Gooley A., Britton W. J., Infect. Immun. 61, 4834 (1993).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_15_2","doi-asserted-by":"crossref","first-page":"3137","DOI":"10.1073\/pnas.93.7.3132","volume":"93","author":"Philipp W. J.","year":"1996","unstructured":"Philipp W. J., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 3137 (1996).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"2349","DOI":"10.1111\/j.1365-2958.1992.tb01410.x","volume":"6","author":"Joliff G.","year":"1992","unstructured":"Joliff G., et al., Mol. Microbiol. 6, 2349 (1992).","journal-title":"Mol. Microbiol."},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.63.8.2846-2853.1995"},{"key":"e_1_3_1_18_2","unstructured":"Protein obtained by precipitation with a 40% saturated solution of (NH 4 ) 2 SO 4 was dialyzed against storage buffer [10 mM KH 2 PO 4 (pH 7.5) 1 mM EDTA 1 mM DTT] and 38 mg was applied to a column (1 cm by 10 cm) of Phenyl Sepharose (Pharmacia Biotech Uppsala Sweden). The column was washed with three volumes of storage buffer at a flow rate of 1 ml\/min which eluted most of the proteins while leaving the Ag85 complex bound to the Phenyl Sepharose matrix. The individual proteins of the Ag85 complex were eluted with 30 ml of buffer A [10 mM tris-HCl (pH 8.6) 1 mM DTT 1 mM EDTA] followed by a linear gradient composed of 100% buffer A to 100% buffer B [10 mM tris-HCl (pH 8.6) 1 mM DTT 1 mM EDTA 50% ethylene glycol] over a 40-ml volume followed by 10 ml of 100% buffer B."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/0003-2697(81)90783-1","volume":"117","author":"Morrissey J. H.","year":"1981","unstructured":"Morrissey J. H., Anal. Biochem. 117, 307 (1981).","journal-title":"Anal. Biochem."},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1128\/iai.59.1.372-382.1991","volume":"59","author":"Nagai S.","year":"1991","unstructured":"Nagai S., Wiker H. G., Harboe M., Kinomoto M., Infect. Immun. 59, 372 (1991).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1016\/0167-7799(94)90022-1","volume":"12","author":"Kazlauskas R. J.","year":"1994","unstructured":"Kazlauskas R. J., Trends Biotechnol. 12, 464 (1994).","journal-title":"Trends Biotechnol."},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1007\/BF00280552","volume":"79","author":"Young L. J. S.","year":"1988","unstructured":"Young L. J. S., et al., Hum. Genet. 79, 137 (1988).","journal-title":"Hum. Genet."},{"key":"e_1_3_1_23_2","unstructured":"A 1.2-kb DNA fragment in pBluescript II SK\u2212 and containing fbpC was used as the template along with primers P 3 (CATATGTTCTCTAGGCCCGGTCTT) and P 4 (CTCGAGAGCGGCCGGAGCAGCAGG) to amplify by PCR the portion of fbpC encoding mature leaderless Ag85C. The resulting 0.9-kb DNA fragment was ligated into the Sma I site of pBluescript II SK\u2212 amplified in E. coli DH5\u03b1 isolated by digestion with Nde I and Xho I and directionally ligated into the E. coli expression vector pET23b (Novagen Madison WI) to form a COOH-terminal His fusion product. This recombinant plasmid was designated pCSB9. Site-directed mutagenesis resulting in a T to G mutation of nucleotide 373 of the cloned fbpC fragment was accomplished by the PCR amplification of overlapping 390\u2013 and 531\u2013base pair (bp) fragments with pCSB9 as the template. The 390-bp fragment was amplified with the primers P 3 and P 5 (5\u2032-TCCGCCTGACATCG[C]AAGACCCACCGC-3\u2032) a primer that overlaps nucleotide 373 and substitutes C for A at this position. The 531-bp fragment was generated with the primers P 4 and P 6 (5\u2032-GCGGTGGGTCTT[G]CGATGTCGGGC- GGT-3\u2032) which also overlapped with nucleotide 373 and substituted a G for T at this position. The overlapping 390- and 531-bp fragments were mixed and a full-length 894-bp fragment was generated by overlap extension PCR (28) with the P 3 and P 4 primers. This product was ligated into pET23b as described for the nonmutated fbpC fragment to yield the recombinant plasmid pCSB9sa. Fidelity of the pCSB9 and pCSB9sa constructs was determined by sequence analysis."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","first-page":"29545","DOI":"10.1074\/jbc.271.47.29545","volume":"47","author":"Liu J.","year":"1996","unstructured":"Liu J., Barry C. E., Besra G. S., Kaido H. N., J. Biol. Chem. 47, 29545 (1996).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_25_2","doi-asserted-by":"crossref","first-page":"2235","DOI":"10.1128\/AAC.39.10.2235","volume":"39","author":"Chung G. A.","year":"1995","unstructured":"Chung G. A., Aktar Z., Jackson S., Duncan K., Antimicrob. Agents. Chemother. 39, 2235 (1995).","journal-title":"Antimicrob. Agents. Chemother."},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","unstructured":"S. Hanessian and P. Lavall\u00e9e J. Antibiotics 25 683 (1972).","DOI":"10.7164\/antibiotics.25.683"},{"key":"e_1_3_1_27_2","unstructured":"Control and ADT-treated cultures were grown to an absorbance at 600 nm of \u223c0.2 before the addition of ADT (100 \u03bcg\/ml) and preincubated at 37 o C for 4 hours before the addition of [1 2- 14 C]acetate (Na salt; 110 mCi\/mmol) at 1 \u03bcCi\/ml to both control and ADT-treated cultures and further incubated with agitation for 20 hours at 37\u00b0C. Carbon-14\u2013labeled lipids were isolated as described (29) and \u223c50 000 cpm of both control and ADT-treated extracts were applied to 2D TLC plates. Radioactive products resolved by 2D TLC were marked extracted and counted. The residual 14 C-labeled delipidated cells rich in the bound mycolate esters of arabinogalactan were subjected to alkaline hydrolysis methylation TLC autoradiography and counting (29)."},{"key":"e_1_3_1_28_2","first-page":"251","volume":"15","author":"Horton R. M.","year":"1993","unstructured":"Horton R. M., Methods Mol. Biol. 15, 251 (1993).","journal-title":"Methods Mol. Biol."},{"key":"e_1_3_1_29_2","doi-asserted-by":"crossref","first-page":"2813","DOI":"10.1128\/AAC.40.12.2813","volume":"40","author":"Slayden R. A.","year":"1996","unstructured":"Slayden R. A., et al., Antimicrob. Agents Chemother. 40, 2813 (1996).","journal-title":"Antimicrob. Agents Chemother."},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/227680a0"},{"key":"e_1_3_1_31_2","doi-asserted-by":"crossref","unstructured":"D. G. Higgins A. J. Bleasby R. Fuchs Comput. Appl. Biosci. 8 189 (1992).","DOI":"10.1093\/bioinformatics\/8.2.189"},{"key":"e_1_3_1_32_2","unstructured":"We thank M. Sonnenberg for technical support M. Hein for preparation of the manuscript and C. Marander for graphics. Supported by grants AI-18357 AI-35220 and AI-38087 from the National Institute of Allergy and Infectious Diseases (NIAID) NIH and SmithKline Biochem. and contract AI-25147 from NIAID."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.276.5317.1420","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T03:58:39Z","timestamp":1705118319000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.276.5317.1420"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,5,30]]},"references-count":37,"journal-issue":{"issue":"5317","published-print":{"date-parts":[[1997,5,30]]}},"alternative-id":["10.1126\/science.276.5317.1420"],"URL":"https:\/\/doi.org\/10.1126\/science.276.5317.1420","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,5,30]]}}}