{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T16:29:51Z","timestamp":1791304191398,"version":"4.3.1"},"reference-count":34,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5369","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,6,5]]},"abstract":"<jats:p>\n            Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated\n            <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n            was shown to consist of a covalent complex of INH, AcpM, and a \u03b2-ketoacyl acyl carrier protein synthase, KasA. Amino acid\u2013altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.\n          <\/jats:p>","DOI":"10.1126\/science.280.5369.1607","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:50:55Z","timestamp":1027763455000},"page":"1607-1610","source":"Crossref","is-referenced-by-count":316,"title":["Inhibition of a\n            <i>Mycobacterium tuberculosis<\/i>\n            \u03b2-Ketoacyl ACP Synthase by Isoniazid"],"prefix":"10.1126","volume":"280","author":[{"given":"Khisimuzi","family":"Mdluli","sequence":"first","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard A.","family":"Slayden","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"YaQi","family":"Zhu","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Srinivas","family":"Ramaswamy","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xi","family":"Pan","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Mead","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deborah D.","family":"Crane","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James M.","family":"Musser","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Clifton E.","family":"Barry","sequence":"additional","affiliation":[{"name":"K. Mdluli, R. A. Slayden, Y. Zhu, D. Mead, D. D. Crane, C. E. Barry III, Tuberculosis Research Unit, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases (NIAID), National Institutes of Health, Hamilton, MT 59840, USA."},{"name":"S. Ramaswamy, X. Pan, J. M. Musser, Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1164\/ajrccm.149.5.8173779","volume":"149","author":"Bass J. 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(Springer-Verlag Berlin 1979) pp. 98\u2013119.","DOI":"10.1007\/978-3-642-46403-4_6"},{"key":"e_1_3_1_13_2","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1111\/j.1365-2958.1995.tb02238.x","volume":"15","author":"Heym B.","year":"1995","unstructured":"Heym B., Alzari P. M., Honore N., Cole S. T., Mol. Microbiol.15, 235 (1995).","journal-title":"Mol. Microbiol."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1093\/infdis\/173.1.196","volume":"173","author":"Musser J. M.","year":"1996","unstructured":"Musser J. M., et al., J. Infect. Dis.173, 196 (1996).","journal-title":"J. Infect. 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Barry III unpublished observations."},{"key":"e_1_3_1_24_2","unstructured":"Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr."},{"key":"e_1_3_1_25_2","first-page":"269","volume":"36","author":"Prescott D. J.","year":"1972","unstructured":"Prescott D. J., Vagelos P. R., Adv. Enzymol.36, 269 (1972).","journal-title":"Adv. Enzymol."},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","first-page":"9389","DOI":"10.1021\/bi00029a015","volume":"34","author":"Summers R. G.","year":"1995","unstructured":"Summers R. G., Ali A., Shen B., Wessel W. A., Hutchinson C. R., Biochemistry34, 9389 (1995).","journal-title":"Biochemistry"},{"key":"e_1_3_1_27_2","unstructured":"The 10 000 g supernatant from a cell lysate of M. tuberculosis H37Rv that had been treated with 1 to 5 \u03bcg\/ml INH was brought to 80% saturation with solid ammonium sulfate. The supernatant from centrifugation at 10 000 g was acidified with acetic acid to pH 3.9 centrifuged and the pellet was redissolved in distilled water. Further purification was accomplished by preparative native gel electrophoresis followed by electroelution."},{"key":"e_1_3_1_28_2","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1016\/S0021-9258(18)52372-3","volume":"266","author":"Post-Beittenmiller D.","year":"1991","unstructured":"Post-Beittenmiller D., Jaworski J. G., Ohlrogge J. B., J. Biol. Chem.266, 1858 (1991).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_29_2","unstructured":"We added 10 \u03bcg\/ml INH for 30 min to 6 liters of mid\u2013log phase M. tuberculosis H37Rv in 7H9 ADC and Tween-80. We then added 7 mCi of [ 35 S]methionine and after 4 hours the bacteria were harvested by centrifugation and resuspended in an equal volume of water. This was added to 10 g of 0.1-mm glass beads and homogenized at 4\u00b0C for three 1-min pulses by bead-beating and centrifuged at 10 000 g for 15 min. The 70 and 80% ammonium sulfate pellets were resuspended in 10 ml of 50 mM sodium phosphate (pH 6.0) containing 0.75 M ammonium sulfate and loaded onto an HP1610 Phenyl-HIC column (Pharmacia). This column was eluted from a concentration of 0.75 M ammonium sulfate until it reached 0 M. Fractions containing the 80-kD protein determined by protein immunoblot of unlabeled samples or radioactivity were combined and dialyzed against 10 mM MES (pH 6.0) before loading onto a Resource Q column equilibrated in the same buffer. After a 2-column volume (CV) wash the column was eluted over 10 CV to 36% 1 M NaCl in 10 mM MES (pH 6.0) (B) and then to 42% B over another 20 CV and finally to 100% B over another 10 CV. Appropriate fractions were pooled and concentrated."},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac950914h"},{"key":"e_1_3_1_31_2","unstructured":"The intact gene cluster was cloned as a Kpn I to Bsp HI fragment into pMV206H (14) which had been cut with Kpn I and Nco I. Transformation of this construct into either M. smegmatis or M. tuberculosis H37Rv and analysis of the resulting transformants by SDS\u2013polyacrylamide gel electrophoresis (PAGE) showed that none of the encoded proteins appeared significantly up-regulated. acpM was cloned individually as an Sph I to Bam HI fragment into pMV261H and the orientation was determined by restriction mapping. Expression in this construct is driven by the mycobacterial hsp60 promoter. Transformation of this construct into either M. smegmatis or M. tuberculosis produced no colonies (<1 colony\/\u03bcg of DNA) while transformation of the same gene in the opposite orientation produced between 1 \u00d7 10 3 and 1 \u00d7 10 6 colonies\/\u03bcg of DNA. kasA was polymerase chain reaction\u2013amplified using Pwo polymerase and was cloned into pMH29H in which transcription is driven by a synthetic mycobacterial promoter sequence. Transformation of this construct into M. smegmatis and M. tuberculosis produced very small slowly growing colonies. These colonies overproduced KasA protein as demonstrated by SDS-PAGE and protein immunoblotting using affinity-purified antisera to a peptide segment of the KasA primary sequence. It was not possible to accurately assess the INH susceptibility of these colonies because of their impaired growth but they did not appear to be significantly more resistant to INH than were controls."},{"key":"e_1_3_1_32_2","doi-asserted-by":"crossref","first-page":"22229","DOI":"10.1074\/jbc.270.38.22229","volume":"270","author":"Keating D. H.","year":"1995","unstructured":"Keating D. H., Carey M. R., Cronan J. E., J. Biol. Chem.270, 22229 (1995).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_33_2","unstructured":"DNA was isolated from recent clinical isolates from the collection of J.M.M. HN113 was recovered from the sputum of a patient in Houston TX was resistant to INH at >10 \u03bcg\/ml and had the KatG Ser 315 \u2192 Thr 315 mutation. HN113 had nine copies of IS6110 and belonged to major genetic group 1 (33). Strain HN335 was recovered from a patient in Houston and showed INH resistance at a level of 1 to 2 \u03bcg\/ml. This strain had no mutations in KatG 14 copies of IS6110 and belonged to group 2. Strain TB029 was recovered from a patient in Japan and was resistant to >10 \u03bcg\/ml of INH. It contained a KatG Ser 315 \u2192 Asn 315 mutation four copies of IS6110 and belonged to group 1. Strain HN93 was recovered from a lymph-node biopsy of a Houston patient and showed INH resistance at 1 to 2 \u03bcg\/ml. This strain had a single copy of IS6110 and also belonged to group 1. None of these strains had alterations in the inhA locus or upstream of ahpC."},{"key":"e_1_3_1_34_2","doi-asserted-by":"crossref","first-page":"9869","DOI":"10.1073\/pnas.94.18.9869","volume":"94","author":"Sreevatsan S.","year":"1997","unstructured":"Sreevatsan S., et al., Proc. Natl. Acad. Sci. U.S.A.94, 9869 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_35_2","unstructured":"We thank H. Caldwell for proofreading the manuscript and the NIH-AIDS Research and Reference Reagent Program of NIAID for providing radioactive INH. Supported by USPHS grant AI37004 to J.M.M."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.280.5369.1607","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T04:10:31Z","timestamp":1705119031000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.280.5369.1607"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,6,5]]},"references-count":34,"journal-issue":{"issue":"5369","published-print":{"date-parts":[[1998,6,5]]}},"alternative-id":["10.1126\/science.280.5369.1607"],"URL":"https:\/\/doi.org\/10.1126\/science.280.5369.1607","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,6,5]]}}}