{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T19:54:21Z","timestamp":1790625261179,"version":"4.1.0"},"reference-count":29,"publisher":"American Society for Microbiology","issue":"12","license":[{"start":{"date-parts":[[2004,12,1]],"date-time":"2004-12-01T00:00:00Z","timestamp":1101859200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Antimicrob Agents Chemother"],"published-print":{"date-parts":[[2004,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:italic>Bacillus anthracis<\/jats:italic>\n            is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for\n            <jats:italic>B. anthracis<\/jats:italic>\n            Sterne is &gt;2,048 but \u22644,096 \u03bcg\/ml. A putative DHFR sequence was amplified from\n            <jats:italic>B. anthracis<\/jats:italic>\n            Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7\/CT-TOPO vector, followed by transformation into\n            <jats:italic>Escherichia coli<\/jats:italic>\n            TOP10F\u2032 chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into\n            <jats:italic>E. coli<\/jats:italic>\n            BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with\n            <jats:italic>E. coli<\/jats:italic>\n            BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of\n            <jats:italic>B. anthracis<\/jats:italic>\n            . These results confirm the natural resistance of\n            <jats:italic>B. anthracis<\/jats:italic>\n            to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.\n          <\/jats:p>","DOI":"10.1128\/aac.48.12.4643-4649.2004","type":"journal-article","created":{"date-parts":[[2004,11,24]],"date-time":"2004-11-24T03:00:46Z","timestamp":1101265246000},"page":"4643-4649","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":28,"title":["Functional Cloning of\n            <i>Bacillus anthracis<\/i>\n            Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim"],"prefix":"10.1128","volume":"48","author":[{"given":"Esther W.","family":"Barrow","sequence":"first","affiliation":[{"name":"Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip C.","family":"Bourne","sequence":"additional","affiliation":[{"name":"Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William W.","family":"Barrow","sequence":"additional","affiliation":[{"name":"Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.41.11.2406"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1080\/00365540210147679"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0924-8579(01)00464-2"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.46.7.2307-2309.2002"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.41.5.1134"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.46.12.3843-3845.2002"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.43.1.141"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1996.0770"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.39.9.1920"},{"key":"e_1_3_2_11_2","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1111\/j.1432-1033.1987.tb10664.x","volume":"162","year":"1987","unstructured":"Flensburg, J., and O. Skold. 1987. Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim. Eur. J. Biochem.162:473-476.","journal-title":"Eur. J. Biochem."},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1080\/1120009X.1993.11741083"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(67)90179-0"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.39.2.279"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1001\/jama.281.18.1735"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature01582"},{"key":"e_1_3_2_17_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/0378-1119(88)90476-3","volume":"64","year":"1988","unstructured":"Iwakura, M., M. Kawata, K. Tsuda, and T. Tanaka. 1988. Nucleotide sequence of the thymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon. 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