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Antimicrobial Agents and Chemotherapy, December 2004, p. 4643-4649, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4643-4649.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Cloning of Bacillus anthracis Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim

Esther W. Barrow, Philip C. Bourne, and William W. Barrow*

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma

Received 9 March 2004/ Returned for modification 31 May 2004/ Accepted 30 July 2004

Bacillus anthracis 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 B. anthracis Sterne is >2,048 but ≤4,096 µg/ml. A putative DHFR sequence was amplified from B. anthracis Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into Escherichia coli TOP10F' 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 E. coli 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 E. coli 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 B. anthracis. These results confirm the natural resistance of B. anthracis 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.

* Corresponding author. Mailing address: Department of Veterinary Pathobiology, 250 McElroy, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078. Phone: (405) 744-1842. Fax: (405) 744-5275. E-mail: barrowb{at}okstate.edu.

Antimicrobial Agents and Chemotherapy, December 2004, p. 4643-4649, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4643-4649.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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