Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim

Abstract

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 < or =4,096 microg/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.

FIG. 1.

Part of the contig obtained from the TIGR database (http://www.tigr.org) by BLAST search (BLAST hit start of 2088847 and BLAST hit end of 2089326 >contig:6611:b_anthracis). The B. anthracis DHFR gene sequence (asterisks), start and stop codons (boldface type), and the putative Shine-Dalgarno sequence (double underline) are indicated. The locations of the left primer (>>>>) and right primer (<<<<) are indicated.

FIG. 2.

ORF obtained with ORF Finder (NCBI) using the contig presented in Fig. 1. Start and stop codons are shown in boldface type.

FIG. 3.

SDS-12.5% polyacrylamide gel showing steps involved in purification of B. anthracis Sterne rDHFR. Lanes 1 and 6 contain Novagen Perfect Protein markers (15 to 150 kDa ladder). Lane 2 contains cell lysate from E. coli BL21 Star (DE3) carrying plasmids pLysS and pCRT7/CT-TOPO-Sterne rDHFR with the six-histidine tag (precolumn) (lysate filtered through a 0.22-μm-pore-size filter). Lane 3 contains pooled Sterne rDHFR with the six-His tag (enzyme purified by passage through a nickel column). Lane 4 contains FPLC-purified Sterne rDHFR with the six-His tag, and lane 5 contains FPLC-purified Sterne rDHFR with the six-His tag removed.

FIG. 4.

Alignment of the B. anthracis Sterne DHFR sequence (GenBank accession number AY569129) (BanthSterne) with the DHFR sequences from B. anthracis Ames, Western N. America GT3 Group A1a (West), and Kruger B GT87 Group B1 (Kruger) and from B. cereus (Bcer), B. subtilis (Bsubt), and M. avium (Mavium). Identical residues are shown as white letters on black background. Alignment was accomplished with MacVector 7 (Accelrys, San Diego, Calif.). Gaps introduced to maximize alignment are shown as dashes.