Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in approximately 4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg(2+) binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg(2+)directly, can nonetheless inhibit Mg(2+) binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg(2+) dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.