Insertions in xanthene dehydrogenase genes suppress cell death due to stabilization of topoisomerase IA covalent complex

Abstract

Bacterial type IA or IIA topoisomerases modify DNA topology by transiently cleaving and rejoining either one or two strands of DNA respectively. When topoisomerases cleave the DNA, a covalent complex between the cleaved DNA and the enzyme is formed. Stabilization of the type IIA topoisomerase complex by antibacterial drugs such as fluoroquinolones leads to decrease in DNA rejoining, accumulation of DNA strand breaks which in turn leads to cell death. There is an urgent need for new antibiotics due to the emerging drug resistance and topoisomerase IA is a promising target. It has been shown that a recombinant type IA Yersinia pestis topoisomerase with a conserved glycine residue in the TOPRIM motif mutated to serine accumulates the covalent complex and causes rapid bacterial cell killing. The main goal of this work is to study the mechanism of cell killing resistance for the stabilized type IA topoisomerase covalent complex. Insertional mutagenesis was used to identify and characterize the genes in Escherichia coli that influence the cell killing by the mutant Y. pestis topoisomerase I. We observed that insertions in the xanthene dehydrogenase operon (xdhABC) and homolog xdhD resulted in resistance to topoisomerase IA covalent complex mediated cell killing by ~4 log10 fold. Currently, studies are being conducted to elucidate the effect of these insertions on SOS response and other DNA damaging agents.