Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation

Abstract

c Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcriptiondriven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response. Microbial pathogens resistant to current antibiotics are becoming an increasingly urgent public health crisis. For example, quinolones are widely used broad-spectrum antimicrobial agents that are highly effective for rapid bactericidal outcomes (1), but quinolone resistance can be acquired rapidly by the bacterial pathogens via a number of mechanisms (2, 3). The bacterial SOS response system has been shown to contribute to the increase in antibiotic resistance (4). There are many factors that can potentially lead to the increased resistance in response to the SOS-inducing antimicrobials. These factors include elevations in the expression levels of error-prone DNA polymerases (5) or plasmidmediated antibiotic resistance determinants (6, 7), higher rates of transfer of resistance determinants (8, 9), and increases in the numbers of persistors in the bacterial population (10).The transcriptome in the bacterial cell must adjust rapidly to the antibiotic challenge for survival. Due to resistance to the rotational motion of the transcription ensemble around the DNA during transcription, the transcription loci can accumulate positive supercoils ahead of the RNA polymerase (RNAP)-nascent RNA complex and negative supercoils behind the comple...

Investigating direct interaction between Escherichia coli topoisomerase I and RecA

“…More recently, a role of topoisomerase I was observed in E. coli SOS response (Liu et al, 2011; Yang et al, 2015), which prompted us to verify the possibility of a direct physical interaction between these proteins. Purified His-EcTOP1 and RecA were incubated together in the presence of ATP, and pulled-down with Cobalt agarose resin.…”

“…Pull-down assays were carried out to establish the physical interactions of proteins in solution (Yang et al, 2015). An assay involving the incubation of purified RecA and topoisomerase I was carried out to study the direct physical interactions between these proteins.…”

“…It remains unclear whether this stimulatory effect is due to direct protein-protein interaction between E. coli RecA and topoisomerase I, or is only due to the effect of E. coli RecA on DNA conformation. More recent results showed that mutations in E. coli topA gene coding for topoisomerase I can diminish the E. coli SOS response to DNA damage and antibiotics treatment (Yang et al, 2015). The interaction between RecA and topoisomerase I may influence the increase in antibiotic resistance (Hastings et al; Beaber et al, 2004; Thi et al, 2011) and persistence shown to be associated with the SOS response (Dörr et al, 2009).…”

“…Herein, we present evidence for a direct physical interaction between E. coli RecA, and topoisomerase I in solution by pull-down assays (Yang et al, 2015) as well assess the influence of ATP, and the domains of topoisomerase I involved in the protein-protein interaction with RecA. We further investigated the inter-protein interaction between E. coli RecA and topoisomerase I by using surface plasmon resonance (SPR) and molecular docking.…”

Targeting Bacterial Topoisomerases: How to Counter Mechanisms of Resistance

“…Perturbation of E. coli topoisomerase I interactions with RNA polymerase has been shown to increase susceptibility to antibacterial compounds and suppress the SOS-dependent elevation of mutation frequency associated with antibiotic resistance development [102]. M. tuberculosis topoisomerase I is likely to play a similar Targeting bacterial topoisomerases -how to counter mechanisms of resistance Review important role in response to stress from host defense and TB drug action.…”