An Analysis of ICE R391 Pathogen-encoded Rum DNA Polymerase in Generating Mutations Driving Rapid Acquisition of Antibiotic Drug Resistance in Diverse Recipient Bacterial Species - PROJECT SUMMARY The acquisition of resistance to antibiotics (Abs) in pathogenic bacteria has grown into a worldwide pandemic, with around 3 million cases and roughly 35,000 deaths in 2019, according to the CDC. Bacteria can acquire Ab resistance by the horizontal transfer of specific Ab resistance genes, and by the de novo occurrence of mutations caused by exposure to Abs or to a wide variety of naturally occurring external stressors, e.g., UV radiation, environmental pollutants. Our grant proposal is centered on the acquisition of Ab resistance in bacteria caused by mutations resulting from the transfer of a highly error-prone DNA polymerase, Rum pol, encoded by the integrative conjugative element R391. R391 is found ubiquitously in nature. We have previously shown that the pathogen-encoded Rum pol, in order to be enzymatically active in vitro and active mutagenically in multiple types of recipient bacteria, must be assembled into a mutasomal complex requiring the stable presence of each individual homolog RecA recombinase and ATP, Rum Mut = Rum pol-RecA-ATP. This proposal will test our hypothesis that by generating a broad mutational landscape, Rum pol is responsible for driving the rapid acquisition of Ab resistance in its bacterial recipient cells. We further hypothesize that a single methionine at position 197 in RecA (RecA M197), which is conserved in RecA recipient cell homologs, serves as a “master regulator” for Rum pol mutagenesis and for the acquisition of Ab and multi-Ab resistance. Our recent published data provide strong support for this hypothesis, and new preliminary data strongly reinforce this hypothesis. We will use ICE R391 “donor” and Escherichia coli and Vibrio cholerae recipient bacteria as our model system. There are two in vivo microbiology and two mechanistic based specific aims. Aim 1 investigates the role of Rum pol in rapid acquisition of Ab resistance; Aim 2 maps regions of Ab- and external stressor-induced genomic instability (ssDNA) globally in genomic DNA, maps the location of Rum pol on the genome, and identifies the all of the mutations using NG sequencing; Aim 3 visualizes the assembly Rum pol into Rum Mut at single molecule resolution in real-time using TIRF-FRET microscopy; Aim 4 will obtain high- resolution structures of Activated Rum Mut using Cryo-electron microscopy. An important objective is to identify specific sites in Rum Mut that can serve as targets for the design and discovery of small molecule inhibitors that prevent assembly of Rum pol into mutagenically active Rum Mut or that inhibit the activity of activated Rum Mut.
