Project Summary Multi-drug resistant bacteria such as the ESKAPE pathogens are difficult to treat; finding new drug targets and alternate methods of interfering with cell growth are crucial to infectious disease control. A Gram-negative, nosocomial ESKAPE pathogen, Acinetobacter baumannii, has multiple antibiotic-resistance genes and a mutagenic response to DNA damage that antibiotics can cause. The PI’s undergraduate research team has uncovered a novel DNA damage response (DDR) system in this pathogen that uses a non- canonical repressor, UmuDAb, and a small protein coregulator, DdrR, to repress mutagenic error-prone polymerases. Typical DDR regulators, namely the LexA repressor of SOS genes and the SulA cell division inhibitor, are not encoded by the non-enteric Acinetobacter genus, yet they possess a checkpoint response of filamentation after DNA damage. There is increasing interest in targeting cell division and control checkpoints to control bacterial infections, and novel mechanisms of cell division inhibition are recent refinements to Escherichia coli and Bacillus DDR models. Staphylococcus aureus (also an ESKAPE pathogen) and other Gram-positive and non-enteric bacteria that lack SulA instead use small proteins lacking inter- genus homologies to inhibit cell division after DNA damage. Our team’s evidence suggests that the DdrR component of SOS regulation mimics the actions of SulA and these Gram-positive growth inhibitors after DNA damage: ddrR mutants have growth sensitivity to DNA damage and cannot inhibit cell division after it. This project’s long-term goal is to construct a new model of inhibiting cell division in Gram-negative Gammaproteobacteria such as A. baumannii, which could help find non-antibiotic means of inhibiting its growth and treating infectious disease. Our objectives are to identify how UmuDAb and DdrR directly and indirectly control error-prone polymerase production and cell division checkpoints as part of the Acinetobacter DDR. We hypothesize that the UmuDAb and DdrR regulators physically interact to control this LexA-deficient pathogen's SOS response. Our objectives will be achieved using complementary approaches to determine whether their phenotype of cell division inhibition results from directly interacting with divisome proteins, or occurs through co-regulatory actions. Specifically, we will: (Aim 1) use biochemical methods to identify UmuDAb-DdrR interactions; (Aim 2) establish these proteins' roles in inhibiting cell division with fluorescence microscopy localization experiments, two-hybrid and growth inhibition assays; and (Aim 3) examine previously obtained transcriptome data for targets of DdrR and UmuDAb regulatory activities that may control DNA damage checkpoints and growth after DNA damage. The PI’s previously demonstrated mutant strains, purified proteins, and expertise in microbiology, molecular biology, biochemistry, and bioinformatics will allow her team of undergraduate students to complete thes...