Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

The DdrR Coregulator of the Acinetobacter baumannii Mutagenic DNA Damage Response Potentiates UmuDAb Repression of Error-Prone Polymerases.

Acinetobacter baumannii strain 17978 is an opportunistic pathogen with a unique DNA damage repair response that lacks the LexA repressor but induces ~150 genes after DNA damage. It uses the UmuD homolog UmuDAb and the small protein DdrR, unique to Acinetobacter, to repress multiple horizontally acquired umuDC error-prone polymerase genes through an unknown mechanism. We used reverse transcription-quantitative PCR and immunoblotting to elucidate UmuDAb regulatory requirements and DdrR contributions to the corepression of this specialized regulon. Mutations in the putative UmuDAb helix-turn-helix (HTH) domain could not repress the expression of the UmuDAb/DdrR regulon. A ddrR insertion mutation in these HTH mutant backgrounds produced even greater derepression of the regulon, suggesting that DdrR exerts an additional level of control over this mutagenic response. These ddrR HTH mutant A. baumannii cells overexpressed UmuDAb, cleaving it after treatment with the DNA-damaging agent mitomycin C. This showed that DdrR was not required for UmuDAb self-cleavage and that UmuDAb repression and self-cleavage actions were independent. An uncleavable umuDAb mutant with an A-to-Y change at position 83 (A83Y) could neither induce the UmuDAb/DdrR regulon nor conduct SOS mutagenesis. However, a prophage-encoded umuDrumB operon was still partially induced after DNA damage in this mutant. Surprisingly, that prophage's putative repressor was dispensable for prophage-encoded umuDrumB induction, implying another repressor's involvement. This study revealed that UmuDAb behaves like LexA, requiring an N-terminal HTH motif for repression and C-terminal self-cleavage for gene induction and subsequent SOS mutagenesis, and DdrR cooperates with it to exert an additional level of repressive control on this pathogen's mutagenic response to DNA damage. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that acquires antibiotic resistance genes through conjugative transfer and carries out a robust mutagenic DNA damage response. After exposure to conditions typically encountered in health care settings, such as antibiotics, UV light, and desiccation, this species induces error-prone UmuD'2C polymerases. This mutagenic capability increases A. baumannii survival and virulence and is regulated by the UmuDAb/DdrR corepressor system unique to the Acinetobacter genus. Our study has revealed that the DdrR protein provides an additional layer of control in preventing mutagenic polymerase expression by enhancing UmuDAb repression actions. Understanding these repressors could lead to new drug targets, as multidrug resistance in hospital-acquired infections has decreased treatment options, with limited new drugs being developed.