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Homodimerization and heterodimerization requirements of Acinetobacter baumannii SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses.

ABSTRACT: The multidrug-resistant pathogen Acinetobacter baumannii displays unusual control of its SOS mutagenesis genes, as it does not encode a LexA repressor, but instead employs the UmuDAb repressor and a small protein, DdrR, that is uniquely found in Acinetobacter species. We used bacterial adenylate cyclase two-hybrid analyses to determine if UmuDAb and DdrR coregulation might involve physical interactions. Neither quantitative nor qualitative assays showed UmuDAb interaction with DdrR. DdrR hybrid proteins, however, demonstrated modest head-to-tail interactions in a qualitative assay. The similarity of UmuDAb to the homodimer-forming polymerase manager UmuD and LexA repressor proteins suggested that it may form dimers, which we observed. UmuDAb homodimerization required a free C terminus, and either small truncations or addition of a histidine tag at the C terminus abolished this homodimerization. The amino acid N100, crucial for UmuD dimer formation, was dispensable if both C termini were free to interact. However, mutation of the amino acid G124, necessary for LexA dimerization, yielded significantly less UmuDAb dimerization, even if both C termini were free. This suggests that UmuDAb forms dimers like LexA does, but may not coregulate gene expression involving a physical association with DdrR. The homodimerization of these coregulators provides insight into a LexA-independent, coregulatory process of controlling a conserved bacterial action such as the mutagenic DNA damage response.

SUBMITTER: Cook D

PROVIDER: S-EPMC8113343 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Homodimerization and heterodimerization requirements of Acinetobacter baumannii SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses.

Cook Deborah D Carrington Jordan J Johnson Kevin K Hare Janelle J

Canadian journal of microbiology 20201112 5

The multidrug-resistant pathogen Acinetobacter baumannii displays unusual control of its SOS mutagenesis genes, as it does not encode a LexA repressor, but instead employs the UmuDAb repressor and a small protein, DdrR, that is uniquely found in Acinetobacter species. We used bacterial adenylate cyclase two-hybrid analyses to determine if UmuDAb and DdrR coregulation might involve physical interactions. Neither quantitative nor qualitative assays showed UmuDAb interaction with DdrR ...[more]

PMID: 33180570

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Project description: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.

Project description:The SOS response to DNA damage that induces up to 10% of the prokaryotic genome requires RecA action to relieve LexA transcriptional repression. In Acinetobacter species, which lack LexA, the error-prone polymerase accessory UmuDAb is instead required for ddrR induction after DNA damage, suggesting it might be a LexA analog. RNA-Seq experiments defined the DNA damage transcriptome (mitomycin C-induced) of wild type, recA and umuDAb mutant strains of both A. baylyi ADP1 and A. baumannii ATCC 17978. Of the typical SOS response genes, few were differentially regulated in these species; many were repressed or absent. A striking 38.4% of all ADP1 genes, and 11.4% of all 17978 genes, were repressed under these conditions. In A. baylyi ADP1, 66 genes (2.0% of the genome), including a CRISPR/Cas system, were DNA damage-induced, and belonged to four regulons defined by differential use of recA and umuDAb. In A. baumannii ATCC 17978, however, induction of 99% of the 152 mitomycin C-induced genes depended on recA, and only 28 of these genes required umuDAb for their induction. 90% of the induced A. baumannii genes were clustered in three prophage regions, and bacteriophage particles were observed after mitomycin C treatment. These prophages encoded esvI, esvK1, and esvK2, ethanol-stimulated virulence genes previously identified in a Caenorhabditis elegans model, as well as error-prone polymerase alleles. The induction of all 17978 error-prone polymerase alleles, whether prophage-encoded or not, was recA dependent, but only these DNA polymerase V-related genes were de-repressed in the umuDAb mutant in the absence of DNA damage. These results suggest that both species possess a robust and complex DNA damage response involving both recA-dependent and recA-independent regulons, and further demonstrates that although umuDAb has a specialized role in repressing error-prone polymerases, additional regulators likely participate in these species' transcriptional response to DNA damage.

Dataset Information

Homodimerization and heterodimerization requirements of Acinetobacter baumannii SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses.

Publications

Homodimerization and heterodimerization requirements of <i>Acinetobacter baumannii</i> SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses.

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