In the presence of DNA damage, the multi-drug resistant bacteria Acinetobacter baumannii employs the proteins UmuDAb and DdrR to repress the expression of error-prone polymerases.https://scholarworks.moreheadstate.edu/celebration_posters_2022/1010/thumbnail.jp

Candra, Belinda

Cook, Deborah

Hare, Janelle

Li, Xiaohong

Rouchka, Eric

English

Morehead State University

Results of RT-qPCR validationGenes involved in cell division in Acinetobacter baumannii are coregulated by UmuDAb and DdrRBelinda Candra1, Xiaohong Li2, Eric Rouchka2, Deborah Cook1 & Janelle Hare11Department of Biology and Chemistry, Morehead State University2Department of Biochemistry and Molecular Genetics, University of Louisville AcknowledgementsConclusion References• Identify cell division genes that are dysregulated in A. baumannii ATCC 17978 after DNA damage• Perform an in silico analysis of A. baumannii RNA-Seq transcriptome data in the wild-type (WT), ddrR-mutant (DR-), and umuDAb- mutant (UD-) strains, particularly in regard to cell division genes. • Use RT-qPCR to validate the expression levels of cell division genes ObjectivesTo examine the expression requirements of the bacteria’s cell division genes, we re-analyzed our previously acquired RNA-Seq data from wild-type, DR-, and UD- strains1,2. 1. We analyzed Cuffnorm- or HTSeq- normalized counts of the expression of 35 putative cell division genes (obtained from the KEGG database) using the Cuffdiff and DESeq2 pathways, respectively.2. RNA-Seq data was sorted using Excel based on the following criteria: Materials & MethodsResults of RNA-Seq analysisIntroductionCrystal structure of catalytic core of A. baumannii TOPO IV (ParE-ParC fusion truncate). PDB: 2XKJIn the presence of DNA damage, the multi-drug resistant bacteria Acinetobacter baumannii employs the proteins UmuDAb and DdrR to repress the expression of error-prone polymerases. RNA-Seq data analysisRT-qPCR validationTo confirm the gene dysregulation suggested by the RNA-Seq data• Dysregulation of parE and xerC was observed in both UD- and DR- mutants through the results of RNA-Seq analysis and RT-qPCR • RNA-Seq results of parE were not consistent with RT-qPCR results. However, a different trend was still observed in WT vs mutant strains• To follow up on these results, we are constructing mutants with disruptions in these genes to observe their resulting cell division and growth phenotypes. These results may also help test whether DdrR and/or UmuDAb regulate these genes.Repression InductionGene Expression after DNA Damagelog2FC value < -1 & q-value < 0.05log2FC value > 1 & q-value < 0.05Summer Transcriptome Data Results RT-qPCR Data ResultsGene 17978 WT ddrR null umuDAb null 17978 WT ddrR null umuDAb nullA1S_3359 (parE)A1S_2629 (xerC)Comparison of expression pattern of A1S_3359 and A1S_2629 in each strain based on the RNA-seq data results vs RT-qPCR data results. Genes of Interest related to Cell Division parE (A1S_3359)• A topoisomerase IV subunit B: separates the physical linkage of two daughter chromosomal DNA at the end of replication • Toxin of Type II parE-parD TA systemxerC (A1S_2629)• A site-specific tyrosine recombinase • Forms a cyclic heterotetramericcomplex with XerD, which interacts via their C-terminal regiondiagram created in https://biorender.comC-terminal domain of XerD recombinase in complex with gamma domain of FtsK. PDB: 5DCF• Derepression of site-specific tyrosine recombinase xerC in both mutants• Induction of topoisomerase IV/Type II toxin(/antitoxin) parE in both mutants• Derepression of septal ring lytic transglycosylase rlpA in the ddrRmutant, but not the other strainsXerC and ParE function at a DNA damage-associated filamentation locus in Escherichia coli. XerC and Topoisomerase IV also interact with cell division protein FtsK.This work was funded by NIH grant 2R15GM085722-03A1 and NIH grant 2P20RR016481-09.We would also like to thank the Morehead State Honors Program for continued support for our endeavors .*This was a different umuDAb mutant than the one which with we had performed the initial RNA-SeqThese coregulators may also affectthephenotypes ofcell division and growth sensitivity after DNA damage. ddrR mutant cells do not form filaments after DNA damage.= induction = repression = unchanged1. Hare JM, Ferrell JC, Witkowski TA, Grice AN. 2014. Prophage induction and differential RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacter baumannii and Acinetobacter Baylyi. PLoS ONE 9.2. Peterson MA, Grice AN, Hare JM. 2020. A Corepressor participates in LexA-independent regulation of error-prone polymerases in Acinetobacter. Microbiology 166:212–226. 10100100010000100000xerC parE rlpA2-ΔCqAverage normalized expression level of genes of interest in each strainWT MMC- WT MMC+ dR MMC- dR MMC+ uDAb MMC- uDAb MMC+MOREHEAD STATE UNIVERSITY 801 60 18 -E :1. ■ - 15 ddrR mutants .I: • +' C) C 10 (U • - ••• --(U (.) 5 • • ol...::§:i:~~-==±~::::.._~===..=•~.:::§:i:~•~ MMC - + - + • • • •w.~I • . .. . . . . .... -MMC +MMC ■ . . . . . . . . -. . . . • • • .. . . . • • • •• • • • ■ • • . • • • •' •• • • - + - + E1.4X K-~ ddtRJTnKf . . ' . . . . ... . . • • • , . . ... .. . . . ' • , • • •• • • . '• •• .. . \· . - I. L I I • ~A... -· .. • ■ ■ ■ ■ A. baumannii strains: WT, umuDAb·, ddrR-Amplification Threshold No amplification Copies per reaction (Ct) no MMC ,---+ treatment +MMC .___ ... treatment Analysis RNA ~ synthesis ~ ,/ ~) I cDNA t synthesis ~ / ~J .. ······ ··· ' .. • t t t .. • .. • ! .. • .. • ! .. • .. • t 

Genes involved in cell division in Acinetobacter baumannii are coregulated by UmuDAb and DdrR

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Genes involved in cell division in Acinetobacter baumannii are coregulated by UmuDAb and DdrR

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