Investigating the Role of Acinetobacter baumannii umuD A1S_1389 Gene in Mutagenesis

Abstract

Acinetobacter baumannii contains 2 umuDC operons, 2 unlinked umuCs and a single umuD gene (umuD1389), thus encoding multiple DNA Polymerase V. This is the most error-prone DNA polymerase known and we have evidence supporting the hypothesis that more copies of these genes result in high mutagenesis. The aim of this project is to develop a umuD1389 gene knockout in A. baumannii. To do so we are constructing, via Splicing by Overlapping Extension PCR, a DNA fragment with a Kanamycin gene in the middle of umuD1389. This fragment will be ligated into an appropriate E. coli vector, which then will be introduced by transformation into E. coli competent cells to maintain the plasmid. This will be isolated from E. coli to introduce by electroporation into A. baumannii, thus allowing the disrupted umuD1389::Kan construct to crossover onto the chromosome, since the E. coli plasmid cannot replicate in A. baumannii. The success of the crossover will be assessed by plating strains on LB Agar plates with kanamycin. This research should result in the creation of an A. baumannii strain containing an inactivated umuD1389::Kan gene. The significance of this experiment is two-fold: (1) it would knockdown the unlinked umuC gene copies in Acinetobacter reducing thus the total number of active DNA Pol Vs, which should result in decreased mutagenesis; (2) it would demonstrate that UmuD1389 is not the DNA damage response repressor in Acinetobacter as LexA is in E. coli since deletion of the latter on its own in E. coli is lethal.