Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.
Bottom Line: Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques.Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products.This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships.
Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.Show MeSH
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Mentions: To establish the relevance of this recombineering system in characterizing gene-phenotype relationships, we first focused on the well-studied response of bacteria to oxidative stress. Response to oxidative stress is important in order for bacteria to adapt and survive in the presence of biocides and antibiotics (15, 16). In E. coli, hydrogen peroxide triggers activation of the transcription factor OxyR, which induces expression of oxidative stress protection genes (17, 18). Deletion of oxyR in E. coli results in increased sensitivity to hydrogen peroxide. The protein A1S_0992 from A. baumannii strain 17978 shows 29% identity (E value, 2e−34) to E. coli OxyR by BLAST bioinformatics analysis, suggesting that A1S_0992 may play an OxyR-like role in A. baumannii. Consistent with studies of OxyR in E. coli, deletion of A1S_0992 using our recombineering system produced an A. baumannii strain that displays increased sensitivity to hydrogen peroxide (Fig. 3, top). Ectopic expression of A1S_0992 (oxyR) from a plasmid complements the mutant phenotype, demonstrating that A1S_0992 is very likely an OxyR homolog that plays an important role in oxidative stress protection in A. baumannii (Fig. 3, bottom).
Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.