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Project 1

Genetic analysis of new resistance functions of Acinetobacter baumannii


Principal Investigator: Colin Manoil

Goals and Objectives

The overall goal of the project is to identify genes of unknown function contributing to seven resistance traits of A. baumannii and to characterize the genes using a variety of genetic and genomic methods. The first step is to identify such genes using both unbiased screens of transposon mutants for strains with increased sensitivity using Tn-seq and targeted screens of mutants of conserved genes needed for resistance in other bacteria. Gene-phenotype associations identified using Tn-seq will be confirmed and quantified using individual mutants retrieved from a defined transposon mutant library. A subset of genes of unknown function with strong mutant phenotypes will be subjected to a battery of tests to help define their molecular functions. These tests will include phenotypic microarray analysis of target gene mutants to identify additional phenotypes, synthetic lethal analysis using Tn-seq to identify genes functioning with the targeted genes, RNA-seq to identify transcriptional changes associated with loss of target gene function, and protein complex analysis to identify polypeptides physically interacting with target gene products. This information should make it possible to hypothesize molecular functions for many of the genes. We will test such hypotheses using assays dictated by the functions predicted.


Many bacterial genes identified by sequence cannot be assigned molecular functions using standard annotation methods. Specifying the activities of such genes is a fundamental challenge for achieving a comprehensive understanding of bacterial biology, including that of pathogens. This project is discovering, verifying and assigning functions to conserved genes of unknown function in A. baumannii, an understudied pathogen infamous for its resistance to antibiotics, biocides and desiccation. The project is focused on protein-coding genes that contribute to any of seven clinically relevant resistance traits.