Screening the Keio Collection Library for Nitric Oxide Sensitive Mutants

Abstract

Nitric oxide (NO•) and its intracellular metabolites constitute potent antimicrobial agents that cause nitrosative stress in bacteria by damaging iron-sulfur groups, modifying cysteines, damaging thiols, and causing double stranded breaks in DNA [1]. Bacteria have evolved nitric oxide stress mechanisms in order to cope with nitric oxide. Among these include transcriptional factors that sense nitric oxide and regulate genes that produce proteins involved in the major nitric oxide detoxification pathways [2]. Nitric oxide stress responses in E. coli have not yet been fully characterized. In order to identify novel genes involved in the nitric oxide stress response pathways, the Keio collection of E. coli single gene deletion mutants was screened for nitric oxide sensitive mutants. First, growth assays were performed on four Keio collection plates, which contained more than 300 E. coli mutants. These growth assays led to the identification of two positive hits for potential nitric oxide sensitive mutants: BW25113 ΔhyaD and BW25113 ΔyidZ. After further experimentation, it was determined that BW25113 ΔhyaD was a false positive, while BW25113 ΔyidZ displayed slight nitric oxide growth sensitivity whose magnitude was strain dependent. To design a more robust screen for nitric oxide sensitive mutants, a prototype competition assay was developed that uses nitric oxide and various antibiotics to select for sensitive mutants. Treatment of a co-culture of MG1655 wild type and the known nitric oxide sensitive mutant, MG1655 Δhmp, with nitric oxide and ampicillin led to increased survivability of MG1655 Δhmp over wild type for an extended period of time. This assay may provide investigators with an additional means of determining genes involved in the nitric oxide stress response pathways, which could be potential targets of direly needed novel antimicrobial therapeutics.