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Abstract

Nitric oxide (NO) is a small biomolecule produced by macrophages during the immune response. Macrophages engulf foreign intruders such as bacteria and deposit them into a phagosome. The phagosomes release NO as an immune response to prevent infection. NO can react with oxygen and superoxide to form harmful compounds such as N2O3, NO2 and ONOO-, that can cause severe damage to essential biomolecules such as DNA and proteins. However, bacteria such as Escherichia coli have developed defense mechanisms that degrade NO into a less harmful form. The mechanisms of this phenomenon have been investigated by the Brynildsen Group. DksA, a RNA polymerase (RNAP) regulator was found to play a key role in the E.coli NO detoxification system during nitrosative stress. DksA binds to the secondary channel of RNAP to regulate transcription. This study sought to investigate the specific role of DksA on E.coli’s NO detoxification system. ppGpp, DksA’s binding partner has two binding sites on RNAP that were assessed for their impact on this system. Site 2 essentiality was assessed by using a DksA mutant with compromised ppGpp binding. In addition, the transcription elongation factors GreA and GreB that bind to the secondary channel of RNAP were assessed because of their functional relationship to DksA. Preliminary results from this study show that loss of ppGpp binding site 1 or 2 do not have an essential role in the NO detoxification system. Preliminary data shows that the DksA mutant with disrupted site 2 binding has impaired growth but normal NO clearance times. Loss of greA or greB does not have an observable impact on the NO detoxification system. However, preliminary data with the double mutant ΔgreAΔgreB provide evidence of large impact on the system leading to approximately a two-fold increase in clearance time of NO when compared to the wild-type control.