The aim of this project was to investigate the molecular basis of the interaction between regulators that respond to nitric oxide and oxygen with DNA. The intention was to gain a further insight into how pathogenic bacteria are able to respond to these key environmental cues in a coordinated response of gene expression to changing conditions.
Bacteria can sense gases in their environment. In this project we investigated how a bacterium which causes meningitis is able to recognise and respond to oxygen and a second gas called nitric oxide which is produced in the body as part of the immune system, and also as a signalling molecule under normal conditions.
We identified that NO dependent repressor NsrR controls a core set of genes all intimately involved in NO regulation, and that NO controls transcription via both NsrR and FNR, but not the iron sensitive repressor FUR, which has been implied to have a role in nitric oxide dependent regulation in other organisms.
We showed that DNA binding impacts on O2 sensitivity of the O2 sensing regulator FNR. This finding was an element of the project to determine the mechanism by which four different regulators sense environment to appropriately control the same promoter (in preparation).
Training, career progression and collaborations. The post-doc from this project has gone on to a job as a university lecturer, which followed directly from the experience he had gained in application of biochemical and biophysical methods in this project. The project has enabled stronger interactions between JM and mathematical modellers, enabling more engagement with systems biology.
Achievement of Objectives.
1. Characterisation of NsrR protein and its cofactor. Partially. Identified that NsrR contains a nitric oxide sensitive iron sulphur cluster, and that it is also sensitive to oxygen in vitro.
2. The interaction of NsrR with DNA. Partially. We were able to show that NsrR binds a specific DNA motif, but oxygen sensitivity and instability of protein meant we could not fully quantify this. Other regulators of denitrification (FNR, FUR, NarP) all purified and DNA binding specificity was quantified (over and above original expectations in the grant).
3. Control of NsrR repressible transcripts. Yes.
4. Control of the aniA promoter in response to oxygen by FNR and NsrR. Yes.
5. The NO regulon. Yes
6. The relative impact of NO on NsrR, FNR and FUR. Yes.