The transcriptional response to nitric oxide in Neisseria meningitidis

We are interested in how a bacterium which causes the disease meningitis works. This bacterium, called Neisseria meningitidis, lives in the human nose and throat as its sole habitat. Here it is exposed to all sorts of stresses, one of the most important of which is a toxic radical compound called nitric oxide. The microbe has special mechanisms to remove this nitric oxide, and thus protect itself from being killed. What we are interested in here is how the organism detects this radical and how it then makes its response. We have found evidence for a particular protein which is part of the Neisseria meningitidis bacterial cell that binds to the nitric oxide and directs the cell to make another protein that causes the breakdown of the nitric oxide. Here we intend to try to find out how this process works. The experiments we are proposing to do will allow us to ask a fundamental question about the way cells are organised: is the response of the cell to the environmental cue nitric oxide a simple linear process in which a single protein carries out the physiologically relevant detection and response to nitric oxide, or is the cell acting in a more complex way in which multiple regulators are affected by nitric oxide, and the final cell behaviour emerges from the action of a complex network of these regulatory events. We intend to investigate the biological phenomenon of nitric oxide regulation by investigating the properties of the candidate nitric oxide responsive regulators as proteins, and investigating how these proteins interact with DNA in the test tube. Furthermore, we will use genetic approaches to investigate the roles of these proteins in responding to nitric oxide in the whole cell using specific gene probes and global approaches that allow us to investigate the expression of all of the N. meningitidis genes simultaneously.

Neisseria meningitidis is exposed to nitric oxide that it generates internally by its own metabolic processes, and externally from NO generated in its human host. Its ability to deal with this potentially toxic free radical impacts on its own survival and on the host's signalling processes. We have identified a key regulator of nitric oxide metabolism from this organism, and analysis of the function of this protein is a central focus of this proposed study. In addition to investigating the specific properties of this regulator, we wish to investigate the properties of nitric oxide as a regulatory cue more broadly. NO is a highly reactive free radical gas that is capable of binding to metal centres of proteins. Is the physiological response to nitric oxide executed via a single specific regulatory protein, or is it the result of nitric oxide interacting with several regulatory proteins? NsrR is a repressor which controls the NO-responsive expression of the NO reductase in N. meningitidis. NsrR also acts as a regulator of nitrite reductase expression, but it does this in an NO-independent manner. The purified NsrR protein can be reconstituted to contain a cofactor that has the properties of an iron-sulfur protein, the spectra of which are sensitive to nitric oxide binding. To investigate the functionality of NsrR and the role of NO as a regulator acting via NsrR and other metal-containing regulatory proteins in N. meningitidis, we will investigate the biochemical properties of the NsrR with respect to NO and other potential physiological factors compared with other regulators FNR and FUR, both of which have been shown to be reactive with NO in other microorganisms. The impact of varying NO concentrations on cofactor binding, DNA binding and gene expression will be followed. We will also investigate the sensitivity of FNR to oxygen, and investigate how FNR and NsrR combine to allow effective regulation of gene expression in response to microoxia.