Nitric oxide (NO) is a water-soluble free-radical gas that is toxic in biological systems by virtue of its reactivity towards proteins, metal ions, lipids and DNA. Eukaryotic phagocytic cells exploit this toxicity by synthesizing NO as one of the arsenal of poisonous molecules that are used to kill invading pathogens. Successful intra-cellular pathogens (such as Salmonella and Mycobacterium species) are able to resist phagocyte killing mechanisms. There is increasing evidence that the ability to detoxify NO is required by some pathogens for survival inside host cells.
NO is also synthesized by Bacteria as an intermediate or by-product of normal respiratory processes. Specifically, nitrite can be used as an electron acceptor for anaerobic respiration by the denitrifying Bacteria, which reduce nitrite to NO, and also use NO as an electron acceptor, reducing it to nitrous oxide. The enteric Bacteria reduce nitrite to ammonia, but also catalyze the reduction of nitrite to NO, such that NO is made at a low concentration as a by-product of nitrite respiration (Fig. 1). Escherichia coli has three enzymes that reduce or oxidize NO to less toxic compounds, and we speculate that one or more of these enzymes has a role in protecting the cell against the NO that is made endogenously from nitrite. The same enzymes may allow pathogens such as Salmonella to detoxify the NO made by host cells.
Fig. 1. Pathways for NO synthesis and consumption in Escherichia coli and Salmonella enterica, and regulation of expression of the enzymes involved in NO consumption. Under anaerobic conditions, nitrate is reduced to nitrite by nitrate reductase (NarA, NarZ or Nap), and nitrite is reduced to ammonia by a respiratory or NADH-linked nitrite reductase (Nrf and NirB, respectively). Maximal rates of nitrate and nitrite reduction occur in cultures grown anaerobically in the presence of nitrate or nitrite (the regulatory mechanisms involved are not shown, but involve FNR and the two-component systems NarXL and NarPQ). Nitrite can be converted to NO by biological reduction (by nitrate and/or nitrite reductase) or by disproportionation. Under anaerobic conditions, NO is reduced to ammonia by Nrf, or to nitrous oxide by the flavorubredoxin (FlRd). In the presence of oxygen, NO is oxidized to nitrate by flavohaemoglobin (Hmp). The relevant regulators are shown along with their signals. Positive regulation is denoted by arrows, negative regulation by perpendicular lines. Hcy, homocysteine.
All three NO detoxification systems are up-regulated by exposure to nitrite or NO, and our major interest is to characterize the regulatory mechanisms involved, using E. coli as a model system. We study a transcriptional activator called NorR, which controls expression of the genes encoding a flavorubredoxin that reduces NO to nitrous oxide under anaerobic conditions. We have defined the mechanism of NO sensing by NorR and charaterized the cis-acting regulatory sequences required for transcriptional regulation. Future work will continue to probe structure-function relationships in the NorR protein, and to identify any additional genes that are regulated by NorR.
We recently discovered a transcriptional repressor, NsrR, that regulates expression of a flavohemoglobin, which oxidizes NO to nitrate. NsrR regulates at least three other genes, the products of which have poorly defined roles in mediating NO resistance. An important goal now is to study the biochemistry of NsrR, with a view to understanding the mechanism by which repression is relieved by NO. We have recently used chromatin immunoprecipitation and microarray analysis (ChIP-on-chip) to define NsrR binding sites across the entire E. coli genome. This study has revealed that there are many more targets for NsrR regulation than we previously suspected, most of which do not have obvious roles in NO detoxification. Future work will use genetic, molecular and physiological approaches to probe the roles of NsrR-regulated genes.
Fig. 3. NsrR regulates motility in E. coli. Soft agar motility assays show that over-expression of NsrR inhibits motility in lab (MG1655) and pathogenic (CFT073) strains. Addition of a source of NO to the medium reverses the effect of NsrR over-expression. From Partridge et al. (2009) Molecular Microbiology 73: 680-694.
In a broader sense, the lab is interested in defining the enzymatic source(s) of NO during nitrite respiration. We are interested to identify the proteins that provide protection against endogenously generated NO, and to identify the major cellular targets for the low concentrations of NO that are made during nitrite respiration.