We previously elucidated the global transcriptional responses of Escherichia coli to the nitrosating agent S-nitrosoglutathione (GSNO) in both aerobic and anaerobic chemostats, demonstrated the expression of nitric oxide (NO)-protective mechanisms, and obtained evidence of critical thiol nitrosation. The present study was the first to examine the transcriptome of NO-exposed E. coli in a chemostat. Using identical conditions, we compared the GSNO stimulon with the stimulon of NO released from two NO donor compounds {3-[2-hydroxy-1-(1-methyl-ethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7)} simultaneously and demonstrated that there were marked differences in the transcriptional responses to these distinct nitrosative stresses. Exposure to NO did not induce met genes, suggesting that, unlike GSNO, NO does not elicit homocysteine S nitrosation and compensatory increases in methionine biosynthesis. After entry into cells, exogenous methionine provided protection from GSNO-mediated killing but not from NO-mediated killing. Anaerobic exposure to NO led to up-regulation of multiple Fnr-repressed genes and down-regulation of Fnr-activated genes, including nrfA, which encodes cytochrome c nitrite reductase, providing strong evidence that there is NO inactivation of Fnr. Other global regulators apparently affected by NO were IscR, Fur, SoxR, NsrR, and NorR. We tried to identify components of the NorR regulon by performing a microarray comparison of NO-exposed wild-type and norR mutant strains; only norVW, encoding the NO-detoxifying flavorubredoxin and its cognate reductase, were unambiguously identified. Mutation of norV or norR had no effect on E. coli survival in mouse macrophages. Thus, GSNO (a nitrosating agent) and NO have distinct cellular effects; NO more effectively interacts with global regulators that mediate adaptive responses to nitrosative stress but does not affect methionine requirements arising from homocysteine nitrosation.Nitric oxide (NO) is a key component of the host immune response and is encountered by pathogenic bacteria during their lives outside and within hosts. In particular, phagocytic cells of a host produce the antimicrobial radical NO at micromolar concentrations through the activity of inducible NO synthase (20).Enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, use two major mechanisms to detoxify NO (Fig. 1) (60), the flavohemoglobin Hmp and the flavorubredoxin NorV. The former enzyme, using an electron from NAD(P)H delivered via the flavin protein domain, catalyzes either an O 2 -dependent denitrosylase ("dioxygenase") reaction converting NO to the nitrate ion or an anoxic reductive reaction forming NO Ϫ (63). The flavorubredoxin NorV along with its cognate reductase, NorW, however, catalyzes the reductive detoxification of NO only under microaerobic or anaerobic conditions (25). The synthesis of NorV and NorW is positively regulated at the transcriptional level by the NorR...

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K m ‫؍‬ 0.8 M) and a V max of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K m value of 40 nM and a V max of 6 to 9 nmol/mg/s. Lowtemperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.

A. actinomycetemcomitans, F. nucleatum and P. gingivalis were present in significantly different quantities and proportions in subgingival plaque, according to periodontal disease status. No significant differences were identified between the subgingival microbiota of type 2 diabetes mellitus patients compared with nondiabetic subjects.