Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparing Escherichia coli MG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO 4 concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularly zntA, zraP, and hydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations of cusF (involved in Ag and Cu efflux), ais (an Al-inducible gene), asr (encoding an acid shock-inducible periplasmic protein), cpxP (a periplasmic chaperone gene), and basR. Five upregulated genes, basR and basS [encoding a sensor-regulator implicated in Salmonella in Fe(III) sensing and antibiotic resistance], fliM (flagellar synthesis), and ycdM and yibD (both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since the mdt operon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions.

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 M GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant upregulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress. Nitric oxide (NO)1 is recognized to be one of the most important small molecules in biology. It is a lipophilic radical that has the ability to diffuse across biological membranes and through the cytoplasm. At high concentrations NO is viewed as a toxic molecule, capable of reacting with all major classes of biomolecules. Of particular interest is its ability to react with thiols and transition metal centers, often altering the functions of the proteins that contain such groups, including terminal oxidases and other heme proteins that bind dioxygen (1-3). Nitric oxide is also inhibitory to iron-sulfur centers in dehydratases such as aconitase (4). These reactions with biomolecules underpin the use of NO as a powerful weapon in the armory of mammalian cells to combat bacterial infection. Because NO is an extremely reactive molecule, it leads to the production of other reactive nitrogen species in biological systems (reviewed in Ref. 5). Peroxynitrite, formed during the oxidative burst of macrophages by the reaction of NO with superoxide is the most highly reactive and potentially cytotoxic of all the reactive nitrogen species (6). Nitrosation is the transfer of an NO ϩ group to a nucleophilic center, usually to a sulfur or nitrogen lone ...

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...

SummaryThe Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the s N -dependent enhancer binding protein, NIFA, via the formation of a NIFL±NIFA protein complex. The NIFA protein contains three domains: an Nterminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the s N -RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNAbinding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions.

The effective reduction of medical errors depends on an environment of safety for patients in both clinically-based and systems-oriented arenas. Formal teamwork training is proposed as a systems approach that will achieve these ends. In a study conducted by Dynamics Research Corporation, weaknesses and error patterns in Emergency Department teamwork were assessed, and a prospective evaluation of a formal teamwork training intervention was conducted. Improvements were obtained in five key teamwork measures, and most importantly, clinical errors were significantly reduced.