Transcriptional Responses of Escherichia coli to S-Nitrosoglutathione under Defined Chemostat Conditions Reveal Major Changes in Methionine Biosynthesis

Abstract

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 ...

Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coli – Implications for urinary tract infection

“…The effect of NO on respiration in J96∆norV was similar to the effects observed in J96wt suggesting that NorV activity is not important for NO detoxification in UPEC during the present conditions. In line with our findings, deletion of norV showed little effect on NO consumption in a non-pathogenic E. coli in aerobic conditions [6,19] Previously, a DNA-based microarray has shown that hmp was among the top 50 genes up-regulated in UPEC strain CFT073 in a mouse UTI-model [44]. Moreover, increased transcription of hmp and norV was reported in an asymptomatic bacteriuria E. coli strain from three patients based on microarray analysis [45].…”

“…Respiratory inhibition in response to NO is well established in non-pathogenic E. coli [3,6,43]. Respiration of UPEC was also significantly inhibited by NO and the inhibition was initiated momentarily upon NO addition and resumed after the NO levels decreased.…”

“…In approximately 80 % of the community-acquired urinary tract infections (UTIs) the cause of the infection is a uropathogenic Escherichia coli (UPEC) strain. Flavohemoglobin (Hmp) and flavorubredoxin (NorV) are the two main NO-detoxifying systems in E. coli and are up-regulated in response to NO and nitrosating agents such as S-nitrosoglutathione [5,6,7,8]. The main function of Hmp is to catalyse the conversion of NO to nitrate [9,10], but Hmp can also operate in anaerobic conditions reducing NO to nitrous oxide (N 2 O) [11].…”

The yjeB ( nsrR ) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

“…Several regulatory proteins have been shown to be sensitive to NO in vitro or in vivo, or both, and so may mediate adaptive responses to NO exposure. These include SoxR, OxyR, FNR, MetR, and Fur (5,6,10,12,17), though in each case the principal function of the regulator is to sense another signal (superoxide, hydrogen peroxide, oxygen, homocysteine, and iron, respectively). The only regulator known to serve exclusively as an NO sensor in E. coli is NorR, which activates transcription of the norVW genes encoding the flavorubredoxin and associated flavoprotein that together reduce NO to nitrous oxide (7,13,14,16,18).…”

“…Three studies have made use of microarrays to evaluate the response of the E. coli transcriptome to exposure to NO or other reagents that cause nitrosative stress (12,21,27). These experiments differed in their choice of medium (rich or defined), growth conditions (aerobic or anaerobic, batch or continuous culture) and the reagents used to impose nitrosative stress (S-nitrosoglutathione, acidified nitrite, or aqueous NO), which may in part account for the different gene sets identified in each case.…”