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

Janet Flatley,Jason Barrett,Steven T Pullan,Martin N Hughes,Jeffrey Green,Robert K Poole

doi:10.1074/jbc.m410393200

Janet Flatley, Jason Barrett + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m410393200

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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 microm 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 up-regulation, 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.

Highlights

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction
A concentration of 200 ␮M was chosen from previous studies, which determined the maximal expression of hmp in response to varying concentrations of GSNO.2
The responses of steady-state chemostat-cultured E. coli to the nitrosating agent, GSNO, under both aerobic and anaerobic growth conditions are reported

Summary

GSNO Stress and Methionine Biosynthesis

Sulfoxide reductase, MsrA, has been demonstrated in the defense of E. coli to peroxynitrite-mediated damage [18]. In addition to specific NO defense mechanisms, there are two known regulons in E. coli that are thought to respond primarily to redox stresses but which can be activated by NO and reactive nitrogen species. These are the SoxRS and OxyR regulons, both of which mediate the expression of a number of genes involved in oxidative and nitrosative stress responses. The genome-wide response of E. coli to GSNO and acidified sodium nitrite in aerobic batch cultures has been reported [22] This revealed that NorR and Fur are major regulators of the nitrosative defense system under the conditions tested. In a medium with adequate supplies of chelated metal ions, Fur is not involved in responses to nitrosative stress

EXPERIMENTAL PROCEDURES

Inhibition of respiration by NO

RESULTS

Aerobic codB ydcC yeaD nac Anaerobic bcp rpmB rpmG

Multidrug transporter

DISCUSSION