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Journal of Bacteriology, March 2007, p. 1845-1855, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01354-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Nitric Oxide in Chemostat-Cultured Escherichia coli Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation{triangledown} ,{dagger}

Steven T. Pullan,1,{ddagger} Mark D. Gidley,1 Richard A. Jones,1 Jason Barrett,1,§ Tania M. Stevanin,2 Robert C. Read,2 Jeffrey Green,1 and Robert K. Poole1*

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN,1 Division of Immunity and Infectious Diseases, University of Sheffield Medical School, Royal Hallamshire Hospital, Sheffield S10 2RX, United Kingdom2

Received 25 August 2006/ Accepted 5 December 2006

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.


* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom. Phone: (44) 114 222 4447. Fax: (44) 114 222 2800. E-mail: r.poole{at}sheffield.ac.uk.

{triangledown} Published ahead of print on 22 December 2006.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

§ Present address: Bioline, 16 The Edge Business Centre, Humber Road, London NW2 6EW, United Kingdom.


Journal of Bacteriology, March 2007, p. 1845-1855, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01354-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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