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

Abstract

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...

Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli

“…Nevertheless, ''physiological'' amounts of NO do not stimulate the SOS response in Salmonella enterica because RecA is not essential to prevent NO-dependent bacterial DNA fragmentation (32). Moreover, several transcriptomic analysis of E. coli demonstrated that expression of the genes of the SOS response was not enhanced following exposure to NO (18)(19)(20)(21). Additionally, we observed that stx2 mRNA expression was not modulated when bacteria were treated at the same time with mitomycin C and NOR-4.…”

“…Cellular production of NO requires the enzyme NO synthase that oxidizes L-arginine as a substrate; the calciumindependent inducible NO synthase (iNOS) isoform is expressed in numerous cells, e.g., enterocytes, in response to type 1 cytokines (16). High output of iNOS-derived NO is cytotoxic for pathogenic bacteria (17) or may induce transcriptomic changes (18)(19)(20)(21) by interacting with different NO sensors such as the nitrite-sensitive repressor (NsrR), a key regulator of the nitrosative stress in enterobacteria (22,23). NsrR DNA-binding activity is suppressed by NO, yielding to the expression of various genes involved in NO detoxification.…”

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

“…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].…”

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli

“…The suf operon is subject to complex regulation, with roles for OxyR, Fur, IscR and IHF in controlling activity of the suf promoter (Lee et al, 2003(Lee et al, , 2004(Lee et al, , 2008Yeo et al, 2006). There is evidence that the suf genes are upregulated by exposure to NO (Justino et al, 2005;Pullan et al, 2007), and of an NsrR binding site in the sufA promoter (Partridge et al, 2009). Our microarray data showed modest de-repression of suf genes in an nsrR mutant, although the statistical cut-off was not met for the DnsrR/ AUG nsrR comparison.…”