The exploitation of the ability of a multicopy plasmid to titrate out NsrR and hence relieve NsrR repression enabled us to demonstrate that NsrR is a global regulator of the response to RNS in E. coli
). This approach is similar to the ferric uptake regulator titration assay (42
), with the inclusion of a microarray analysis to identify the genes derepressed by NsrR titration. Some of the small increases in transcript abundance detected when NsrR is active were almost certainly (but it was not independently confirmed) due to secondary effects, but the primary effects of NsrR as a repressor of operons involved in the relief of reactive nitrogen stress were largely confirmed by independent evidence. Previous studies have reported that hmpA
transcription is induced during NO- or S
-nitrosoglutathione-induced stress (18
), and NO also induces synthesis of the di-iron protein YtfE (4
). We recently predicted that there is a previously undocumented nitrite-responsive transcription factor that regulates the expression of the nrf
operon encoding the periplasmic nitrate reductase, as well as genes of unknown function that include ytfE
). All of these transcripts were more abundant in the transformant in which the pool of NsrR was depleted than in the control transformant, validating our repressor titration approach and indicating that NsrR is the additional transcription factor.
One interesting result from the microarray analysis was the observation that the complete periplasmic pathway for nitrate reduction to ammonia is regulated by NsrR. Pathogenic bacteria must be able to defend themselves from RNS originating from four sources: products of their own metabolism; products of other bacteria that share their ecological niche (for example, NO generated by lactic acid bacteria in the gastrointestinal tract); NO generated as part of host defense mechanisms; and products of nonspecific chemical reactions. Most of the above threats originate outside enteric bacteria, so it is appropriate that they can be neutralized by the NO reductase activity previously documented for the periplasmic nitrite reductase Nrf (36
). If so, how does E. coli
protect itself against RNS that enter or are made in the cytoplasm?
A major protection mechanism against NO in the cytoplasm is provided by flavorubredoxin and its reductase, NorVW, which are synthesized in response to NO activation of the transcription activator NorR (15
). The three operons most strongly up-regulated in response to NsrR titration are hmpA
, and hcp-hcr
(Table ). Both Hmp and YtfE are clearly established as cytoplasmic components of the RNS response (25
), suggesting that the same might be true for HCP. Microarray analysis of the E. coli
FNR, NarXL, and NarQP regulons and supporting transcription fusion data revealed that hcp
expression is regulated in parallel with the cytoplasmic NADH-dependent nitrite reductase Nir (11
). We inferred that this implied a function for HCP in detoxifying a product generated when the NarXL two-component system is activated, possibly an RNS generated as a side product of nitrite reduction to ammonia by Nir. The hcr
product has been shown to be an NADH-dependent HCP reductase that presumably functions to provide electrons for the reduction of the HCP substrate (45
), which was tentatively identified to be hydroxylamine (46
). However, there is a hydroxylamine reductase activity associated with the cytoplasmic NADH-dependent nitrite reductase NirBD that is at least as effective as that of HCP (23
). While it is possible that HCP is a back-up mechanism to provide protection against hydroxylamine toxicity, conditions under which its role is significant remain to be revealed, so other physiological roles for HCP must be considered. Even when it is expressed from a single chromosomal copy of the hcp
gene, HCP accumulates as an abundant protein (45
). Possibly HCP simply binds hydroxylamine stoichiometrically to prevent it from inhibiting bacterial metabolism until it can be reduced by Nir to ammonia (23
). A precedent for such a detoxification mechanism is cytochrome c
′, which protects pathogenic neisseria by binding nitric oxide (21
). A further possibility meriting consideration is that HCP repairs NO- or hydroxylamine-induced damage to iron-sulfur centers, for example, the Fe-S centers of NirB.
Recently an entirely different role was proposed for HCP, namely, that it functions as a peroxidase (1
). This suggestion was based upon observations that HCP is oxidized by hydrogen peroxide, that an hcp
mutant is more sensitive to hydrogen peroxide than its parent, and that hcp
transcription is regulated by OxyR. However, other links between RNS and the OxyR regulon have been reported (18
), and the phenotype of the hcp
mutant was rather weak. We therefore suggest that the physiological substrate reduced by HCP remains to be determined but is more likely a reactive nitrogen compound (other than hydroxylamine) than a reactive oxygen species.
The microarray analysis confirmed our previous suggestion that transcription of the two-gene operon yeaR-yoaG
is subject to NsrR repression. This operon is therefore another candidate for encoding proteins that protect E. coli
against RNS, especially in anaerobic environments where FNR might be inactivated by severe NO damage (11
). Future studies must focus on the biochemical functions of YeaR, YoaG, YtfE, and yet again, HCP.