Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium

Abstract

The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N₂O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N₂O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N₂O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N₂O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N₂O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N₂O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N₂O production, and this can account for up to 20% of the nitrate catabolized.

Production and consumption of nitrous oxide in nitrate-ammonifying Wolinella succinogenes cells

“…The ability of nitrate-ammonifying cells to produce N 2 O in pure culture has been reported before (Bleakley & Tiedje, 1982;Rowley et al, 2012;Stremiń ska et al, 2012). In Salmonella enterica serovar Typhimurium, 20 % of nitrate-N was released as N 2 O under nitrate-sufficient conditions when nitrite had accumulated to millimolar levels (Rowley et al, 2012).…”

“…In Salmonella enterica serovar Typhimurium, 20 % of nitrate-N was released as N 2 O under nitrate-sufficient conditions when nitrite had accumulated to millimolar levels (Rowley et al, 2012). This phenotype is envisaged to result from NO production from nitrite by the Nar-type nitrate reductase (see above) and subsequent anoxic NO detoxification to N 2 O.…”

Mechanism of [4Fe-4S](Cys)4 Cluster Nitrosylation Is Conserved among NO-responsive Regulators

“…It is suggested that if these dedicated NO detoxification systems are unable to lower the NO concentration sufficiently to counteract the ensuing nitrosative stress, FNR will become nitrosylated (30), leading to lowered expression of the nar, nir, nrf, and nap operons (that require [4Fe-4S] FNR for activation) and derepression of hmp expression. We note that NarG is the most important source of endogenously derived NO during nitrate/ nitrite respiration (7,(41)(42)(43). Thus, nitrosylation of FNR would serve to protect the cell by down-regulating the principal source of endogenous NO and by up-regulating Hmp, an important NO-detoxifying enzyme.…”

The Production of Ammonia by Multiheme Cytochromes c

“…To our knowledge, this is an as yet largely unaccounted feature in globalscale models of ammonia production in habitats such as anaerobic soil or marine oxygen minimum zones [29,30,[106][107][108]. The situation is reminiscent to the discovery that bacterial ammonifiers of nitrate and nitrite, i.e., NrfA-containing microorganisms, might significantly contribute to atmospheric N 2 O production, in addition to the denitrifying microbial community [109][110][111][112]. In this case, any NO formed either enzymatically or abiotically from nitrite is apparently detoxified under anaerobic conditions to N 2 O by a variety of NO-reactive enzymes.…”