A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12

Abstract

Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3=diaminonaphthaIene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJ/ operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply t o that of an electron donor in the bacterial reduction of nitrite t o nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.

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.…”

Evidence for Mutagenesis by Nitric Oxide during Nitrate Metabolism in Escherichia coli

“…Hypoxia rather than anaerobiosis is sufficient for the induction of nitrate and nitrite reductase activities (18). It is possible that when the cell is growing in the presence of nitrate under hypoxic conditions, nitrate and nitrite reductase activities may be sufficiently induced, and there may still be sufficient oxygen present, so that NO· and N 2 O 3 are formed.…”

Nitrosative stress in Escherichia coli: reduction of nitric oxide

“…After 5 h of growth, the concentration of nitrate in the medium had decreased. It has been reported previously that, at this low nitrate concentration, NarG starts to reduce nitrite to NO instead of reducing its cognate substrate, nitrate [14][15][16][17][18]. The build up of NO in the mutant strain lacking NirB, NrfA, NorVW and Hmp would be toxic, and hence cause a growth defect.…”