coli cytoplasm, possibly because it is reduced as it crosses the periplasm or cytoplasmic membrane. To estimate the maximum possible rate of NO generation from nitrite, the residual rate of nitrite reduction click here by a strain defective in both of the E. coli nitrite reductases, NrfA and NirB, was determined after anaerobic growth in the presence of nitrate. This rate was between 1 and 2 nmol of nitrite reduced min−1 (mg of bacterial dry mass)−1. This was an order of magnitude less than the rate of NO reduction by this

strain measured using an NO-sensitive electrode, which was 15 or 25 nmol of NO reduced min−1 (mg of bacterial dry mass)−1, depending on whether the bacteria had been grown in the presence of nitrite or nitrate (see also Vine & Cole, 2011). One possible explanation why externally added NO did not induce Phcp::lacZ transcription was that it is reduced by an active NO reductase located either in the periplasm or in the cytoplasmic membrane. An obvious candidate for such NO reductase activity is NrfAB, which despite its high Km for NO has been proposed to fulfil this role with high catalytic efficiency (Poock et al., C646 manufacturer 2002; van Wonderen et al., 2008). Cultures of the

parent strain and the nrfA mutant were therefore supplemented every 30 min with NO to a final concentration of 20 μM and compared with unsupplemented control cultures (Table 1). Loss of NrfAB function did not increase the transcription response of Phcp to NO, indicating that NrfAB is not the enzyme responsible for elimination of externally added NO. The aminophylline NarL-activated narGHJI operon is strongly induced during anaerobic growth in the presence of high concentrations of nitrate, whereas the nrf operon is repressed by nitrate-activated NarL, but induced by nitrite- or nitrate-activated NarP. Synthesis of the cytoplasmic nitrite reductase, NirBD, is induced by both NarP and NarL during anaerobic growth in the presence of nitrate or nitrite. The β-galactosidase assay was used to compare the response of mutants defective in each of these

enzymes with the parent strain during growth in the presence of nitrite. Deletion of nrfA or nirB resulted in increased responses to nitrite, suggesting that these nitrite reductases primarily decrease NO accumulation in the cytoplasm, and therefore protect bacteria against nitrosative stress (Table 2). In contrast, the narG mutant responded poorly to the addition of nitrite, consistent with nitrite reduction by NarGHI being the major source of NO in the cytoplasm. The residual response to nitrite by the NarG mutant indicates that there are additional sources of cytoplasmic NO. To investigate whether this residual induction was due to NO formation by the periplasmic nitrate reductase, NapA, a mutant was constructed that is defective in the nitrate reductases, NarG and NapA. Transcription at Phcp was still induced in this strain during anaerobic growth in the presence of nitrite (Table 2). Cruz-Ramos et al.