Culture samples were centrifuged at 13 000 g for 2 min, and the concentration ABT-199 nmr of nitrite in the supernatant was assayed as described by Pope & Cole (1984). As a reporter system, we used a plasmid from which β-galactosidase synthesis is dependent upon relief of NsrR repression of the promoter of the hcp gene in response to cytoplasmic NO (Filenko et al., 2007; Chismon et al., 2010). In this and other earlier work, nitrite was used as a source of NO to study NsrR repression at a range of promoters (Kim et al., 2003; Constantinidou et al., 2006; Vine & Cole, 2011). In initial experiments, duplicate cultures of E. coli strain RK4353 transformed with the Phcp::lacZ fusion plasmid were grown anaerobically

to mid-exponential phase in the absence of nitrite, and 2.5 mM nitrite was then added to Src inhibitor one culture. Transcription from Phcp was strongly activated in the presence of nitrite, but not in its absence (Table 2). The experiments were then repeated using an nsrR mutant as the host strain. As expected, high activities were detected both in the presence and absence of nitrite (Table 2). This was consistent with the expectation that

the response of the NsrR+ culture to nitrite was dependent upon inactivation of the repressor activity of NsrR by NO that had accumulated in the cytoplasm. However, the response to NO generated from nitrite was far smaller than the effect of an nsrR deletion mutation. Various sources of NO have been used in different laboratories to study its effects on gene regulation and metabolism, mainly because NO reacts rapidly with oxygen in aerobic cultures. In the absence

of oxygen, NO is stable, and so it is possible to avoid using S-nitrosoglutathione, nitrite or other sources of NO as a surrogate for NO. First, an NO-sensitive electrode was used to confirm that NO was stable in the absence of bacteria for long periods under conditions used for subsequent experiments. The effect on bacterial growth of sequential additions of P-type ATPase various concentrations of NO at 30 min intervals was then determined (Fig. 1). Growth was totally inhibited at concentrations above 10 μM NO, which is well above the range encountered by bacteria in vivo, and was also inhibited by sequential additions of 5 μM (not shown) or 10 μM NO, but not by 1 μM NO (not shown). As NsrR responds to sub-μM concentrations of NO, 5–20 μM NO was used in subsequent experiments. To determine optimal growth conditions for transcription activation at Phcp, further cultures were supplemented with either 10 mM sodium nitrite, 20 mM sodium nitrate, or oxygen-free, NO-saturated water added to a final concentration of 10 μM. Transcription of hcp::lacZ was induced far less by nitrate than by nitrite, but there was even less response to externally added NO, even when supplementation with NO was repeated at 30 min intervals (Fig. 2).