The effect of nitrite stress induced in Paracoccus denitrificans PD1222 was examined using
additions of sodium nitrite to an aerobic bacterial culture. Nitrite generates a strong stress
response in P. denitrificans, causing growth inhibition. This is dependent on both the
concentration of nitrite present and the pH. The pH dependent effect of nitrite growth
inhibition is likely a result of nitrite and free nitrous acid (FNA; pKa = 3.16) and subsequent
reactive nitrogen oxides, generated from the intracellular passage of FNA into P.
denitrificans. A flavohemoglobin (fhb; Pd1689) and its associated NsrR family,
transcriptional regulator (Pd1690), were transcribed above a ≥2 fold expression filter (p ≤
0.05) at 95% significance in qRT-PCR and a type II microarray transcriptional analysis at
12.5 mM nitrite in batch culture. Additionally, >25 fold expression of the flavohemoglobin
was confirmed by qRT-PCR in continuous culture with nitrite. A deletion mutant determined
fhb to be involved in conveying nitrite resistance at high nitrite concentrations and is linked
to a stimulation of biomass generated by the presence of nitrite. The cytochrome ba3 oxidase
was found to be associated with nitrite in transcriptional analysis, suggesting the uncoupling
of the protonmotive force caused by the transport of FNA across the membrane, and
subsequent dissociation in the cytoplasm were reduced by a method of counterbalance. No
nitrate accumulation was seen and nitrous oxide levels were above that observed for
atmospheric background levels. The microarray analysis was used to confirm that in batch
growth at 12.5 mM nitrite addition, P. denitrificans shows an overall stress response
associated with protein, DNA and lipid repair, with the addition of fhb detoxification and
action of the cytochrome ba3 oxidase. It is therefore suggested that nitrite presents a pHdependent
stress response in P. denitrificans, likely due to the production of associated
reactive nitrogen species such as NO from the internalisation of FNA and the uncoupling of
the protonmotive force
