The Flavohemoglobin Hmp and Nitric Oxide Reductase Restrict Initial nir Expression in the Bet-Hedging Denitrifier Paracoccus denitrificans by Curtailing Hypoxic NO Signalling

In denitrifying bacteria, nitric oxide (NO) is an electron acceptor and a free intermediate produced during anaerobic respiration. NO is also a signal for transcriptional regulation of the genes encoding nitrite (Nir), nitric oxide (Nor) and nitrous oxide reductases (N₂OR). We hypothesise that the timing and strength of the NO signal necessary for full nir expression are key factors in the bet-hedging strategy of Paracoccus denitrificans, and that systems scavenging NO under hypoxia reduce the probability of nir induction. We show that the flavohemoglobin Hmp scavenges NO in aerobic cultures and that hmp is regulated by an NsrR-type repressor. Using a strain with an mCherry-nirS fusion, we found a clear, negative effect of Hmp on initial nir expression. Deletion of norCB eliminated bet-hedging, but the elevated NO levels in co-cultures with the wild type did not abolish bet-hedging in the wild type cells. Our results demonstrate clear roles for Hmp and Nor in regulating the expression of nirS through NO scavenging, while suggesting that the trigger for nir induction is not NO itself, but rather an intracellularly generated derivative. Our findings have important implications for understanding the regulatory network controlling the transition to anaerobic respiration.