Preparation for Denitrification and Phenotypic Diversification at the Cusp of Anoxia: a Purpose for NO Reductase Multiple Roles of O.

Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed "bet-hedging," where all cells synthesize the NO reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O or NO. To test this, cells were exposed to gradual O depletion or sudden anoxia and subsequent spikes of O and NO. The synthesis of NirS in single cells was monitored by using an fusion replacing the native , and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e-acceptor deprivation and show that O spiking leads to bet-hedging, while NO spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the NO spike had much lower respiration rates than those rescued by the O spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O. Our results bring into relief a fitness advantage of pairing restrictive expression with universal NosZ synthesis in energy-limited systems. Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their NO emissions because all cells express NosZ, reducing NO to N, while a minority express NirS + Nor, reducing NO to NO. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O or NO. We confirm such entrapment and the rescue of all cells by an NO spike but only a fraction by an O spike. The results shed light on the role of O repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of NO, which contribute to anthropogenic climate forcing.