Denitrifying bradyrhizobia retain strong N2O reduction during periods of starvation

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also have to survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2 tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well-known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O− over NO3−-reduction in bradyrhizobia is retained under carbon limitation. Here we show that starved cultures of a Bradyrhizobium strain with respiration rates 1-18% of well-fed cultures, reduced all available N2O before touching provided NO3−. Proteomics showed similar abundance of Nap (periplasmic NO3− reductase) and NosZ (N2O reductase), suggesting that competition between electron pathways to Nap and NosZ favoured N2O reduction also in starved cells, similar to well-fed cultures. This contrasts the general notion that NosZ activity diminishes under carbon limitation. The results suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions and that this criterion should be considered in the development of biofertilizers. Importance Novel biotechnological approaches are needed to curb the escalating emissions of the greenhouse gas N2O from agricultural soils. One possibility is inoculation of N2O−reducing organisms into soil. Rhizobial inoculants are used at large scale to enhance N2-fixation of legumes. The genus Bradyrhizobium encompasses N2-fixing symbionts of economically important legumes. Most bradyrhizobia can also denitrify, but the commercially available inoculants generally lack the last denitrification step, reduction of N2O. Recent results revealed that bradyrhizobia with complete denitrification strongly favor N2O over nitrate, making them potential strong sinks for N2O. In those studies the cuItures received ample amounts of substrate, while bacteria in soil generally starve. Here we demonstrate that bradyrhizobia retain their strong preference for N2O also under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing new bradyrhizobial legume inoculants with the dual capacity to optimize N2-fixation and minimize N2O emission.