Denitrification by Bradyrhizobia under Feast and Famine and the Role of the bc1 Complex in Securing Electrons for N 2 O Reduction.

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also must 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 O 2 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 N 2 O- over NO 3 - reduction in bradyrhizobia is retained under carbon limitation. Here, we show that starved cultures of a Bradyrhizobium strain with respiration rates 1 to 18% of well-fed cultures reduced all available N 2 O before touching provided NO 3 - . These organisms, which carry out complete denitrification, have the periplasmic nitrate reductase NapA but lack the membrane-bound nitrate reductase NarG. Proteomics showed similar levels of NapA and NosZ (N 2 O reductase), excluding that the lack of NO 3 - reduction was due to low NapA abundance. Instead, this points to a metabolic-level phenomenon where the bc1 complex, which channels electrons to NosZ via cytochromes, is a much stronger competitor for electrons from the quinol pool than the NapC enzyme, which provides electrons to NapA via NapB. The results contrast the general notion that NosZ activity diminishes under carbon limitation and suggest that bradyrhizobia carrying NosZ can act as strong sinks for N 2 O under natural conditions, implying that this criterion should be considered in the development of biofertilizers. IMPORTANCE Legume cropped farmlands account for substantial N 2 O emissions globally. Legumes are commonly inoculated with N 2 -fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging to Bradyrhizobium , the microsymbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N 2 O reductase and will be N 2 O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N 2 O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here, we demonstrate that bradyrhizobia retain their strong preference for N 2 O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N 2 fixation and minimize N 2 O emission.