Levels of Periplasmic Nitrate Reductase during Denitrification are Lower in Bradyrhizobium japonicum than in Bradyrhizobium diazoefficiens

Abstract

Soybean plants host endosymbiotic dinitrogen (N₂)-fixing bacteria from the genus Bradyrhizobium. Under oxygen-limiting conditions, Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum perform denitrification by sequentially reducing nitrate (NO₃^-) to nitrous oxide (N₂O) or N₂. The anaerobic reduction of NO₃^- to N₂O was previously shown to be lower in B. japonicum than in B. diazoefficiens due to impaired periplasmic nitrate reductase (Nap) activity in B. japonicum. We herein demonstrated that impaired Nap activity in B. japonicum was due to low Nap protein levels, which may be related to a decline in the production of FixP and FixO proteins by the cbb₃-type oxidase.

Keywords: Bradyrhizobium; cbb3 oxidase; competitive growth; denitrification; periplasmic nitrate reductase.

Fig. 1.

Detection of Nap in Bradyrhizobium diazoefficiens USDA 110 (Bd) and Bradyrhizobium japonicum USDA 6 (Bj) in HMMN medium under microaerobic and anaerobic conditions. (A) Immunoblotting analysis of NapA in soluble fractions (10‍ ‍μg). (B) Heme staining of membrane fractions (10‍ ‍μg). In panels A and B, the B. diazoefficiens USDA 110 ΔnapA mutant grown in HMMN medium under microaerobic conditions is shown as the control.

Fig. 2.

Average number of cells in co-cultures (1:1) of Bradyrhizobium diazoefficiens USDA 110 tagged with GFP (white bars) and Bradyrhizobium japonicum USDA 6 tagged with DsRed (grey bars) under anaerobic, microaerobic, and aerobic conditions in HMMN medium. Error bars indicate SE. * Values significantly different between USDA 110 and USDA 6 (t-test, P<0.05; n=3).