Short Communication The cbbL Gene is Required for Thiosulfate-Dependent Autotrophic Growth of Bradyrhizobium japonicum

Bradyrhizobium japonicum, a nitrogen-fixing endosymbiont of soybean nodules (6), is a facultative chemoautotroph utilizing thiosulfate (15), H2 (5, 8, 13) and CO (11, 14) as an electron donor and CO2 as a carbon source. However, the genes relevant to CO2 fixation during the chemoautotrophic growth of B. japonicum have yet to be identified. There are four major pathways for CO2 fixation; the Calvin-Benson-Bassham (CBB) cycle, reductive tricarboxylic acid (rTCA) cycle, 3-hydroxypropionate (3-HP) cycle and reductive acetyl coenzyme A (acetyl-CoA) pathway, in bacteria and archaea (17). A search for the genes related to CO2 fixation pathway on the genome of B. japonicum strain USDA110 revealed the presence of structural genes for the CBB cycle and rTCA cycle. Activity of ribulose 1,5-bisphosphate carboxylase (RuBisCO) was biochemically detected in a crude cell extract of B. japonicum strain USDA122 grown chemoautotrophically with H2 as an electron donor under a gas mixture (v/v) of 84% N2, 5% CO2, 1% O2 and 10% H2 (13). RuBisCO was also purified from USDA122 cells (18). In addition, the expression of cbbLS encoding RuBisCO was enhanced with H2 as a sole electron donor, as compared with that under heterotrophic conditions in B. japonicum strain USDA110 (5). Recently, Masuda et al. (15) found that B. japonicum USDA110 is able to fix ambient CO2 during chemoautotrophic growth using thiosulfate at quite low concentrations of CO2 (0.03–0.07% [v/v]) in contrast with previous reports of H2-dependent chemoautotrophic growth (5% [v/v] CO2) (5, 8, 13). Generally, RuBisCO enzymes have low affinity for CO2 and require higher CO2 concentrations (1). Therefore, we examined whether cbb encoding RuBisCO is required for thiosulfate-dependent chemoautotrophic growth at ambient concentrations of CO2 in the air. The bacterial strains and plasmids used in this study are listed in Table 1. B. japonicum strains were cultured aerobically at 30°C in HM salt medium (2) supplemented with 0.1% (w/v) arabinose and 0.025% (w/v) Difco TM yeast