Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids

Abstract

pestle, with 0.15 M sodium phosphate buffer, pH 7.5, added in 2-ml portions to a final volume of 2 ml of buffer per g of nodules. The homogenate was filtered through two Miracloth disks in a 50-ml syringe into a 30-ml centrifuge tube. The tube was capped tightly and centrifuged at 6,000 x g for 10 min. The tube was opened in the glove box, the supernatant was discarded, and the pellet was washed twice by pipetting 1 ml of the buffer over it. The pellet was resuspended, and the mixture was again filtered through Miracloth disks to remove any unsuspended bacteroids and adjusted to a final concentration of 2 ml of buffer per g of nodules. This (16), and 0.3 ml of 0.15 M sodium phosphate buffer, pH 7.5. The tubes were flushed with N2 and sealed with a serum stopper with an attached cup containing a filter paper wick impregnated with 70 p.l of 10% KOH. A sample (1.4 ml) was removed from the gas phase and replaced with 1.4 ml of , giving a final concentration of 2% 02 in the gas phase. Buffered substrate (0.5 ml; 1 ,umol at 1 ,uCi/,umol) was then injected into the reaction mixture, and the tubes were shaken in a rotary shaker at 250 rpm. Incubation was terminated by placing the tubes in an ice bath. The stoppers were quickly removed, and the tubes were centrifuged at 25,000 x g for 10 min. The supernatant was discarded, and the pellet was washed twice by pipetting 1 ml of the cold buffer over it and discarding the washes. The

Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions

“…The I4CO2 evolved is also an underestimation, because the dissolved C 0 2 is not accounted for. The 14C02 release is, however, proportional to the total C 0 2 respired and shows a linear increase after a lag period (Salminen & Streeter, 1987).…”

“…However, we found that, when Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with [ U-14C]succinate or [U-14C]malate under microaerobic conditions, 20 to 40% of the label taken up was converted to glutamate (Salminen & Streeter, 1987). When only the 14C remaining in bacteroids is considered ( 4C02 subtracted), glutamate accounted for 75 % of the radioactivity recovered.…”

“…The feeding of 14C-labelled substrates and the methods used for the analysis of labelling of individual metabolites has been described previously (Salminen & Streeter, 1987).…”

“…The accumulation of label in glutamate in bacteroids (Ohyama & Kumazawa, 1980;Salminen & Streeter, 1987) probably reflects a relatively large pool size, and quantitative data indicate that glutamate is a major metabolite in B. japonicum (Kouchi & Yoneyama, 1986;Streeter, 1987). Glutamate can readily be synthesized from 2-oxoglutarate either by glutamate dehydrogenase (GDH) or by the combined action of glutamine synthetase (GS) and glutamine : 2-oxoglutarate amino-S. 0.…”

“…japonicum bacteroids can absorb and respire glutamate (Bergersen & Turner, 1988;Salminen & Streeter, 1987), and some workers have even suggested that glutamate may be an important carbon source for bacteroids (Kahn et af., 1985). However, glutamate transport through the peribacteroid membrane may be restricted (Udvardi et al, 1988), so it would appear that glutamate accumulation in bacteroids may be unrelated to glutamate imported from the host cytoplasm.…”