Bacteroids of B. japonicum from nodules of soybean roots were isolated using differential centrifugation (the standard bench method) and density gradient centrifugation methods (either sucrose- or Percoll-) under anaerobic conditions in which N2 fixation was preserved. The relationships between N2 fixation and respiration, O2 supply, O2 demand, substrate (mainly malate) transport and metabolism in bacteroids were investigated using the flow chamber system. In related experiments, the primary products of N2 fixation which leave the bacteroids were investigated using a 15N-labelling technique in a closed shaken system and other biochemical methods.¶
In the flow chamber experiments, the rates at which O2 was supplied to bacteroids in the chamber were varied by (a) changing the flow rate of reaction medium through the chamber; (b) by changing the [O2 free] in the inflowing reaction medium by using either 3-5% (v/v) or 100% air in the gas mixture above the stirred reaction medium in two reservoir flasks; (c) by successively withdrawing bacteroids from the chamber, thus increasing the supply of O2 per bacteroid to those remaining in the chamber. The results showed that the rate of O2 supply regulates respiratory demand for O2 by bacteroids rather than the O2 concentration present in the reaction system. Respiration is always coupled to N2 fixation. ¶
Uptake of malate by bacteroids withdrawn from the flow chamber was measured under microaerobic conditions. Malate uptake by these N2-fixing bacteroids was lower than that by bacteroids isolated under aerobic conditions, which eliminate N2 fixation of bacteroids, but is closely correlated with bacteroid respiration rates. When respiration was increased by an increase in O2 supply, malate uptake by bacteroids was also increased. This suggested that transport of malate through the bacteroid membrane is also regulated by O2 supply, but indirectly. Higher uptake by bacteroids under aerobic conditions was observed because respiration was enhanced by the high availability of O2, but the fast uptake of malate by bacteroids driven by the abnormal respiration rates may not reflect the reality of malate demand in vivo by bacteroids when N2 fixation by bacteroids is fully coupled. ¶
The results of 15N labelling experiments and other biochemical assays once again demonstrated that ammonia is the principal significant 15N labelled product of N2 fixation accumulated during 30 min in shaken assays with 0.008-0.01 atm O2. Alanine although sometimes found in low concentrations in the flow chamber reactions, was not labelled with 15N in shaken closed system experiments. No evidence could be obtained from the other biochemical assays, either. Therefore, it is concluded that these and earlier results were not due to contamination with host cytosolic enzymes as suggested by Waters et al. (Proc. Natl. Aca. Sci. 95, 1998, pp 12038-12042). ¶
Malate transported into bacteroids is oxidized in a modified TCA cycle present in bacteroids. The results of flow chamber experiments with a sucA mutant (lacking a-ketoglutarate dehydrogenase) showed that respiratory demand for O2 by the mutant bacteroids is regulated by O2 supply in the same way as the wild-type. Despite differences in other symbiotic properties, rates of nitrogen fixation by the mutant bacteroids, based on the bacteroid dry weight, appeared to be the same as in the wild-type. Also N2 fixation was closely coupled with respiration in the same manner in both mutant bacteroids and wild type bacteroids. These results and other supporting data, strongly support the conclusion that there is an alternative pathway of the TCA cycle in bacteroids, which enables the missing step in the mutant to be by-passed with sufficient activity to support metabolism of transported malate.
| Identifer | oai:union.ndltd.org:ADTP/216753 |
| Date | January 2003 |
| Creators | Li, Youzhong, Youzhong.Li@health.gov.au |
| Publisher | The Australian National University. Faculty of Science |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.anu.edu.au/legal/copyrit.html), Copyright Youzhong Li |
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