Energisation of nitrogen fixation in the Rhizobium-legume symbiosis

Bacteria are simple single celled organisms that lack the membrane bound structures found in higher cells of plants and animals. However, while bacteria may have a less complex cellular organisation they carry out a huge range of chemical reactions not found in plants and animals. Bacteria are responsible for the cycling of many nutrients such as N2 (N2 is also known as nitrogen gas and consists of two nitrogen atoms bound by a strong triple bond), which is a very inert atmospheric gas. N2 makes up 78% of the atmosphere but is very unreactive and cannot be used directly as a source of nitrogen, which is needed for amino acid, protein and DNA synthesis. However, a small number of bacteria can reduce (add hydrogen) to N2 and convert it into ammonia (NH3), which is readily incorporated into amino acids and then all the other building blocks of life, by a wide range of organisms including bacteria and plants. In many parts of the world the limitation to growth of plants, which in turn support animal life, is the supply of nitrogen as ammonia or related compounds. Since up to 65% of available nitrogen (eg ammonia) comes from bacteria this makes them essential for life on earth. Within the bacteria, most of the nitrogen is actually produced by one family known as the Rhizobiacea. This remarkable group of bacteria form a symbiotic association (both partners benefit) with plants of the legume family, that results in the formation of root nodules (on pea plants these are 2-3 mm bulbs that can easily be seen by pulling up a plant and inspecting its roots). The rhizobia are held inside the nodules where the plant provides them with an ideal environment (low O2 and lots of energy) in which they can reduce N2 to ammonia. The ammonia is supplied to the plant as its nitrogen source so this is why this is known as a symbiotic interaction. It means that the plant does need any nitrogen in the soil and enables rapid growth. The purpose of this research is to understand the type of fuel provided by the plant to power the fixation of N2 to ammonia by the bacteria. Questions include how is the fuel delivered to the bacteria and how do they metabolise (break it down) it to simpler compounds. Finally, we want to know whether, apart from ammonia, the bacteria secrete other compounds to the plant.

The Aap and Bra are broad specificity amino acid transporters that are essential for productive N2-fixation in peas (Hosie et al., 2001; Hosie et al., 2002; Lodwig et al., 2003; Prell & Poole, 2006; White et al., 2007) and in recent ground-breaking work we have concluded they secrete alanine as part of a GABA-alanine-cycle operating in nodules. The oxo-acid generated by this cycle is succinate semialdehyde, which our calculations reveal has one of the lowest redox potentials of any compound in intermediary metabolism. Its midpoint potential is less than that of ferredoxin and is therefore ideally suited for the generation of reductant for nitrogenase. Furthermore, the GABA-alanine cycle bypasses a large part of the TCA-cycle, suggesting that central metabolism may be completely changed in legume bacteroids; explaining why many TCA-cycle mutants are able to fix nitrogen in planta. It is crucial to determine whether the GABA-alanine- and TCA-cycles function alone or in combination to drive N2-fixation in Rhizobium-legume symbioses to underpin the global nitrogen cycle. We therefore propose to determine how GABA is metabolised to balance bacteroid metabolism and fuel N2-fixation, and how this is integrated with the TCA-cycle. The very low midpoint potential of succinate semialdehyde is an extraordinarily powerful reason for bacteroid metabolism to use semialdehyde oxidation and may be important in generating a global redox switch for N2-fixation in legume nodules. Thus, the GABA-alanine-cycle may explain both how carbon and nitrogen are exchanged between the plant and bacterial symbionts, as well as provide a powerful chemical rationale for how reduction of N2 is achieved.