Rhizobia-legume symbiosis and nitrogen fixation
Introduction
Nitrogen is one of the most fundamental elements necessary for all life forms. Nitrogen is a major component of amino acids, the most basic building blocks of various proteins that sustain the proper functioning of a living organism; DNA, the fundamental biochemical unit of heredity that stores information in living organisms, also requires nitrogen to build up. Though nitrogen is highly abundant in the atmosphere in the form of dinitrogen gas, this molecular form of nitrogen is inert in that the triple-bonding between the two nitrogen atoms makes the molecule extremely stable at normal temperature and pressure. Given the contribution of nitrogen to sustaining life on Earth, natural biological pathways that convert nitrogen gas into bio-accessible forms are of great ecological and evolutionary significance.
Rhizobium bacteria represent one of the groups that perform the service of biological nitrogen fixation (1). The genus Rhizobium, commonly known as rhizobia, includes species of various gram-negative alphaproteobacteria and betaproteobacteria that inhabit the root nodules of leguminous plants (2). Rhizobium was first discovered and named at the end of the 19th century when scientists began to notice that atmospheric nitrogen was assimilated into the root nodules of legumes: the german agricultural chemist Hermann Hellriegel first discovered that leguminous plants took in atmospheric nitrogen and turned it into ammonium; later, the Dutch microbiologist Beijerinck explored the mechanisms by which nitrogen is fixed through legume root-nodules and identified the bacteria responsible for this function, the rhizobia (3). Today, the pathways through which rhizobia fix nitrogen and the genetic and ecological regulations that control the process have been thoroughly studied.
Rhizobia-legume Symbiosis: substances exchange
The most characteristic feature of a rhizobium is its symbiotic relationship with the host plant. Once infecting a host, the rhizobium elicits the formation of nodules in the host’s roots where the bacterium inhabits and fixes nitrogen; within the nodule, rhizobia are modified into bacteroids and compartmented into symbiosomes surrounded by symbiosome membranes (6). This symbiotic relationship between rhizobia and legumes is considered mutualistic since there are clear evolutionary advantages for both sides: while the bacteria provide the plant with a valuable nitrogen source, the plant in exchange feeds the rhizobium with sugar; this process has been understood on a molecular level. Within the root nodules, rhizobia reduce N2 into NH3 via the nitrogenase enzyme complex (4). A great challenge of the enzyme nitrogenase is its strict requirement of an anaerobic condition. Rhizobia perform aerobic respiration; this suggests that special adaptations are needed to maintain the efficiency of nitrogen fixation within the cell. Several mechanisms protect the enzyme from oxygen: the inner cortex of the nodule serves as a diffusion barrier that limits the movement of oxygen into the rhizobium cell (5); the cellular respiration of rhizobia within the nodule is enhanced by the high-oxygen-affinity terminal oxidase which leads to a greater rate of oxygen consumption (5); and finally, the leghaemoglobin, a specific protein that binds to oxygen, is produced in high concentrations by the roots colonized by rhizobia in order to buffer the oxygen concentration within root nodules (4)(5).
A functional nitrogenase carries out the energy-intensive process of nitrogen fixation that produces ammonia to be delivered to the host. In exchange, the host plant provides the bacteroids with carbon sources. In the legume cells infected by rhizobia-derived bacteroids within the nodule, the photosynthetic product of sucrose, which is cleaved by the reversible enzyme sucrose synthase, is the primary source of carbon for symbiotic metabolism going on inside nodules (5). One study that used mutant peas with a low level of sucrose synthase activities showed that the enzyme is indispensable for the proper functioning of symbiotic nitrogen fixation; reduced levels of sucrose synthase decreased the leghemoglobin level by 80% which almost completely inhibited nitrogenase functioning (7). Since rhizobia inhabit root cells that do not have access to sunlight, carbon must be transported within the host plant in order to deliver sucrose to the infected region. Research showed that the cells uninfected by bacteroids exhibited different metabolic activities from the infected cells where bacteroids perform nitrogen fixation: a series of transporter-regulated pathways translocate sucrose symplastically to the infected cells (8). Within the infected region of root nodules, sucrose gets further processed. Under normal conditions, the cleaved sugar then subsequently feeds into glycolysis. However, in cells infected by rhizobia, the subsequent sugar metabolism exhibits alternative pathways. Evidence suggests that dicarboxylate, especially malate, is the final source of carbon that the host feeds to rhizobia; in root nodules, enzymes of the infected cells such as phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase are greatly upregulated which divert carbon intermediates away from the glycolytic pathways and into malate synthesis (5)(9). The malate then gets transported across the symbiosome membrane via specialized membrane dicarboxylate transporters and finally reaches the bacteroids; it is interesting that, besides carbon, the bacteroid also receives amino acids from the host via the symbiosome membrane as necessary building blocks of enzymes before nitrogen fixation begins (5). Malate could be used as the sole source of energy in bacteroids; once the malate is provided, the bacteroid breaks it down through malic enzymes that decarboxylate malate into pyruvate (9). Pyruvate is further processed into Acetyl-CoA, which feeds into the TCA cycle common to all forms of metabolism. This series of biochemical adaptations operated within both the host and the rhizobium provide a source of energy for the energy-intensive nitrogen fixation process.
Just as the host delivers carbon to its symbiotic partner, rhizobia need to transport the product of nitrogen fixation to the host to maintain the mutualistic relationship. The process of nitrogen fixation generates ammonia inside bacteroids; the ammonia will not be assimilated into organic forms but instead transported across the symbiosome membrane into the infected cell. Several mechanisms exist for this transmembrane ammonia transportation: since ammonia is a small weak acid molecule that exists in both charged and uncharged states, it can diffuse across the symbiosome membrane either through simple diffusion or via protein channels unspecific to ammonia; it can also be transported through active cation channels in the form of ammonium (5). Ammonia is toxic when accumulated in great abundance, hence the cell needs to reprocess ammonia into alternative nitrogen forms to be transported within the host’s tissues; the form of nitrogen exported from the nodule to the rest of the host plant varies among species. For example, research suggested that purine synthesis is one of the pathways many legumes use to assimilate nitrogen fixed by rhizobia (10). In tropical legumes such as soybeans and cowpeas, fixed nitrogen in forms of ammonia and ammonium is delivered completely to the purine pathway, which leads to the end product of ureides, the dominant form of stored nitrogen within the tissues of these legumes.
Section 2
Include some current research, with at least one figure showing data.
Section 3
Include some current research, with at least one figure showing data.
Section 4
Conclusion
References
Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2022, Kenyon College
