David W. Emerich

Symbiotic nitrogen fixation requires the transfer of fixed organic nitrogen compounds from the symbiotic bacteria to a host plant, yet the chemical nature of the compounds is in question. Bradyrhizobium diazoefficiens bacteroids were isolated anaerobically from soybean nodules and assayed at varying densities, varying partial pressures of oxygen, and varying levels of l-malate. Ammonium was released at low bacteroid densities and high partial pressures of oxygen, but was apparently taken up at high bacteroid densities and low partial pressures of oxygen in the presence of l-malate; these later conditions were optimal for amino acid excretion. The ratio of partial pressure of oxygen/bacteroid density of apparent ammonium uptake and of alanine excretion displayed an inverse relationship. Ammonium uptake, alanine and branch chain amino acid release were all dependent on the concentration of l-malate displaying similar K_0.5 values of 0.5 mM demonstrating concerted regulation. The hyperbolic kinetics of ammonium uptake and amino acid excretion suggests transport via a membrane carrier and also suggested that transport was rate limiting. Glutamate uptake displayed exponential kinetics implying transport via a channel. The chemical nature of the compounds released were dependent upon bacteroid density, partial pressure of oxygen and concentration of l-malate demonstrating an integrated metabolism.

The form and physiology of Bradyrhizobium diazoefficiens after the decline of symbiotic nitrogen fixation has been characterized. Proteomic analyses showed that post-symbiotic B. diazoefficiens underwent metabolic remodeling as well-defined groups of proteins declined, increased or remained unchanged from 56 to 119 days after planting, suggesting a transition to a hemibiotrophic-like lifestyle. Enzymatic analysis showed distinct patterns in both the cytoplasm and the periplasm. Similar to the bacteroid, the post-symbiotic bacteria rely on a non-citric acid cycle supply of succinate and, although viable, they did not demonstrate the ability to grow within the senescent nodule.

The transcriptional activity of Bradyrhizobium diazoefficens isolated from soybean nodules was monitored over the period from symbiosis to late plant nodule senescence. The bacteria retained a near constant level of RNA throughout this period, and the variation in genes demonstrating increased, decreased, and/or patterned transcriptional activity indicates that the bacteria are responding to the changing environment within the nodule as the plant cells progress from an organized cellular structure to an unorganized state of internal decay. The transcriptional variation and persistence of the bacteria suggest that the bacteria are adapting to their environment and acting similar to hemibiotrophs, which survive both as saprophytes on live plant tissues and then as necrophytes on decaying plant tissues. The host plant restrictions of symbiosis make B. diazoefficiens a highly specialized, restricted hemibiotroph.

Symbiosomes are a unique structural entity that performs the role of biological nitrogen fixation, an energy-demanding process that is the primary entryway of fixed nitrogen into the biosphere. Symbiosomes result from the infection of specific rhizobial strains into the roots of an appropriate leguminous host plant forming an organ referred to as a nodule. Within the infected plant cells of the nodule, the rhizobia are encased within membrane-bounded structures that develop into symbiosomes. Mature symbiosomes create an environment that allows the rhizobia to differentiate into a nitrogen-fixing form called bacteroids. The bacteroids are surrounded by the symbiosome space, which is populated by proteins from both eukaryotic and prokaryotic symbionts, suggesting this space is the quintessential component of symbiosis: an inter-kingdom environment with the single purpose of symbiotic nitrogen fixation. Proteins associated with the symbiosome membrane are largely plant-derived proteins and are non-metabolic in nature. The proteins of the symbiosome space are mostly derived from the bacteroid with annotated functions of carbon metabolism, whereas relatively few are involved in nitrogen metabolism. An appreciable portion of both the eukaryotic and prokaryotic proteins in the symbiosome are also ‘moonlighting’ proteins, which are defined as proteins that perform roles unrelated to their annotated activities when found in an unexpected physiological environment. The essential functions of symbiotic nitrogen fixation of the symbiosome are performed by co-operative interactions of proteins from both symbionts some of which may be performing unexpected roles.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean ( Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2‐D PAGE using narrow range IPG strips. More than 1200 proteins were detected, of which nearly 500 proteins were analysed by MALDI‐TOF and 310 spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free‐living B. japonicum. A comparative analysis of proteomes of free‐living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states.

As a prelude to using functional proteomics towards understanding the process of symbiotic nitrogen fixation between the legume soybean and the soil bacteria Bradyrhizobium japonicum , we examined the total protein expression pattern of the nodule bacteria, often referred to as bacteroids. A partial proteome map was constructed by separating the total bacteroid proteins using high‐resolution 2‐DE. Of the several hundred protein spots analyzed using PMF, 180 spots were tentatively identified by searching the available database for B. japonicum , ( http://www.kazusa.or.jp/index.html ). The data showed that the bacteroid expressed a dominant and elaborate protein network for nitrogen and carbon metabolism, which is closely dependent on the plant supplied metabolites, and seems aptly supported by a selective group of bacteroid transporter proteins. However, they seem to lack a defined fatty acid and nucleic acid metabolism. Interestingly, the proteins related to protein synthesis, scaffolding and degradation were among the most predominant spots of the bacteroid proteome. In addition, several proteins, which showed fairly good expression, were identified to be involved with cellular detoxification, stress regulation and signaling communication components. This preliminary proteomic data matches very well with several biochemical and genetic reports, and clearly shows the inter‐connection between several metabolic pathways that meet the needs of the bacteroid. It is expected that in the future this will allow us to develop testable hypotheses about the roles of several of these proteins in context to the metabolic pathway connections and metabolite fluxes.