Key role of bacterial NH(4)(+) metabolism in Rhizobium-plant symbiosis.

Symbiotic nitrogen fixation is carried out in specialized organs, the nodules, whose formation is induced on leguminous host plants by bacteria belonging to the family Rhizobiaceae: Nodule development is a complex multistep process, which requires continued interaction between the two partners and thus the exchange of different signals and metabolites. NH(4)(+) is not only the primary product but also the main regulator of the symbiosis: either as ammonium and after conversion into organic compounds, it regulates most stages of the interaction, from the production of nodule inducers to the growth, function, and maintenance of nodules. This review examines the adaptation of bacterial NH(4)(+) metabolism to the variable environment generated by the plant, which actively controls and restricts bacterial growth by affecting oxygen and nutrient availability, thereby allowing a proficient interaction and at the same time preventing parasitic invasion. We describe the regulatory circuitry responsible for the downregulation of bacterial genes involved in NH(4)(+) assimilation occurring early during nodule invasion. This is a key and necessary step for the differentiation of N(2)-fixing bacteroids (the endocellular symbiotic form of rhizobia) and for the development of efficient nodules.

Organogenesis of legume root nodules.

The N(2)-fixing nodules elicited by rhizobia on legume roots represent a useful model for studying plant development. Nodule formation implies a complex progression of temporally and spatially regulated events of cell differentiation/dedifferentiation involving several root tissues. In this review we describe the morphogenetic events leading to the development of these histologically well-structured organs. These events include (1) root hair deformation, (2) development and growth of infection threads, (3) induction of the nodule primordium, and (4) induction, activity, and persistence of the nodular meristem and/or of foci of meristematic activities. Particular attention is given to specific aspects of the symbiosis, such as the early stages of intracellular invasion and to differentiation of the intracellular form of rhizobia, called symbiosomes. These developmental aspects were correlated with (1) the regulatory signals exchanged, (2) the plant genes expressed in specific cell types, and (3) the staining procedures that allow the recognition of some cell types. When strictly linked with morphogenesis, the nodulation phenotypes of plant and bacterial mutants such as the developmental consequence of the treatment with metabolic inhibitors, metabolic intermediates, or the variation of physical parameters are described. Finally, some aspects of nodule senescence and of regulation of nodulation are discussed.

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.

Glutamine utilization by Rhizobium etli.

We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli. Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase. The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln). We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult. Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress. Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.