Characterization of two inducible phosphate transport systems in Rhizobium tropici.

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (Pi). To better understand phosphorus movement between the bacteroid and the host plant, Pi transport was characterized in R. tropici. We observed two Pi transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The Km and Vmax values for the low-affinity system were estimated to be 34 6 3 m MP i and 118 6 8 nmol of Pi z min 21 z mg (dry weight) of cells 21 , respectively, and the Km and Vmax values for the high-affinity system were 0.45 6 0.01 m MP i and 86 6 5 nmol of Pi z min 21 z mg (dry weight) of cells 21 , respectively. Both systems were inducible by Pi starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but Pi transport through both systems was eliminated by the ATPase inhibitor N,N*-dicyclohexylcarbodiimide; the Pi transport rate was correlated with the intracellular ATP concentration. Also, Pi movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both Pi transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity. Nitrogen fixation in legume nodules involves a complex exchange of nutrients between the plant and bacteroids. This exchange involves transport across the bacteroid membrane and the plant-derived envelope surrounding the bacteroid, the peribacteroid membrane. In its simplest terms, this symbiosis is often viewed as an exchange of reduced carbon for reduced nitrogen. However, it is clear that optimum nodule function also involves a balanced flow of other nutrients (33). One nutrient that has been shown to be important for this symbiosis is phosphorus. Low phosphorus availability in soils is common and limits legume production worldwide; however, phosphorus metabolism in this plant-microbe interaction has not been well characterized. Given the significant metabolic activity of bacteroids, the phosphorus supply may be critical for optimum symbiotic functioning of bacteroids, and understanding the mechanisms by which bacteroids acquire phosphorus should provide useful information concerning phosphorus exchange between the symbionts and phosphorus flow in the symbiosis. Phosphate (Pi) uptake has been investigated in various bacteria. In some microorganisms only a single transport system has been found. This is the case for Micrococcus lysodeikticus (19) and for several Rhizobium species (41). In other bacteria, two Pi transport systems have been found. Examples of such bacteria include Escherichia coli (34, 53), Acinetobacter johnsonii (48), and Pseudomonas aeruginosa (26). In each of the latter bacteria, a constitutively expressed low-affinity transport system and a Pi-repressible high-affinity permease have been