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 (P-i), To better understand phosphorus movement between the bacteroid and the host plant, P-i transport was characterized in R, tropici, We observed two P-i 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 K-m and V-max values for the low-affinity system were estimated to be 34 +/- 3 mu M P-i and 118 +/- 8 nmol of P-i . min(-1) . mg (dry weight) of cells(-1), respectively, and the K-m and V-max values for the high-affinity system were 0.45 +/- 0.01 mu M P-i and 86 +/- 5 nmol of P-i . min(-1) . mg (dry weight) of cells(-1), respectively. Both systems were inducible by P-i 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 P-i transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P-i transport rate was correlated with the intracellular ATP concentration. Also, P-i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the mild-type strain or the mutant. These properties suggest that both P-i 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.