Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Abstract

ABSTRACT 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 Tn 5 B22 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 μM 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 μM 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 wild-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.