The Pha Gene Cluster of Rhizobium Meliloti Involved in PH Adaptation and Symbiosis Encodes a Novel Type of K+ Efflux System

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf-/Fix-) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6kb genomic region in which mutations resulted in both Inf-/Fix- and K+-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.