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 alf alfa 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 o pen reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G, The putative PhaABC protein s exhibit homology to the subunits of a Na+/H+ antiporter from an alka lophilic Bacillus strain, Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxi doreductase respectively, Computer analysis suggests that all seven pr oteins are highly hydrophobic with several possible transmembrane doma ins, Some of these domains were confirmed by generating active alkalin e phosphatase fusions, ion transport studies on phaA mutant cells reve aled a defect in K+ efflux at alkaline pH after the addition of a memb rane-permeable amine, These results suggest that the pha genes of R. m eliloti encode for a novel type of K+ efflux system that is involved i n pH adaptation and is required for the adaptation to the altered envi ronment inside the plant.