A Novel Regulatory Pathway for K + Uptake in the Legume Symbiont Azorhizobium caulinodans in Which TrkJ Represses the kdpFABC Operon at High Extracellular K + Concentrations

Abstract

ABSTRACT Bacteria have multiple K + uptake systems. Escherichia coli , for example, has three types of K + uptake systems, which include the low-K + -inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata , also has three types of K + uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH , designated trkI and trkJ . We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI , trkJ , and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δ kup and Δ kup Δ kdpA mutants formed Fix – nodules, while the Δ kup Δ trkA Δ trkI Δ trkJ mutant formed Fix + nodules, suggesting that with the additional deletion of Trk system genes in the Δ kup mutant, Fix + nodule phenotypes were recovered. kdpFABC of the Δ kup Δ trkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K + conditions. However, kdpFABC of the Δ kup Δ trkA Δ trkI Δ trkJ mutant was highly expressed even under high-K + conditions. The cytoplasmic K + levels in the Δ kup Δ trkA Δ trkI mutant, which did not express kdpFABC under high-K + conditions, were markedly lower than those in the Δ kup Δ trkA Δ trkI Δ trkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K + concentrations and that the TrkAI system is unable to function in stem nodules. IMPORTANCE K + is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K + uptake systems to control the cytoplasmic K + levels. In many bacteria, the K + uptake system KdpFABC is expressed under low-K + conditions. For years, many researchers have argued over how bacteria sense K + concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K + concentrations, the detailed mechanism of K + sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K + concentration and that high extracellular K + concentrations repress the expression of KdpFABC via TrkJ.