Cloning of the trkAH gene cluster and characterization of the Trk K+-uptake system of Vibrio alginolyticus

Abstract

K+-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, orf1, whose function is not clear. Products of V. alginolyticus trkA and orf1 were detected in E. coli minicells. trkA and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli Delta trkA and an E. coli Delta trkG Delta trkH strain, respectively, suggesting that these V. alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli Delta sapABCDF (Delta trkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K+-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.