N-Terminal Quarter Part of Tetracycline Transporter from pACYC184 Complements K⁺ Uptake Activity in K⁺ Uptake-Deficient Mutants of Escherichia coli and Vibrio alginolyticus

Abstract

In an attempt to clone a gene encoding the K⁺ uptake system from Vibrio alginolyticus, two plasmids, pKT2 and pKT4, were derived from pACYC184. These plasmids allowed the growth of K⁺ uptake-deficient mutant strains of Escherichia coli TK420 and V. alginolyticus FS181 in a low K⁺ medium. The pKT2 and pKT4 had an insertion about 7 and 6kb, respectively, from the genome of V. alginolyticus. We prepared deletion plasmids from both plasmids and found that the site of genes inserted in the two was not identical and that the active locus corresponded to the structural gene encoding the N-terminal quarter part of tetA (C) gene. The N-terminal region of tetA (C) gene was ligated in another vector plasmid pHG165 to produce pHGK23. pHGK23 complemented the growth of TK420 in the low K⁺ medium. It contained only 62bp from the genome of V. alginolyticus, and the open reading frame was composed of 98 amino acid residues from the N-terminal quarter part of tetA (C) and 5 amino acid residues attached by gene fusion. Using the Na⁺-loaded cells of TK420, pHGK23 was found to increase the activity of K⁺ uptake. These results show that the N-terminal side of tetA (C) gene product functions as a K⁺ uptake system.