Structure and function of the class C tetracycline/H⁺antiporter

Abstract

The class C tetracycline/H+ antiporter, TetA(C), confers nine distinct phenotypes in Escherichia coli: resistance to tetracycline, reduced culture density at stationary phase (growth yield), increased supercoiling of plasmid DNA, delayed growth in succinate minimal medium, complementation of potassium uptake defects, increased susceptibility to cadmium, increased susceptibility to fusaric acid, increased susceptibility to bleomycin and increased susceptibility to several classes of cationic aminoglycoside antibiotics. These nine phenotypes were resolved into three 'linkage' groups based on their patterns of suppression by mutations of the tetA(C) gene of plasmid pBR322. Group I includes resistance to tetracycline, increased susceptibility to cadmium and reduced growth yield. Group II includes delayed growth in succinate minimal medium and complementation of potassium uptake defects. Group III includes increased supercoiling of plasmid DNA and increased susceptibilities to fusaric acid, bleomycin and cationic aminoglycosides. Phenotypes of Groups II and III, but not Group I, also were conferred by a chimeric gene encoding a fusion between the N-terminal 34 residues of TetA(C) and the C-terminal 429 residues of a structurally-similar protein, the E. coli galactose/H+ symporter, GalP. In contrast, none of these phenotypes was conferred by a chimeric gene encoding a fusion between the N-terminal 34 residues of TetA(C) and a structurally-dissimilar protein, TEM beta-lactamase. These results demonstrate that the three groups of linked phenotypes are dependent on different elements of the TetA(C) amino acid sequence, implying that TetA(C) confers these phenotypes by at least three independent mechanisms.

Reengineering a Natural Riboswitch by Dual Genetic Selection

“…The TPP riboswitch encoded in the 5′ UTR of Escherichia coli thiM 3a was subcloned in the 5′ UTR of tetA which encodes the class C tetracycline/H + antiporter derived from the plasmid pBR322. 4 A riboswitch plasmid library was constructed by incorporating up to 30 degenerate bases between the TPP aptamer and the Shine-Dalgarno (SD) sequence using polymerase chain reaction (PCR) (Figure 1A, also see Supporting Information) and transformed into E. coli TOP10 cells, which yielded approximately 75 000 unique riboswitch clones.…”

Divergent Directed Evolution of a TetR-type Repressor Towards Aromatic Molecules

“…Fluorescence activated cell-sorting has been used extensively for dual selection 53,56 , and genes that encode multiple phenotypes such as TolC 57 and thymidine kinases 58 have been used as dual selection markers. To this end, we employ the E. coli TetA antiporter as a dual selection marker to couple aTF activity to cell growth 38,39,59 , which requires tetracycline and NiCl 2 for positive and negative selection, respectively 60 . We extended the reporter module by inserting TetA downstream of GFP for polycistronic expression (Fig.…”

Evolutionary innovation using EDGE, a system for localized elevated mutagenesis

“…the TetA efflux pump as the target gene. Expression of active tetA increases E. coli sensitivity to the antibiotic apramycin [25], and loss of tetA function allows those variants to grow on plates containing apramycin. Following IPTG induction of EDGE, we observe a seven-fold increase in the number of apramycin resistant mutants than without IPTG, (34.7 versus 5 mutants /10 5 cells, Fig 2b).…”