Abstract
The transposon Tn10-encoded tetracycline resistance protein TetA is an integral membrane protein responsible for the export of tetracycline from the cytoplasmic to the periplasmic side of the inner membrane of Gram-negative bacteria. From a plot of the average hydrophobicity along the sequence of this protein, a two-dimensional membrane topology with 12 transmembrane domains may be predicted. Using plasmid-bearing Escherichia coli maxicells we specifically radiolabeled the TetA protein. The amino terminus of this membrane protein was shown not to be processed, and its location on the inner side of the cytoplasmic membrane was demonstrated by a newly developed use of a chemical method. Spheroplasts and inside-out vesicles of the TetA protein synthesizing maxicells were subjected to limited digestion by proteases of different specificities. The TetA protein was not accessible to proteases from the periplasmic side. On the inner side of the cytoplasmic membrane, the carboxyl terminus and four sites accessible to endoproteases could be identified. The cleavage sites are proposed to be localized between amino acid residues 60-70, 110-130, 180-200, and at amino acid 327. These results allow the definition of a model for the two-dimensional topology of the TetA protein.
Highlights
Resistance appears to be based on a reduced intracellular concentration of the antibiotic [8,9]
A genetic approach, fusion of the E. coli alkaline phosphatase gene phoA to sites within a gene encoding a membrane protein, has been developed, which may allowthe localization of domains of the integral membrane protein that are located on the outside of the cytoplasmic membrane [20]
To characterize the topology of the tetracycline resistance protein within the cytoplasmic membrane, we have analyzed its susceptibility to digestion by various proteases from inside as well as outside
Summary
Plasmid pCB113 was constructed by insertion of a 2.8-kilobase EglII fragment from TnlO, containing genes tetA and tetR [24], into the singular BglII site of pFD51 [25]. Subsequent deletion of the DNA between a singular EcoRI site within the HindIII fragment and a singular EcoRI site within the polylinker of the vector plasmid resulted in plasmid pCB301 In this way the distal half of the tetA gene was deleted. Bovine serum albumin was added to 200 pglml, and the proteins were precipitated with trichloroacetic acid, washed once with acetone, and subjected to SDS-PAGE. After washing the gel slices in HzO theywere incubated with 30% hydrochloric acid for 1 h a t 100 "C under nitrogen This resulted in the cleavage of the cyanate-modified firstamino acid as a hydantoin derivative. The dried plates were sprayed with 20% 2,5-diphenyloxazole dissolved in dimethyl sulfoxide and used for fluorography
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