Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes.

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance.

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.

Reduced expression of Tn 10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon.

In Escherichia coli a single copy of Tn10 confers high-level resistance to tetracycline. Resistance itself results from expression of three distinct mechanisms which normally act together (Shales et al., 1980). In cells containing two copies of Tn10, the level of resistance to tetracycline was reduced. This was not due to overproduction of the repressor which controls the resistance genes, because strains diploid for an operator-constitutive allele of Tn10 also exhibited reduced expression of resistance. The negative gene dosage effect resulted from decreased expression of two mechanisms (1 and 2) consequent on enhanced expression of the third mechanism. The net result of increasing the copy number was a decrease in resistance because mechanism 3 was less efficient than mechanisms 1 and 2 in protecting the cell against tetracycline. The DNA sequence responsible for the reduced expression of resistance was contained in the internal BglII fragment of Tn10. This sequence, which is probably unique to Tn10, may encode the protein which mediates mechanism 3. Elevated levels of this protein probably cause decreased expression of mechanisms 1 and 2.

Evidence for more than one mechanism of plasmid-determined tetracycline resistance in Escherichia coli.

The basis of tetracycline resistance mediated by TetA determinants and joint resistance to tetracycline and minocycline coded by TetB determinants was investigated. The TetA class of determinants was represented by those carried on plasmids pSC101, RP1 and pIP7 and TetB by Tn10. The relationships between expression of tetracycline and minocycline resistance and accumulation of these antibiotics suggest that there are three mechanisms of plasmid-determined resistance conferring (1) about a 10- to 20-fold increase in resistance to tetracycline that is not associated with decreased antibiotic accumulation, (2) a 4- to 7-fold increase in resistance to tetracycline that is associated with decreased drug accumulation, and (3) about a 2- to 3-fold increase in resistance to both tetracycline and minocycline that is not associated with decreased accumulation of either antibiotic. Mechanism 1 was coded by the tetracycline resistance determinant of pSC101 (TetA), mechanisms 1 and 2 by the determinants in RP1 and pIP7 (TetA) and all three mechanisms by Tn10 (TetB).

Comparison of the polypeptide composition of Escherichia coli outer membranes prepared by two methods.

Escherichia coli outer membranes were prepared by centrifugation to equilibrium in sucrose gradients and then treated with Sarkosyl in the presence of ethylenediaminetetraacetate. The polypeptide profiles of the two outer membrane preparations were compared by two-dimensional polyacrylamide gel electrophoresis. The patterns obtained were not identical, and Sarkosyl removed several minor proteins from the outer membrane.

Litter accumulation in woodlands contaminated by Pb, Zn, Cd and Cu.

Close to a primary lead-zinc-cadmium smelter the standing crop of litter in woodlands was found to be elevated relative to more distant sites. The total litter accumulation is similar to that from contaminated sites reported by other authors but in this case the concentrations of heavy metals are considerably lower than those reported for other sites. Evidence is provided to support the hypothesis that within the woodlands studied, litter accumulation is not closely pH dependent, but is clearly related to both cadmium and zinc concentrations in litter. Litter accumulation occurs in certain particle size ranges and fractionation shows that the weight of accumulated litter in these size ranges is highly correlated to cadmium concentrations. These results are discussed in relation to the reported possible long term effects of metal contamination on decomposition processes and the possibility of adaptation to these adverse effects.