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.

Characterization of beta-lactamase-deficient (bla) mutants of the R plasmid R1 in Escherichia coli K-12 and comparison with similar mutants of RP1.

Thirty-eight mutants of R1, an R plasmid specifying the type IIIa (TEM) beta-lactamase, were isolated; these mutants are partially or totally unable to synthesize the type IIIa beta-lactamase. The loss of beta-lactamase activity was associated with a reduction in the level of penicillin resistance conferred by the mutants upon their host strain. At least two of the mutants synthesized a beta-lactamase with altered substrate specificity. These properties are compared with those of two beta-lactamase-deficient mutants of plasmid RP1. The results suggest that, for both R plasmids, penicillin resistance is entirely attributable to the presence of beta-lactamase activity. The properties of two R1 derivatives, pUB251 and pUB252, which have phenotypes similar to that of RP1, support this conclusion.

Quantitative correlation between penicillin resistance and beta-lactamase activity specified by the R plasmids R1, R1 bla-45, and RP1 in Escherichia coli K-12.

A mutant of the R plasmid R1 which synthesizes a beta-lactamase with altered kinetic characteristics was isolated. The level of penicillin resistance specified by this plasmid was correctly predicted from the properties of the wild-type R1 according to a simple theoretical model published by Zimmermann and Rosselet (Antimicrob. Agents Chemother. 12:368--372, 1977). The model also accounts for the high level of penicillin resistance specified by the R plasmid RP1.

Translocation of the tetracycline resistance determinant from R100-1 to the Escherichia coli K-12 chromosome.

Pairs of normally incompatible derivatives of R100-1 (one ChlS TetR, the other ChilR TetS) were forced to coexist in a recA host by selection for ChlR TetR cells. After many generations stable derivatives were isolated. The analysis of none independent stabilization experiments showed that in each case TetR was translocated from the plasmid to the chromosome of the host. No evidence for the joint integration of other plasmid genes (those controlling transfer, antibiotic resistance, incompatibility, or origin of transfer replication) was obtained. One of the chromosomal TetR determinants was mapped close to metE.

Male-specific bacteriophage MS2 propagation in fluorophenylalanine-resistant Escherichia coli K12.

Mutation of Escherichia coli K12 HfrH to resistance to fluorophenylalanine resulted in changes in the plaque morphology of bacteriophage MS2 on this strain and led to an increased efficiency of propagation of the phage in liquid cultures. Evidence was obtained that the mutation resulted in inhibition of early lysis in infected cells and that lysis involved the production of a lysozyme. Genetic studies suggested that the observed pleiotropy of the resistance mutation was due to informational suppression.

Deletion map of the chloramphenicol resistance region of R1 and R100-1.

Recombination between single-site and multisite chloramphenicol-sensitive mutants of the F-like R factors R1 and R100-1 indicates that the chloramphenicol resistance region is a single structural gene coding for the 20,000-molecular weight subunit of chloramphenicol acetyltransferase.

Purification of sex pili from Escherichia coli carrying a derepressed F-like R factor.

A procedure for the purification of sex pili is described. Escherichia coli K-12 carrying Rldrd19 was grown in nutrient broth and blended at the time of peak sex pilus production. The cells were removed by centrifugation, and the supernatant fraction was concentrated, dialyzed, and clarified in an ultrafiltration system. After an additional blend and a clearing spin, the material was centrifuged in a CsCl gradient, and the fractions containing the sex pili were subjected to isoelectric focusing. About 5 mg of intact pili of approximately 98% purity were obtained by this method from about 100 g (wet weight) of cells.