ABSTRACT
Transformation of uropathogenic Escherichia coli strains with plasmid DNA was in general unsuccessful or very inefficient. Transformation was much more efficient when galE mutants of such strains, in which the lipopolysaccharide chains appeared shorter, were used as recipients.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Lipopolysaccharides/physiology , Plasmids , Transformation, Bacterial , Escherichia coli/pathogenicity , Galactose/metabolism , Lipopolysaccharides/analysis , Mutation , Urinary Tract/microbiology , VirulenceABSTRACT
Cloning techniques make it possible to accommodate bacterial genes on vector DNA molecules. On that basis the investigation of bacterial structures and functions got new impetus. The potentials of molecular genetics for detailed analysis of bacterial structures are illustrated in this paper for the gene cluster involved in the expression of F72 fimbriae associated with a uropathogenic Escherichia coli O6:K2:H1:F7 strain.
Subject(s)
Escherichia coli/genetics , Fimbriae, Bacterial , Genes, Bacterial , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , DNA, Bacterial/analysis , DNA, Recombinant/analysis , Nucleic Acid Hybridization , NucleotidesABSTRACT
Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.
