Osmolability of Escherichia coli and modification of [¹²⁵I]ampicillin-binding by competence induction for uptake of transforming DNA

Abstract

The regimen conferring competence for uptake of transforming DNA is shown to render Escherichia coli osmolabile. Three different K-12 strains were exposed to the standard procedure of competence induction, i.e. incubation in the presence of 0.1 M Ca²⁺ or Mg²⁺ for 50 min at 0°C, interrupted by a heat shock for 5 min at 37°C. Upon osmotic challenge of competent cells formation of protoplasts was observed in approximately 2% of the treated cells. Incubation of competent cells of strain W1485 in phosphate-buffered saline for 1, 2, and 3 h reduced the viable counts to 67, 58, and 41%, respectively. Competence induction with divalent cations altered the affinity of penicillin-binding proteins (PBPs) for [¹²⁵I]ampicillin. In isolated cell envelopes the presence of Ca²⁺ and Mg²⁺ stimulated the binding of [¹²⁵I]ampicillin to PBPs 1, 3, 4, 5, and 6, whereas the binding to PBP 2 remained unchanged. The binding to PBP 1 C was inhibited by 0.23 M Ca²⁺. In living cells the binding to PBPs 1, 3, and 4 was enhanced, while the binding to PBP 8 was inhibited. Newly [¹²⁵I]ampicillin-labelled proteins of M _r 55,000 and 45,000 were apparent, especially after competence induction with Ca²⁺. Interaction of divalent cations with PBPs is suggested to contribute to osmolability of competent cells. Disintegration of the cell wall may be necessary for uptake of transforming DNA.