Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence

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Abstract
Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in H. influenzae was accompanied by the generation on the cell surface of membrane extensions (blebs) 80-100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+ or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (> 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

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