At one time DNA uptake was thought to be mediated by membrane-bound vesicles called transformasomes (Goodgal and Smith lab papers). These structures appear as surface 'blebs' in electron mocrographs of competent H. influenzae and H. parainfluenzae and to a lesser extent on non-competent cells. Blebs often contain DNA which is inaccessible both to externally added nucleases and to the restriction enzymes present in the cell's cytoplasm. They appear to be internalized on DNA uptake (only in H. para?), and are shed into the medium by some competence mutants and when normal cells lost competence. Purified blebs bind DNA tightly and with the same specificity as intact cells but do not internalize it (???).
The name transformasomes resulted from the interpretation that these blebs were structural adaptations for DNA uptake. However microbiologists have subsequently realized that similar blebs are often produced when gram negative bacteria are stressed (Beveridge papers), and they are no longer thought to be specialized for DNA uptake. Rather, they are thought to contain DNA uptake machinery only because this is located in thecell envelope from which the blebs form.
However, it is still possible that those parts of the envelope containing the DNA uptake machinery are particularly likely to form blebs. The proposal will describe using competence mutants to test this. Even if blebs are not enriched for DNA uptake machinery relative to the cell envelope, they lack all of the cytoplasmic proteins and this may be a valuable tool for characterizing competence-specific proteins and protein-DNA interactions. Their ability to bind DNA tightly without taking it up will be especially useful.
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