Investigation of the DNA uptake machinery in naturally competent bacteria

Natural transformation is a mode of horizontal gene transfer in bacteria which involves uptake of naked DNA from the environment. The acquired DNA can be incorporated into the bacterial genome and confer useful traits, such as antibiotic resistance and increased virulence. The mechanistic details of how specialised transport proteins allow passage of DNA trough the cell envelope remain elusive. The membrane protein ComEC forms a channel for DNA transport into the cytoplasm but the molecular details of its structure and function are unknown. An extensive expression screen identified ComEC from Moorella thermoacetica as a candidate for structural and biochemical analysis. Low resolution structural data suggest that the two extramembranous domains of ComEC are located on the same side of the membrane and that they interact. Biophysical and biochemical studies of the C-terminal soluble domain of ComEC showed that it has manganese(II)-dependent nuclease activity. It was shown that the nuclease activity of the C-terminal domain is required for efficient transformation in B. subtilis. It is likely that the C-terminal domain degrades the non-transforming strand during DNA uptake. Attempts at isolating DNA uptake complexes for biochemical and structural analysis from competent Bacillus subtilis cells revealed that the competence machinery components are present at very low levels. No native DNA uptake complexes could be isolated. ComFA is a cytoplasmic membrane-associated ATPase which is thought to power DNA uptake through the ComEC channel. A thermophilic homologue of ComFA was expressed, purified and shown to bind both single- and double-stranded DNA. ComFA was also shown to have ATPase activity. A unique N-terminal extension domain was identified in the thermophilic ComFA, but its function remains unknown. Together these data contribute to our understanding of DNA uptake across the cytoplasmic membrane.