Meningococcal inner membrane proteins and their role in transformation
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AbstractNeisseria meningitidis, the meningococcus, is one of the main causes of meningitis and septicaemia in children worldwide. Horizontal gene transfer, such as transformation, transduction and conjugation, generate much of the genetic diversity observed in bacterial species. The main source of DNA taken up by meningococci is transformation, however, the distinct steps of the transformation pathway are poorly understood.
The expression of filamentous appendages, pili, is a prerequisite for DNA uptake by transformation in the meningococcus. The pilus fibre is a dynamic structure moving in and out through the PilQ complex in the bacterial outer membrane. Since pilus expression and DNA uptake through transformation are so tightly linked, it has been hypothesized that DNA is taken up into the bacterial cell during pilus retraction.
The purpose of the present thesis was to identify and characterize neisserial DNA binding components and investigate their role in transformation. The main goal was to study inner membrane (IM) proteins, since DNA uptake through the outer membrane PilQ complex has previously been described. DNA binding proteins in enriched inner membranes were identified by South-western assays and band-shift analysis. A number of DNA binding candidates were detected, and the corresponding knock-out mutants were tested for competence for transformation. The IM pilus biogenesis protein PilG was found to possess considerable DNA binding activity and was shown to interact with PilQ, suggesting a direct role for PilG in transformation. Furthermore, the lipoprotein ComL interacted with DNA and PilG, thus ComL might mediate transport of DNA over the neisserial membranes. The characterization of these novel DNA binding components sheds new light on the fundamental transformation process.
List of publications
I Emma Lång, Kristine Haugen, Burkhard Fleckenstein, Håvard Homberset, Stephan A. Frye, Ole Herman Ambur, and Tone Tønjum. Identification of neisserial DNA binding components. Microbiology. 2009 Mar;155(Pt 3):852-862
II Afsaneh V. Benam, Emma Lång, Kristian Alfsnes, Håvard Homberset, Burkhard Fleckenstein, Alexander D. Rowe, Ole Herman Ambur, Stephan A. Frye, and Tone Tønjum. The competence lipoprotein ComL directly interacts with DNA. Manuscript in preparation.
III Emma Lång, Seetha Balasingham, Håvard Homberset, Alexander D. Rowe, Burkhard Fleckenstein, Ole Herman Ambur, Stephan A. Frye, and Tone Tønjum. The neisserial inner membrane protein PilG directly interacts with DNA and pilus biogenesis components in transformation. Manuscript in preparation.