title: Structure-function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation.
abstract: Neisseria meningitidis, the meningococcus, is naturally competent for transformation throughout its growth cycle. The uptake of exogenous DNA into the meningococcus cell during transformation is a multi-step process. Beyond the requirement for type IV pilus expression for efficient transformation, little is known about the neisserial proteins involved in DNA binding, uptake and genome integration. This study aimed to identify and characterize neisserial DNA binding proteins in order to further elucidate the multi-factorial transformation machinery. The meningococcus inner membrane and soluble cell fractions were searched for DNA binding components by employing 1D and 2D gel electrophoresis approaches in combination with a solid-phase overlay assay with DNA substrates. Proteins that bound DNA were identified by MS analysis. In the membrane fraction, multiple components bound DNA, including the neisserial competence lipoprotein ComL. In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant. The DNA binding activity of the recombinant ComL and SSB proteins purified to homogeneity was verified by electromobility shift assay, and the ComL-DNA interaction was shown to be Mg²+-dependent. In 3D models of the meningococcus ComL and SSB predicted structures, potential DNA binding sites were suggested. ComL was found to co-purify with the outer membrane, directly interacting with the secretin PilQ. The combined use of 1D/2D solid-phase overlay assays with MS analysis was a useful strategy for identifying DNA binding components. The ComL DNA binding properties and outer membrane localization suggest that this lipoprotein plays a direct role in neisserial transformation, while neisserial SSB is a DNA binding protein that contributes to the terminal part of the transformation process.
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Afsaneh V
authors.initials: AV
authors.lastname: Benam
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Emma
authors.initials: E
authors.lastname: Lång
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Kristian
authors.initials: K
authors.lastname: Alfsnes
authors.affiliation: Centre for Immune Regulation, Institute of Immunology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Fleckenstein
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Rowe
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Eirik
authors.initials: E
authors.lastname: Hovland
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Ole Herman
authors.initials: OH
authors.lastname: Ambur
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Frye
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Oslo University Hospital (Rikshospitalet), NO-0027 Oslo, Norway.|Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.
authors.forename: Tone
authors.initials: T
authors.lastname: Tønjum
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D009345:Neisseria meningitidis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008074:Lipoproteins