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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.
Highlights
IntroductionThe meningococcus, is a common inhabitant of the mucosal surface of the oro- and nasopharynx in humans
Neisseria meningitidis, or the meningococcus, is a common inhabitant of the mucosal surface of the oro- and nasopharynx in humans
The primary concern regarding meningococcus colonization is the sudden occurrence of systemic meningococcal disease that can occur in
Summary
The meningococcus, is a common inhabitant of the mucosal surface of the oro- and nasopharynx in humans. The primary concern regarding meningococcus colonization is the sudden occurrence of systemic meningococcal disease that can occur in. The mechanisms that allow some meningococcus strains to disseminate from their local oro-pharyngeal niche and cause acute systemic disease are poorly understood. Most cases of meningococcal disease are caused by clonal complexes of related sequence types (STs), the so-called hyperinvasive lineages (Yazdankhah et al, 2004). These lineages are underrepresented in healthy carriers, and significant numbers of individuals are colonized with carriage isolates belonging to a set of STs that rarely cause disease (Caugant, 2008). Meningococcus cells exhibit abundant antigenic diversity due to frequent recombination, random mutational events, phase variation and high frequencies of horizontal gene transfer
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