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Structure-function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation.

Benam AV, Lång E, Alfsnes K, Fleckenstein B, Rowe AD, Hovland E, Ambur OH, Frye SA, Tønjum T - Microbiology (Reading, Engl.) (2011)

Bottom Line: In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.

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.

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Identification of DNA binding components by a 1D solid-phase overlay approach. 1D Solid-phase overlay analysis of proteins isolated from the inner membrane and soluble fractions of neisserial strains. (a) 1D Coomassie-blue-stained SDS-PAGE of the inner membrane fraction of meningococcus strain H44/76. (b) Identification of DNA binding components in the inner membrane fraction of meningococcus strain H44/76. (c) 1D Coomassie-blue-stained SDS-PAGE of meningococcus soluble fractions. (d) Identification of SSB as a DNA binding component. Lanes: 1, H44/76; 2, MC58; 3, Z2491. The DNA substrate used in the assays depicted was ssDNA containing the 10 bp DUS. Positions of the size standards (kDa) are shown on the left and arrows on the right indicate the position of the proteins identified by MS analysis.
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f1: Identification of DNA binding components by a 1D solid-phase overlay approach. 1D Solid-phase overlay analysis of proteins isolated from the inner membrane and soluble fractions of neisserial strains. (a) 1D Coomassie-blue-stained SDS-PAGE of the inner membrane fraction of meningococcus strain H44/76. (b) Identification of DNA binding components in the inner membrane fraction of meningococcus strain H44/76. (c) 1D Coomassie-blue-stained SDS-PAGE of meningococcus soluble fractions. (d) Identification of SSB as a DNA binding component. Lanes: 1, H44/76; 2, MC58; 3, Z2491. The DNA substrate used in the assays depicted was ssDNA containing the 10 bp DUS. Positions of the size standards (kDa) are shown on the left and arrows on the right indicate the position of the proteins identified by MS analysis.

Mentions: Proteins in the enriched meningococcus membranes were assessed with regard to their DNA binding activity in a solid-phase overlay assay. Among the multiple DNA binding bands detected in cellular fractions from meningococcus strains representing the major serogroups and one gonococcus strain, a 29 kDa band was the most predominant (Fig. 1a and b). This and four additional reproducibly detected bands were selected for identification. The experiment was performed with ssDNA and dsDNA substrates, with or without DUS. The 29 kDa DNA binding component was shown to interact with both ssDNA and dsDNA substrates, and the DNA binding observed was not enhanced by the presence of DUS.


Structure-function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation.

Benam AV, Lång E, Alfsnes K, Fleckenstein B, Rowe AD, Hovland E, Ambur OH, Frye SA, Tønjum T - Microbiology (Reading, Engl.) (2011)

Identification of DNA binding components by a 1D solid-phase overlay approach. 1D Solid-phase overlay analysis of proteins isolated from the inner membrane and soluble fractions of neisserial strains. (a) 1D Coomassie-blue-stained SDS-PAGE of the inner membrane fraction of meningococcus strain H44/76. (b) Identification of DNA binding components in the inner membrane fraction of meningococcus strain H44/76. (c) 1D Coomassie-blue-stained SDS-PAGE of meningococcus soluble fractions. (d) Identification of SSB as a DNA binding component. Lanes: 1, H44/76; 2, MC58; 3, Z2491. The DNA substrate used in the assays depicted was ssDNA containing the 10 bp DUS. Positions of the size standards (kDa) are shown on the left and arrows on the right indicate the position of the proteins identified by MS analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3140584&req=5

f1: Identification of DNA binding components by a 1D solid-phase overlay approach. 1D Solid-phase overlay analysis of proteins isolated from the inner membrane and soluble fractions of neisserial strains. (a) 1D Coomassie-blue-stained SDS-PAGE of the inner membrane fraction of meningococcus strain H44/76. (b) Identification of DNA binding components in the inner membrane fraction of meningococcus strain H44/76. (c) 1D Coomassie-blue-stained SDS-PAGE of meningococcus soluble fractions. (d) Identification of SSB as a DNA binding component. Lanes: 1, H44/76; 2, MC58; 3, Z2491. The DNA substrate used in the assays depicted was ssDNA containing the 10 bp DUS. Positions of the size standards (kDa) are shown on the left and arrows on the right indicate the position of the proteins identified by MS analysis.
Mentions: Proteins in the enriched meningococcus membranes were assessed with regard to their DNA binding activity in a solid-phase overlay assay. Among the multiple DNA binding bands detected in cellular fractions from meningococcus strains representing the major serogroups and one gonococcus strain, a 29 kDa band was the most predominant (Fig. 1a and b). This and four additional reproducibly detected bands were selected for identification. The experiment was performed with ssDNA and dsDNA substrates, with or without DUS. The 29 kDa DNA binding component was shown to interact with both ssDNA and dsDNA substrates, and the DNA binding observed was not enhanced by the presence of DUS.

Bottom Line: In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology and Neuroscience, Institute of Microbiology, University of Oslo, NO-0027 Oslo, Norway.

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.

Show MeSH
Related in: MedlinePlus