Limits...
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.

Show MeSH

Related in: MedlinePlus

N. meningitidis ComL 3D model. The N. meningitidis MC58 ComL hypothetical 3D structure generated by the phyre threading server is shown in cartoon form (centre) with the concurrent surface representation overlaid. The charge distribution, calculated using APBS, is shown on the surface (blue = positive, red = negative). The ComL surface is shown in four further images (in corners) rotated progressively around a horizontal axis in order to give a complete overview of the charge distribution. The large areas of positive charge may indicate that they play a role in DNA binding.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3140584&req=5

f6: N. meningitidis ComL 3D model. The N. meningitidis MC58 ComL hypothetical 3D structure generated by the phyre threading server is shown in cartoon form (centre) with the concurrent surface representation overlaid. The charge distribution, calculated using APBS, is shown on the surface (blue = positive, red = negative). The ComL surface is shown in four further images (in corners) rotated progressively around a horizontal axis in order to give a complete overview of the charge distribution. The large areas of positive charge may indicate that they play a role in DNA binding.

Mentions: Predicted 3D models for the structure of ComL and SSB were generated using the phyre service (Bennett-Lovsey et al., 2008). The sequence identity of ComL relative to known structures was below the 30 % level required to generate a confident homology model, with the best results returning between 11 and 18 % sequence identity. However, the e-values of these alignments were very low (<1e−19), which allowed for the generation of a hypothetical structure by threading. The most compatible structures returned by the server were characterized due to the presence of a tetratricopeptide repeat (TPR) structure; these findings included Pseudomonas aeruginosa PilF (Kim et al., 2006), which was also the component closest in length to ComL. Since sequence-based DNA binding predictions with regard to ComL were uniformly negative, the threading model structure was used as a basis to investigate the possible distribution of positive charges on the surface, thereby yielding a possible explanation for the DNA binding observed. A 3D model structure was generated and the charge on the molecular surface was calculated by using the Adaptive Poisson–Boltzmann Solver (Fig. 6) (Baker et al., 2001). Charge distribution on the ComL surface is shown for a representative range of orientations, demonstrating that there are several regions of positive charge (blue), which may function in specific or non-specific DNA binding.


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)

N. meningitidis ComL 3D model. The N. meningitidis MC58 ComL hypothetical 3D structure generated by the phyre threading server is shown in cartoon form (centre) with the concurrent surface representation overlaid. The charge distribution, calculated using APBS, is shown on the surface (blue = positive, red = negative). The ComL surface is shown in four further images (in corners) rotated progressively around a horizontal axis in order to give a complete overview of the charge distribution. The large areas of positive charge may indicate that they play a role in DNA binding.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: N. meningitidis ComL 3D model. The N. meningitidis MC58 ComL hypothetical 3D structure generated by the phyre threading server is shown in cartoon form (centre) with the concurrent surface representation overlaid. The charge distribution, calculated using APBS, is shown on the surface (blue = positive, red = negative). The ComL surface is shown in four further images (in corners) rotated progressively around a horizontal axis in order to give a complete overview of the charge distribution. The large areas of positive charge may indicate that they play a role in DNA binding.
Mentions: Predicted 3D models for the structure of ComL and SSB were generated using the phyre service (Bennett-Lovsey et al., 2008). The sequence identity of ComL relative to known structures was below the 30 % level required to generate a confident homology model, with the best results returning between 11 and 18 % sequence identity. However, the e-values of these alignments were very low (<1e−19), which allowed for the generation of a hypothetical structure by threading. The most compatible structures returned by the server were characterized due to the presence of a tetratricopeptide repeat (TPR) structure; these findings included Pseudomonas aeruginosa PilF (Kim et al., 2006), which was also the component closest in length to ComL. Since sequence-based DNA binding predictions with regard to ComL were uniformly negative, the threading model structure was used as a basis to investigate the possible distribution of positive charges on the surface, thereby yielding a possible explanation for the DNA binding observed. A 3D model structure was generated and the charge on the molecular surface was calculated by using the Adaptive Poisson–Boltzmann Solver (Fig. 6) (Baker et al., 2001). Charge distribution on the ComL surface is shown for a representative range of orientations, demonstrating that there are several regions of positive charge (blue), which may function in specific or non-specific DNA binding.

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