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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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N. meningitidis SSB 3D model. The N. meningitidis MC58 SSB predicted 3D structure modelled by homology using the swiss-model service is shown here as a dimer, substituted into the crystal structure for E. coli SSB bound to ssDNA. This illustrates a functional mode for ssDNA binding by SSB.
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f7: N. meningitidis SSB 3D model. The N. meningitidis MC58 SSB predicted 3D structure modelled by homology using the swiss-model service is shown here as a dimer, substituted into the crystal structure for E. coli SSB bound to ssDNA. This illustrates a functional mode for ssDNA binding by SSB.

Mentions: In contrast, neisserial SSB had 33.6 % sequence identity with a known 3D crystal structure from Thermus aquaticus (Fedorov et al., 2006), which allowed the automated generation of a homology model by means of the swiss-model server pipeline (Arnold et al., 2006). An additional structural SSB homologue from E. coli has been crystallized in its multimeric form, bound to ssDNA (Raghunathan et al., 2000). The 3D model structure for neisserial SSB has been substituted as a dimer into this structure in order to illustrate the predicted meningococcus SSB mode of binding for ssDNA (Fig. 7).


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 SSB 3D model. The N. meningitidis MC58 SSB predicted 3D structure modelled by homology using the swiss-model service is shown here as a dimer, substituted into the crystal structure for E. coli SSB bound to ssDNA. This illustrates a functional mode for ssDNA binding by SSB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: N. meningitidis SSB 3D model. The N. meningitidis MC58 SSB predicted 3D structure modelled by homology using the swiss-model service is shown here as a dimer, substituted into the crystal structure for E. coli SSB bound to ssDNA. This illustrates a functional mode for ssDNA binding by SSB.
Mentions: In contrast, neisserial SSB had 33.6 % sequence identity with a known 3D crystal structure from Thermus aquaticus (Fedorov et al., 2006), which allowed the automated generation of a homology model by means of the swiss-model server pipeline (Arnold et al., 2006). An additional structural SSB homologue from E. coli has been crystallized in its multimeric form, bound to ssDNA (Raghunathan et al., 2000). The 3D model structure for neisserial SSB has been substituted as a dimer into this structure in order to illustrate the predicted meningococcus SSB mode of binding for ssDNA (Fig. 7).

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