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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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ComL co-purifies with the outer membrane and is expressed at high levels. (a) Analysis of cellular fractions after separation of outer and inner membranes from N. meningitidis M1080 by sucrose-gradient centrifugation. Samples from each fraction were separated by SDS-PAGE and stained with Coomassie blue (top panel) or detected by immunoblotting using antibodies against ComL, PilG, PilP, PilQ and PilW (lower panels). (b) Quantitative immunoblotting of a defined amount of N. meningitidis M1080 cellular suspension using the same antibodies as in (a).
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f3: ComL co-purifies with the outer membrane and is expressed at high levels. (a) Analysis of cellular fractions after separation of outer and inner membranes from N. meningitidis M1080 by sucrose-gradient centrifugation. Samples from each fraction were separated by SDS-PAGE and stained with Coomassie blue (top panel) or detected by immunoblotting using antibodies against ComL, PilG, PilP, PilQ and PilW (lower panels). (b) Quantitative immunoblotting of a defined amount of N. meningitidis M1080 cellular suspension using the same antibodies as in (a).

Mentions: In order to determine the subcellular localization of ComL, meningococcus outer and inner membranes were separated by sucrose density gradient centrifugation. Fractions from the sucrose gradient were analysed by immunoblotting using antisera against ComL, PilG, PilP, PilQ and PilW (Fig. 3a). PilQ and PilP have previously been shown to reside in the outer and inner membrane, respectively (Balasingham et al., 2007), while PilW resides in the outer membrane, interacting with PilQ (Carbonnelle et al., 2005; Trindade et al., 2008). The main amounts of ComL and PilW peaked with PilQ in the higher-density outer membrane fractions, while PilG and the lipoprotein PilP were concentrated in the inner membrane gradient fractions with very low levels detected in the higher-density fractions, demonstrating that ComL co-purifies with the outer membrane. ComL was shown to be expressed at levels as high as the secretin PilQ (Fig. 3b).


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)

ComL co-purifies with the outer membrane and is expressed at high levels. (a) Analysis of cellular fractions after separation of outer and inner membranes from N. meningitidis M1080 by sucrose-gradient centrifugation. Samples from each fraction were separated by SDS-PAGE and stained with Coomassie blue (top panel) or detected by immunoblotting using antibodies against ComL, PilG, PilP, PilQ and PilW (lower panels). (b) Quantitative immunoblotting of a defined amount of N. meningitidis M1080 cellular suspension using the same antibodies as in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: ComL co-purifies with the outer membrane and is expressed at high levels. (a) Analysis of cellular fractions after separation of outer and inner membranes from N. meningitidis M1080 by sucrose-gradient centrifugation. Samples from each fraction were separated by SDS-PAGE and stained with Coomassie blue (top panel) or detected by immunoblotting using antibodies against ComL, PilG, PilP, PilQ and PilW (lower panels). (b) Quantitative immunoblotting of a defined amount of N. meningitidis M1080 cellular suspension using the same antibodies as in (a).
Mentions: In order to determine the subcellular localization of ComL, meningococcus outer and inner membranes were separated by sucrose density gradient centrifugation. Fractions from the sucrose gradient were analysed by immunoblotting using antisera against ComL, PilG, PilP, PilQ and PilW (Fig. 3a). PilQ and PilP have previously been shown to reside in the outer and inner membrane, respectively (Balasingham et al., 2007), while PilW resides in the outer membrane, interacting with PilQ (Carbonnelle et al., 2005; Trindade et al., 2008). The main amounts of ComL and PilW peaked with PilQ in the higher-density outer membrane fractions, while PilG and the lipoprotein PilP were concentrated in the inner membrane gradient fractions with very low levels detected in the higher-density fractions, demonstrating that ComL co-purifies with the outer membrane. ComL was shown to be expressed at levels as high as the secretin PilQ (Fig. 3b).

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