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 recombinant ComL and SSB exhibit DNA binding activity. The DNA binding abilities of purified recombinant full-length ComL and SSB proteins were assessed by EMSA. (a) ComL binds to ssDNA substrate without the DUS. A protein concentration between 0.66 and 1.3 µM is required to shift 50 % of the ssDNA substrate, indicating an apparent dissociation constant of 0.66–1.3 µM. (b) ComL binds to dsDNA substrate without DUS. A protein concentration between 0.33 and 0.66 µM is required to shift 50 % of the dsDNA substrate, indicating an apparent dissociation constant of 0.33–0.66 µM. A 50 ng sample of recombinant meningococcus Fpg was used as a positive control. (c, d) The same results were obtained with ssDNA and dsDNA substrates with the 12 bp DUS. (e) SSB binds to both ssDNA and dsDNA, though with a strong preference for ssDNA. A protein concentration between 0.25 and 0.5 µM is required to shift 50 % of the ssDNA substrate, and between 10 and 100 µM is required to shift 50 % of the dsDNA substrate. This indicates an approximately 150-fold higher affinity for ssDNA than for dsDNA. atDUS, 12 bp DUS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: N. meningitidis recombinant ComL and SSB exhibit DNA binding activity. The DNA binding abilities of purified recombinant full-length ComL and SSB proteins were assessed by EMSA. (a) ComL binds to ssDNA substrate without the DUS. A protein concentration between 0.66 and 1.3 µM is required to shift 50 % of the ssDNA substrate, indicating an apparent dissociation constant of 0.66–1.3 µM. (b) ComL binds to dsDNA substrate without DUS. A protein concentration between 0.33 and 0.66 µM is required to shift 50 % of the dsDNA substrate, indicating an apparent dissociation constant of 0.33–0.66 µM. A 50 ng sample of recombinant meningococcus Fpg was used as a positive control. (c, d) The same results were obtained with ssDNA and dsDNA substrates with the 12 bp DUS. (e) SSB binds to both ssDNA and dsDNA, though with a strong preference for ssDNA. A protein concentration between 0.25 and 0.5 µM is required to shift 50 % of the ssDNA substrate, and between 10 and 100 µM is required to shift 50 % of the dsDNA substrate. This indicates an approximately 150-fold higher affinity for ssDNA than for dsDNA. atDUS, 12 bp DUS.

Mentions: Native ComL and SSB were identified as neisserial DNA binding proteins by using 1D and 2D solid-phase overlay assays in combination with MS analysis (Figs 1 and 2) (Lång et al., 2009). The DNA binding activity of recombinant ComL and SSB was verified by EMSA (Fig. 4). The ComL DNA binding activity observed was dependent on the presence of Mg2+ and was abolished when EMSA buffers containing EDTA or no additive were used (Fig. 5). Similar results were obtained with all DNA substrates employed (Supplementary Table S2). The DNA binding activities of recombinant ComL and SSB were not DUS-specific (Fig. 4).


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 recombinant ComL and SSB exhibit DNA binding activity. The DNA binding abilities of purified recombinant full-length ComL and SSB proteins were assessed by EMSA. (a) ComL binds to ssDNA substrate without the DUS. A protein concentration between 0.66 and 1.3 µM is required to shift 50 % of the ssDNA substrate, indicating an apparent dissociation constant of 0.66–1.3 µM. (b) ComL binds to dsDNA substrate without DUS. A protein concentration between 0.33 and 0.66 µM is required to shift 50 % of the dsDNA substrate, indicating an apparent dissociation constant of 0.33–0.66 µM. A 50 ng sample of recombinant meningococcus Fpg was used as a positive control. (c, d) The same results were obtained with ssDNA and dsDNA substrates with the 12 bp DUS. (e) SSB binds to both ssDNA and dsDNA, though with a strong preference for ssDNA. A protein concentration between 0.25 and 0.5 µM is required to shift 50 % of the ssDNA substrate, and between 10 and 100 µM is required to shift 50 % of the dsDNA substrate. This indicates an approximately 150-fold higher affinity for ssDNA than for dsDNA. atDUS, 12 bp DUS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: N. meningitidis recombinant ComL and SSB exhibit DNA binding activity. The DNA binding abilities of purified recombinant full-length ComL and SSB proteins were assessed by EMSA. (a) ComL binds to ssDNA substrate without the DUS. A protein concentration between 0.66 and 1.3 µM is required to shift 50 % of the ssDNA substrate, indicating an apparent dissociation constant of 0.66–1.3 µM. (b) ComL binds to dsDNA substrate without DUS. A protein concentration between 0.33 and 0.66 µM is required to shift 50 % of the dsDNA substrate, indicating an apparent dissociation constant of 0.33–0.66 µM. A 50 ng sample of recombinant meningococcus Fpg was used as a positive control. (c, d) The same results were obtained with ssDNA and dsDNA substrates with the 12 bp DUS. (e) SSB binds to both ssDNA and dsDNA, though with a strong preference for ssDNA. A protein concentration between 0.25 and 0.5 µM is required to shift 50 % of the ssDNA substrate, and between 10 and 100 µM is required to shift 50 % of the dsDNA substrate. This indicates an approximately 150-fold higher affinity for ssDNA than for dsDNA. atDUS, 12 bp DUS.
Mentions: Native ComL and SSB were identified as neisserial DNA binding proteins by using 1D and 2D solid-phase overlay assays in combination with MS analysis (Figs 1 and 2) (Lång et al., 2009). The DNA binding activity of recombinant ComL and SSB was verified by EMSA (Fig. 4). The ComL DNA binding activity observed was dependent on the presence of Mg2+ and was abolished when EMSA buffers containing EDTA or no additive were used (Fig. 5). Similar results were obtained with all DNA substrates employed (Supplementary Table S2). The DNA binding activities of recombinant ComL and SSB were not DUS-specific (Fig. 4).

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