The overall structure of Sgo0707N.

<p>A: Ribbon representation of Sgo0707N. The N1-domain is coloured pink and the N2-domain purple. B: Topology diagram of Sgo0707N1. C: Topology diagram of Sgo0707N2. β-strands are depicted as triangles and α-helices as circles. The N1- and N2-domains are shown in pink and purple respectively.</p

Data collection, refinement and model quality statistics for Sgo0707.

a<p>Values in parentheses indicate statistics for the highest resolution shell.</p>b<p><i>R</i>_sym = Σ<i>_hkl</i> Σ<i>_i</i> |<i>I</i>_i<i>(hkl)</i> - <<i>I</i>(<i>hkl</i>)>|/Σ<i>_hkl</i> Σ<i>_i I</i>_i (<i>hkl</i>), where <i>I_i</i>(<i>hkl</i>) is the intensity of the <i>i</i>th observation of reflection <i>hkl and </i> is the average over of all observations of reflection <i>hkl.</i>^c<i>R</i>_work = Σ | |<i>F</i>_obs| - | <i>F</i>_calc| |/Σ | <i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure factor amplitudes, respectively. <i>R</i>_free is <i>R</i>_work calculated using 5% of the data, randomly omitted from refinement.</p

Role of Sgo0707 in <i>S. gordonii</i> binding to type-1 collagen, saliva, serum and oral keratinocytes.

<p>A: Fluorescence micrographs showing adherence of wild-type and ΔSgo0707 strains to protein-coated surfaces and to oral keratinocytes. In panels 1–3 bacteria were flowed over surfaces coated with type-1 collagen, 25% saliva or 10% serum for 2 h, as described, and adhered bacteria stained green using the LIVE/DEAD® <i>Bac</i>light™ Bacterial Viability stain. Images were obtained using CLSM and the surface coverage determined by image analysis using the <i>bio</i>Image_L software package. In panel 4, bacteria were flowed over keratinocyte layers for 1 h, stained using the LIVE <i>Bac</i>Light™ Bacterial Gram Stain Kit and viewed with CLSM. The mean number of bacteria (stained red)/1000 keratinocytes (stained green) was determined by manual image analysis. B: Graphs showing the adherence of the wild-type (grey bars) and ΔSgo0707 (white bars) strains to different surface coatings. For type-1 collagen, saliva and serum, adherence is expressed as surface coverage (arbitrary units) whereas for the keratinocytes, binding is expressed as number of bacteria/1000 keratinocytes. The mean and standard error of three independent experiments is shown (* indicates a significant difference p<0.05).</p

Silver-stained 2DE gels of cell wall proteins from wild-type and ΔSgo0707 strains of <i>S.gordonii</i>.

<p>Proteins prepared as described in materials and methods were subjected to isoelectric focussing at pH 4–7 followed by SDS-PAGE in 7% gels. The migration positions of molecular mass markers are shown.</p

Sgo0707 collagen docking.

<p>A model of collagen docked to the Sgo0707 crystal structure using Firedock. A: The collagen triple helix is docked into the binding pocket of the N1-domain. B: Collagen is docked in the groove formed by the interface between the N1- and N2-domains.</p

DNA binding analysis of VicR with wildtype and mutated promoter regions of <i>gcrR</i>.

<p>(A) EMSA analysis of <i>gcrR</i> 77 bp variants. VicR at increasing concentrations was incubated with labeled wildtype and mutated <i>gcrR</i> probe. The S above the fifth lane indicates that the <i>gcrR</i> substrate was incubated in the absence of VicR. F indicates free DNA, B indicates bound DNA. (B) DNaseI footprint analysis of a <i>gcrR</i> 77 bp wildtype (WalR consensus TGTTATagaacTGTAAT) and a <i>gcrR</i> double DR mutant (WalR mutated consensus GACGGC agaacGACGGG). VicR at increasing concentrations was incubated with labeled probe. The S above the fifth lane indicates that the substrate was incubated in the absence of VicR. The solid bars indicate the match to the WalR consensus and the mutated sequence. The dashed line indicates the region protected by VicR. (C) DNaseI footprint analysis of individual <i>gcrR</i> DR mutants. VicR at increasing concentrations was incubated with labeled probe. The S above the fifth lane indicates that the substrate was incubated in the absence of VicR. The solid bars indicate the match to the WalR consensus and the mutated sequences. The dashed lines indicate the regions protected by VicR.</p

Inhibition of binding of <i>S. gordonii</i> DL1 to type-1 collagen coated surfaces using recombinant Sgo0707_36–455 protein.

<p>Flow-cell surfaces were coated with type-1 collagen followed by different concentrations of recombinant Sgo0707_36–455 protein. Bacteria were flowed over the surfaces for 2 h in the presence of the same concentrations of recombinant Sgo0707_36–455 and adhered bacteria were visualized using the Baclight Live/Dead stain and analysed with <i>bio</i>Image_L software package. The mean and standard error of three independent experiments is shown (*** indicates a significant difference p<0.001 from control).</p

A Biochemical Characterization of the DNA Binding Activity of the Response Regulator VicR from <i>Streptococcus mutans</i>

<div><p>Two-component systems (TCSs) are ubiquitous among bacteria and are among the most elegant and effective sensing systems in nature. They allow for efficient adaptive responses to rapidly changing environmental conditions. In this study, we investigated the biochemical characteristics of the <i>Streptococcus mutans</i> protein VicR, an essential response regulator that is part of the VicRK TCS. We dissected the DNA binding requirements of the recognition sequences for VicR in its phosphorylated and unphosphorylated forms. In doing so, we were able to make predictions for the expansion of the VicR regulon within <i>S. mutans</i>. With the ever increasing number of bacteria that are rapidly becoming resistant to even the antibiotics of last resort, TCSs such as the VicRK provide promising targets for a new class of antimicrobials.</p></div

Protein melting temperatures when supplemented with metal ion solutions.

<p>Protein melting temperatures when supplemented with metal ion solutions.</p

DNaseI footprint analysis of <i>gcrR</i> helical phasing mutants.

<p>Labeled probe was incubated with no (S above the fifth lane) or increasing amounts of VicR. The solid bars indicate the match to the WalR consensus and the mutated sequences. The dashed lines indicate the regions protected by VicR. The +1 spacer is a 82 bp substrate and the +2 spacer is a 87 bp substrate.</p