CapA1B1/CapA2B2 sequence comparison.

<p>The sequences of the chimera CapA1B1 and CapA2B2 have been aligned using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075958#pone.0075958-Chenna1" target="_blank">[32]</a>. The conserved secondary structure elements as well as the 3₁₀-helix η1 only observed in the CapA1B2 structure (PDB IDs 2VED and 3BFV) are indicated at the top of the alignment. using Esprit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075958#pone.0075958-Gouet1" target="_blank">[33]</a>. Residue numbering of CapA1B1 is indicated with CapA1 residues numbered from V194a to N222a and CapB1 residues numbered from M1 to E228. The N-terminal methionine of CapB1 and CapB2 is highlighted by a blue arrow. The active site residues involved in nucleotide binding (including the Walker A motif and the catalytic lysine K55) are highlighted by green stars. The conserved interface residues including the conserved EX₂RX₂R motif in helix α2 are highlighted by orange dots. The first residue of the Y-cluster is indicated by a red arrow.</p

Functional and structural characterization of CapA1B1. A

<p>- CapB1 (left panel) and CapB2 (right panel) activation assays using either purified full length 6xHis-CapA1-FL (6xHis-CapA1-FL) or membrane fractions enriched with full-length 6xHis-CapA1-FL. An increasing concentration of CapA1 was used as indicated. CapB1 and CapB2 autophosphorylation was determined after incubation with radioactive ATP, SDS-PAGE separation and autoradiography. <b>B</b> - Phosphorylation of CapA1B1 and CapA1B2 during overexpression in <i>E. coli</i>. CapA1B1 and CapA1B2 purified from <i>E. coli</i> were analyzed by SDS-PAGE and transferred onto a PVDP membrane. Their phosphorylation was revealed by immunoblotting using the anti-phosphotyrosine antibody 4G-10 and the secondary antibody HRP conjugate after direct film exposure. <b>C</b> - Electron density of the ADP-Mg molecule bound in the CapA1B1 active site. (2Fo-Fc) map contoured at 1 σ. The bound nucleotide and the residues involved in nucleotide and Mg²⁺ binding are shown in sticks. <b>D</b> - Stability of CapA1B1. Differential Scanning Calorimetry of CapA1B1 compared with CapA1B2.</p

The CapA1B2 octameric ring.

<p>The octameric structure of the non-phosphorylated CapA1B2(K55M) mutant protein in complex with ADP-Mg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075958#pone.0075958-Lee1" target="_blank">[16]</a> (PDB ID 2VED) is represented as cartoon. In each subunit, the αA-βA elements from CapA1 are colored in grey and the CapB2 moiety is colored by spectrum from blue to red. The bound nucleotides are highlighted as black sticks.</p

CapA2B2 phosphorylation state analysis.

<p>Electrophoretic profile of CapA1B2 and CapA2B2 directly after purification (− ATP) or after 4h incubation at 37°C with 200 µM ATP-Mg (+ATP). The proteins were loaded on a 12.5% non-denaturing polyacrylamide gel and stained with Coomassie Brilliant Blue. The five observed bands were labeled according to the mass spectrometry analysis previously performed on CapA1B2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075958#pone.0075958-Lee1" target="_blank">[16]</a>: the upper band corresponds to the non-phosphorylated form of the protein (0P), and the lower band to the fully phosphorylated form (4P).</p

Bacterial strains, plasmids and primers used in this study.

a<p>Forward and reverse primers are represented by plus (+) or minus (−), respectively.</p>b<p>restriction sites are italicized.</p>c<p>The bases mutated from those present in the wild type are bold.</p

Morphology of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells.

<p>(A) Phase contrast microscopy (lower panel) and FM4–64 membrane staining (upper panel) images of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> exponentially growing cells at 37°C in THY medium. Scale bar, 5 µm. (B) Scanning electron micrograph of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells. Scale bar, 1 µm. (C) Transmission electron micrograph of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells. Scale bar, 1 µm. Asterisks indicate defective septal initiations in staggered rows in the Δ<i>gpsB</i> cell.</p

Interplay of GpsB, DivIVA and StkP.

<p>(A) Western immunoblot of whole-cell lysates from the wild type (WT), Δ<i>stkP</i>, <i>gpsB::gfp-gpsB</i>, Δ<i>gpsB</i>, Δ<i>divIVA</i> and Δ<i>gpsB</i>Δ<i>divIVA</i> cells grown in THY at 37°C probed with anti-phosphothreonine antibodies. The same amounts (25 µg) of proteins were loaded in all gel lanes. Arrow indicates the signal observed around 15 kDa. The phosphorylation signal for DivIVA and StkP are indicated. (B) Western immunoblot of whole-cell lysates from wild type (WT) or <i>gpsB::gfp-gpsB</i> cells probed with anti-GFP antibodies. Purified GFP is used as control. Arrow indicates the signal observed for GFP-GpsB. (C) StkP localization using a GFP N-terminal fusion in WT, Δ<i>gpsB</i>, Δ<i>divIVA</i> and Δ<i>gpsB</i>Δ<i>divIVA</i> cells. GFP (green) and phase-contrast (grey) images were taken from a typical field of exponentially grown cells in THY at 37°C. Merged pictures (lower panels) show the overlay of StkP (green) and phase contrast images (red). Scale bar, 5 µm. (D) Cell morphology of <i>stkP-K42M</i> cells deficient for DivIVA or GpsB expression. Cells producing a kinase dead-form of StkP (<i>stkP-K42M</i>, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Fleurie1" target="_blank">[14]</a>) were deleted either for <i>divIVA</i> or <i>gpsB</i> resulting thus in Δ<i>divIVA</i>-<i>stkP-K42M</i> and Δ<i>gpsB</i>-<i>stkP-K42M</i> strains, respectively. Phase contrast microscopy (upper row) and FM4–64 membrane staining (lower row) images of Δ<i>divIVA</i>-<i>stkP-K42M</i> (left panel) and Δ<i>gpsB</i>-stkP-K42M (right panel) exponentially growing cells at 37°C in THY medium. Scale bar, 5 µm.</p

Localization of PBP2x, PBP2b, FtsW and RodA in WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells.

<p>Chromosomal copy of either <i>pbp2x</i>, <i>pbp2b</i>, <i>ftsW</i> or <i>rodA</i> were substituted for a <i>gfp</i>-fused gene in WT (A) or Δ<i>gpsB</i> (B), or Δ<i>divIVA</i> (C), or Δ<i>divIVA</i>Δ<i>gpsB</i> (D) cells. Cells were grown in THY medium at 37°C. GFP (green) and phase-contrast (grey) images were taken from a typical field of exponentially growing cells. Merged pictures show the overlay of GFP fluorescence (green) and phase contrast images (red). Arrows show helical organization of GFP-PBP2x, GFP-PBP2b, FtsW-GFP and RodA-GFP. Scale bar, 5 µm. All fusion proteins are the only source of PBP2X, PBP2b, FtsW or RodA in the cells.</p

Alignment for GpsB and DivIVA proteins from several bacteria.

<p>(A) Multiple sequence alignments of GpsB and DivIVA sequences from streptococci and Gram-positive bacteria. Protein sequences similar to that of pneumococcus GpsB and DivIVA were identified by BLAST searches and aligned using CLUSTALW. Spn: <i>S. pneumoniae</i>; Sag: <i>S. agalactiae</i>, Bsu: <i>B. subtilis</i>; Sta: <i>S. aureus</i>, Mtb: <i>M. tuberculosis</i>; Sco: <i>S. coelicolor</i>. Yellow highlights the potential coiled-coil motifs retrieved from UniProtKB/Swiss-Prot:Q8CWP9 and UniProtKB/Swiss-Prot:C1CIN3 entry annotations for Spn-DivIVA (residues 34–135 and 199–236) and Spn-GpsB (36–63) respectively. The PF05103 PFAM DivIVA family signatures are mapped as green open boxes for DivIVA and GpsB. When identified, phosphorylation sites are red boxed. The <i>S. coelicolor</i> DivIVA phosphopeptide containing unidentified phosphorylation sites are highlighted in orange letters. Identical residues are in pink letters and positions showing conservation of similar residues are in blue. Dots indicate gaps introduced in sequences during alignment computation. The figure was rendered with the ESPript server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Gouet1" target="_blank">[54]</a>. (B) Multiple sequence alignments of GpsB sequences from streptococci. Protein sequences were aligned using CLUSTALW. Spy: <i>S. pyogenes</i>; Sag: <i>S. agalactiae</i>, Smu: <i>S. mutans</i>; Sth: <i>S. thermophylus</i>; Ssa: <i>S. salivarius</i>; Spn: <i>S. pneumoniae</i>; Smi: <i>S. mitis</i>, Sgo: <i>S. gordonii</i>. The PFAM PF05103 DivIVA family signatures are mapped as green boxes. Yellow highlights the potential coiled-coil motifs retrieved from UniProtKB/Swiss-Prot:C1CIN3 entry annotations for Spn-GpsB (36–63). The phosphothreonine identified for <i>S. agalactiae</i> GpsB is red boxed. Glutamic acids possibly mimicking threonine phosphorylation are black boxed with white letters. Identical residues are in pink letters and positions showing conservation of similar residues are in blue. Dots indicate gaps introduced in sequences during alignment computation. The figure was rendered with the ESPript server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Gouet1" target="_blank">[54]</a>.</p

Models for PG synthesis in <i>S. pneumoniae</i>.

<p>In this model, a large membrane PG assembly complex (Yin Yang circle) contains both the septal (red) and the peripheral (orange) PG assembly machineries. The two transpeptidases PBP2x and PBP2b (noted 2x and 2b) and the two lipid-flippases FtsW and RodA (noted W and A) are indicated in green and blue, respectively. Non-phosphorylated forms of DivIVA and other StkP substrates are required for cell elongation and thus peripheral PG synthesis. GpsB is not <i>per se</i> involved in the production of the cross-wall, but is required at the septum to localize StkP (light green oval), to allow the phosphorylation of StkP substrates including DivIVA and to favor production of septal PG by down-regulating peripheral PG synthesis. The paralogs GpsB (pink oval) and DivIVA (purple oval) constitute a molecular switch that connects, together with EzrA (green oval), the Z-ring with the PG assembly complex. StkP kinase activity, counterbalanced by the phosphatase PhpP (yellow oval) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Beilharz1" target="_blank">[15]</a> and triggered by GpsB, modulates the function of a set of proteins (dashed ovals) including DivIVA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Fleurie1" target="_blank">[14]</a>. The StkP/DivIVA/GpsB triad is thus proposed to orchestrate and to finely tune production of septal and peripheral peptidoglycan synthesis responsible for the ovoid-shape of pneumococcus.</p