Fig 1.
No pigmentation of W50WbaP mutant.
Pigmentation of WT and W50WbaP mutant on blood agar plates.
Fig 2.
Cell envelope analysis of wild type and W50ABK*WbaP mutant.
(A) CE proteins from WT (W50), W50ABK*WbaP, W50WbaP and ECR353 (rgpA- rgpB- kgp-) were separated on SDS-PAGE. The gel was cut into segments and the proteins were identified by mass spectrometry. (B) A plot of Mascot scores of identified CTD proteins in the CE from WT, W50ABK*WbaP, W50WbaP and ECR353. When the same protein was identified from multiple gel segments the Mascot scores were summed (S1 Table).
Fig 3.
Absence of A-LPS in W50ABK*WbaP and W50WbaP mutants.
Immunoblot analysis of whole cell culture lysates with anti-APS (mAb 1B5) antibody. (A) WT and W50ABK*WbaP mutant, (B) WT, W50ABK mutant original stock and ECR353 (another ABK mutant in W50), (C) WT and W50WbaP mutant. Images on the right of each immunoblot (A-C) shows the corresponding commassie stained gel to show a loading control. (D) Silver stained LPS profiles from WT and W50WbaP.
Fig 4.
Absence of EDSL on W50WbaP and substantially reduced EDSL on ECR353 (W50ABK).
Representative cryo-EM micrographs of (A) Wild type, (B) ECR353 (W50ABK mutant) and (C) W50WbaP. A magnified section is shown on each right bottom corner. Arrows point to the electron-dense surface layer (EDSL) that is absent in W50WbaP and much reduced in ECR353.
Fig 5.
Accumulation of CTD proteins in the CCF of W50ABK*WbaP and W50WbaP mutants.
(A) CCF from WT, W50ABK*WbaP, ECR353 and W50WbaP was subjected to SDS-PAGE analysis. The gel was cut into segments and proteins were identified by mass spectrometry. (B) A plot of Mascot scores of identified CTD proteins in CCF of WT, W50ABK*WbaP, ECR353 and W50WbaP. When the same protein was identified from multiple gel segments the Mascot scores were summed (S2 Table).
Fig 6.
The CTD proteins in W50ABK*WbaP mutant are processed with their C-terminal domains cleaved off.
2-Dimensional gel electrophoresis of CCF from (A)W50ABK*WbaP mutant and (B) W50WbaP mutant. The protein spots were identified by mass spectrometry and selected proteins are listed in Table 1.
Table 1.
Identification of CTD proteins in the 2-D gel of W50ABK*WbaP and W50WbaP CCF by MALDI-TOF/TOF MS.
Table 2.
Identification of cleaved CTDs and their putative cleavage sites.
Fig 7.
MS analysis of intact CTD proteins shows heterogenous modification of these proteins in W50ABK*WbaP.
(A) CPG70, P59 and P27 were purified from the CF of W50ABK*WbaP and analysed by ESI-TOF for accurate molecular mass determination. Circled in blue are the expected molecular mass for each protein, except for P59. (B) W50ABK*WbaP was grown overnight in the presence of 0.2 M glycine. CPG70, P59 and P27 were purified from CF and analysed on ESI-TOF. Circled in blue are the expected molecular mass and circled in red are the expected molecular mass plus glycine (57 Da).
Fig 8.
Modification of the C-terminal peptide of Pro-CPG70 with peptides or glycine.
Purified Pro-CPG70 from W50ABK*WbaP was subjected to in-solution digest with trypsin and the tryptic fragments were analysed with LC-MS/MS (Orbitrap). (A) An example MS/MS spectrum of C-terminal peptide of mature Pro-CPG70 showing modification with ‘SLT’ at the C-terminus in the absence of glycine, see Table 3 for more modifications. (B) An example MS/MS spectrum of C-terminal peptide of mature Pro-CPG70 showing modification with ‘G’ at the C-terminus in the presence of 0.2 M glycine in the growth medium.
Table 3.
Modified C-terminal peptides identified from CPG70 tryptic digests using the Orbitrap MS with and without the inclusion of 0.2 M glycine in the growth medium.
CPG70 was purified from W50ABK*WbaP.
Fig 9.
The C-terminal peptide of P59 is modified with glycine from the growth medium.
W50WbaP was grown overnight in the (A) absence or (B) presence of 0.2 M glycine. The proteins in the CCF were subjected to SDS-PAGE separation and the band corresponding to P59 was excised and in-gel digestion was performed. Tryptic fragments were analysed by LC-MS/MS (Ion trap). Extracted ion chromatograms were plotted for m/z 729.4 (IVWSDTQWTHAN) and m/z 757.85 (IVWSDTQWTHANG). These peaks were identified as C-terminal peptide of P59 using Mascot search. * Peak corresponding to another protein.
Fig 10.
Modification of CTD proteins isolated from wild type W50 with the 648 Da linker.
Modified CTD proteins were semi-purified, deglycosylated with TFMS, digested in-gel with trypsin and analysed by LC-MS/MS in positive ion mode using the ion trap MS. Each panel shows the MS/MS spectrum obtained for a C-terminal trytic peptide modified with the 648 Da linker. (A) CPG70, peptide sequence = KAEDYIEVILDD. (B) P59, peptide sequence = IVWSDTQWTHAN. (C) RgpB, peptide sequence = VEGT. (D) PG0553, peptide sequence = GSGISN. The b-ions and their corresponding sequence assignments are shown in blue with masses (104, 198 and 346) corresponding to components of the linker highlighted yellow. The y-ions are labeled such that the ion corresponding to the modification alone is y-1. Y-ions beginning with the intact (648 Da) modification are in red, and are labeled y 1, y 2 etc. Y-ions beginning with the 302 Da component of the linker are in brown and are labeled y 1**, y 2** etc. Y-ions beginning with the 105 Da component of the linker are in green and are labeled y 1*, y 2* etc. The intense peaks labeled with a red star are doubly charged ions that have lost the 346 Da component of the linker. N* denotes partial deamidation.
Fig 11.
Release of the 648 Da linker by proteinase K cleavage.
Modified CTD proteins were semi-purified, deglycosylated with TFMS, digested with proteinase K and analysed by LC-MS/MS in positive ion mode using the ion trap MS. A singly charged peak of m/z 649.3 was obtained and MS/MS analysis showed a major loss of 346 Da to produce the m/z 303.1 ion. An additional fragment ion at m/z 199 corresponded to a further loss of 104 Da.
Fig 12.
Model of sortase-like mechanism in P. gingivalis.
CTD proteins translocate to the cell surface via the T9SS. (1) On the surface, proposed sortase PG0026 cleaves the CTD peptide, (2) resulting in the formation of a thioester enzyme intermediate and release of cleaved CTD peptide into the culture fluid. (3) In the absence of A-LPS, nucleophilic attack from the free amino group of peptides in the growth media resolves the acyl-enzyme intermediate and releases CTD protein modified with peptide into the culture fluid. (4) In the presence of A-LPS, nucleophilic attack from the free amino group on the A-LPS at the thioester bond resolves the acyl-enzyme intermediate and synthesizes an amide bond between CTD proteins and A-LPS resulting in the anchorage of CTD proteins to the cell surface. (5) Active site of PG0026 is regenerated.