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. 2015 Sep 25;10(9):e0138103. doi: 10.1371/journal.pone.0138103

Fig 4. Role of putative N-glycosylated asparagyl residues in PilA stability.

Fig 4

(A) Structural modeling of PilA (blue ribbon) compared to the S. pneumoniae homolog RrgA (orange ribbon). Domain organization (D1 to D4) appears to be conserved, position of in silico putative predicted N-glycosylated aspargyl residues N427 and N597 in domains D3 and D2 are indicated (blue circle). (B) Proteins anchored to the cell-wall were isolated from S. agalactiae strain NEM316 and its isogenic pilA mutants harvested at both exponential and stationary growth phase, separated on 4%–12% Criterion XT SDS-PAGE gel, and detected by immunoblotting with specific anti-PilA antiserum. Equivalent amount corresponding to 500 μl of the initial culture was loaded in each well. The PilA monomer is indicated by a black arrow. The high-molecular-weight species correspond to PilA polymers while the lower band at 50 kDa is most likely a degradation product. (C) Immuno-electron-microscopy (IEM) analyses of the pilus subunits PilB. S. agalactiae wild-type strain NEM316 (WT) and its isogenic pilus mutants (ΔVWA2, and PilAN427Q-N597Q) were incubated with rabbit polyclonal antibody raised against PilB. Antibodies were conjugated to 10 nm gold particles.