Journal: Frontiers in Cellular and Infection Microbiology
Article Title: The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae
doi: 10.3389/fcimb.2021.808601
Figure Lengend Snippet: Competence pilus assembly and transformability depends on the pneumococcal minor pilins. (A) Schematic representation of the comG operon in S. pneumoniae R6 strain encoding comGA and comGB (white), the major pilin gene comGC (black) and the minor pilin genes comGD, comGE, comGF and comGG (grey). The invariant Glu5 residue, present in all pneumococcal pilins except ComGG, is highlighted (E5). (B) ComGC was detected by immunoblotting in bacterial whole cell lysates and sheared pili of mutants lacking Δ comGC , Δ comGG , Δ comGFG , Δ comGEFG . The WT strain was included as a positive control and GAPDH as loading control. (C) Transformation frequency of WT , comG mutants and R6Δ comGDEFG complemented strains. The detection limit (dl) of the assay was 2.39e -9 and the error bars represent the standard deviation of a minimum of three independent experiments (n=3). (D) Western blotting analysis of ComGC in bacterial whole cell lysates and sheared pili of Δ comGDEFG strains expressing individual minor pilins, comGDEF or comGDEFG at the ectopic bgaA locus. The WT strain was included as a positive control and GAPDH as loading control. (E) Immunofluorescence microscopy (IF) of competence-induced pili in WT R6 and R6Δ comGDEFG complemented with comGG, comGDEF or comGDEFG. Bacteria were visualized by bright field (BF) microscopy and competence pili were labelled with antisera specific for ComGC (red). Scale bars represent 2 µm.
Article Snippet: For this, PCR fragments flanking the upstream and downstream regions of the target genes were amplified from the S. pneumoniae R6 genomic DNA using Fusion Flash polymerase (Thermo Fisher Scientific).
Techniques: Residue, Western Blot, Positive Control, Control, Transformation Assay, Standard Deviation, Expressing, Immunofluorescence, Microscopy, Bacteria
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