Figures and data
Schematic overview of PrgB domain organization and function. A) Updated schematic overview of the domain organization of PrgB. SS: Signal sequence, COI: Coiled-coil, PAD: Polymer adhesin domain, CSA: adhesin isopeptide-forming adherence domain, CSC: cell-surface antigen C-terminal domain, LPXTG: cell wall anchor sequence. B) In a setting with a donor cell (green) and multiple recipient cells (brown), PrgB is produced and sits on the cell wall. There it enhances cellular aggregation and/or biofilm formation, either by directly binding LTA from the cell-wall of a recipient or first to eDNA to. PrgB compacts the eDNA, and thereby likely pulls the recipient cells closer. Once close enough, PrgB binds to the LTA of the recipient bacteria and facilitates mating-pair formation and conjugation.
Structure of Ig-like domains of PrgB. A) The Ig-like domains are arranged as tandem pairs (CSA1-CSC1 and CSA2-CSC2). B) Each Ig-like domain has an internal isopeptide bond (indicated by the striped circle in panel A) strengthening the structural integrity of the domain. Each of the four isopeptide bonds is between a lysine and an asparagine and further stabilized by an aspartic acid residue (highlighted residues are shown in stick representation). C) A conserved metal binding site is situated in the CSA2 domain (highlighted by a striped box in panel A), here modelled with a Mg2+ (green sphere).
Crystal structures of the PrgB polymer adhesin domain and Ig-like domains in cartoon representation docked into the volumes acquired by single particle cryo-EM. A) The PrgB188-1233 model shows the polymer adhesin domain folded back onto the Ig-like domains. B) In the DNA-bound PrgB188-1233 model the polymer adhesin domain has undergone a large conformation change away from the Ig-like domain.
In vivo phenotypes of PrgB variants. PrgB variants are expressed from the pMSP3545S-MCS vector in the OG1RF pCF10ΔprgB background for phenotypic analysis with three assays: A) cellular aggregation B) biofilm formation and C) conjugation assays. For all assays OG1RF pCF10 carrying an empty vector serves as positive control and OG1RF pCF10ΔprgB with an empty vector as negative control. The value of each column represents the average of three independent experiments and the error bars represent the standard error of the mean (SEM).
In vivo phenotypes of PrgB variants with point mutation(s) in the conserved site of the polymer adhesin domain. PrgB variants are expressed from the pMSP3545S-MCS vector in the OG1RF pCF10ΔprgB or OG1RF pCF10 strain and analyzed in: A) cellular aggregation, B) biofilm formation, and C) conjugation assays. OG1RF pCF10 carrying the empty vector serves as positive control and OG1RF pCF10ΔprgB with the empty vector as negative control. The height of each column represents the average of three independent experiments and the error bars indicate the standard error of the mean (SEM).
The RGD motifs play a role in the structural integrity of PrgB. A) Close up view of the RGD motif in the CSA1 domain (purple). Most of the important interactions, mainly hydrogen bonds, formed by this motif is to residues on the CSC1 domain (green). B) Close up view of the RGD motif in the CSA2 domain (sand colored). Most of the important interactions, mainly hydrogen bonds, formed by this motif is to residues on the CSC2 domain (dark red). Potential hydrogen bonds are marked by lines and the surface of the protein indicated by transparent gray.
