Figure 6.

LpoA/LpoB and their docking domains in PBP1A/PBP1B have recently evolved together. A. Schematic representation of PBP1A and PBP1B, illustrating the conserved TPase and TGase domains of both proteins, as well as the newly evolved UB2H domain in PBP1B and the comparably sized insertion region ODD in PBP1A. B. Phylogenetic distribution of Lpo proteins and PBP1A/PBP1B with or without the docking regions. STRING (Jensen et al., 2009) was used for assessing protein and domain conservation over >400 bacterial species. ODD and LpoA are limited to γ-proteobacteria (red and yellow lines) and UB2H and LpoB are further restricted to enterobacteria (yellow lines); stringent cutoffs were used to assess conservation of LpoA and LpoB (100 bits), and of UB2H and ODD domains within the class A PBPs (35% amino acid sequence identity). Note that exceptions exist for some large bacterial clades depicted here; for example in the Firmicutes phylum, Mycoplasmae and Ureoplasma have no class A PBP, whereas staphylococci have only one class A PBP that has similar levels of homology to PBP1A and PBP1B. C. UB2H is the PBP1B docking domain of LpoB. LpoB does not interact with a PBP1B variant that lacks the UB2H (PBP1BΔUB2H). In vivo cross-linking/co-immunoprecipitation of LpoB with anti-PBP1B was performed as in Fig. 2G. D. ODD is the PBP1A docking region of LpoA. Overexpression of ODD with an N-terminal signal sequence for periplasmic localization (pssODD) leads to lysis in cells that depend on a functional PBP1A-LpoA complex [mrcB⁻ (green diamonds) and lpoB⁻ (blue circles)], but does not affect wildtype cells (black squares). Note that the OD axis is in log₁₀ and there is a ~25% drop in cell culture density for mrcB⁻ and lpoB⁻ cells, leading to clear formation of cellular debris. Overexpression of LpoA together with pssODD averts lysis (inset panel).