Fig 5.

PBP3 interacts with components of the polar divisome. (a) Bacterial two-hybrid analysis of interactions between PBP3 and other PG synthases or components of the polar divisome. The β-galactosidase activity was normalized to the negative control. N-terminal T18 or T24 fusion to PBP3 was paired with reciprocal N-terminal fusions to the indicated proteins. Data are from three technical replicates. (b) The schematic shows interactions detected in the bacterial two-hybrid analyses. Components of the predicted polar divisome are indicated. PBP1 may also be a part of the polar divisome based on co-immunoprecipitation analyses using SpoVD-FLAG₃ as bait. (c and d) Co-immunoprecipitations performed on cells sampled from sporulation-inducing plates after 12 h of growth. (c) PBP3-FLAG₃ was used as bait in the ∆pbp3/pbp3-FLAG₃ strain background; (d) SpoVD-FLAG₃ was used as bait in the ∆spoVD/spoVD-FLAG₃ (WT) and ∆spoVD∆pbp3/spoVD-FLAG₃ (∆pbp3) strain backgrounds. ∆pbp3/pbp3 and ∆spoVD/spoVD strains were used as negative controls (no tag). The presence of SpoVD, PBP3, and PBP1 in the pull-downs was probed using antibodies against the indicated proteins and Western blotting. The FLAG-tagged proteins were detected using an anti-FLAG antibody. SpoIID was used as a control protein because it is also a PG-associated transmembrane protein localized to the forespore membrane, but it is not predicted to be a part of the polar divisome.