The PASTA domains of Bacillus subtilis PBP2B stabilize the interaction of PBP2B with DivIB

Abstract

23Bacterial cell division is mediated by a protein complex known as the divisome. Many protein-24 protein interactions in the divisome have been characterized. In this report, we analyse the role 25 of the PASTA (Penicillin binding protein And Serine Threonine kinase Associated)-domains 26 of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the 27 PBP2B PASTA domains results in impaired cell division and a heat sensitive phenotype. This 28 resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two hybrid 29 and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is 30 weakened when the PBP2B PASTA domains are removed. Combined, our results show that 31 the PBP2B PASTA domains are required to stabilize the interaction between PBP2B and 32DivIB. 33 34 Protein stability 129Membranes from strains 4132 and 4133 grown at 30°C on CH with 0.2% (w/v) xylose were 130 isolated. Cells were grown until exponential phase and spun down (3 000 rpm, 7 min, 4 °C). 131Pellets were washed in PBS and then cells were lysed by sonication. Membranes were collected 132 by centrifugation (45,000 rpm, 4°C, 50 min) and resuspended in PBS. The protein 133 concentration was equalised for the two strain samples and aliquots of membrane material of 134 the same volume were prepared. Aliquots were incubated at 30 or 48 °C for 5 min, 20 min, 1 135 hr, 2 hr and 14 hr. Then, Bocillin 650/665 (5 µg/ml) was added to each sample, and samples 136 were further incubated at RT for 10 min. After incubation, sample buffer was added to each 137 sample to stop further protein degradation, and samples were run in SDS (10 %) gel. GFP and 138Bocillin were detected using a Typhoon FLA950 (GE Healthcare). For GFP, the 473 nm laser 139 and the LPB (Long Pass Blue) filter were used, and for Bocillin the 635 nm laser and the LPR 140 (Long Pass Red) were used. 141After imaging, the same gels were used for immunoblotting. Proteins were transferred to a 142 PVDF membrane. Primary antibodies were anti-GFP (Thermofisher). Anti-Rabbit IgG alkaline 143 phosphatase conjugated secondary antibodies (Sigma Aldrich) were used. Blots were 144 developed using CDP-Star (Roche) and chemiluminescence was detected using a Fujifilm LAS 145 4000 imager (GE Healthcare).

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

“…DivIB/FtsQ interacts with PG through its extracellular PASTA domain and is required as a cell division checkpoint based on studies in S. aureus . The extracellular portion of DivIB/FtsQ is also known to interact with PBPs, such as PBP2b in B. subtilis . It appears that the DivIB/DivIC/FtsL (FtsQ/FtsB/FtsL) complex links the early intracellular stages of cell division with PG remodeling, which is predominantly an extracellular process .…”