Phosphorylation-dependent activation of the cell wall synthase PBP2a in
            <i>Streptococcus pneumoniae</i>
            by MacP

Fenton, Andrew K.; Manuse, Sylvie; Flores-Kim, Josué; Garcia, Pierre; Chryslène, Mercy; Grangeasse, Christophe; Bernhardt, Thomas G.; Rudner, David Z.

doi:10.1073/pnas.1715218115

Cited by 58 publications

(61 citation statements)

References 40 publications

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“…Interestingly, it was shown that aberrant PBP1a activity can be detected outside the midcell zone in pneumococci lacking MreC or CozE, supporting the model that PBP1a can function autonomously (32). PBP2a, on the other hand, interacts with and is regulated by MacP, a substrate of the global cell cycle regulator StkP (34). The interplay between the two PBPs and their respective partners appears to be specific, as interactions between CozE/PBP2a and MacP/PBP1a have not been detected (32,34).…”

Section: Discussionmentioning

confidence: 71%

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Straume

Piechowiak

Olsen

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis. class A PBPs | CbpD | peptidoglycan | Streptococcus pneumoniae

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Section: Discussionmentioning

confidence: 71%

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Straume

Piechowiak

Olsen

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been reported that PBP1a forms a complex with CozE, MreC and MreD 40 , and that it coimmunoprecipitates with the cell cycle protein GpsB 41 . Furthermore, it has been shown that PBP2a is regulated by MacP, a substrate of the global cell cycle regulator StkP 42 . The interplay between the two PBPs and their respective partners appears to be specific, as interactions between CozE/PBP2a and MacP/PBP1a have not been detected 40,42 .…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, it has been shown that PBP2a is regulated by MacP, a substrate of the global cell cycle regulator StkP 42 . The interplay between the two PBPs and their respective partners appears to be specific, as interactions between CozE/PBP2a and MacP/PBP1a have not been detected 40,42 . Presumably, the specific partners of PBP1a and PBP2a are important for the precise spatiotemporal regulation of their activity.…”

Section: Discussionmentioning

confidence: 99%

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Straume

Piechowiak

Olsen

et al. 2019

Preprint

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In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class BPBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question.Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

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“…Notably, we did not detect a PBP2B self-interaction. Also, we only found positive results when the PBP2B variants were expressed from the pKT25 plasmid (Figure S4) – this is probably due to the difference in copy numbers between the two plasmids used in the assay and not uncommon in BACTH screens of interactions between PBPs and other proteins (29). We also analysed the interactions using a β-galactosidase assay (Figure 4B), which has the added benefit of providing a quantitative result which can give a hint about the strength of the interaction.…”

Section: Resultsmentioning

confidence: 96%

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

Angeles

Macia-Valero

Bohorquez

et al. 2019

Preprint

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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).

show abstract

Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP

Cited by 58 publications

References 40 publications

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

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

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