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Conditional deletion of PBP3 also caused a defect in cell division and increased susceptibility to β-lactams. The exception was PBP3, where cell growth occurred only when the protein was conditionally expressed on an integrated plasmid. Disruptions of the transpeptidase domains of most HMM PBPs, including double disruptions, had only minimal effects on cell growth. aeruginosa, we performed gene knockouts of all the high-molecular-mass (HMM) PBPs and determined the impacts on cell growth and morphology, susceptibility to β-lactams, peptidoglycan structure, virulence, and pathogenicity. To identify potential drug targets among the PBPs in P. The pathogen Pseudomonas aeruginosa poses a particular risk to immunocompromised and cystic fibrosis patients, and infections caused by this pathogen are difficult to treat due to antibiotic resistance. As the well-known drug targets for β-lactam antibiotics, the physiological functions of PBPs and whether they are essential for growth are of significant interest.
![pbp3 is essential pbp3 is essential](https://www.researchgate.net/publication/289602044/figure/fig2/AS:560874802298880@1510734381534/Overall-structure-of-PBP3-and-electron-density-maps-A-Rainbow-coloured-cartoon.png)
In contrast to FtsW, MurJ (0.5–5 μM) has no effect on the polymerase activity of PBP1b.Penicillin-binding proteins (PBPs) function as transpeptidases, carboxypeptidases, or endopeptidases during peptidoglycan synthesis in bacteria. The effects of FtsW and FtsW-PBP3 on PBP1b’s PBP domain parallel the effects on GTase in A, consistent with the coupling of both domains. Examples of the corresponding HPLC chromatograms are depicted in Supplemental Figure S1. Values are the mean ± SD of three experiments. Total TPase activity is the sum of activities derived from the TPase domain of PBP1b, including peptide cross-linking and carboxypeptidase activities. The percentage of muropeptide products detected by HPLC analysis of PG produced in vitro by PBP1b in the presence of the proteins indicated beneath each bar (endpoint assay). ( D) Effect of FtsW and the FtsW-PBP3 complex on TPase activity of PBP1b. ( C) PBP1b activation by LpoB (200 nM) does not suppress the inhibitory effect of FtsW (5 μM). ( B) In contrast to FtsW (5 μM), the polymerase activity of PBP1b is not inhibited by the FtsW-PBP3 complex (5 μM) and slightly decreases in the presence of PBP3 (5 μM). inhibition of lipid II polymerization by FtsW from E. The fluorescence decreases over time upon polymerization of dansyl-lipid II by PBP1b. ( A– C) Continuous fluorescence assay to measure the polymerization (GT activity) with dansyl-lipid II. In panel E, the flow-through fraction (FT) was analyzed by immunoblotting using antibodies against the epitope HA to detect FtsW HA. FtsW* indicates an FtsW degradation product. The bands of PBP1b, PBP3 and FtsW are indicated on the right side of the gels. Lanes E depict elution fractions from the Ni-affinity columns and FT the flow-through. 3, 1b and W3 indicate lanes with purified PBP3, PBP1b and FtsW-PBP3, respectively (size controls). The gels were first subjected to fluorescence imaging followed by Coomassie blue staining. The eluted fractions or flow-through fractions were labeled by fluorescent ampicillin and analyzed by SDS-PAGE. The cytoplasmic membranes from cells expressing the indicated proteins were solubilized with detergent followed by a purification step over a Ni-affinity column. The expressed protein cannot be detected in the total extract but only after affinity purification. The co-expressed proteins are indicated below each panel. coli cells one contains a His-Tag and was used as bait while the other proteins are untagged. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.
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All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. The divisome controls septal PG synthesis and separation of daughter cells. Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division.