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Penicillin-binding protein 2x of Streptococcus pneumoniae: the mutation Ala707Asp within the C-terminal PASTA2 domain
Inga Schweizer1, Katharina Peters, Christoph Stahlmann
1Department of Microbiology, University of Kaiserslautern , Kaiserslautern, Germany .
Abstract:
Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.
Insights
A mutation in Streptococcus pneumoniae PBP2x’s PASTA2 domain disrupts its division site localization and stability. This finding impacts understanding bacterial cell wall synthesis and potential therapeutic targets.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is crucial for bacterial cell wall synthesis.
- PBP2x localization to the division site depends on its PASTA domains.
Purpose of the Study:
- To investigate the cause of defective PBP2x localization observed in a mutant strain.
- To characterize the functional and stability consequences of a specific PBP2x mutation.
Main Methods:
- Isolation and analysis of a mutant strain with mislocalized GFP-PBP2x.
- Detailed characterization of the mutant PBP2x protein in live cells.
- Assessment of beta-lactam binding, functionality, and protease susceptibility.
Main Results:
- A point mutation in the C-terminal PASTA2 domain was identified as the cause of mislocalization.
- The mutation significantly reduced full-length GFP-PBP2x levels.
- The mutant PBP2x exhibited increased susceptibility to the protease HtrA.
Conclusions:
- The PASTA2 domain is essential for PBP2x localization and stability.
- The identified mutation severely impacts PBP2x function and cellular processing.
- This research provides insights into the regulation of bacterial cell division machinery.