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Published on: October 23, 2017
Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus
Mihoko Ishibashi1, Kenji Kurokawa, Satoshi Nishida
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Abstract:
Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.
Insights
Staphylococcus aureus MurC protein is essential for bacterial growth and antibiotic development. Mutations affecting its L-alanine binding pocket reveal new insights into enzyme function and potential drug targets.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacterial peptidoglycan biosynthesis is a crucial pathway for novel antibiotic development.
- Enzymes within this pathway represent significant targets for antimicrobial therapies.
- Staphylococcus aureus is a significant Gram-positive pathogen with a well-studied cell wall structure.
Purpose of the Study:
- To identify and characterize essential genes involved in Staphylococcus aureus peptidoglycan biosynthesis.
- To investigate the function of the MurC enzyme, UDP-N-acetylmuramic acid:L-alanine ligase, in S. aureus growth.
- To elucidate the role of specific amino acid residues in MurC function, particularly in L-alanine binding.
Main Methods:
- Screening of 750 temperature-sensitive (TS) mutants of Staphylococcus aureus.
- Complementation analysis using the murC gene.
- Characterization of single amino acid substitutions and their phenotypic effects.
- Assessment of phenotypic suppression under high osmotic stress.
- Allele-specific suppression studies using the aapA gene encoding an alanine transporter.
Main Results:
- Six TS mutants were complemented by the murC gene, indicating its essential role.
- TS phenotypes were suppressed by high osmotic stress, suggesting MurC's involvement in cell wall integrity.
- The G222E substitution led to a decrease in viable cell count at restrictive temperatures, confirming MurC's essentiality.
- The H343Y mutation, located in the putative alanine-binding pocket, was suppressed by increased aapA gene dosage, implicating H343 in L-alanine binding.
Conclusions:
- Staphylococcus aureus MurC protein is essential for bacterial cell growth.
- The H343 residue of MurC plays a critical role in high-affinity L-alanine binding in vivo.
- Understanding MurC's active site and substrate interactions can inform the design of new antibiotics targeting peptidoglycan synthesis.
