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Published on: May 20, 2020
Identification of a functionally important loop in Salmonella typhimurium ArnT.
Nicholas A Impellitteri1, Jacqueline A Merten, Lynn E Bretscher
1Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Biochemistry
|December 2, 2009
Summary
ArnT protein modification confers polymyxin antibiotic resistance in bacteria like Salmonella typhimurium. Researchers identified key residues essential for ArnT function and protein stability.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Gram-negative bacteria possess an outer membrane containing lipid A.
- Lipid A modification by ArnT confers resistance to polymyxin antibiotics.
- Previous work characterized Salmonella typhimurium ArnT structure and function.
Purpose of the Study:
- To investigate the role of cysteines in S. typhimurium ArnT.
- To identify residues critical for ArnT function and protein stability through mutagenesis.
- To further characterize ArnT structure and function.
Main Methods:
- Cysteine-specific modification with mPEG-mal.
- Circular dichroism spectroscopy.
- In vivo functional growth assays.
- Site-directed mutagenesis and functional analysis.
Main Results:
- All eight native cysteines in S. typhimurium ArnT are reduced and not involved in disulfide bonds.
- Cysteine-free ArnT is structurally and functionally intact.
- 14 residues essential for ArnT transferase function were identified.
- 3 residues were found to disrupt protein folding or membrane insertion.
Conclusions:
- ArnT's function in polymyxin resistance is independent of disulfide bonds.
- Specific residues within a putative loop are crucial for ArnT activity and proper localization.
- This study provides critical insights into the mechanism of antibiotic resistance in bacteria.
