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The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
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Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity
Mark T Anderson1, Lindsay A Mitchell1, Harry L T Mobley2
1University of Michigan Medical School, Department of Microbiology and Immunology, Ann Arbor, Michigan, USA.
Journal of Bacteriology
|June 1, 2017
Summary
Cysteine biosynthesis, regulated by CysE, is essential for Serratia marcescens phospholipase A secretion and flagellar motility. Supplementing cysteine restores these functions and PhlA gene transcription.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Serratia marcescens is a pathogen causing opportunistic infections with high antibiotic resistance.
- PhlA phospholipase is a secreted enzyme contributing to S. marcescens virulence.
- Understanding PhlA secretion mechanisms is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To identify genes regulating the production and secretion of the PhlA phospholipase in S. marcescens.
- To investigate the role of the cysteine biosynthesis pathway in PhlA secretion and bacterial motility.
- To elucidate the regulatory mechanisms underlying PhlA phospholipase activity and flagellar function.
Main Methods:
- Screening of a transposon insertion library for phospholipase-deficient mutants.
- Utilizing a fluorogenic substrate to confirm phospholipase activity.
- Assessing gene transcription levels via quantitative PCR.
- Investigating swarming motility defects in mutant strains.
Main Results:
- Mutations in cyaA, crp, fliJ, and fliP implicated flagellar apparatus in PhlA secretion.
- A cysE mutant showed significantly reduced phospholipase activity, dependent on cysteine availability.
- CysE deficiency led to decreased phlA transcript levels and impaired swarming motility.
- Restoration of cysteine levels normalized PhlA secretion and flagellar gene transcription.
Conclusions:
- Cysteine biosynthesis is critical for extracellular phospholipase activity in S. marcescens.
- Cysteine availability regulates both PhlA secretion and flagellar-dependent motility.
- CysE plays a key role in the transcriptional regulation of virulence factors and motility in S. marcescens.
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