Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas
Yeshi Yin, F Heath Damron, T Ryan Withers
1Department of Biochemistry and Microbiology, Joan C, Edwards School of Medicine at Marshall University, Huntington, WV 25755, USA. yuh@marshall.edu.
Background:
Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon.
Results:
In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.
Conclusions:
Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.
Insights
MucE overproduction triggers alginate overproduction in Pseudomonas aeruginosa, a key factor in cystic fibrosis lung infections. This study reveals a positive feedback loop between MucE and AlgU, suggesting MucE acts as a cell wall stress sensor.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Alginate overproduction (mucoidy) in Pseudomonas aeruginosa is a poor prognostic marker for cystic fibrosis (CF) patients.
- A previously constructed strain overexpressed MucE, activating protease AlgW, which degraded MucA, releasing AlgU (σ(22)) to initiate alginate synthesis.
Purpose of the Study:
- To investigate the regulatory relationship between MucE and AlgU in Pseudomonas aeruginosa.
- To identify proteins affected by MucE overexpression and AlgU dependence.
Main Methods:
- Transcriptional analysis of the mucE gene.
- Shotgun proteomic analysis of bacterial cell lysates.
- Genotypic analysis of CF isolates.
Main Results:
- P(mucE) activity was dependent on AlgU and induced by triclosan and sodium dodecyl sulfate.
- MucE-mediated mucoidy in CF isolates depended on MucA size and algU genotype.
- Nine AlgU-dependent and two AlgU-independent proteins were identified as affected by MucE overexpression.
Conclusions:
- A positive feedback loop exists between MucE and AlgU.
- MucE likely functions as a signal transduction component sensing cell wall stress in P. aeruginosa.
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