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Conditional mutations in OutE and OutL block exoenzyme secretion across the Erwinia carotovora outer membrane
J N Housby1, J D Thomas, S D Wharam
1University of Cambridge, Department of Biochemistry, UK.
Abstract:
The phytopathogen Erwinia carotovora subspecies carotovora secretes pectinases and cellulase via the general secretory pathway, a process requiring at least 13 proteins encoded by the out gene cluster. By exploiting delta::Tn5, a generalised transducing phage (psi KP) and localised mutagenesis of the out gene cluster, we have produced a histidine auxotroph and 19 new secretory mutants, including two (HJN1003 and HJN1004) which were conditional (temperature sensitive) for secretion. All of the mutants accumulated pectinases and cellulase in the periplasm, but in the case of HJN1003 and HJN1004, only at the restrictive temperature. HJN1003 and HJN1004 were complemented by the outE and outL wild-type genes, respectively, and both mutant alleles were cloned and sequenced to reveal single missense substitutions. HJN1003 carries an Arg166 to His alteration in OutE and HJN1004 carries a Pro159 to Leu alteration in OutL. Topology mapping of OutL using a beta-lactamase probe confirmed that OutL is a type II bitopic trans-inner membrane protein and that the mutated Pro159 residue in HJN1004 is located in the cytoplasmic domain of OutL. Hence, the secretion of exoenzymes across the outer membrane is critically dependent on the conformation of secretory components located at the cytoplasmic face of the inner membrane.
Insights
Erwinia carotovora subspecies carotovora secretes exoenzymes using the general secretory pathway. Mutations in OutE and OutL proteins disrupt this secretion, highlighting the importance of inner membrane protein conformation for bacterial virulence.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Erwinia carotovora subspecies carotovora is a phytopathogen that secretes pectinases and cellulase.
- Secretion occurs via the general secretory pathway, involving at least 13 proteins encoded by the out gene cluster.
Purpose of the Study:
- To identify and characterize new mutants in the out gene cluster of Erwinia carotovora subspecies carotovora.
- To elucidate the role of specific secretory proteins in the general secretory pathway.
Main Methods:
- Utilized delta::Tn5 and localized mutagenesis to generate mutants.
- Employed complementation studies and DNA sequencing to analyze mutations.
- Used beta-lactamase fusion probes for protein topology mapping.
Main Results:
- Generated 19 new secretory mutants, including two conditional temperature-sensitive mutants (HJN1003 and HJN1004).
- Mutants accumulated pectinases and cellulase in the periplasm.
- HJN1003 (OutE Arg166His) and HJN1004 (OutL Pro159Leu) mutations were identified and characterized.
- OutL was confirmed as a type II bitopic inner membrane protein with the mutation in its cytoplasmic domain.
Conclusions:
- The conformation of secretory components at the cytoplasmic face of the inner membrane is critical for exoenzyme secretion across the outer membrane.
- Specific residues in OutE and OutL play essential roles in the general secretory pathway of Erwinia carotovora subspecies carotovora.