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Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain
Vellaichamy Adaikkalam1, Sanjay Swarup
1Department of Biological Sciences, National University of Singapore, Singapore.
Canadian Journal of Microbiology
|May 28, 2005
Summary
The Pseudomonas putida copABCD operon plays a minor role in copper homeostasis compared to the cueAR operon. This study identifies copABCD as a second copper homeostasis operon in this strain.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Copper homeostasis is crucial for bacterial survival.
- Pseudomonas putida PNL-MK25 possesses a P1-type ATPase (cueAR) for copper management.
- The role of other copper homeostasis systems in this strain was unexplored.
Purpose of the Study:
- To characterize the copABCD operon in Pseudomonas putida PNL-MK25.
- To assess the contribution of copABCD to copper tolerance and homeostasis.
- To compare the function of copABCD with the cueAR operon.
Main Methods:
- Mini-Tn5::gfp mutagenesis to identify copper-responsive mutants.
- Sequencing to identify transposon insertion sites.
- Gene expression analysis to determine induction levels.
- Copper-sensitivity assays to evaluate tolerance.
Main Results:
- Three copper-sensitive mutants with transposon insertions in the copABCD operon were identified.
- The P. putida copABCD operon is inducible by copper starting at 3 micromol/L.
- Mutants exhibited only a marginal reduction in copper tolerance (0.16-fold).
- The cueAR mutant showed a significantly greater reduction in copper tolerance (6-fold).
Conclusions:
- The copABCD operon has a minimal role in copper homeostasis in P. putida PNL-MK25 compared to other strains and the cueAR operon.
- Reduced function may be due to fewer metal-binding domains in the encoded proteins.
- The cueAR operon is the primary determinant of copper tolerance in this strain.