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Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase
Abstract:
Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot- blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22.
Insights
Researchers identified the algA gene responsible for phosphomannose isomerase activity in Pseudomonas aeruginosa. Overexpression of this gene confirmed its role in producing the enzyme essential for alginate synthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Alginate production in mucoid Pseudomonas aeruginosa is crucial for biofilm formation and pathogenesis.
- The enzyme phosphomannose isomerase plays a key role in the mannose metabolic pathway, potentially linked to alginate synthesis.
- Previous studies indicated undetectable phosphomannose isomerase activity in mucoid P. aeruginosa strains.
Purpose of the Study:
- To identify and characterize the gene responsible for phosphomannose isomerase activity in Pseudomonas aeruginosa.
- To confirm the functional role of the identified gene in phosphomannose isomerase activity and its potential link to alginate production.
- To investigate the transcriptional regulation of the phosphomannose isomerase gene.
Main Methods:
- Gene cloning and overexpression of a candidate P. aeruginosa gene in a broad-host-range vector (pMMB22) under the control of the tac promoter.
- Enzyme assays to measure phosphomannose isomerase activity in P. aeruginosa and Escherichia coli manA mutant.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to visualize protein expression.
- RNA-DNA dot blot hybridization to assess gene transcription levels.
Main Results:
- Overexpression of the P. aeruginosa gene in P. aeruginosa and an E. coli manA mutant restored phosphomannose isomerase activity.
- The enzyme catalyzed the interconversion of fructose 6-phosphate and mannose 6-phosphate, confirming its identity.
- A 56,000-dalton polypeptide was observed upon induction, corresponding to the overexpressed enzyme.
- Transcription of the algA gene was undetectable in chromosomal DNA of mucoid and non-mucoid P. aeruginosa but was highly detectable when expressed from the pMMB22 vector.
Conclusions:
- The cloned P. aeruginosa gene, designated algA, codes for phosphomannose isomerase.
- The algA gene is essential for phosphomannose isomerase activity, which is likely involved in alginate biosynthesis.
- Transcriptional analysis suggests that algA expression is tightly regulated in P. aeruginosa, with potential for inducible expression.