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Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa
J L Rae1, J F Cutfield, I L Lamont
1Department of Biochemistry and Centre for Gene Research, University of Otago, Dunedin, New Zealand.
Abstract:
A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH.
Insights
A Pseudomonas aeruginosa mutant deficient in pyoverdine production was identified as an aceB mutant, lacking a functional pyruvate dehydrogenase complex (PDH). This defect causes pyruvic acid accumulation and medium acidification, explaining the pyoverdine deficiency.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- A Pseudomonas aeruginosa mutant (OT2100) was previously isolated with a defect in pyoverdine production.
- Transposon mutagenesis was used to identify the mutated gene responsible for this phenotype.
Purpose of the Study:
- To identify and characterize the gene mutated in Pseudomonas aeruginosa OT2100.
- To elucidate the molecular basis for the pyoverdine-deficient phenotype.
Main Methods:
- DNA sequencing to identify the transposon insertion site.
- Enzyme assays to assess pyruvate dehydrogenase (PDH) activity.
- Transcriptional analysis to study gene expression.
Main Results:
- The mutated gene, aceB, encodes the E2 component of PDH, crucial for complex assembly.
- The mutant OT2100 showed defective PDH activity, which was partially restored by adding an E1 component.
- A second gene, aceA (encoding the E1 component), was identified upstream of aceB.
- aceA and aceB are co-transcribed as a polycistronic mRNA, with aceB also having a separate transcript.
Conclusions:
- Mutant OT2100 is an aceB mutant defective in the dihydrolipoamide acetyltransferase (E2) subunit of PDH.
- The pyoverdine deficiency is likely a secondary effect of medium acidification caused by pyruvic acid accumulation due to non-functional PDH.