Summary auto-generated
This study describes the cloning and characterization of genes encoding components of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa. The researchers isolated a 3.2 kb genomic DNA fragment containing the cytochrome c550 gene (exaB) and identified adjacent genes: a truncated quinoprotein ethanol dehydrogenase gene (exaA), a NAD+-dependent acetaldehyde dehydrogenase gene (exaC), and genes related to PQQ biosynthesis (pqqA and pqqB). The cytochrome c550 protein was heterologously expressed in E. coli and shown to be functional. Through genetic complementation studies using mutagenized strains, they demonstrated that cytochrome c550 is essential for ethanol utilization—a strain with an inactivated exaB gene could not grow on ethanol. The gene organization differs from the well-characterized methanol oxidation system in methylotrophic bacteria, and the cytochrome c550 sequence shows only moderate similarity to other bacterial cytochromes.
Key findings
- Cytochrome c550 is essential for ethanol oxidation in P. aeruginosa, as exaB mutants cannot grow on ethanol
- A gene cluster containing exaA (QEDH), exaB (cytochrome c550), exaC (acetaldehyde dehydrogenase), pqqA, and pqqB was identified and sequenced
- Heterologously expressed cytochrome c550 in E. coli is functional and mediates electron transfer from QEDH to ferricyanide
- The genetic organization of the ethanol oxidation system differs from the analogous methanol oxidation system in methylotrophs
- Cytochrome c550 shows sequence similarity to algal cytochrome c6 and acetic acid bacteria quinohaemoproteins, but not to methanol oxidation cytochromes
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Abstract
Pseudomonas aeruginosa ATCC 17933 grown aerobically on ethanol produces a soluble cytochrome c550 together with a quinoprotein ethanol dehydrogenase. A 3.2 kb genomic DNA fragment containing the gene encoding cytochrome c550 was cloned and sequenced. Two other complete and two truncated ORFs were also identified. A truncated ORF encoding the quinoprotein ethanol dehydrogenase (exaA) was found upstream of the cytochrome c550 gene (exaB) and in reverse orientation. An ORF encoding a NAD(+)-dependent acetaldehyde dehydrogenase (exaC) was located downstream of the cytochrome c550 gene and in the same orientation. Another ORF showed similarity to the pqqA gene and a truncated ORF similarity to the pqqB gene, both involved in the biosynthesis of the prosthetic group PQQ. The organization of these genes was found to be different from the well-studied methanol oxidation system in methylotrophic bacteria. The deduced amino acid sequence of cytochrome c550 from P. aeruginosa showed some similarity to cytochrome c6 of the alga Chlamydomonas reinhardtii and the haem domain of quinohaemoprotein alcohol dehydrogenases of acetic acid bacteria, but no similarity to the soluble cytochrome cL of the quinoprotein methanol oxidation system of methylotrophs could be detected. A mutant of P. aeruginosa with an interrupted cytochrome c550 gene was unable to grow on ethanol, which proves that cytochrome c550 is an essential component of the ethanol oxidation system in this organism.