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The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

Petasch J, Disch EM, Markert S, Becher D, Schweder T, Hüttel B, Reinhardt R, Harder J - BMC Microbiol. (2014)

Bottom Line: An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes.CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase.Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, Bremen D-28359, Germany. jharder@mpi-bremen.de.

ABSTRACT

Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.

Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.

Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

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Anaerobic growth of C. defragrans 65Phen on limonene and putative metabolites. Limonene (A), perillyl alcohol (B), perillyl aldehyde (C) and perillic acid (D) were tested as growth substrate, each with a concentration of 3 mM and nitrate limitation (10 mM).
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Figure 1: Anaerobic growth of C. defragrans 65Phen on limonene and putative metabolites. Limonene (A), perillyl alcohol (B), perillyl aldehyde (C) and perillic acid (D) were tested as growth substrate, each with a concentration of 3 mM and nitrate limitation (10 mM).

Mentions: The geraniol dehydrogenase GeoA is an allyl-alcohol dehydrogenase with the highest catalytic activity on perillyl alcohol [26]. The high expression of GeoA and GeoB in α-phellandrene-grown cells questioned the utilization of the cyclic monoterpene alcohol by C. defragrans. The strain 65Phen grew on perillyl alcohol, perillyl aldehyde and perillic acid (Figure 1B-D). During active denitrification, perillyl aldehyde accumulated transiently in the culture growing on perillyl alcohol (Figure 1B). Perillic acid accumulated in cultures growing on the alcohol and the aldehyde. When the electron acceptor nitrate was depleted, the cells disproportionated perillyl aldehyde into perillyl alcohol and perillyl acid (Figure 1B, C). During growth on limonene (Figure 1A), the perillyl derivatives were not detected suggesting a rate-limitation in the pathway by the initial limonene-transforming enzyme.


The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

Petasch J, Disch EM, Markert S, Becher D, Schweder T, Hüttel B, Reinhardt R, Harder J - BMC Microbiol. (2014)

Anaerobic growth of C. defragrans 65Phen on limonene and putative metabolites. Limonene (A), perillyl alcohol (B), perillyl aldehyde (C) and perillic acid (D) were tested as growth substrate, each with a concentration of 3 mM and nitrate limitation (10 mM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109377&req=5

Figure 1: Anaerobic growth of C. defragrans 65Phen on limonene and putative metabolites. Limonene (A), perillyl alcohol (B), perillyl aldehyde (C) and perillic acid (D) were tested as growth substrate, each with a concentration of 3 mM and nitrate limitation (10 mM).
Mentions: The geraniol dehydrogenase GeoA is an allyl-alcohol dehydrogenase with the highest catalytic activity on perillyl alcohol [26]. The high expression of GeoA and GeoB in α-phellandrene-grown cells questioned the utilization of the cyclic monoterpene alcohol by C. defragrans. The strain 65Phen grew on perillyl alcohol, perillyl aldehyde and perillic acid (Figure 1B-D). During active denitrification, perillyl aldehyde accumulated transiently in the culture growing on perillyl alcohol (Figure 1B). Perillic acid accumulated in cultures growing on the alcohol and the aldehyde. When the electron acceptor nitrate was depleted, the cells disproportionated perillyl aldehyde into perillyl alcohol and perillyl acid (Figure 1B, C). During growth on limonene (Figure 1A), the perillyl derivatives were not detected suggesting a rate-limitation in the pathway by the initial limonene-transforming enzyme.

Bottom Line: An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes.CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase.Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, Bremen D-28359, Germany. jharder@mpi-bremen.de.

ABSTRACT

Background: The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.

Results: Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.

Conclusions: The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

Show MeSH
Related in: MedlinePlus