Isoprene Oxidation by the Gram-Negative Model bacterium Variovorax sp. WS11

Abstract

Plant-produced isoprene (2-methyl-1,3-butadiene) represents a significant portion of global volatile organic compound production, equaled only by methane. A metabolic pathway for the degradation of isoprene was first described for the Gram-positive bacterium Rhodococcus sp. AD45, and an alternative model organism has yet to be characterised. Here, we report the characterisation of a novel Gram-negative isoprene-degrading bacterium, Variovorax sp. WS11. Isoprene metabolism in this bacterium involves a plasmid-encoded iso metabolic gene cluster which differs from that found in Rhodococcus sp. AD45 in terms of organisation and regulation. Expression of iso metabolic genes is significantly upregulated by both isoprene and epoxyisoprene. The enzyme responsible for the initial oxidation of isoprene, isoprene monooxygenase, oxidises a wide range of alkene substrates in a manner which is strongly influenced by the presence of alkyl side-chains and differs from other well-characterised soluble diiron monooxygenases according to its response to alkyne inhibitors. This study presents Variovorax sp. WS11 as both a comparative and contrasting model organism for the study of isoprene metabolism in bacteria, aiding our understanding of the conservation of this biochemical pathway across diverse ecological niches.

Sphingopyxis sp. Strain OPL5, an Isoprene-Degrading Bacterium from the Sphingomonadaceae Family Isolated from Oil Palm Leaves

“…Optimal growth conditions for Sphingopyxis strain OPL5 were used (30 °C, 160 rpm, pH 6.5, 1 mL per liter vitamins v10 solution, and air flow 2.4 L/min). Isoprene was supplied by bubbling air (1 mL min −1 ) through a small volume of liquid isoprene contained in a 30-mL vial, held on ice (as described in [ 26 ]). Cells were harvested three times, at an optical density (540 nm) of 1.0, 2.5, and 3.1, respectively ( Figure S7 ) by centrifuging at 7000× g for 20 min and resuspending in 50 mM HEPES, pH 6.5, before snap-freezing and storing at −80 °C.…”

“…Frozen cells were thawed and resuspended to an OD 540 of 2.0 in 3 mL of 0.5 M phosphate buffer (pH 6.5) and equilibrated to 30 °C for 2–3 min with stirring. Substrate-induced rates of oxygen depletion were calculated for concentrations of isoprene from 3 μM to 30 μM ( Figure S8 ) as detailed in [ 26 ].…”

“…The best studied is Rhodococcus strain AD45, isolated from freshwater sediment, which has become a model bacterium for the study of isoprene metabolism [ 23 , 24 ]. There are fewer cultivated Gram-negative bacteria that have been reported to degrade isoprene [ 25 , 26 ]. The most well-characterized of these is Variovorax strain WS11, originally isolated from soil [ 25 , 26 , 27 ].…”

“…There are fewer cultivated Gram-negative bacteria that have been reported to degrade isoprene [ 25 , 26 ]. The most well-characterized of these is Variovorax strain WS11, originally isolated from soil [ 25 , 26 , 27 ]. All isoprene degraders characterized to date contain a multi-subunit isoprene monooxygenase, encoded by the genes isoABCDEF , which catalyzes the oxidation of isoprene to epoxyisoprene, and the gene cluster isoGHIJ encoding a glutathione transferase and enzymes catalyzing further steps in the isoprene oxidation pathway [ 23 ].…”

“…The isoprene monooxygenase (IsoMO) can be distinguished from related soluble di-iron monooxygenases (SDIMOs, [ 28 ]) that are involved in metabolism of alkenes, as well as alkanes and aromatic compounds (reviewed in [ 18 ]). In Variovorax strain WS11 and Rhodococcus strain AD45, iso genes are carried on megaplasmids [ 24 , 26 ]. New understanding of the physiology, biochemistry, and molecular biology of isoprene metabolism has provided the tools for designing functional gene primers and probes based on isoA, encoding a key component of the IsoMO [ 19 , 29 ].…”