Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Analysis of Essential Isoprene Metabolic Pathway Proteins in Variovorax sp. Strain WS11.

Isoprene monooxygenase (IsoMO, encoded by isoABCDEF) initiates the oxidation of the climate-active gas isoprene, with the genes isoGHIJ and aldH nearly always found adjacent to isoABCDEF in extant and metagenome-derived isoprene degraders. The roles of isoGHIJ and aldH are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, Rhodococcus sp. strain AD45 and Variovorax sp. strain WS11. The genes isoHIJ and aldH from Variovorax and aldH from Rhodococcus were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione S-transferases from Variovorax, IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of Variovorax is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from Variovorax and Rhodococcus were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An in silico analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in Variovorax, is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. IMPORTANCE Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes isoABCDEF. In isoprene-degrading bacteria, a second gene cluster, isoGHIJ, is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera Rhodococcus and Variovorax.

Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.

Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.

Author Correction: DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton.

In the version of this Letter originally published, the Methods incorrectly stated that all phytoplankton cultures were sampled in mid-exponential phase. The low-nitrogen cultures were sampled in early stationary phase and at the point at which Fv/Fm values decreased, to indicate that cultures were experiencing low-nitrogen conditions. All other phytoplankton cultures were sampled in exponential phase. Growth and Fv/Fm data are provided here on high- and low-nitrogen cultures (Figs 1, 2 and 3) to clarify and support this correction. The Methods also stated that cell counting was done using a Beckman Multisizer 3 Coulter Counter, but a CASY Model TT Cell Counter was used.

DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton.

Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1-3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton 4 , and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified 5 . However, eukaryotic phytoplankton probably produce most of Earth's DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.

Identification and characterisation of isoprene-degrading bacteria in an estuarine environment.

Approximately one-third of volatile organic compounds (VOCs) emitted to the atmosphere consists of isoprene, originating from the terrestrial and marine biosphere, with a profound effect on atmospheric chemistry. However, isoprene provides an abundant and largely unexplored source of carbon and energy for microbes. The potential for isoprene degradation in marine and estuarine samples from the Colne Estuary, UK, was investigated using DNA-Stable Isotope Probing (DNA-SIP). Analysis at two timepoints showed the development of communities dominated by Actinobacteria including members of the genera Mycobacterium, Rhodococcus, Microbacterium and Gordonia. Representative isolates, capable of growth on isoprene as sole carbon and energy source, were obtained from marine and estuarine locations, and isoprene-degrading strains of Gordonia and Mycobacterium were characterised physiologically and their genomes were sequenced. Genes predicted to be required for isoprene metabolism, including four-component isoprene monooxygenases (IsoMO), were identified and compared with previously characterised examples. Transcriptional and activity assays of strains growing on isoprene or alternative carbon sources showed that growth on isoprene is an inducible trait requiring a specific IsoMO. This study is the first to identify active isoprene degraders in estuarine and marine environments using DNA-SIP and to characterise marine isoprene-degrading bacteria at the physiological and molecular level.