Poplar phyllosphere harbors disparate isoprene-degrading bacteria.

The climate-active gas isoprene (2-methyl-1,3-butadiene) is released to the atmosphere in huge quantities, almost equaling that of methane, yet we know little about the biological cycling of isoprene in the environment. Although bacteria capable of growth on isoprene as the sole source of carbon and energy have previously been isolated from soils and sediments, no microbiological studies have targeted the major source of isoprene and examined the phyllosphere of isoprene-emitting trees for the presence of degraders of this abundant carbon source. Here, we identified isoprene-degrading bacteria in poplar tree-derived microcosms by DNA stable isotope probing. The genomes of isoprene-degrading taxa were reconstructed, putative isoprene metabolic genes were identified, and isoprene-related gene transcription was analyzed by shotgun metagenomics and metatranscriptomics. Gram-positive bacteria of the genus Rhodococcus proved to be the dominant isoprene degraders, as previously found in soil. However, a wider diversity of isoprene utilizers was also revealed, notably Variovorax, a genus not previously associated with this trait. This finding was confirmed by expression of the isoprene monooxygenase from Variovorax in a heterologous host. A Variovorax strain that could grow on isoprene as the sole carbon and energy source was isolated. Analysis of its genome confirmed that it contained isoprene metabolic genes with an identical layout and high similarity to those identified by DNA-stable isotope probing and metagenomics. This study provides evidence of a wide diversity of isoprene-degrading bacteria in the isoprene-emitting tree phyllosphere and greatly enhances our understanding of the biodegradation of this important metabolite and climate-active gas.

Complete Genome of Isoprene Degrading Nocardioides sp. WS12.

Isoprene is a climate-active gas whose wide-spread global production stems mostly from terrestrial plant emissions. The biodegradation of isoprene is carried out by a number of different bacteria from a wide range of environments. This study investigates the genome of a novel isoprene degrading bacterium Nocardioides sp. WS12, isolated from soil associated with Salix alba (Willow), a tree known to produce high amounts of isoprene. The Nocardioides sp. WS12 genome was fully sequenced, revealing the presence of a complete isoprene monooxygenase gene cluster, along with associated isoprene degradation pathway genes. Genes associated with rubber degradation were also present, suggesting that Nocardioides sp. WS12 may also have the capacity to degrade poly-cis-1,4-isoprene.

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

The volatile secondary metabolite, isoprene, is released by trees to the atmosphere in enormous quantities, where it has important effects on air quality and climate. Oil palm trees, one of the highest isoprene emitters, are increasingly dominating agroforestry over large areas of Asia, with associated uncertainties over their effects on climate. Microbes capable of using isoprene as a source of carbon for growth have been identified in soils and in the tree phyllosphere, and most are members of the Actinobacteria. Here, we used DNA stable isotope probing to identify the isoprene-degrading bacteria associated with oil palm leaves and inhabiting the surrounding soil. Among the most abundant isoprene degraders of the leaf-associated community were members of the Sphingomonadales, although no representatives of this order were previously known to degrade isoprene. Informed by these data, we obtained representatives of the most abundant isoprene degraders in enrichments, including Sphingopyxis strain OPL5 (Sphingomonadales), able to grow on isoprene as the sole source of carbon and energy. Sequencing of the genome of strain OPL5, as well as a novel Gordonia strain, confirmed their pathways of isoprene degradation and broadened our knowledge of the genetic and taxonomic diversity of this important bacterial trait.

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

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.

Novel Isoprene-Degrading Proteobacteria From Soil and Leaves Identified by Cultivation and Metagenomics Analysis of Stable Isotope Probing Experiments.

Isoprene is a climate-active gas and one of the most abundant biogenic volatile organic compounds (BVOC) released into the atmosphere. In the terrestrial environment, plants are the primary producers of isoprene, releasing between 500 and 750 million tons per year to protect themselves from environmental stresses such as direct radiation, heat, and reactive oxygen species. While many studies have explored isoprene production, relatively little is known about consumption of isoprene by microbes and the most well-characterized isoprene degrader is a Rhodococcus strain isolated from freshwater sediment. In order to identify a wider range of bacterial isoprene-degraders in the environment, DNA stable isotope probing (DNA-SIP) with 13C-labeled isoprene was used to identify active isoprene degraders associated with soil in the vicinity of a willow tree. Retrieval by PCR of 16S rRNA genes from the 13C-labeled DNA revealed an active isoprene-degrading bacterial community dominated by Proteobacteria, together with a minor portion of Actinobacteria, mainly of the genus Rhodococcus. Metagenome sequencing of 13C-labeled DNA from SIP experiments enabled analysis of genes encoding key enzymes of isoprene metabolism from novel isoprene degraders. Informed by these DNA-SIP experiments and working with leaves and soil from the vicinity of tree species known to produce high amounts of isoprene, four novel isoprene-degrading strains of the genera Nocardioides, Ramlibacter, Variovorax and Sphingopyxis, along with strains of Rhodococcus and Gordonia, genera that are known to contain isoprene-degrading strains, were isolated. The use of lower concentrations of isoprene during enrichment experiments has revealed active Gram-negative isoprene-degrading bacteria associated with isoprene-emitting trees. Analysis of isoprene-degradation genes from these new isolates provided a more robust phylogenetic framework for analysis of isoA, encoding the α-subunit of the isoprene monooxygenase, a key molecular marker gene for cultivation-independent studies on isoprene degradation in the terrestrial environment.

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment.

BACKGROUND: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. RESULTS: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. CONCLUSION: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas.