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Abstract Background Isoprene accounts for about half of total biogenic volatile organic compound emissions globally, and as a climate active gas it plays a significant and varied role in atmospheric chemistry. Terrestrial plants are the largest source of isoprene, with willow (Salix) making up one of the most active groups of isoprene producing trees. Bacteria act as a biological sink for isoprene and those bacteria associated with high isoprene-emitting trees may provide further insight into its biodegradation. Results A DNA-SIP experiment incubating willow (Salix fragilis) leaves with 13C-labelled isoprene revealed an abundance of Comamonadaceae, Methylobacterium, Mycobacterium and Polaromonas in the isoprene degrading community when analysed by 16S rRNA gene amplicon sequencing. Metagenomic analysis of 13C-enriched samples confirmed the abundance of Comamonadaceae, Acidovorax, Polaromonas, Variovorax and Ramlibacter. Mycobacterium and Methylobacterium were also identified after metagenomic analysis and a Mycobacterium metagenome-assembled genome (MAG) was recovered. This contained two complete isoprene degradation metabolic gene clusters, along with a propane monooxygenase gene cluster. Analysis of the abundance of the alpha subunit of the isoprene monooxygenase, isoA, in unenriched DNA samples revealed that isoprene degraders associated with willow leaves are abundant, making up nearly 0.2% of the natural bacterial community. Conclusions Analysis of the isoprene degrading community associated with willow leaves using DNA-SIP and focused metagenomics techniques enabled recovery of the genome of an active isoprene-degrading Mycobacterium species and provided valuable insight into bacteria involved in degradation of isoprene on the leaves of a key species of isoprene-emitting tree in the northern hemisphere.

Abstract Background Isoprene is the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those of methane. Despite its importance in atmospheric chemistry and climate, little is known about the biological degradation of isoprene in the environment. The largest source of isoprene is terrestrial plants, and oil palms, the cultivation of which is expanding rapidly, are among the highest isoprene-producing trees. Results DNA stable isotope probing (DNA-SIP) to study the microbial isoprene-degrading community associated with oil palm trees revealed novel genera of isoprene-utilising bacteria including Novosphingobium, Pelomonas, Rhodoblastus, Sphingomonas and Zoogloea in both oil palm soils and on leaves. Amplicon sequencing of isoA genes, which encode the α-subunit of the isoprene monooxygenase (IsoMO), a key enzyme in isoprene metabolism, confirmed that oil palm trees harbour a novel diversity of isoA sequences. In addition, metagenome-assembled genomes (MAGs) were reconstructed from oil palm soil and leaf metagenomes and putative isoprene degradation genes were identified. Analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere. Conclusion This study greatly expands the known diversity of bacteria that can metabolise isoprene and contributes to a better understanding of the biological degradation of this important but neglected climate-active gas. Video abstract.

Background: Isoprene accounts for about half of total biogenic volatile organic compound emissions globally, and as a climate active gas it plays a significant and varied role in atmospheric chemistry. Terrestrial plants are the largest source of isoprene, with willow (Salix) making up one of the most active groups of isoprene producing trees. Bacteria act as a biological sink for isoprene and those bacteria associated with high isoprene-emitting trees may provide further insight into its biodegradation. Results: A DNA-SIP experiment incubating willow (Salix fragilis) leaves with 13C-labelled isoprene revealed an abundance of Comamonadaceae, Methylobacterium, Mycobacterium and Polaromonas in the isoprene degrading community when analysed by 16S rRNA gene amplicon sequencing. Metagenomic analysis of 13C-enriched samples confirmed the abundance of Comamonadaceae, Acidovorax, Polaromonas, Variovorax and Ramlibacter. Mycobacterium and Methylobacterium were also identified after metagenomic analysis and a Mycobacterium metagenome-assembled genome (MAG) was recovered. This contained two complete isoprene degradation metabolic gene clusters, along with a propane monooxygenase gene cluster. Analysis of the abundance of the alpha subunit of the isoprene monooxygenase, isoA, in unenriched DNA samples revealed that isoprene degraders associated with willow leaves are abundant, making up nearly 0.2% of the natural bacterial community. Conclusions: Analysis of the isoprene degrading community associated with willow leaves using DNA-SIP and focused metagenomics techniques enabled recovery of the genome of an active isoprene-degrading Mycobacterium species and provided valuable insight into bacteria involved in degradation of isoprene on the leaves of a key species of isoprene-emitting tree in the northern hemisphere. [ABSTRACT FROM AUTHOR]

Abstract 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.

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.

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. [ABSTRACT FROM AUTHOR]

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 is a highly abundant biogenic volatile organic compound (BVOC) that is emitted to the atmosphere in amounts approximating to those of methane. The effects that isoprene has on Earth’s climate are both significant and complex, however, unlike methane, very little is known about the biological degradation of this environmentally important trace gas. Here, we review the mechanisms by which bacteria catabolise isoprene, what is known about the diversity of isoprene degraders in the environment, and the molecular tools currently available to study their ecology. Specifically, we focus on the use of probes based on the gene encoding the α-subunit of isoprene monooxygenase, isoA, and DNA stable-isotope probing (DNA-SIP) alone or in combination with other cultivation-independent techniques to determine the abundance, diversity, and activity of isoprene degraders in the environment. These parameters are essential in order to evaluate how microbes might mitigate the effects of this important but neglected climate-active gas. We also suggest key aspects of isoprene metabolism that require further investigation in order to better understand the global isoprene biogeochemical cycle.

[Display omitted] • Novel isoprene degrading Arthrobacter sp. strain BHU FT2 was sequenced. • Potential degradation pathway enzymes were identified. • Molecular docking gave high affinity of pathway enzymes for respective substrates. • Novel high sequence identity monooxygenase with known isoprene monooxygenase obtained. • In-vitro analysis on hydrocarbons displayed high specificity of isolate for isoprene. The bacterial degradation of isoprene is important for maintaining its atmospheric concentration in unpolluted environment. It may be possible to use natural isoprene degrading bacteria in engineered systems to eliminate or limit isoprene emissions from various sources. Biodegradation of isoprene by Arthrobacter sp. strain BHU FT2 was investigated. The genome was found to contain 4151545 bp long chromosome having 3747 coding genes, and coded potential isoprene degrading enzymes. The molecular docking of monooxygenases with isoprene displayed a higher binding energy (−4.59 kcal/mol) for WP_015938387.1 monooxygenase. Analysis of the identified monooxygenases with the known isoprene monooxygenases revealed 67% sequence identity of WP_015938387.1 (Locus tag JHV56_10705) monooxygenase of the considered strain with the OPX16961.1 monooxygenase of Gordonia sp. i37 isoprene degrading starin. These results provided a strong evidence for the high isoprene degrading potential of the Arthrobacter sp. BHU FT2 which could be efficiently exploited for isoprene degradation in large scale bio-filtration units. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Isoprene is a highly abundant biogenic volatile organic compound (BVOC) that is emitted to the atmosphere in amounts approximating to those of methane. The effects that isoprene has on Earth's climate are both significant and complex, however, unlike methane, very little is known about the biological degradation of this environmentally important trace gas. Here, we review the mechanisms by which bacteria catabolise isoprene, what is known about the diversity of isoprene degraders in the environment, and the molecular tools currently available to study their ecology. Specifically, we focus on the use of probes based on the gene encoding the α-subunit of isoprene monooxygenase, isoA, and DNA stable-isotope probing (DNA-SIP) alone or in combination with other cultivation-independent techniques to determine the abundance, diversity, and activity of isoprene degraders in the environment. These parameters are essential in order to evaluate how microbes might mitigate the effects of this important but neglected climate-active gas. We also suggest key aspects of isoprene metabolism that require further investigation in order to better understand the global isoprene biogeochemical cycle. [ABSTRACT FROM AUTHOR]