Bacterial Degradation of Isoprene in the Terrestrial Environment

Isoprene is a climate active gas emitted from natural and anthropogenic sources in quantities equivalent to the global methane flux to the atmosphere. 90 % of the emitted isoprene is produced enzymatically in the chloroplast of terrestrial plants from dimethylallyl pyrophosphate via the methylerythritol pathway. The main role of isoprene emission by plants is to reduce the damage caused by heat stress through stabilizing cellular membranes. Isoprene emission from microbes, animals, and humans has also been reported, albeit less understood than isoprene emission from plants. Despite large emissions, isoprene is present at low concentrations in the atmosphere due to its rapid reactions with other atmospheric components, such as hydroxyl radicals. Isoprene can extend the lifetime of potent greenhouse gases, influence the tropospheric concentrations of ozone, and induce the formation of secondary organic aerosols. While substantial knowledge exists about isoprene production and atmospheric chemistry, our knowledge of isoprene sinks is limited. Soils consume isoprene at a high rate and contain numerous isoprene-utilizing bacteria. However, Rhodococcus sp. AD45 is the only terrestrial isoprene-degrading bacterium characterized in any detail. A pathway for isoprene degradation involving a putative soluble monooxygenase has been proposed. In this study, we report the isolation of two novel isoprene-degrading bacteria and characterization of the isoprene gene clusters in their draft genomes. Using marker exchange mutagenesis, transcription assays and proteomics analyses, we provide conclusive evidence that isoprene is metabolized in Rhodococcus sp. AD45 through the induced activity of soluble isoprene monooxygenase, a close relative to well known soluble diiron center monooxygenase enzymes. Metabolic gene PCR assays based on a key component of isoprene monooxygenase were also developed to detect isoprene degraders in the environment. The diversity of active isoprene degraders in the terrestrial environment was investigated using DNA-stable isotope probing experiments combined with 454 pyrosequencing

Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle

Emissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation. Here, we report the genome of an isoprene degrading isolate, Rhodococcus sp. AD45, and, using mutagenesis shows that a plasmid-encoded soluble di-iron centre isoprene monooxygenase (IsoMO) is essential for isoprene metabolism. Using RNA sequencing (RNAseq) to analyse cells exposed to isoprene or epoxyisoprene in a substrate-switch time-course experiment, we show that transcripts from 22 contiguous genes, including those encoding IsoMO, were highly upregulated, becoming among the most abundant in the cell and comprising over 25% of the entire transcriptome. Analysis of gene transcription in the wild type and an IsoMO-disrupted mutant strain showed that epoxyisoprene, or a subsequent product of isoprene metabolism, rather than isoprene itself, was the inducing molecule. We provide a foundation of molecular data for future research on the environmental biological consumption of this important, climate-active compound

Draft genome sequences of three terrestrial isoprene-degrading Rhodococcus strains

Isoprene is produced in abundance by plants and constitutes a carbon source for microbes. The genomes of three isoprene degraders isolated from tree leaves or soil from the campus of the University of East Anglia were sequenced. These high-GC-content isolates are actinobacteria belonging to the genus Rhodococcus

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

Prospective randomized trial of iliohypogastric-ilioinguinal nerve block on post-operative morphine use after inpatient surgery of the female reproductive tract

<p>Abstract</p> <p>Objective</p> <p>To determine the impact of pre-operative and intra-operative ilioinguinal and iliohypogastric nerve block on post-operative analgesic utilization and length of stay (LOS).</p> <p>Methods</p> <p>We conducted a prospective randomized double-blind placebo controlled trial to assess effectiveness of ilioinguinal-iliohypogastric nerve block (IINB) on post-operative morphine consumption in female study patients (<it>n </it>= 60). Patients undergoing laparotomy via Pfannenstiel incision received injection of either 0.5% bupivacaine + 5 mcg/ml epinephrine for IINB (Group I, <it>n </it>= 28) or saline of equivalent volume given to the same site (Group II, <it>n </it>= 32). All injections were placed before the skin incision and after closure of rectus fascia via direct infiltration. Measured outcomes were post-operative morphine consumption (and associated side-effects), visual analogue pain scores, and hospital length of stay (LOS).</p> <p>Results</p> <p>No difference in morphine use was observed between the two groups (47.3 mg in Group I vs. 45.9 mg in Group II; <it>p </it>= 0.85). There was a trend toward lower pain scores after surgery in Group I, but this was not statistically significant. The mean time to initiate oral narcotics was also similar, 23.3 h in Group I and 22.8 h in Group II (<it>p </it>= 0.7). LOS was somewhat shorter in Group I compared to Group II, but this difference was not statistically significant (<it>p </it>= 0.8). Side-effects occurred with similar frequency in both study groups.</p> <p>Conclusion</p> <p>In this population of patients undergoing inpatient surgery of the female reproductive tract, utilization of post-operative narcotics was not significantly influenced by IINB. Pain scores and LOS were also apparently unaffected by IINB, indicating a need for additional properly controlled prospective studies to identify alternative methods to optimize post-surgical pain management and reduce LOS.</p

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

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

Diversity of isoprene-degrading bacteria in phyllosphere and soil communities from a high isoprene-emitting environment: a Malaysian oil palm plantation

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

Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome. In Rhodococcus strain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEF and isoGHIJ. Sequence analysis of the isoA gene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria

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