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Bacterial isoprene metabolism: a missing link in a key global biogeochemical cycle

Periodic Reporting for period 2 - IsoMet (Bacterial isoprene metabolism: a missing link in a key global biogeochemical cycle)

Reporting period: 2018-05-01 to 2019-10-31

Isoprene is an important biogenic volatile organic compound (BVOC) which is released into the environment in similar amounts as methane, making it the second most abundant organic trace gas after methane. Although we know a great deal about the biological cycling of methane, we know virtually nothing about how isoprene is recycled in the biosphere before it can escape to the atmosphere. Isoprene is an important building block for the isoprenoid family of compounds which include rubber, cholesterol and other steroids, vitamin A, carotenoids, monoterpenes and a component of compounds such as chlorophyll and vitamin K. There is a natural biogeochemical cycle for isoprene, with around 500-750 million tonnes of isoprene (about 1/3 of all BVOCs) being released into the environment by terrestrial plants per year. This represents 1-2% of net primary productivity by land plants re-emitted to the atmosphere as Isoprene. Isoprene has important effects on atmospheric chemistry. Isoprene in the atmosphere is oxidised by hydroxyl radicals which can remove the global warming gas methane from the atmosphere. Therefore more isoprene in the atmosphere indirectly contributes to global warming. Isoprene can also affect cloud formation by forming particles in the atmosphere and under certain circumstances can act as a global cooling gas. Fluctuating emissions of isoprene may have important consequences for biosphere function, atmospheric chemistry and climate. It also affects the amount of ozone in our atmosphere and so it is really important to know what happens to the isoprene that is emitted by plants and how it is removed from our biosphere. Some plants such as willow and oil palm are now being grown in huge amounts for the production of biofuels. These are very high emitters of isoprene and so removal of native trees and planting and growing these high isoprene-emitters in their place may have substantial consequences on air quality in these regions. It is important therefore that society needs to able to understand the global isoprene cycle. For example, w now know through microbiology research a lot about the methane cycle and how microbes both produce and consume methane. This allows us to be able to manage our global ecosystem and mitigate the effects of methane release to the atmosphere, for example through management of landfill, wetlands, rice paddies which are major systems that produce the greenhouse gas methane

It has been known for some time that bacteria might degrade isoprene in our environment but compared to our knowledge of methane oxidising bacteria we know virtually nothing about isoprene degradation. The overall aim of the IsoMet project is therefore to obtain a critical, fundamental understanding of the metabolism and ecological importance of biological isoprene degradation and to test the hypothesis that isoprene degrading bacteria play a role in isoprene cycling in the environment.
Key Objectives are to:
Determine the biological mechanisms by which isoprene is metabolised, both in model bacteria in the laboratory and in the environment.
Use novel methods to study isoprene degradation in the environment.
Elucidate at the mechanistic level how isoprene cycling by microbes is regulated in the environment.

These three broad objectives require a coordinated multidisciplinary approach and use of a wide range of innovative and leading edge techniques and so the specific aims of the project are:

a) Isolate and characterise bacteria that metabolise isoprene.
b) Elucidate the pathways of isoprene metabolism and their regulation.
c) Characterise the enzymes catalysing key steps in isoprene degradation.
d) Identify genes encoding isoprene-degrading enzymes and regulatory mechanisms.
e) Develop functional gene probes for the detection of isoprene degraders in the environment.
f) Determine the diversity and activity of isoprene degraders in the environment.
g) Assess the contribution that bacteria make to isoprene cycling in the en
We have characterised eight species of isoprene degrading bacteria from a variety of soils and leaves proving the widespread nature of these types of bacteria in the environment. We have shown that they are abundant in environments where there are high isoprene-emitting trees such as polar, willow and oil palm. In depth analysis of several of these isoprene degraders, including a gram positive Rhodococcus and a gram negative Variovorax has identified the pathways of isoprene degradation. They all use a soluble diiron centre monooxygenase called isoprene monooxygenase and a glutathione transferase enzyme, followed by two dehydrogenases to metabolise isoprene. We have shown that this is a common pathway found in all isolates. The genes encoding these enzymes are all clustered on the genome of isoprene degraders and are found on large plasmids in several of them. These isoprene gene clusters are evolutionarily related and are regulated by isoprene and its oxidation products. Genetic systems have been developed for Rhodococcus and Variovorax and we have shown that iso genes are switched on in response to oxygen. Detailed transcriptional analysis and the mechanisms of expression/regulation are being investigated. A knowledge of the regulation and promoters has allowed us to develop reporter strains to study isoprene degradation on plant leaf surfaces. We have purified the isoprene monooxygenase from Rhodococcus and it consists of a three component oxygenase, a reductase, a Rieske-like ferredoxin and a coupling protein. All are essential for activity in vitro. This novel and complex multicomponent enzyme that oxygenates isoprene to an epoxide. This metabolite is toxic but the bacteria are smart and use another enzyme, a glutathione transferase, to detoxify the epoxide and then assimilate its carbon into cellular material before it kills the bacterium. The isoprene monooxygenase is a versatile enzyme with a relatively high affinity for isoprene; it can also cooxidise a range of alkenes. We have discovered that octyne is a potent inhibitor which allows us to validate isoprene oxidation by this system in vivo.

We have developed functional gene probes to capture isoprene monooxygenase genes directly from the environment. This enables us to carry out cultivation-independent studies on these bacteria directly in the environment without the need to cultivate them in the laboratory. Using functional gene probes we have discovered that isoprene degrading bacteria can be detected in many different soil environments and especially on the leaves of isoprene-emitting trees such as willow, poplar and oil palm. A quantitative PCR assay be developed has allowed us to show that they are abundant in soils near the vicinity of isoprene emitting trees and we are currently investigating isoprene degradation on leaves of isoprene emitting plants using our gfp reporter strains which fluoresce when isoprene is used.

DNA stable isotope probing has been used in conjunction with metagenomics to determine the distribution and diversity of active isoprene degrading bacteria in soils from the vicinity of isoprene degrading trees and on their leaf surfaces, including willow and poplar (from the UK) and oil palm from Malaysia. These are hotspots of isoprene production, especially oil palm which is the highest known producer of isoprene and is grown in huge areas of the tropics. Our work has shown that isoprene degraders thrive on the leaves of these trees. Surveys also indicate their abundance depends on the occurrence of isoprene in the environment. We have rescued the genomes of key players in isoprene degradation from plants, including Variovorax, Rhodococcus, Gordonia, Ramlibacter, Sphingopyxsis, Nocardioides. They all contain the isoprene monooxygenase and isoprene pathway genes. Using this DNA sequence information and targeted enrichment strategies, we have now isolated these new isoprene degraders, investigation mechanisms of regulation of isoprene met
In the IsoMet project we have made significant progress in understanding the fundamental mechanisms of isoprene degradation by bacteria and how isoprene degrading bacteria survive and thrive in the environment. We can isolate and grow these bacteria in the laboratory in bioreactors using continuous culture techniques, which to our knowledge has not been achieved before. Using these techniques we can study the physiology of these bacteria and their regulation at the molecular level. We have not adapted existing methods to purify the key enzyme isoprene monooxygenase, developed several assays to monitor the activity of the enzyme. We use a Fast Isoprene Sensor built and modified for us by a company in Colorado which is providing an excellent real time monitor of bacterial isoprene consumption with sensitivity much greater than exisiting gas chromatography methods. This is the first time that this method has been employed by researchers to study isoprene metabolism.
We are developing and exploiting a novel molecular toolbox to estimate the distribution, diversity and activity of isoprene degrading bacteria in the environment. This includes making functional gene probes which are specific for these bacteria and detect them from a background of thousands of different bacteria in environmental samples. We exploit DNA and Protein Stable Isotope Probing methods in conjunction with genomics and metagenomics to examine the genomes and proteomes of isoprene degraders directly from environmental samples. With gets around the need to cultivate any bacteria which may be difficult to grow in the laboratory. We also develop single cell detection methods, reporter gene strains and use Raman microspectroscopy to visualise isoprene degraders directly in environmental samples and to determine if they are active in these environments.

Expected results in the future stages of the project include extensive knowledge about the physiology, biochemistry and molecular genetics/biology of novel isoprene degrading bacteria, how their isoprene-degrading activity is regultated in model bacteria in the laboratory and in the environment. The will be able to understand the exact molecular mechanisms by which isoprene is degraded and the roles that microbes play in the degradatation of isoprene in the environment and how they respond to increasing levels of isoprene in environments where there is increasing isoprene in the biosphere. This will in turn allow a synthesis of the role of microbes in regulation trace gas production in the environment and the importance of microbes in maintaining a healthy environment.
The global isoprene cycle
Genes and enzymes of the isoprene degradation pathway
Microscope image of an isoprene-degrading Rhodococcus