Poplar phyllosphere harbors disparate isoprene- degrading bacteria Andrew T. Crombiea,1, Nasmille L. Larke-Mejiab, Helen Emeryb, Robin Dawsonb, Jennifer Pratscherc, Gordon P. Murphyd, Terry J. McGenityd, and J. Colin Murrellb aSchool of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; bSchool of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; cLyell Centre, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom; and dSchool of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved November 1, 2018 (received for review July 24, 2018) The climate-active gas isoprene (2-methyl-1,3-butadiene) is re- Plants produce isoprene in the chloroplast and release it to the leased to the atmosphere in huge quantities, almost equaling that atmosphere from the abaxial surface of leaves via the stomata of methane, yet we know little about the biological cycling of (12). Although isoprene production is not a universal trait isoprene in the environment. Although bacteria capable of growth among plants, it protects against heat and oxidative stress (13, on isoprene as the sole source of carbon and energy have previously 14) and has roles in plant/insect signaling and plant energy been isolated from soils and sediments, no microbiological studies dynamics (5, 15). High-isoprene–emitting trees worldwide in- have targeted the major source of isoprene and examined the clude oil palm (Elaeis guineensis), eucalyptus (Eucalyptus spp.) phyllosphere of isoprene-emitting trees for the presence of degraders and, in temperate regions, English oak (Quercus robur), poplar of this abundant carbon source. Here, we identified isoprene- (Populus spp.), and willow (Salix spp.), with reported emissions −1 −1 degrading bacteria in poplar tree-derived microcosms by DNA stable of 77 (English oak) and 175 (oil palm) μg·g (dry leaves) h isotope probing. The genomes of isoprene-degrading taxa were (16). Due to its short atmospheric lifetime, isoprene concen- reconstructed, putative isoprene metabolic genes were identified, trations vary from a few parts per billion by volume (ppbv) – and isoprene-related gene transcription was analyzed by shotgun (17), to tens of ppbv in high-isoprene emitting forests (18). metagenomics and metatranscriptomics. Gram-positive bacteria of Soils acted as a biological sink, both in closed chambers (19, the genus Rhodococcus proved to be the dominant isoprene de- 20) and in continuous systems using isoprene concentra- MICROBIOLOGY – graders, as previously found in soil. However, a wider diversity of tions of 2 200 ppbv (21), suggesting that soil microbes act as a isoprene utilizers was also revealed, notably Variovorax, a genus significant sink for isoprene, consuming up to 4% of global not previously associated with this trait. This finding was confirmed emissions. Variovorax Several isoprene-consuming bacterial strains have been iso- by expression of the isoprene monooxygenase from in a – heterologous host. A Variovorax strain that could grow on isoprene lated from soils and marine sediments (6, 22 25), and recently several strains have been characterized biochemically and ge- as the sole carbon and energy source was isolated. Analysis of its – genome confirmed that it contained isoprene metabolic genes with netically (26 30). Before this work, all strains characterized by sequence data were members of the Actinobacteria, and all an identical layout and high similarity to those identified by DNA- stable isotope probing and metagenomics. This study provides evi- dence of a wide diversity of isoprene-degrading bacteria in the Significance isoprene-emitting tree phyllosphere and greatly enhances our under- standing of the biodegradation of this important metabolite and Isoprene is a climate-active gas, produced in huge amounts by climate-active gas. trees, yet we know little about its biogeochemical cycle. Bac- teria able to grow on isoprene have been isolated from soils isoprene | microbiology | plant–microbe interactions | DNA-SIP | and sediments, but the phyllosphere, the principal isoprene phyllosphere source, has remained unexplored. Using targeted cultivation- independent techniques, we show that the phyllosphere of an soprene (2-methyl-1,3-butadiene) is emitted to the atmosphere isoprene-emitting tree contains a diverse and active isoprene- − at a rate of ∼500 Tg·y 1, on a par with methane and approxi- degrading population. We reconstruct the genome of an I Variovorax mately one-third of the total volatile organic compound (VOC) isoprene-degrading strain and show that it contains emissions (1, 2). The vast majority originates from terrestrial a functional isoprene monooxygenase. This detailed study tar- − plants (400–600 Tg·y 1), with a small but uncertain flux from gets isoprene degraders from the phyllosphere, applies metao- − mics to isoprene degradation, and isolates and sequences an marine algae (0.1–12 Tg·y 1)(2–4), bacteria, fungi, and animals isoprene-degrading member of the Proteobacteria. (5, 6). A reactive diene, isoprene is rapidly photochemically ox- idized (1), with a significant and complex effect on global climate Author contributions: A.T.C., N.L.L.-M., G.P.M., T.J.M., and J.C.M. designed research; (7). Hydroxyl (OH) and nitrate (NO3) radicals and ozone (O3)in A.T.C., N.L.L.-M., H.E., R.D., and G.P.M. performed research; A.T.C., N.L.L.-M., H.E., J.P., the atmosphere react with isoprene depending on prevailing and G.P.M. analyzed data; and A.T.C., T.J.M., and J.C.M. wrote the paper. conditions (1). In pristine environments, isoprene reacts directly The authors declare no conflict of interest. with ozone and hydroxyl radicals, resulting in ozone depletion. This article is a PNAS Direct Submission. However, the high NOx levels typical of urban environments This open access article is distributed under Creative Commons Attribution-NonCommercial- result in the formation of tropospheric ozone, with important NoDerivatives License 4.0 (CC BY-NC-ND). negative effects on human health and on yields of ozone- Data deposition: The genome sequences of Variovorax sp. WS11 and megaplasmid-cured sensitive crops (8). Globally, these reactions result in a net ra- Rhodococcus AD45-ID have been deposited at DNA Data Bank of Japan/European Nucle- · −2 otide Archive/GenBank under accession nos. PXZZ00000000 and PYHL00000000. Versions diative forcing of 0.09 W m , with an additional indirect effect described here are PXZZ01000000 and PYHL0100000, respectively. Sequence reads have since depletion of OH radicals increases the atmospheric life- been deposited at GenBank Sequence Read Archive (accession no. SRP101805). time of methane (9). The isoprene oxidation products form 1To whom correspondence should be addressed. Email: [email protected]. secondary organic aerosols and cloud condensation nuclei with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. implications for planetary albedo, air quality, and climate 1073/pnas.1812668115/-/DCSupplemental. (10, 11). Published online November 29, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1812668115 PNAS | December 18, 2018 | vol. 115 | no. 51 | 13081–13086 Downloaded by guest on September 29, 2021 terrestrial examples were rhodococci. They all contain six genes incubated in minimal medium with 13C-labeled or unlabeled (isoABCDEF) encoding a multicomponent isoprene mono- isoprene (headspace concentration ∼500 ppmv). Added isoprene oxygenase (IsoMO) (Fig. 1) with four additional genes, isoGHIJ, was consumed rapidly, and cells were harvested after 7 or 8 d (SI just upstream. van Hylckama Vlieg et al. (26, 27) showed that Appendix,Fig.S1). Extraction, fractionation (based on incorpo- isoI encodes a glutathione S-transferase that conjugates gluta- ration of 13C label), and quantification of DNA showed that thione with the epoxide product of IsoMO, and that isoH en- labeled DNA from the 13C-isoprene incubations migrated to codes a dehydrogenase that acts on the product of IsoI. IsoG and “heavy” and “light” regions of the ultracentrifuge tubes depending IsoJ are involved in the subsequent metabolic pathway but have on incorporation of label, whereas DNA from 12C-isoprene in- yet to be characterized. In Rhodococcus AD45, the entire cluster cubations was restricted (>99%) to light fractions (SI Appendix, isoGHIJABCDEF is cotranscribed as an operon (28). Signifi- Fig. S2A). Analysis of 16S rRNA gene amplicons showed that cantly, all previously characterized isoprene gene clusters include the unenriched bacterial community, highly consistent across six the glutathione biosynthesis genes gshAB, suggesting a specific replicates, was composed mainly of Proteobacteria [Sphingomonas role for glutathione in isoprene metabolism. This small thiol is (mean relative abundance (RA) 36%), Pseudomonas (17%), not usually found in Gram-positive bacteria (31), although a role members of the Oxalobacteraceae (8%), Methylobacterium in styrene metabolism has also been suggested recently (32). (2%) and members of the Comamonadaceae (2%)], Bacter- The plant microbiome plays an essential role in plant health oidetes [Hymenobacter (17%) and Sphingobacteriaceae (1%)], and development (33), and the phyllosphere, although an un- and Actinobacteria (4%) (Fig. 2 and SI Appendix, Fig. S3), in stable environment, constitutes an extensive habitat for micro- agreement with previous studies of plant-associated bacteria (45,