Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal

Abstract

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.

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Fig. 1: Attraction of springtails to Streptomyces headspace, geosmin or 2-MIB.
Fig. 2: Antennal responses to Streptomyces headspace components in F. candida.
Fig. 3: Developmental regulation of geosmin and 2-MIB biosynthetic genes.
Fig. 4: Dispersal of S. coelicolor spores mediated by springtails.

Data availability

The transcriptional profiling data from Affymetrix arrays and the ChIP–seq data in Fig. 3 have been deposited at the ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5853 for the whiH mutant array data, E-MTAB-2716 for the bldM mutant array data, E-MTAB-6702 for the WhiH ChIP–seq data and E-MTAB-2698 for the BldM ChIP–seq data). Source data for Figs. 1a,b,e,f,g and 4b,c and Extended Data Figs. 1a,b,c, 4, 5 and 6a,b,c are included in this letter and its Supplementary Information files. Other data that support the findings of the present study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank O. Gustafsson for assistance with electron microscopy, M. Holmstrup for helping us to establish F. candida, M. Karlsson for confirming species identity of F. candida, T. Verschut for discussions, G. Birgersson for technical support (statistics@slu) and F. Jüttner for comments on the manuscript. This work was funded by a PlantLink seed money grant to P.G.B. and K.F., BBSRC grants (nos. BB/H006125/1 and BB/I002197/1) to M.J. Buttner, BBSRC Institute Strategic Programme grants (nos. BB/J004561/1 and BB/P012523/1) to the John Innes Centre, grants from the Crafoord Foundation and support through the SLU Centre for Biological Control to P.G.B., and grants from the Swedish Research Council (grant nos. 2010-4463 and 2015-05452) to K.F. B.P.M. was supported by the New National Excellence Program of the Ministry of Human Capacities (ÚNKP-19-4) and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

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Contributions

M.J. Buttner, P.G.B. and K.F. designed the research. V.V. performed the field trap experiments and statistical analyses. V.V. and P.G.B. did the work involving springtails and analyses of volatiles; together with E.B. and K.F. they performed the spore dispersal assays. B.P.M. performed the GC–EAD experiments. M.J. Bibb and M.M.A.-B. performed the time-resolved transcriptional profiling, M.J. Bibb, M.J. Bush and M.M.A.-B. performed the ChIP–seq experiments. G.C. analysed the ChIP–seq data. M.J. Bibb and M.J. Bush constructed mutants and plasmids. E.B. and K.F. identified WhiH as a regulator of geoA. L.S. and G.L.C. performed initial GC–MS analyses of the Streptomyces mutants. P.G.B. and K.F. wrote the manuscript together with V.V. and M.J. Buttner. All the authors discussed the results and commented on the manuscript.

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Correspondence to Paul G. Becher or Klas Flärdh.

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Extended data

Extended Data Fig. 1 Effects and production of volatiles from Streptomyces coelicolor.

Captures of insects (a) and arachnids (b) on the ground of a field site using sticky traps baited with Streptomyces coelicolor cultured on agar medium, compared to control traps that were kept without bait or held a fresh, non-inoculated plate of agar culture medium (n=19). The boxplots show the medians, lower- and upper quartiles as well as the span of data points that are within 1.5 times the interquartile range. Electroantennographic responses (c) towards 2-MIB or geosmin puffed into an air stream passing over antennae of the springtails Folsomia candida. The average responses to all tested stimuli (n=8 per stimulus) significantly differed from the antennal response to charcoal filtered air. Statistical difference is labelled by different letters above the columns; error bars show the standard errors (X25, 48 =629.54, P<0.001). GC-MS chromatograms (d) of S. coelicolor wild type and two biosynthetic mutants show that geosmin or geosmin and 2-MIB, respectively, are missing from the geoA and geoA mibAB mutants (strains J3003 and J2192, respectively). The headspace collection for the specific strains was repeated three times with similar results. Source data

Extended Data Fig. 2

Absolute amounts (ng) of geosmin and 2-methylisoborneol (2-MIB) tested as individual compounds or as components of Streptomyces headspace in the Y-tube choice assay.

Extended Data Fig. 3 Folsomia candida springtails feed on Streptomyces coelicolor mycelium.

(a) Folsomia candida springtails feeding on sporulating Streptomyces coelicolor. The bacteria were grown as lawns on agar covered with cellophane membranes, and then scraped off and transferred to F. candida kept on a Petri dish lined with gypsum plaster. The photo shows the dark bacterial biomass visible inside the guts of the animals as well as faecal pellets covering the plaster. (b, c) Agar plugs with developed colonies of S. coelicolor strain M145 were placed in containers with autoclaved potting soil, either without springtails (b), or with 10 F. candida individuals (c). After 6 days of incubation, the colonies in soil without springtails remained intact, while the colonies in soil with springtails were consumed by grazing of the animals. Control experiments showed that the animals did not graze on uninoculated agarose plugs (not shown). Scale bars, 10 mm. Similar observations were made in three independent experiments. In addition to the grazing, the ability of springtails to disperse S. coelicolor was clearly reflected in the large number of microcolonies (seen as white spots in the photographs) developing throughout the microcosms containing F. candida individuals.

Extended Data Fig. 4 Folsomia candida springtails are positively affected by being fed Streptomyces coelicolor mycelium.

Survival, moulting and oviposition of female F. candida kept for 10 days on sporulating cultures of S. coelicolor (n=31, grey solid lines) or SFM agar controls (n=23, light grey dashed lines). Survival on S. coelicolor was as high as on SFM control (a, X21, 54 =1.7, P=0.195). Exposure to S. coelicolor had a positive effect on the initiation of moulting (b, X21, 54 =11, P<0.001***) and oviposition (c, X21, 54 =6.4, P=0.011*) with more eggs being laid (d, X21, 54 =6.064, P=0.0138*; the boxplot shows the medians, lower- and upper quartiles as well as the span of data points that are within 1.5 times the interquartile range). Source data

Extended Data Fig. 5 Effects of developmental regulatory genes bldM and whiH on production of geosmin and 2-methylisoborneol (2-MIB) in Streptomyces venezuelae.

(a) Gas chromatograms analysed for the presence of 2-MIB, geosmin and germacrene D in Streptomyces venezuelae headspace sampled from wild-type, whiH, bldM mutants or a system control. The chromatograms and the quantification of Streptomyces metabolites (b; n=3; error bars give the standard deviation of the mean) illustrate that in comparison to wild-type, geosmin and germacrene D were reduced in the whiH mutant. None of the three Streptomyces metabolites was detected (n.d.) in the bldM mutant or the control. C12 –C14 hydrocarbons were used for calculation of retention indices. Octyl acetate was introduced with the internal standard heptyl acetate (not shown), which was used for quantification. Source data

Extended Data Fig. 6 Assays of spore adherence and dispersal.

To compare dispersal of sporulating and non-sporulating strains, individuals of F. candida (n=12) were fed for 3 days with developed mycelium of S. coelicolor strain M145 or its congenic non-sporulating whiG mutant J2400. CFUs that could be washed off (0.05% Tween-20) from individual animals were quantified by plating on SFM agar. Significantly more spores than hyphae had adhered to the springtail body (a, two-tailed Mann Whitney test, P<0.001 **). To monitor the release of viable cells by defaecation, each washed springtail was allowed to roam for 20 h on MM agar before colonies were allowed to develop. Significantly more CFU were excreted from springtails that had been feeding on the sporulating Streptomyces strain (b, two-tailed Mann Whitney test, P<0.001 **). The boxplots show the medians, lower- and upper quartiles as well as the span of data points (smallest to largest). In a dispersal assay (c), S. coelicolor strains J2192/pIJ10646 (geoA mibAB mutant with geoA and mibAB genes provided in trans on integrated plasmid) and J2192/pIJ10770 (geoA mibAB mutant with empty vector) were allowed to develop and sporulate (5 days) in small spots in center of MM agar plates. Two springtails (F. candida) (n=13) were released for 2 h on each plate. Animals were then removed, plates incubated, and Streptomyces colonies counted. The presence of geoA and mibAB genes (strain J2192/pIJ10646) resulted in significantly more CFU being dispersed by springtails (c, two-tailed Mann Whitney test, P=0.022 *). Error bars show the standard error of the mean. Volatiles from cultures of these strains were collected for 24 h on an air filter, eluted with hexane, and analysed by GC-MS. Chromatograms (d) show that geosmin and 2-MIB were present in strain J2192/pIJ10646 but not detectable (n.d.) in the non-complemented J2192/pIJ10770 strain. The analyses were independently repeated three times with similar results. Source data

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Supplementary Information

Supplementary Discussion, Table 1 and references.

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Supplementary Video

QuickTime video showing springtails (F. candida) feeding on S. coelicolor biomass. The S. coelicolor strain M145 was grown on SFM agar medium covered with a cellophane membrane to allow abundant sporulation (8 d, 27 °C), scraped off from the plate, and placed on a Petri dish lined with gypsum plaster on which springtails were kept. Similar observations were made in three independent experiments.

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Becher, P.G., Verschut, V., Bibb, M.J. et al. Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal. Nat Microbiol 5, 821–829 (2020). https://doi.org/10.1038/s41564-020-0697-x

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