Biological and chemical strategies for exploring inter- and intra-kingdom communication mediated via bacterial volatile signals


Airborne chemical signals emitted by bacteria influence the behavior of other bacteria and plants. We present an overview of in vitro methods for evaluating bacterial and plant responses to bacterial volatile compounds (BVCs). Three types of equipment have been used to physically separate the bacterial test strains from either other bacterial strains or plants (in our laboratory we use either Arabidopsis or tobacco plant seedlings): a Petri dish containing two compartments (BI Petri dish); two Petri dishes connected with tubing; and a microtiter-based assay. The optimized procedure for the BI Petri dish system is described in this protocol and can be widely used for elucidation of potential function in interactions between diverse microbes and those plant and chemical volatiles emitted by bacteria that are most likely to mediate bacterial or plant responses to BVCs. We also describe a procedure for metabolome-based BVC profiling via dynamic (i.e., continuous airflow) or static headspace sampling using solid-phase microextraction (SPME). Using both these procedures, bacteria–bacteria communications and bacteria–plant interactions mediated by BVCs can be rapidly investigated (within 1–4 weeks).

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Figure 1: Bacterial volatiles promote plant growth and induce systemic resistance.
Figure 2: Bacterial volatiles affect bacterial motility and antibiotic resistance.
Figure 3: KEIO collection screening and assessment of antibiotic resistance using microtiter plates.
Figure 4: Representative images showing the effect of BVCs on plant growth and immunity, as well as on bacterial motility and antibiotic resistance, in the BI plate system.
Figure 5: Proposed outline for BVC collection from bacteria.
Figure 6: Two-Petri-dish assay.
Figure 7: A metabolome (SPME-GC–MS)-based analysis protocol that identifies and analyzes volatile compounds in the headspace above a live bacterial culture.


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This research was supported by grants from the BioNano Health-Guard Research Center, funded by the Ministry of Science, ICT and Future Planning of Korea as a Global Frontier Project (Grant H-GUARD_2013M3A6B2078953); the Woo Jang-Choon Project (PJ01093904) of the Rural Development Administration (RDA); and KRIBB Initiative Program South Korea to C.-M.R. B.A. and J.-M.G. are supported by grant no.: ANR-10-LABX-62-IBEID (Investissement d'Avenir Program) and by Fondation pour la Recherche Médicale grant Equipe FRM DEQ20140329508. M.A.F. acknowledges funding received from the Alexander von Humboldt Foundation, Germany, and Cairo University Research Grant no. 31.

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C.-M.R. and M.A.F. designed the experiments; M.A.F., G.C.S., S.L., B.A. and J.-M.G. performed the experiments; and M.A.F., C.-M.R., Y.-S.P., J.-M.G., B.A. and J.W.K. wrote the paper.

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Correspondence to Choong-Min Ryu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Chromatographic profiles of volatiles released from B. subtilis PGPR strain GBO3 (A), and uninoculated media (B)

Compounds positively identified include acetoin (1), 2,3-butanediol (2), decanal (3), decane (4), tetramethyl pyrazine (5), and undecane (6). Highlighted in blue is the earlier elution region where most identified bioactive volatiles are eluted including 1, acetoin and 2, 2,3-butanediol. Peaks commonly released from media include some short chain hydrocarbons and tetramethylpyrazine (s).

Supplementary information

Supplementary Figure 1

Chromatographic profiles of volatiles released from (a) B. subtilis PGPR strain GBO3 and (b) uninoculated medium. (PDF 130 kb)

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Farag, M., Song, G., Park, Y. et al. Biological and chemical strategies for exploring inter- and intra-kingdom communication mediated via bacterial volatile signals. Nat Protoc 12, 1359–1377 (2017).

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