The smell of War: Weaponizing Volatiles to Inhibit Competitor Biofilms from a Far

Published in Microbiology
The smell of War: Weaponizing Volatiles to Inhibit Competitor Biofilms from a Far

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Despite the widely held view of bacteria as unicellular organisms that struggle for individual survival, in nature, bacteria establish complex communities, referred to as biofilms, that are stimulated by chemical communication 1. A biofilm can be viewed as a differentiated community, where the inhabitant cells are held together by an extracellular matrix. Bacterial biofilms are ubiquitous and are of high significance in agricultural, industrial, environmental, and clinical settings1. In our recent paper, we inquire the role of airborne molecules in microbial warfare

A key example for biofilm development are the motile Bacillus subtilis cells, which organize themselves into conspicuous multicellular structures that carry out specialized tasks2. This Gram-positive bacterium is a genetically manipulatable model organism for biofilm development and for beneficial environmental activities of bacteria. Like other bacteria, B. subtilis produces a wide repertoire of volatile compounds (VCs) - biologically active airborne molecules 3. These volatiles allow bacteria to interact with their environment, and were first identified as cross-kingdom signals influencing survival and behavior of fungi, plants and vertebrates. For B. subtilis, ammonia 4 and acetic acid 5 produced by B. subtilis floating biofilms stimulated neighboring pellicle formation. However, collectively, previous results suggest that in nature, the role of VCs is highly context-dependent, and that additional studies are needed to understand the mechanisms mediating the effects of VCs produced by biofilms during ecological microbial interactions6.  In this paper we explored the biofilm colony models to investigate the dose-dependent activity of VCs in inter- and intra-species interaction between biofilms. First, we found when bacterial communities reach a critical biomass, B. subtilis biofilms can use a limited repertoire of VCs as a specific regulatory signal to inhibit biofilm development of potential competitors.

To our surprise, biomass-dependent inhibition of neighboring biofilms by VCs was conserved in B. subtilis and Echerichia coli. We considered two modes of action for the air-borne inhibition of competing neighbors: a non-specific inhibition of growth and a specific regulation of the biofilm developmental program. Eventually, our results indicate that air-borne biofilm inhibition by competitors was mediated by dysregulation of biofilm transcription program – and that specific VCs produced by biofilms inhibited the expression of genes encoding the extracellular matrix components

Our experimental system generates an interesting prediction for biofilm biology: VCs-dependent inhibition was only evident when VCs producers reached a certain critical biomass, and thus the inhibitory effect described here is by definition a feature of mature biofilms. One appealing scenario for natural settings is that once a critical mass of bacteria is achieved in a given location (pioneers), production of certain species of VCs will prevent the development of competing colonies in proximity, protecting this established community from potential competitors (newcomers).  This new role of bacterial VCs in inhibiting biofilm formation reveals an additional layer of the complex interactions in the competitive natural environments.



1          Elias, S. & Banin, E. Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev, doi:10.1111/j.1574-6976.2012.00325.x (2012).

2          Kovacs, A. T. Bacillus subtilis. Trends Microbiol 27, 724-725, doi:10.1016/j.tim.2019.03.008 (2019).

3          Ryu, C. M. et al. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol 134, 1017-1026, doi:10.1104/pp.103.026583 (2004).

4          Nijland, R. & Burgess, J. G. Bacterial olfaction. Biotechnol J 5, 974-977, doi:10.1002/biot.201000174 (2010).

5          Chen, Y., Gozzi, K., Yan, F. & Chai, Y. Acetic Acid Acts as a Volatile Signal To Stimulate Bacterial Biofilm Formation. MBio 6, e00392, doi:10.1128/mBio.00392-15 (2015).

6          Audrain, B., Farag, M. A., Ryu, C. M. & Ghigo, J. M. Role of bacterial volatile compounds in bacterial biology. FEMS Microbiol Rev 39, 222-233, doi:10.1093/femsre/fuu013 (2015).


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