Microbial secondary metabolites have been exploited by humankind for decades, especially as antibiotics to treat human infectious diseases. Despite their enormous societal importance, little is known of their ecological functions and roles in natural microbial communities. Because of their history as therapeutics, the main perception has been that these unique metabolites with antibiotic activity, also in natural microbiome systems, act as mediators of microbial warfare. However, it was not until recently that research has intensified to understand the myriad of non-antibiotic functions of antimicrobial secondary metabolites and how they influence both the producing microorganisms and the microbiomes they reside in. Antimicrobial secondary metabolites may pose multiple functions in nature, besides being agents of microbial competition. They may also be instrumental in cooperation, signaling, biofilm formation, and microbial population dynamics. In fact, the production itself can be shaped by the surrounding microbial community.
The past years we have studied the impact of antimicrobial and secondary metabolite producing probiotic bacteria in aquaculture applications. Previously, we explored whether biofilm producing antimicrobial marine bacteria could also be used as antifouling agents in ship-paints to reduce biofilm formation on ship hulls. This idea was examined in a project published in 2013. Here, an antimicrobial marine Gamma-proteobacterium was allowed to form biofilm on clean stainless steel surfaces in vitro before it was exposed to planktonic suspensions of a series of well-known primary colonizers isolated from coastal marine surfaces. The initial co-cultivations with the antifouling producing bacteria indicated promising results as the pre-coated surfaces had reduced (almost abolished) the formation of biofilm, as hypothesized. Contrary to expectations, when the biofilm-coated stainless steel surfaces were exposed to seawater microbiomes in situ, we observed increased recruitment to the biofilm from the surrounding seawater rather than preventing it.
From an applied perspective, these observations were not very promising to say the least, however, we did spend quite some time pondering about the findings and the underlying mechanisms. Today, our research revolves around understanding why microbes make these chemically diverse antibiotic secondary metabolites and how they (both the producer and the compounds) impact complex microbial communities in nature, for instance, why they potentially may serve as attractants. Hence, making the approach and discoveries from 2013 quite pertinent for understanding the impact of secondary metabolite producing microbes, specifically in structured heterogeneous communities such as those found in marine biofilms.
In the present study, we made use of the 2013 experimental approach outlined above, to study the impact of the marine Alphaproteobacteria, Phaeobacter spp. and their antimicrobial compound, tropodithietic acid (TDA) on the formation and assembly of a marine biofilm community in an semi-natural model system. Phaeobacter spp. are strong biofilm producers and capable of invading preformed bacterial biofilms, and TDA is believed to be essential for its ability to form biofilm, but also to be a mediator of microbial competition. In this model system we introduced a wild type Phaeobacter and a mutant incapable of producing this particular metabolite, respectively. The TDA producing P. inhibens and the TDA deficient mutant were separately allowed to colonize abiotic surfaces, which were then submerged in natural seawater (Figure 1).
Amplicon sequencing of the 16S rRNA V3-V4 gene region was used to assess differences in the microbial community composition during the microbial community succession forming on top of the biofilm of the TDA-producing wild type and the TDA deficient mutant, respectively. Having studied the antibiotic effects of P. inhibens and its ability to eliminate fish pathogens in aquaculture, we expected that the potential of P. inhibens to produce TDA would strongly affect both biofilm and the surrounding planktonic community assembly patterns. In particular, we expected reduction or even elimination of genera known to be susceptible to TDA in the microbiome associated with the wild type.
Findings from this study, showed that only a minor fraction of the differences in microbial community composition could be attributed to the potential production of TDA, or lack thereof, in the pre-coated biofilms. However, inferred microbial interactions from comparative network analyses revealed that the TDA-producing P. inhibens displayed strong positive associations with specific genera of the Flavobacteriaceae and that P. inhibens became a keystone OTU in the biofilm, and not in the surrounding seawater, exclusively due to its potential to produce TDA. This study, once again, challenges the general perception that microbial secondary metabolites displaying antibiotic activity solely are involved in microbial competition. Hence, we need to think of these molecules in a broader perspective as signals with multiple functions. This obviously has fundamental biological interest and will enable better understanding of microbiome assembly, development and function.
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