Sociomicrobiology of Secondary Metabolites

Microorganisms make up the most numerous and diverse group of organisms on the planet and play a central role in major environmental processes like the carbon cycle, precipitation, and nitrogen fixation. Everywhere, microorganisms interact, cooperate, and compete with each other creating microscopic ecosystems of immense complexity. Many of these interactions are facilitated by accessory compounds, so-called, secondary metabolites (SMs), which are distinguished from primary metabolism by being non-essential to growth and survival of the producer. SMs are often produced from large enzyme complexes encoded in biosynthetic gene clusters (BGCs), which contain the full biosynthetic machinery for production, transport of the SM, and potential immunity systems if the produced SM is toxic. SMs are increasingly coveted as promising therapeutics, biomaterials, food additives, and crop protectants in response to the demand for sustainable alternatives to conventional pesticides and materials and the increasing threat of antimicrobial resistant pathogens.

However, the role of SMs in a natural system is more difficult to define in details due to the emerging interactions between the members of the community. In social interactions, individual microorganisms can compete, cooperate, act spitefully, or even self-sacrifice that is also known as altruism. Cooperation can for example be the costly production of a publicly available good, which benefits the whole population at a production cost for the producer. However, these cooperators are often exploited by cheaters, cells that stop producing the public good but still enjoying the benefits, and thus gain a fitness advantage over the producers. If left unchecked, cheating can lead to complete reduction of producers and loss of cooperation. Many SMs are shared goods, for example, those that allow binding and use of otherwise insoluble ferric iron or other metabolites can lower the surface tension in the environment to allow population-level motility. However, these properties also makes SMs vulnerable to cheating, as the production machinery can be lost while still benefitting from the shared SM.

Deriving from the theorems of social evolution theory, sociomicrobiology attempts to explain the mechanisms that maintain cooperation and exclude cheaters, and how microorganisms have evolved to do so. In this thesis, I outline social evolution theory in the context of microorganisms, and discuss the available literature on cooperative SMs in bacteria. From this foundation, I aimed to identify important environmental factors and cooperation maintenance mechanisms and ascertain how these have affected the evolution of BGCs leading to a theoretical framework for working with SMs in a social context. This theoretical framework is further put to the test focusing on the SMs produced in the soil bacterium Bacillus subtilis, which has been the main workhorse for the experimental work of this project.

This thesis describes three separate experimental studies, each examining the social aspects of a SM in the soil bacteria B. subtilis. The first study was aimed to dissect the role of surfactin, a lipopeptide that is usually associated with cooperative swarming, during formation of a floating biofilm, called a pellicle. Here, surfactin had a significant impact on the timing of pellicle formation, and through promoter fusion constructs for biofilm related genes, we show that this effect was mainly through induction of the eps operon in addition to enhancing growth. In our second study, we identified the BGC that is required for a pigment produced in certain B. subtilis strains. This pigment has been described over a century ago in a Bacillus isolate under the original classification of Bacillus aterrimus. However, the BGC corresponding to pigment production has not been identified using common genome mining tools for SMs. The pigment specific biosynthetic gene cluster featured several genes annotated as enzymes from the primary metabolism, although seemingly diverged in function. This finding is thus valuable for improving our genome mining approaches and additionally provides an example of a, to our knowledge, novel type of BGC. The third study demonstrated protection of non-producers against an invading fungus, Fusarium oxysporum, by the lipopeptide plipastatin. The observed protection was reliant on producer numbers, which in liquid co-cultures were able to eradicate Fusarium spores when in sufficient numbers, thus protecting both producers and non-producers. On solid agar media, the protective effect of plipastatin was reflected in a delay in Fusarium invasion of biofilm colonies, which increased with producer density. However, without the possibility of eradicating the source of the invading Fungus, all colonies on solid agar media were eventually overgrown by Fusarium.

In summary, this thesis presents a theoretical framework for working with SM in social interactions based on current relevant literature. The experimental work within provides insight into the functionality of SMs in regulation and biofilm, and the connection between primary and secondary metabolism. Finally, in discussion of B. subtilis SMs, we identify future prospects for studying of social interactions and SMs.