Control of microbial soil communities by Pseudomonas-produced secondary metabolites

Bacteria and other microbes produce a variety of secondary metabolites, which are highly specialized natural products predominantly known for their antimicrobial properties. However, natural concentrations of secondary metabolites in the nutrient-limited environments of soil are thought to rarely exceed the levels required for their antimicrobial functions demonstrated in vitro. Additionally, an increasing amount of research has shown that subinhibitory concentrations of toxic secondary metabolites can elicit responses in neighboring microorganisms. Thus, the role of these chemicals in natural environments might expand much beyond microbial antagonism.

The focus of this thesis has been on soil-dwelling Pseudomonas spp. of the P. fluorescens group, as they hold an immense potential for secondary metabolite production and are known as key determinants of plant protection towards phytopathogenic microorganisms associated with certain soils. Specifically, we have focused on the metabolites DAPG, pyoluteorin, and orfamide A produced by strains of the biocontrol species, P. protegens. The work presented here describes their role in microbial interactions and vice versa the effects of interactions on their biosynthesis and fate.

We developed two methodologies for accurate and high-throughput differentiation of Pseudomonas spp. in environmental samples. The former, a Pseudomonas-specific amplicon sequencing method, allows for taxonomic identification and relative quantification of Pseudomonas spp., while clearly outperforming the 16S rRNA-based gold standard. Secondly, we engineered a whole-cell biosensor, which enables rapid and effortless screening of isolates for producers of DAPG, compared to PCR-based techniques and analytical chemical methods. These methods allow for detailed investigations of Pseudomonas population dynamics across ecosystems (e.g. different soils and crops).

Additionally, we employed both dual- and multispecies communities in combination with analytical chemistry to investigate the role of Pseudomonas-produced secondary metabolites in microbial interactions. The first study led to the discovery of a dynamic, genus-specific interaction sequentially affecting the production of antimicrobial secondary metabolites (DAPG and pyoluteorin) in P. protegens. This study sheds light on the underlying mechanisms of Pseudomonas specific interactions affecting the biosynthesis of metabolites relevant for biological control of phytopathogenic microorganisms. In the second study, we utilized a four-species synthetic microbial community as a reproducible framework to investigate compositional perturbations caused by secondary metabolites produced by the invading P. protegens. The results from this study revealed a community-level interaction affecting the fate of orfamide A, as the metabolite was enzymatically inactivated and subsequently degraded by separate community members. The outcome of these two studies demonstrates the significance of combining in vitro lab-scale systems and analytical chemistry to reveal and characterize the types of metabolite interactions occurring within dual- and multispecies communities.

The work presented in this thesis provide novel insight into the potential roles of Pseudomonas-produced secondary metabolites, as well as tools for detailed investigations of Pseudomonas-ecology. From a biotechnological perspective, the results from this thesis may assist in discovery and isolation of Pseudomonas strains with agricultural application (e.g. biocontrol), while also contributing to a deeper understanding of metabolite signaling to potentiate the design and assembly of efficient biocontrol consortia with desirable functionality.