Exploring the Ecological Implications of Tropodithietic Acid Production in Phaeobacter piscinae

Microbial secondary metabolites are small, diffusible molecules which, as opposed to primary metabolites, are not required for growth and proliferation. Instead, they play important roles in how microorganisms respond to and interact with their environment by acting as antibiotics, nutrient scavengers, and signals, to name a few. However, we still have a relatively limited understanding of the ecological implications of secondary metabolite production, as we have primarily studied them in relation to their industrial applications. The purpose of this PhD thesis was to investigate the ecological role(s) of secondary metabolites using the multifunctional secondary metabolite tropodithietic acid (TDA), produced by members of the marine Roseobacter group, as a case study.

In Manuscript II, we constructed a scarless deletion mutant devoid of TDA production and found that TDA-production drastically changes the physiology of the producing strain, including global changes in transcriptome, proteome, and proteome. Phenotypically, removing TDA production led to increased motility and decreased cell length. Disruption of TDA production also caused the expression of three putative horizontal gene transfer (HGT) systems; a type IV secretion system, a prophage, and a gene transfer agent (GTA). HGT is generally believed to promote adaptation to novel niches, indicating that TDA production affects adaptation potential of the producer. We proposed a model where TDA acts as a coordinator of colonization and adaptation: Once a TDA-producing population has established itself in a new niche, the accumulation of TDA serves as a signal of successful colonization, prompting a shift to a sessile lifestyle. This would reduce motility and the rate of HGT, while filamentous cells could form the foundation of a biofilm. Furthermore, the antibiotic properties of TDA may inhibit invading competing microorganisms, providing an additional role for TDA. Thus, TDA production may benefit the producer in a number of ways, highlighting the broader ecological importance of secondary metabolites.

GTAs are virus-like particles which packages and transfer random pieces of host DNA and thus, can facilitate HGT in a bacterial population. However, GTA release happens at the expense of the donor cell, which lyses to release the particles. GTA activation is therefore governed by an intricate regulatory network. Given the observation that abolishing TDA production caused GTA activation, we in Manuscript III set out to investigate the regulatory network behind this repression. Based on bioinformatic analysis, we propose that TDA interacts with the quorum sensing (QS) response regulator PgaR to repress expression of GafA, a direct GTA activator, ultimately resulting in repression of GTA activation. Furthermore, TDA may also affect GTA activation through interaction with the CtrA phosphorelay system through an uncharacterized mechanism. These results are the first report of GTA regulation by an endogenously produced secondary metabolite and contribute to the growing understanding of GTA regulation.

A major challenge in microbial secondary metabolite research is the chemical detection of these compounds. As an alternative to direct chemical detection of TDA, we developed a transcriptional and a translational tdaC reporter fusion to investigate TDA production dynamics in Manuscript IV. These systems permit further downstream studies into the model proposed in Manuscript II by studying how TDA production proceeds during colonization and which factors influence TDA production.

The results presented in this thesis contributes to the growing evidence of the importance that secondary metabolites have in modulating bacterial interactions with other organisms and their environment. Furthermore, I conclude that unravelling the ecological role of secondary metabolites requires a holistic and exploratory approach, which may ultimately lead us to novel functions and roles for these compounds.