Molecular mechanisms of microbiome modulation by the eukaryotic secondary metabolite azelaic acid

Reviewer #1 (Public Review):

Summary:
Shibl et al., studied the possible role of dicarboxylate metabolite azelaic acid (Aze) in modulating the response of different bacteria, it was used as a carbon source by Phycobacter and possibly toxic for Alteromonas. The experiments were well conducted using transcriptomics, transcriptional factor coexpression networks, uptake experiments, and chemical methods to unravel the uptake, catabolism, and toxicity of Aze on these two bacteria. They identified a putative Aze TRAP transporter in bacteria and showed that Aze is assimilated through fatty acid degradation in Phycobacter. Meanwhile, in Alteromonas it is suggested that Aze inhibits the ribosome and/or protein synthesis, and that efflux pumps shuttles Aze outside the cytoplasm. Further on, they demonstrate that seawater amended with Aze selects for microbes that can catabolize Aze.

Major strengths:
The manuscript is well written and very clear. Through the combination of gene expression, transcriptional factor co-expression networks, uptake experiments, and chemical methods Shibl et al., showed that Aze has a different response in two bacteria.

Major weakness:
There is no confirmation of the Aze TRAP transporters through mutagenesis.

Impact on the field:
Metabolites exert a significant influence on microbial communities in the ocean, playing a crucial role in their composition, dynamics, and biogeochemical cycles. This research highlights the intriguing capacity of a single metabolite to induce contrasting responses in distinct bacterial species, underscoring its role in shaping microbial interactions and ecosystem functions.

Reviewer #2 (Public Review):

This study explores the breadth of effects of one important metabolite, azelaic acid, on marine microbes, and reveals in-depth its pathway of uptake and catabolism in one model bacterial strain. This compound is known to be widely produced by phytoplankton and plants, and to have complex effects on associated microbiomes.

This work uses transcriptomics to assay the response of two strains that show contrasting responses to the metabolite: one catabolizes the compound and assimilates the carbon, while the other shows growth inhibition and stress response. A highly induced TRAP transporter, adjacent to a previously identified regulator, is inferred to be the specific uptake system for azelaic acid. However the transport function was not directly tested via genetic or biochemical methods. Nevertheless, this is a significant finding that will be useful for exploring the distribution of azelaic acid uptake capability across metagenomes and other bacteria.

The authors use pulse-chase style metabolomics experiments to beautifully demonstrate the fate of azelaic acid through catabolic pathways. They also measure an assimilation rate per cell, though it remains unclear how this measured rate relates to natural systems. The metabolomics approach is an elegant way to show carbon flux through cells, and could serve as a model for future studies.

The study seeks to extend the results from two model strains to complex communities, using seawater mesocosm experiments and soil/Arabidopsis experiments. The seawater experiments show a community shift in mesocosms with added azelaic acid. However, the mechanisms for the shift were not determined; further work is necessary to demonstrate which community members are directly assimilating the compound vs. benefitting indirectly or experiencing inhibition. In my opinion the soil and Arabidopsis experiments are quite preliminary. I appreciate the authors' desire to broaden the scope beyond marine systems, but I believe any conclusions regarding different modes of action in aquatic vs terrestrial microbial communities are speculative at this stage.

This work is a nice illustration of how we can begin to tease apart the effects of chemical currencies on marine ecosystems. A key strength of this work is the combination of transcriptomics and metabolomics methods, along with assaying the impacts of the metabolite on both model strains of bacteria and whole communities. Given the sheer number of compounds that probably play critical roles in community interactions, a key challenge for the field will be navigating the tradeoffs between breadth and depth in future studies of metabolite impacts. This study offers a good compromise and will be a useful model for future studies.