Tropodithietic acid (TDA) mitigates oxidative stress and controls horizontal gene transfer in the producing marine bacterium Phaeobacter piscinae

Some species of the marine bacterial genus Phaeobacter produce tropodithietic acid (TDA), a secondary metabolite with several ecological roles including antibacterial activity, likely driven by proton motive force disruption, signalling, and iron chelation [1]. A knockout mutant of Phaeobacter piscinae deficient in TDA production exhibits increased production of prophages and gene transfer agents (GTAs), underlining the profound effect TDA has on the physiology of the producer [2]. However, the mechanism by which TDA represses these horizontal gene transfer systems in P. piscinae remains unknown.

Here, we applied a general oxidative stress indicator to probe cultures, demonstrating that both the wild-type (WT) and the TDA-deficient mutant (ΔtdaB) strains experienced increased oxidative stress after 72 hours of growth in the presence of 0.5 mM ferric ion (Fe³⁺). The stress levels were significantly higher (2.7-fold) in the ΔtdaB cultures and increased in a dose-dependent manner with the concentration of Fe3+. Furthermore, the ΔtdaB strain was unable to grow at H₂O₂ concentrations above 0.1 mM, unlike the WT strain which still grew in the presence of H₂O₂ at 0.3 mM or higher. Finally, a fluorescent reporter assay based on the promoter of the GTA activation factor A (GafA) gene fused to a promoterless green fluorescent protein (GFP) gene showed a sudden increase in green fluorescence emission after 52 hours of growth, but only in the ΔtdaB cultures and only in medium supplemented with 0.5 mM Fe³⁺.

In conclusion, the increased production of GTAs in the absence of TDA correlates with a heightened oxidative stress response, and both processes seem to be amplified by addition of ferric ions (Fe³⁺) to the culture medium. We therefore hypothesize that TDA mitigates oxidative stress by binding Fe3+, thus preventing it from participating in Fenton reactions with hydrogen peroxide (H₂O₂) to form reactive oxygen species.

This work illustrates how combining ex situ probes with in situ fluorescent reporters makes it possible to study stress responses and gene regulation in bacteria. Such approaches can be applied to diverse microbes and microbiomes to investigate how secondary metabolites shape microbial interactions and horizontal gene transfer.