Re-examining the iron paradox: the influence of growth medium on regulation and functionality of the tropodithietic acid (TDA) biosynthetic pathway in Phaeobacter piscinae.

Tropodithietic acid (TDA) is a secondary metabolite with antimicrobial and iron-binding properties, produced by several marine Roseobacter group bacteria. Although classified as a siderophore, TDA is also produced under iron-replete conditions. To address this paradox, we fused the tdaCDE promoter region of Phaeobacter piscinae S26 with a promoter-less gfp reporter gene to monitor the transcription of TDA biosynthetic genes under different conditions and validated its accuracy using RT-qPCR. In both complex ($\frac{1}{2}$YTIO) and low-complexity (IOCGH) marine media, iron supplementation repressed tdaCDE transcription and reduced iron-chelation activity. However, TDA production responded differently depending on medium composition: iron suppressed TDA production in IOCGH but increased it in $\frac{1}{2}$YTIO. Antimicrobial activity of culture supernatants declined in late-stage iron-limited $\frac{1}{2}$YTIO-based cultures, even though tdaCDE transcription and iron-chelating activity increased. Acidification restored antimicrobial activity and increased detectable TDA levels, while several non-antimicrobial structural analogues-detected by LC-MS in non-acidified samples-diminished upon acidification, suggesting pH-driven interconversion. These findings reveal that TDA biosynthesis is regulated by iron availability but that the antimicrobial output of the producer depends on medium composition and pH-driven chemical transformations between TDA and its related structural analogues.