Abstract Description

The marine environment is a rich reservoir of microbial diversity, where symbiosis underpins ecological stability and drives biochemical innovation. Within this intricate network of interactions, particularly between marine bacteria, their hosts, and neighboring bacteria, lies an unexplored source of sustainable solutions to pharmaceutical and biotechnological challenges. Butyrolactone signaling molecules play a central role in microbial symbiosis by mediating interspecies communication and coordinating cooperative behaviors. These small, diffusible compounds regulate gene expression linked to secondary metabolism, biofilm formation, and mutualistic interactions, enabling microbes to respond dynamically to host and environmental cues. Their involvement in quorum sensing highlights their importance in maintaining balanced and productive symbiotic relationships. Functioning in a concentration-dependent manner, often at micromolar levels, these molecules exemplify biochemical potency. In this study we explored the potential of Thalassomonas actiniarum A5K-106T, a sea anemone symbiont, to produce novel butyrolactone signaling molecules. The genome of T. actiniarum was sequenced and mined for biosynthetic gene clusters (BGCs) using antiSMASH. The biosynthetic genes were cloned into an Escherichia coli ArcticExpressTM . Altered E.coli strains were induced for expression and allowed to ferment. Metabolites were extracted from fermentation broths and purified using a combination of silica gel chromatography and semi-preparative HPLC. Structural elucidation of the target compound was achieved with high-resolution mass spectrometry (HRMS). Purified samples were administered to butyrolactone biosensor strains, as well as to the native host to observe for possible phenotypic changes. Genomic analysis revealed a unique butyrolactone-type operon with low sequence similarity to those predominantly found in terrestrial bacteria. The BSG contains a butyrolactone synthase (TabA: AfsA), haloacid dehydrogenase- like phosphatase (TabB) , aldo/keto reductase (TabC), condensation-domian containing protein (TabD), alpha/beta hydrolase (TabE) and a flavin-dependent monooxygenase (TabF). These genes were cloned and expressed in E. coli ArcticExpressTM in various combinations to elucidate their functional roles. HPLC-MS analysis of the fermentation broths revealed novel masses at 191, 391, 225 and 240 amu. High-resolution mass spectrometry indicated a consistent 87m/z fragment ion, corresponding to the characteristic five membered heterocyclic base structure butyrolactone. Preliminary bioactivity assays demonstrated that the expressed butyrolactones exhibit biofilm altering capabilities. 87 m/z fragment ion, corresponding to the five-membered butyrolactone ring. Preliminary bioactivity assays demonstrated that the expressed butyrolactones possess biofilm-modulating properties, suggesting promising avenues for further functional and therapeutic exploration.