Linking environmental signals to the expression control of biosynthetic gene clusters in Actinomycetota

Abstract

The objective of my master thesis was to advance the understanding of signaling pathways that link environmental cues to the regulation of bacterial specialized metabolite production. The first goal focused on assessing the capability of the COMMBAT software to predict biosynthetic gene clusters (BGCs) regulated by specific transcription factor (TF) and signal pairs, using DmdR1 and iron as a model system. Screening 1,105 Actinomycetota BGCs from the MIBiG database, we identified several siderophore and non-siderophore clusters with highly reliable predicted DmdR1 binding sites. Given that iron-mediated repression of siderophores is well established, experimental validation via mass spectrometry imaging (MSI) prioritized non-siderophore BGCs with diverse bioactivities. Comparative metabolomics confirmed iron-dependent repression for cahuitamycin A, valinomycin, and iminimycins, evidenced by decreased metabolite signals upon iron addition. In contrast, no target metabolites were detected for lenoremycin, aureonuclemycin, oxazolomycin, or skyllamycins, likely due to suboptimal culture conditions, the need for additional elicitors, weak (false positive) DmdR1 binding sites, or limitations in MALDI-MSI detection. Overall, COMMBAT proved to be an effective tool for uncovering novel links between environmental signals and secondary metabolite biosynthesis. The second objective aimed to identify the sugars associated with the LamR transcription factor, predicted to regulate multiple BGCs in Actinomycetota. Regulon prediction revealed a conserved 14-nt palindromic LamR binding site upstream of genes encoding laminarinases and an ABC-type sugar transporter operon (lamEFG), implicating laminaribiose (L2) and laminaritriose (L3), the degradation products of callose/laminarin, as potential environmental signals. To validate this, LamE protein was heterologously expressed and purified, and ligand binding was characterized using differential scanning fluorimetry (DSF). These studies demonstrated high-affinity binding of LamE to L2 and L3 (submicromolar range), with weaker interactions observed for cellobiose and cellotriose. Interestingly, some Streptomyces species lacking lamEFG still metabolize laminarin, suggesting an alternative transporter. The cebEFG operon, responsible for cellobiose and cellotriose uptake, was identified as a likely compensatory system due to the similarity between LamR and CebR transcription factor binding sites. Purified CebE exhibited similarly high-affinity binding to both laminarin-derived and cellobiose sugars, indicating functional overlap. Isothermal titration calorimetry (ITC) further confirmed micromolar affinities and suggested protein dimerization. Collectively, these findings support L2 and L3 as novel environmental signals sensed by LamR and highlight the interplay between LamEFG and CebEFG transporters, positioning these sugars as new regulatory molecules controlling BGC expression via LamR.