Unveiling signalling pathways from plant material to the onset of secondary metabolism in Actinobacteria

Abstract

The World Health Organization (WHO) describes antibiotic resistance as “one of the biggest threats to global health, food security, and development today”, as the number of resistant, multi- and pan-resistant bacteria is rising dangerously. Meanwhile, brute force antibiotic discovery does not yield the same results as it did in the past, and researchers have had to develop creative strategies in order to unravel the hidden potential of microorganisms such as the antibiotic producing Streptomyces species. Indeed, scanning the genomes of this genus has revealed a great number of potential biosynthetic gene clusters, that are probably not expressed under laboratory conditions. In order to activate their expression, we must find out the environmental cues triggering secondary metabolite production through a transcriptional response. Instead of starting at the source of the signal, our approach is based on the assumption that all the information we need is already present on the genome, in the cis-regulatory elements. However, the current knowledge on the cis-trans relationships is relatively low compared to the total number of transcription factors present in each Streptomyces species. This means that the success rate of this strategy is low and the first aim of this master’s thesis was to participate to the effort of filling the database of known cis-acting elements, through the implementation of a methodology for de novo identification of binding-sites of regulatory proteins. To achieve this, these non-coding sequences were scanned for on the genome, and particularly in the upstream regions of LacI transcription factors, who are known to auto-regulate their expression and often be located in the operon they control. This analysis was performed on 68 genomes, of which the LacI-TFs were clustered into orthologous groups. The upstream regions in each COG were aligned in order to identify conserved regulatory motifs, and 62 highly reliable binding sites were detected in total. In the second part of this thesis we aimed to start exploiting the database of already known cis-acting elements using the cellulose utilization repressor CebR as model system. The goal was to determine to which extent a primary metabolism linked regulator could be involved in secondary metabolism in streptomycetes. CebR has been shown to control thaxtomin phytotoxin production in Streptomyces scabies in response to cello-oligosaccharides uptake, hence we investigated if the most abundant polysaccharide on Earth could also be the signal for secondary metabolite production in other streptomycetes. The cbs (CebR binding site) matrix was used to perform a genus-wide regulon prediction on 28 Streptomyces genomes as well as a biosynthetic gene cluster (BGC) prediction. The crossed data revealed that in total, there are more than 200 genes belonging to BGCs that are predicted to be controlled by CebR. One of the predicted pathways was selected for in vitro and in vivo experimental validation and we demonstrated that Streptomyces avermitilis also uses cello-oligosaccharides import to trigger the production of oligomycin in a complex signaling cascade that recruits the SmrAB two-component system, the ECF sigma factor gene sig25, and finally the pathway-specific activator OlmRI.