Structural Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An In Silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production

Abstract

The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279's structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.

Keywords: Antarctic Streptomyces; CRISPR-based activation; biosynthetic gene clusters; molecular dynamics simulation.

Figure 1

Three-dimensional representative structure of the putative TetR/AcrR protein after 200 ns of molecular dynamics. (a) Representative three-dimensional structure of the predicted domains within TetR-279. Regions highlighted correspond to predicted functional structures: residues in orange indicate the tetracycline repressor TetR C-terminal domain, as identified by InterProScan via UniProt; residues in pink represent DNA-binding residues predicted by DP-Bind using the strict consensus criterion. The overall structure is rendered in cartoon style to depict secondary structure elements, while the highlighted regions are shown as backbone traces. The first (N-terminal) and last (C-terminal) residues are labelled for spatial reference. The image was generated using Protein Imager (https://3dproteinimaging.com). (b) Strict consensus prediction of DNA-binding residues obtained using the DP-Bind server. The binary values represent residues where all three classifiers (SVM, KNN, and PNN) concurred in a positive prediction. Pink bars indicate positions of agreement among the classifiers.

Figure 2

Gene expression analysis revealed a significant upregulation of TetR-279 in IMA and ISP4 media, indicating nutrient-dependent activation of the actinomycin D cluster. (a) Relative expression levels of the TetR/AcrR transcription factor within the actinomycin D cluster across three different nutritional environments: IMA (blue bar), ISP4 (light blue bar), and YES (green bar). A significant increase in gene expression was observed in IMA and ISP4 media compared to the M2 (dark bar) control (*, p < 0.05). The APRT gene served as an internal reference for normalisation. The analysis was conducted using three biological replicates and two analytical methods per condition. (b) Summary of transcription factors identified within the actinomycin D biosynthetic gene cluster. Both proteins belong to the TetR/AcrR family, which is commonly associated with transcriptional repression in response to environmental or metabolic signals.

Figure 3

Molecular dynamics revealed that the S166P mutation significantly altered TetR-279 structure and flexibility, while V167A and V167I had minimal impact. Molecular dynamics analysis for overall conformational changes (backbone RMSD) and residue fluctuation in the putative TetR-279 protein. (a) The RMSD plot of the alpha carbons (Cα) as a function of time during a 200 ns molecular dynamics simulation for different variants of the TetR-279 protein: native, S166P, V167A, and V167I. (b) The root mean square fluctuation as a function of residue number for different variants of TetR-279 protein: native, S166P, V167A, and V167I. RMSF measures the flexibility of each residue in the protein structure over time during a molecular dynamics simulation. (c) The figure shows the evolution of SASA over time (in nanoseconds) for different protein variants throughout a 200 ns molecular dynamics simulation. The figure indicates that the S166P mutation has a more significant effect on the protein’s conformation, reducing its solvent-accessible surface area after compensating for proline residue-residue clashes. In contrast, the V167A and V167I mutations have less impact on the overall protein structure.