Beaton, A. D. | bioRxiv Science

bioRxiv2026

Defining the OrrA regulon and its role in development and antibiotic production in Streptomyces venezuelae NRRL B-65442

2.Streptomyces bacteria have complex life cycles involving hyphal growth, sporulation, and the production of diverse specialised metabolites, including antibiotics. In this study, we investigated the role of the highly conserved orphan response regulator OrrA in Streptomyces venezuelae NRRL B-65442. We show that S. venezuelae {Delta}orrA mutants are defective in sporulation and, using ChIP-seq, identify five OrrA binding sites in vivo. Tandem-mass-tag proteomics revealed that OrrA directly activates two of these putative target genes, wblA and vnz_04640, a finding consistent with previous work on OrrA in the distantly related S. coelicolor. We also demonstrate that deleting wblA blocks sporulation and that overexpressing wblA restores sporulation in the {Delta}orrA mutant. Additionally, chloramphenicol biosynthesis is upregulated in both the {Delta}orrA and {Delta}wblA mutants compared with the wild type. Taken together, these results indicate that the primary function of OrrA is to regulate WblA production, and that reduced intracellular WblA levels underlie the phenotypes observed in the {Delta}orrA mutant. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files and via public databases.

microbiology↗

bioRxiv2025

MtrAB activates ectoine production and triggers sporulation in response to osmotic stress in Streptomyces venezuelae

The MtrAB two-component system is a master regulator of antibiotic biosynthesis in Streptomyces species. MtrA is also required for sporulation under certain growth conditions, which means that on some growth media {Delta}mtrA mutant colonies do not produce aerial hyphae or spores. These mutants are referred to as (conditionally) bald because they lack the hairy appearance of wild-type colonies. Here we report that S. venezuelae NRRL B-65442 {Delta}mtrA is bald on R2YE agar but sporulates normally on MYM agar and we demonstrate that this is caused by the presence of 10.2% sucrose in R2YE. Consistent with this, we found that adding 10.2% sucrose to MYM agar also inhibits sporulation of the {Delta}mtrA mutant. Proteomics combined with DNA binding studies revealed that MtrA directly activates the expression of the key developmental regulator genes bldM and whiI on R2YE but not MYM agar. BldM and WhiI work together to activate genes required for aerial hyphae production and sporulation. Crucially, over-expression of bldM-whiI in the {Delta}mtrA mutant restored normal sporulation in the presence of 10.2% sucrose. We hypothesised that MtrAB must sense and respond to osmotic stress and consistent with this we found that MtrA directly activates biosynthesis of the compatible solute and osmoprotectant ectoine on growth media containing 10.2% sucrose. We propose a model in which high concentrations of sucrose induce osmotic stress in Streptomyces species and this activates MtrAB. The response regulator MtrA then directly activates expression of the ectABCD operon to switch on ectoine biosynthesis and expression of bldM and whiI to trigger entry into sporulation. Impact statementStreptomyces species have complex developmental life cycles that start with spore germination and outgrowth of an actively growing substrate mycelium. Environmental stresses including nutrient starvation and osmotic stress trigger the production of aerial hyphae that undergo cell division to form spores. These spores are more resistant to environmental stresses and can stay viable in the soil until conditions improve. Here we show that MtrAB is responsible for sensing and responding to osmotic stress and we demonstrate that it directly controls the biosynthesis of ectoine and the developmental transition to sporulation through direct activation of BldM and WhiI. This work provides new insight into how environmental cues control the development of Streptomyces bacteria. Data summaryThe S. venezuelae NRRL B-65442 genome sequence is available at NCBI (Reference Sequence NZ_CP018074.1) and can be viewed at http://strepDB.streptomyces.org.uk (select vnz chromosome from the drop-down list). The ChIP-seq data (GEO accession number CP018074) used to map MtrA binding sites on the S. venezuelae NRRL B-65442 chromosome and the differential RNA-seq data (accession number GSE81104) used to map global transcript start sites throughout the life cycle were generated for earlier studies (1,2). The tandem mass tag proteomics data are available via ProteomeXchange with identifier PXD069151. The ReDCaT SPR data is included in the manuscript and the supplementary information. Protocols are freely available at http://actinobase.org (3) and strains and plasmids are available from http://streptomyces.org.uk/strepstrains. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗