Biology subjects

Stringer, R.

Publications and source records attributed to Stringer, R..

5 recordsLinked to original sources

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↗

bioRxiv2025

Targeting granule initiation and amyloplast structure to create giant starch granules in wheat

Starch granule size influences the functional, digestive, and processing qualities of starch, but its genetic control in plants is poorly understood. Here, we demonstrate that both space and substrate constraints limit starch granule size in wheat and provide an approach to achieve substantial increases in granule size. Wheat starch typically contains large A-type and small B-type granules. To increase A-type granule size, we explored the effect of mutations in the plastid division component PARALOG OF ARC 6 (PARC6), which increases amyloplast size and therefore the space available for granule growth, and in B-GRANULE CONTENT 1 (BGC1), which reduces the number of granule initiations and thus competition between growing granules for space and substrates. While parc6 and bgc1 single mutants had only modest increases in A-type granule size, the parc6 bgc1 double mutant produced striking giant granules that were more than double the size of typical A-type granules. The increase in granule size in parc6 bgc1 was reproducible in both the glasshouse and field, and had no detectable effect on plant growth, grain size and starch composition or content. We demonstrate that the size increase affects a range of functional properties, including viscosity and pasting temperature. Overall, by targeting both constraints, we created a new class of giant cereal starch that has not been previously observed in nature, with altered physicochemical properties that can be used in food and industrial applications. Significance statementWe provide a major advance in understanding the factors determining starch granule size in plants - a trait that strongly influences starch functionality. Starch granules from cereals are typically smaller than those of most root/tuber crops, rarely exceeding 30 {micro}m. We created a novel cereal starch in wheat that exceeds this size range. By simultaneously increasing amyloplast size and reducing granule initiations, we increased space for granule growth and reduced competition between growing granules for substrates. This resulted in a new class of cereal starch where nearly 50% of the starch volume were in granules greater than 30 {micro}m, and some exceeded 50 {micro}m. These giant granules had altered physicochemical properties that could find novel application in food and industry.

plant biology↗

bioRxiv2025

Primary metabolism underpins the execution of immune responses in different tissues of the same plant

Cell-surface perception of microbes triggers a range of rapid immune responses in plants that include the induction of cell isolation by plasmodesmal closure, the production of reactive oxygen species, and changes to gene expression. Here, we identify that some of these immune response are differentially executed in leaves of different ages in the same plant. We observed that when compared to mature leaves, young, expanding leaves do not close their plasmodesmata, have a reduced transcriptional response to immune elicitors, and are more susceptible to a bacterial pathogen. Disconnecting leaf age from physiology, we determined that both plasmodesmal closure and the magnitude of transcriptional responses are dependent on whether the leaf is a carbon sink or a carbon source. To probe the relevance of differential regulation of plasmodesmata in sink and source tissues, we forced plasmodesmal closure in young sink leaves and found this perturbed the normal outputs of growth and defence. Thus, we propose that sink leaves do not close their plasmodesmata during immune reponses to prioritise carbon use for growth over defence, and consequently that primary metabolism underpins different immune response profiles in different leaves of the same plant.

plant biology↗

bioRxiv2023

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated proto-pyrenoid Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well studied pyrenoid, Chlamydomonas reinhardtii (1). Here we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesised as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, whilst also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and opening novel avenues for engineering starch morphology.

plant biology↗