Biology subjects

Szafran, M. J.

Publications and source records attributed to Szafran, M. J..

4 recordsLinked to original sources

bioRxiv2024

Significance of the CTP-binding motif for the interactions of S. coelicolor ParB with DNA, chromosome segregation and sporogenic hyphal growth.

The segregation of bacterial chromosomes is widely mediated by partitioning proteins (ParAB). While ParB binds DNA specifically by recognising short, palindromic sequences known as parS sites, ParA utilises its ATPase activity to generate the force to translocate ParB-DNA nucleoprotein complexes (segrosomes). The assembly of the segrosome requires the association of ParB with parS, followed by nonspecific spread of the protein along the DNA. To spread on DNA, the ParB dimer must entrap the parS site within the complex, a process triggered by CTP binding to the conserved GERR amino acid motif. In Streptomyces, a genus of soil-dwelling, multigenomic bacteria that have a complex life cycle, ParB-dependent chromosome partitioning is initiated during the growth of sporogenic hyphae. However, the molecular mechanisms underlying segrosome formation in Streptomyces and their ability to coordinate with sporogenic development remain incompletely understood. In this study, we advance the understanding of chromosome segregation in bacteria by exploring the effects of CTP binding and hydrolysis on the formation of the partitioning complex in S. coelicolor. Here, via in vitro approaches, we demonstrate that a conserved GERR motif is essential for CTP binding and hydrolysis by S. coelicolor ParB. Moreover, the motif is crucial for CTP-dependent ParB accumulation on DNA. Using mutant strains, we show the significance of the GERR motif for segrosome complex assembly. Additionally, we provide data showing that the CTP-binding motif contributes to the regulation of the growth of sporogenic cells. Overall, we show that CTP-dependent segrosome assembly impacts the development of S. coelicolor sporogenic cells.

molecular biology↗

bioRxiv2021

Global chromosome topology and the two-component systems in concerted manner regulate transcription in Streptomyces

Bacterial gene expression is controlled at multiple levels, with chromosome supercoiling being one of the most global regulators. Global DNA supercoiling is maintained by the orchestrated action of topoisomerases. In Streptomyces, mycelial soil bacteria with a complex life cycle, topoisomerase I depletion led to elevated chromosome supercoiling, changed expression of significant fraction of genes, delayed growth and blocked sporulation. To identify supercoiling-induced sporulation regulators, we searched for S. coelicolor transposon mutants that were able to restore sporulation despite high chromosome supercoiling. We established that transposon insertion in genes encoding a novel two-component system named SatKR reversed the sporulation blockage resulting from topoisomerase I depletion. Transposition in satKR abolished the transcriptional induction of the genes within the so-called supercoiling-hypersensitive cluster (SHC). Moreover, we found that activated SatR also induced the same set of SHC genes under normal supercoiling conditions. We determined that the expression of genes in this region impacted S. coelicolor growth and sporulation. Interestingly, among the associated products is another two-component system (SitKR), indicating the potential for cascading regulatory effects driven by the SatKR and SitKR two-component systems. Thus, we demonstrated the concerted activity of chromosome supercoiling and a hierarchical two-component signalling system that impacts gene activity governing Streptomyces growth and sporulation. IMPORTANCEStreptomyces, soil bacteria with complex life cycle, are the producers of a broad range of biologically active compounds (e.g. antibiotics). Streptomyces respond to various environmental signals using complex transcriptional regulation mechanism. Understanding regulation of their gene expression is crucial for Streptomyces application as industrial organisms. Here, based on extensive transcriptomics analyses, we describe the concerted regulation of genes crucial for growth and development by global DNA supercoiling and novel two-component system. Our data indicate that regulated genes encode growth and sporulation regulator. Thus, we demonstrate that Streptomyces link the global regulatory strategies to adjusts life cycle to unfavourable conditions.

genomics↗

bioRxiv2020

Spatial rearrangement of the Streptomyces venezuelae linear chromosome during sporogenic development

Depending on the species, bacteria organize their chromosomes with either spatially separated or closely juxtaposed replichores. However, in contrast to eukaryotes, significant changes in bacterial chromosome conformation during the cell cycle have not been demonstrated to date. Streptomyces are unique among bacteria due to their linear chromosomes and complex life cycle. These bacteria develop multigenomic hyphae that differentiate into chains of unigenomic exospores. Only during sporulation-associated cell division, chromosomes are segregated and compacted. In this study, we show that at entry to sporulation, arms of S. venezuelae chromosomes are spatially separated, but they are closely aligned within the core region during sporogenic cell division. Arm juxtaposition is imposed by the segregation protein ParB and condensin SMC. Moreover, we disclose that the chromosomal terminal regions are organized into domains by the Streptomyces-specific protein - HupS. Thus, we demonstrate chromosomal rearrangement from open to close conformation during Streptomyces life cycle.

microbiology↗

bioRxiv2019

Transcriptional response of Streptomyces coelicolor to rapid chromosome relaxation or long-term supercoiling imbalance

Negative DNA supercoiling allows chromosome condensation within a cell and facilitates DNA unwinding, which is required for the occurrence of DNA transaction processes, i.e., DNA replication, transcription and recombination. In bacteria, changes in chromosome supercoiling impact global gene expression; however, the limited studies on the global transcriptional response have focused mostly on pathogenic species and have reported various fractions of affected genes. Furthermore, the transcriptional response to long-term supercoiling imbalance is still poorly understood. Here, we address the transcriptional response to both novobiocin-induced rapid chromosome relaxation or long-term topological imbalance, both increased and decreased supercoiling, in environmental antibiotic-producing bacteria belonging to the Streptomyces genus. During the Streptomyces complex developmental cycle, multiple copies of GC-rich linear chromosomes present in hyphal cells undergo profound topological changes, from being loosely condensed in vegetative hyphae, to being highly compacted in spores. In Streptomyces, chromosomal supercoiling changes may also be triggered by environmental stressors and have been suggested to be associated with the control of antibiotic production. Remarkably, in S. coelicolor, one of model Streptomyces species, topoisomerase I (TopA) is solely responsible for the removal of negative DNA supercoils. Using a S. coelicolor strain in which topA transcription is under the control of an inducible promoter, we generated a long-term supercoiling imbalance, enabling us to identify genes involved in the supercoiling-sensitive transcriptional response. We observed that affected genes are preferentially organized in clusters, and among them, we identified a supercoiling-hypersensitive cluster (SHC) located in the core of the S. coelicolor chromosome. Moreover, using a gyrase inhibitor, we identified the directly affected novobiocin-sensitive genes and established that the AT content in their promoter regions was increased. Notably, genes whose expression was immediately impacted by gyrase inhibition encoded products associated with membrane transport or secondary metabolite synthesis. In contrast to the novobiocin-sensitive genes, the transcripts affected by long-term topological imbalance encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and transcriptional regulators, including multiple developmental regulators. Collectively, our results show that long-term supercoiling imbalance globally regulates gene transcription and has the potential to impact development, secondary metabolism and DNA repair, amongst others.

microbiology↗