Biology subjects

Jakimowicz, D.

Publications and source records attributed to Jakimowicz, D..

9 recordsLinked to original sources

bioRxiv2025

LsrL modulates Lsr2-induced chromatin structure to tune biosynthetic gene cluster regulation in Streptomyces venezuelae

Specialized biosynthetic gene clusters in Streptomyces are subject to complex regulation involving both transcriptional control and chromosome organization. The nucleoid-associated protein Lsr2 silences many of these clusters, yet how it shapes the global chromatin structure and how its conserved paralog LsrL contributes to this process remain poorly understood. In this study, we applied a multi-omics approach, combining transcriptional activity, genome-wide protein-DNA binding profiles, and three-dimensional chromosome conformation to characterize the coordination of Lsr2 and LsrL in exerting transcriptional control and genome architecture in Streptomyces venezuelae. In line with established Lsr2 functions, we find that Lsr2 sets broad transcriptional boundaries, while LsrL acts in a more context-specific manner that depends on the presence of Lsr2 and may function to reinforce or modulate Lsr2-mediated silencing. Loss of Lsr2 reshaped the chromatin landscape genome-wide, relieving its restriction on short-range contacts, triggering strong transcriptional changes and new domain boundaries near de-repressed biosynthetic gene clusters. These findings establish Lsr2 as a dominant but contextually modulated regulator whose interplay with LsrL coordinates specialized metabolism with higher-order chromosome organization.

microbiology↗

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↗

bioRxiv2024

SMC modulates ParB engagement in segregation complexes in Streptomyces

ParB is long established chromosome segregation protein in bacteria. Due to the recently demonstrated CTPase activity of ParB, formation of its nucleoprotein complexes was unrevealed. ParB homodimers bound to CTP are loaded onto DNA at parS sites, where they recruit condensin (SMC), thereby facilitating chromosome organization and segregation. Whether SMC modulates ParB complexes has remained unknown. Here, we generated Streptomyces venezuelae strains producing ParB-HaloTag in the presence or absence of SMC and used single-cell time-lapse fluorescence microscopy, single molecule tracking and fluorescence recovery after photobleaching analysis to explore ParB dynamics. Additionally, we performed chromatin immunoprecipitation to examine ParB interactions with DNA in the presence or absence of SMC. We reveal that SMC modulates ParB complex stability on DNA. We find that the absence of SMC results in faster ParB complex disassembly, and promotes non-specific DNA binding. Additionally, we show that SMC reduces ParB CTPase activity in vitro. Taken together our data provide evidence of SMC positive feedback on the ParB nucleoprotein complex, offering new insight into the nature of ParB complex regulation.

microbiology↗

bioRxiv2024

Roles of PknB and CslA in cell wall morphogenesis of Streptomyces

The bacterial cell wall is essential for maintaining cellular integrity and defining the mode of growth, with different species adopting distinct strategies for cell wall synthesis and remodelling. Streptomyces are filamentous bacteria predominantly found in soil and renowned for producing specialised metabolites, including antibiotics. They grow through tip extension and branching hyphal filaments, forming a multicellular mycelium. New branches are established by forming a new growth zone on the lateral cell wall. Proteins involved in this process are organised into complexes called polarisomes, with DivIVA being the most well-characterised component. To investigate the tip growth requirements in Streptomyces albus we developed a genetic screen utilising toxic DivIVA overproduction and searched for suppressors of its lethality, reasoning that such suppressors would likely encode components functionally linked to DivIVA or the tip growth machinery. Among the identified genes was pknB, encoding a serine/threonine protein kinase implicated in the regulation of cell growth and morphogenesis. We confirmed that deletion of pknB restored the growth phenotype of S. albus following DivIVA overproduction. The phosphoproteome analysis revealed that the absence of PknB alters the phosphorylation state of CslA, a cellulose synthase-like protein. We demonstrate that a phosphoablative mutant of CslA impairs {beta}-glucan synthesis and causes hypersensitivity to lysozyme. Overproduction of CslA restored colony growth defects arising from DivIVA-induced hyperbranching, without however suppressing the hyperbranching phenotype. These findings collectively identify PknB-dependent phosphorylation of CslA as a central regulatory point in Streptomyces cell envelope construction, revealing how modulation of {beta}-glucan synthesis can mitigate the cellular consequences of DivIVA dysregulation.

microbiology↗

bioRxiv2024

The role of two major nucleoid associated proteins in Streptomyces, HupA and HupS, in stress survival and gene expression regulation

Streptomyces are sporulating soil bacteria with enormous potential for secondary metabolites biosynthesis. Regulatory networks governing Streptomyces coelicolor differentiation and secondary metabolites production are complex and composed of numerous regulatory proteins ranging from specific transcriptional regulators to sigma factors. Nucleoid associated proteins (NAPs) are also believed to contribute to regulation of gene expression. Upon DNA binding these proteins impact DNA accessibility. Among NAPs HU proteins are the most widespread and abundant. Unlike other bacteria, the Streptomyces genome encodes two HU homologs: HupA and HupS, differing in structure and expression profile. In this study, we explore whether HupA and HupS affect S. coelicolor growth under optimal and stressful conditions and how they control global gene expression. By testing both single and double mutants we address the question of both HU homologs complementarity. The lack of both hup genes led to growth and sporulation inhibition, as well as increased spore fragility. Our data indicate a synergy between the functions of HupA and HupS during S. coelicolor growth. We also demonstrate, that both HU homologs can be considered global transcription regulators influencing expression of numerous genes encoding proteins linked to chromosome topology, secondary metabolites production and transcription. We identify the independent HupA and HupS regulons as well as genes under the control of both HupA and HupS proteins. Our data indicate some extent of redundancy as well as independent function of both homologs. ImportanceStreptomyces belong to the bacterial family widely used in the production of antibiotics as well as research for new bioactive substances with antimicrobial properties. Gene expression in Streptomyces, and consequently the production of secondary metabolites, is controlled by a vast and complex network of transcriptional regulators. Our data indicate that two proteins, HupA and HupS, involved in the maintenance of chromosome structure, also participate in this regulatory network. Their presence appears to important for S. coelicolors adaptation for survival in unfavorable conditions such as high temperature. The lack of one or both HU proteins affects the expression of many genes, indicating that they act as global transcriptional regulators.

microbiology↗

bioRxiv2023

The interplay between the polar growth determinant DivIVA, the segregation protein ParA and their novel interaction partner PapM controls the Mycobacterium smegmatis cell cycle by modulation of DivIVA subcellular distribution

Bacterial chromosome segregation is facilitated by the ParABS system. The ParB protein binds centromere-like parS sequences and forms nucleoprotein complexes. These nucleoprotein complexes are segregated by the dynamic ATPase ParA. In mycobacteria, ParA also interacts with the polar growth determinant DivIVA (Wag31). This interaction was earlier shown to facilitate the segregation of ParB complexes but also to affect cell extension. Here, we identify an additional partner of ParA in Mycobacterium smegmatis, named PapM. Using E. coli based analysis, we show that PapM likewise interacts with DivIVA and that the tripartite interaction of ParA-PapM-DivIVA is phosphorylation-dependent: ParA binding to DivIVA is diminished, while PapM binding is promoted upon phosphorylation of DivIVA. The presence of PapM enhances the dissociation of ParA from the DivIVA complex upon its phosphorylation. Studies of M. smegmatis mutant strains reveal that altered PapM levels influence chromosome segregation and cell length. The elimination of PapM affects ParA dynamics. Further, ParA and, to a lesser extent, PapM modulates the subcellular distribution of DivIVA. Altogether our studies show that the tripartite interplay between ParA-DivIVA and PapM controls the switch between cell division and cell elongation and in this way affects the mycobacterial cell cycle. ImportanceThe genus of Mycobacteria includes important clinical pathogens (M. tuberculosis). Bacteria of this genus share the unusual features of their cell cycle such as asymmetric polar cell elongation and long generation time. Markedly, control of the mycobacterial cell cycle still remains not fully understood. The main cell growth determinant in Mycobacteria is an essential protein DivIVA, which is also involved in cell division. DivIVA activity is controlled by phosphorylation but the mechanism and significance of this process is unknown. Here, we show how the previously established protein interaction partner of DivIVA in Mycobacteria, the segregation protein ParA, affects the DivIVA subcellular distribution. We also demonstrate the role of a newly identified M. smegmatis DivIVA and ParA interaction partner, a protein named PapM, and we establish how their interactions are modulated by phosphorylation. Demonstrating that the tripartite interplay affects the mycobacterial cell cycle contributes to general understanding of mycobacterial growth regulation.

microbiology↗

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↗