Maintaining an optimal level of chromosomal supercoiling is critical for the progression of DNA replication and transcription. Moreover, changes in global supercoiling affect the expression of a large number of genes and play a fundamental role in adapting to stress. Topoisomerase I (TopA) and gyrase are key players in the regulation of bacterial chromosomal topology through their respective abilities to relax and compact DNA. Soil bacteria such as Streptomyces species, which grow as branched, multigenomic hyphae, are subject to environmental stresses that are associated with changes in chromosomal topology. The topological fluctuations modulate the transcriptional activity of a large number of genes and in Streptomyces are related to the production of antibiotics. To better understand the regulation of topological homeostasis in Streptomyces coelicolor, we investigated the interplay between the activities of the topoisomerase-encoding genes topA and gyrBA. We show that the expression of both genes is supercoiling sensitive. Remarkably, increased chromosomal supercoiling induces the topA promoter but only slightly influences gyrBA transcription, while DNA relaxation affects the topA promoter only marginally but strongly activates the gyrBA operon. Moreover, we showed that exposure to elevated temperatures induces rapid relaxation, which results in changes in the levels of both topoisomerases. We therefore propose a unique mechanism of S. coelicolor chromosomal topology maintenance based on the supercoiling-dependent stimulation, rather than repression, of the transcription of both topoisomerase genes. These findings provide important insight into the maintenance of topological homeostasis in an industrially important antibiotic producer.IMPORTANCE We describe the unique regulation of genes encoding two topoisomerases, topoisomerase I (TopA) and gyrase, in a model Streptomyces species. Our studies demonstrate the coordination of topoisomerase gene regulation, which is crucial for maintenance of topological homeostasis. Streptomyces species are producers of a plethora of biologically active secondary metabolites, including antibiotics, antitumor agents, and immunosuppressants. The significant regulatory factor controlling the secondary metabolism is the global chromosomal topology. Thus, the investigation of chromosomal topology homeostasis in Streptomyces strains is crucial for their use in industrial applications as producers of secondary metabolites.

Negative DNA supercoiling allows chromosome condensation 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. Moreover, changes in chromosomal supercoiling have been suggested to be associated with the control of antibiotic production and environmental stress response. Remarkably, in S. coelicolor , a 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 identified genes involved in the transcriptional response to long-term supercoiling imbalance. The affected genes are preferentially organized in several clusters, and a supercoiling-hypersensitive cluster (SHC) was found to be located in the core of the S. coelicolor chromosome. The transcripts affected by long-term topological imbalance encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and transcriptional regulators, including multiple developmental regulators. Moreover, using a gyrase inhibitor, we identified those genes that were directly affected by novobiocin, and found this was correlated with increased AT content in their promoter regions. In contrast to the genes affected by long-term supercoiling changes, among the novobiocin-sensitive genes, a significant fraction encoded for proteins associated with membrane transport or secondary metabolite synthesis. 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.

10 Negative DNA supercoiling allows chromosome condensation within a cell and facilitates 11 DNA unwinding, which is required for the occurrence of DNA transaction processes, i.e., DNA 12 replication, transcription and recombination. In bacteria, changes in chromosome supercoiling 13 impact global gene expression; however, the limited studies on the global transcriptional response 14 have focused mostly on pathogenic species and have reported various fractions of affected genes. 15 Furthermore, the transcriptional response to long-term supercoiling imbalance is still poorly 16 understood. Here, we address the transcriptional response to both novobiocin-induced rapid 17 chromosome relaxation or long-term topological imbalance, both increased and decreased 18 supercoiling, in environmental antibiotic-producing bacteria belonging to the Streptomyces genus. 19 During the Streptomyces complex developmental cycle, multiple copies of GC-rich linear 20 chromosomes present in hyphal cells undergo profound topological changes, from being loosely 21 condensed in vegetative hyphae, to being highly compacted in spores. In Streptomyces, 22 chromosomal supercoiling changes may also be triggered by environmental stressors and have 23 been suggested to be associated with the control of antibiotic production. Remarkably, in 24 S. coelicolor, one of model Streptomyces species, topoisomerase I (TopA) is solely responsible for 25 the removal of negative DNA supercoils. Using a S. coelicolor strain in which topA transcription is 26under the control of an inducible promoter, we generated a long-term supercoiling imbalance, 27 enabling us to identify genes involved in the supercoiling-sensitive transcriptional response. We 28 observed that affected genes are preferentially organized in clusters, and among them, we 29 identified a supercoiling-hypersensitive cluster (SHC) located in the core of the S. coelicolor 30 chromosome. Moreover, using a gyrase inhibitor, we identified the directly affected novobiocin-31 sensitive genes and established that the AT content in their promoter regions was increased. 32Notably, genes whose expression was immediately impacted by gyrase inhibition encoded 33 products associated with membrane transport or secondary metabolite synthesis. In contrast to 34 the novobiocin-sensitive genes, the transcripts affected by long-term topological imbalance 35 encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and 36 transcriptional regulators, including multiple developmental regulators. Collectively, our results 37show that long-term supercoiling imbalance globally regulates gene transcription and has the 38 potential to impact development, secondary metabolism and DNA repair, amongst others. 39 A bacterial chromosome is highly constrained within the cell, yet remains fully accessible for 41 DNA replication, segregation and transcription. In bacteria, these processes are not separated in 42 space and time, and their co-occurrence significantly impacts chromosome architecture. 43 Chromosome ...