Functional analysis of three topoisomerases that regulate <scp>DNA</scp> supercoiling levels in <scp><i>C</i></scp><i>hlamydia</i>

Orillard, Emilie; Tan, Ming

doi:10.1111/mmi.13241

Cited by 10 publications

(21 citation statements)

References 58 publications

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“…However, the increased relaxation has a weak negative influence on the S. coelicolor topA gene, i.e., the overproduction of TopA slightly inhibits both p1 and p2 activities. Such regulation is remarkably different from the efficient negative-feedback mechanism of topA expression that has been described for other bacterial species ( 18 , 19 , 46 , 49 ). In the closely related species M. smegmatis and M. tuberculosis , topA transcription (also controlled by two promoters) is very efficiently inhibited by chromosomal relaxation induced by novobiocin treatment or TopA overproduction ( 19 ).…”

Section: Discussioncontrasting

confidence: 66%

“…It has been demonstrated that supercoiling conditions affect both topA and gyrase gene transcription in a number of bacterial species ( 11 , 30 , 46 , 49 ), thereby providing a mechanism for maintaining topological balance. We expected that an elevated TopA level should be correlated with decreased gyrase gene transcription.…”

Section: Resultsmentioning

confidence: 99%

“…The phenomenon of relaxation-stimulated transcription of gyrase genes has also been described previously ( 11 , 29 , 46 ). Moreover, it was recently shown that in the Chlamydiae , gyrase genes can be regulated by positive stimulation in response to increasing negative supercoiling ( 49 ). Analysis of topoisomerase gene expression suggested that in S. coelicolor , the main mechanism preventing excessive negative supercoiling employs the induction of TopA and is only slightly dependent on gyrase gene downregulation.…”

Section: Discussionmentioning

confidence: 99%

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The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor

et al. 2016

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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.

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Section: Discussioncontrasting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor

et al. 2016

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“…During this stage of the developmental cycle, when RB-to-EB conversion is occurring, global gene expression profiling supports the hypothesis that about 20% of the genome is upregulated ( 33 , 34 ). Expression of many of these genes has been shown to be regulated by σ 66 , with additional factors, such as DNA topology and supercoiling, expected to contribute to this differential expression mechanism ( 35 – 37 ). σ 28 has been shown to regulate few (but critical) genes during the RB-to-EB conversion process, including those involved in the formation of the condensed DNA structure that is unique to EBs ( 38 – 42 ).…”

Section: Introductionmentioning

confidence: 99%

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

Soules

LaBrie

May

et al. 2020

mBio

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Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ54) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ54 transcriptional activation. CtcC activation of σ54 requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ54 transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ54 promoter elements. Reporter gene analyses using these putative σ54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ54 controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies. IMPORTANCE The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia, sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia. These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells.

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“…For example, early genes are transcribed within the first few hours of EB entry into the host cell (Belland et al, 2003; Hayward et al, 2021; Humphrys et al, 2013). Midcycle genes are transcribed during RB replication and are proposed to be regulated by changes in chlamydial DNA supercoiling (Niehus et al, 2008; Orillard and Tan, 2015). Late genes are transcribed at the time of RB-to-EB conversion and are regulated by a transcription factor EUO (Rosario et al, 2014; Rosario and Tan, 2012).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Chlamydia RsbU phosphatase activity by the enolase product, phosphoenolpyruvate

Rosario

Tan

2022

Preprint

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The intracellular pathogen Chlamydia temporally regulates the expression of its genes but the upstream signals that control transcription are not known. The best studied regulatory pathway is a partner switching mechanism that involves an anti-sigma factor RsbW, which inhibits transcription by binding and sequestering the sigma subunit of RNA polymerase. RsbW is itself regulated by an anti-anti-sigma factor RsbV whose phosyphorylation state is controlled by the phosphatase RsbU. In this study, we showed that Chlamydia trachomatis RsbU requires manganese or magnesium as a cofactor and dephosphorylates RsbV1 and RsbV2, which are the two chlamydial paralogs of RsbV. The gene for RsbU is adjacent to the enolase gene in a number of Chlamydia genomes, and we showed that eno and rsbU are co-transcribed from the same operon. In other bacteria, there is no known functional connection between the Rsb pathway and enolase, which is an enzyme in the glycolytic pathway. We found, however, that Chlamydia RsbU phosphatase activity was inhibited by phosphoenolpyruvate (PEP), the product of the enolase reaction, but not by 2-phosphoglycerate (2PGA), which is the substrate. These findings suggest that the enolase reaction, and more generally glucose metabolism, may provide an upstream signal that regulates transcription in Chlamydia through the RsbW pathway.IMPORTANCEThe RsbW pathway is a phosphorelay that regulates gene expression in Chlamydia but its upsteam signal has not been identified. We showed that RsbU, a phosphatase in this pathway is inhibited by phosphoenolpyruvate, which is the product of the enolase reaction. As enolase is an enzyme in the glycolytic pathway, these results reveal an unrecognized link between glucose metabolism and gene regulation in chlamydiae. Moreover, as these intracellular bacteria acquire gluose from the infected host cell, our findings suggest that glucose availability may be an external signal that controls chlamydial gene expression.

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Functional analysis of three topoisomerases that regulate DNA supercoiling levels in Chlamydia

Cited by 10 publications

References 58 publications

The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor

The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

Regulation of Chlamydia RsbU phosphatase activity by the enolase product, phosphoenolpyruvate

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