Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference
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ABSTRACT:
PROVIDER: PRJNA962055 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24310800_1.fastq.gz | Fastqsanger.gz | |||
| SRR24310800_2.fastq.gz | Fastqsanger.gz | |||
| SRR24310801_1.fastq.gz | Fastqsanger.gz | |||
| SRR24310801_2.fastq.gz | Fastqsanger.gz | |||
| SRR24310802_1.fastq.gz | Fastqsanger.gz |
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Project description:RNAseq analysis was performed for C. trachomatis serovar L2 after knocking down or overexpressing different sigma factors.
2023-09-05 | GSE230645 | GEO
Project description:Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia. Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. IMPORTANCE Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia. Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.
| S-EPMC10597470 | biostudies-literature
Project description:The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintainance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.
2011-12-31 | GSE34089 | GEO
Project description:Abstract of associated manuscript: The Bacillus subtilis extracytoplasmic function (ECF) sigma(M) factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by sigma(M). In many cases, expression is only partially dependent on sigma(M) because of both overlapping promoter recognition with other ECF sigma factors and the presence of additional promoter elements. Genes regulated by sigma(M) have a characteristic pattern of induction in response to cell envelope-acting antibiotics as evidenced by hierarchical clustering analysis. sigma(M) also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by sigma(W), sigma(B) and several two-component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the sigma(M) regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification.
2008-12-05 | GSE13820 | GEO
Project description:In this study, we performed a ChIP-chip experiment to determine the respective regulons of RpoHI and RpoHII in Rhodobacter sphaeroides. We grew R. sphaeroides in aerobic conditions and induced either proteins ectopically and immuno-precipitated the regions of the genomic DNA interacting with the sigma factors.
2012-08-03 | GSE39711 | GEO
Project description:We developed EASI-tag (Easily Abstractable Sulfoxide-based Isobaric tag), a new generation of amine-derivatizing and sulfoxide-containing isobaric labelling reagents, which dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level enable accurate determination of quantitative differences between multiplexed samples. EASI-tag makes isobaric labeling applicable to any bottom up proteomics application and to benchtop mass spectrometers.
2018-06-21 | PXD007165 | Pride
Project description:Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcriptional factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is a laborious process and not scalable. Here, we demonstrate a new technology, RIViTseq, that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViTseq data expanded the transcriptional regulatory network in this bacterium, discovering new regulatory cascades and crosstalk between sigma factors. Implementation of RIViTseq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.
2022-04-22 | GSE184393 | GEO
Project description:Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcriptional factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is a laborious process and not scalable. Here, we demonstrate a new technology, RIViTseq, that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViTseq data expanded the transcriptional regulatory network in this bacterium, discovering new regulatory cascades and crosstalk between sigma factors. Implementation of RIViTseq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.
2022-04-22 | GSE184392 | GEO
Project description:Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis.
2020-11-23 | PXD019393 | Pride
Project description:Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, B, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, A, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription fromB-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between B- and A-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.
2021-03-06 | GSE168393 | GEO