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Expression analysis of Shigella flexneri 2a strain 301

ABSTRACT: Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available.

ORGANISM(S): Shigella flexneri 2a str. 301

PROVIDER: GSE22800 | GEO | 2011/06/27

SECONDARY ACCESSION(S): PRJNA127803

REPOSITORIES: GEO

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Expression analysis of Shigella flexneri 2a strain 301

Project description:Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available. Study using total RNA recovered from five conditions.

2011-06-27 | E-GEOD-22800 | biostudies-arrayexpress

Prediction of Non-Coding sRNAs and Screening of Virulence-Related sRNAs of Salmonella Pullorum

Project description:Purpose: Searching for sRNAs in Salmonella pullorum by RNA sequencing and exploring their functions.Methods: High-throughput sequencing of RNA extracted from Salmonella pullorum under normal growth conditions to detect newly discovered sRNAs, followed by experiments to verify their functions.Results: The proportion of Clean Reads of this sequencing was >65%, and the base Q30s were all above 85%, indicating that the sequencing quality is good and can be used for subsequent analysis. The sRNAscanner software predicted that 148 new sRNAs might exist on the reference genome of Salmonella fowl dysentery, and the reads obtained from sequencing were compared to the genome, and it was found that 110 out of the 148 newly predicted sRNAs could be detected.Conclusions: sRNAs are widely found in bacteria and are involved in many physiological processes. In this study, we detected new sRNAs in Salmonella pullorum by RNA-seq, which lays the foundation for the subsequent investigation of the regulatory functions of sRNAs in bacteria.

2024-06-10 | GSE269156 | GEO

Shigella CGH

Project description:Background Compelling evidence indicates that Shigella species, the etiologic agents of bacillary dysentery, as well as enteroinvasive Escherichia coli, are derived from multiple origins of Escherichia coli and form a single pathovar. To further understand the genome diversity and virulence evolution of Shigella, comparative genomic hybridization microarray analysis was employed to compare the gene content of E. coli K-12 with those of 43 Shigella strains from all serotypes. Results For the 43 strains subjected to CGH microarray analyses, the common backbone of the Shigella genome was estimated to contain more than 1,900 open reading frames, with a mean number of 729 undetectable ORFs. The mosaic distribution of absent regions indicated that insertions and/or deletions have led to the highly diversified genomes of pathogenic strains. Conclusion These results support the hypothesis that by gain and loss of functions, Shigella species became successful human pathogens through convergent evolution from diverse genomic backgrounds. Moreover, we also found many specific differences between different lineages, providing a window into understanding bacterial speciation and taxonomic relationships. Keywords: comparative genomic hybridization

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Identification of Populus small RNAs responsive to mutualistic interactions with mycorrhizal fungi, Laccaria bicolor and Rhizophagus irregularis

Project description:Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant:microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms. In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions. We identified differential expression of sRNAs that were, to a large extent, 1) within the genomic regions lacking annotated genes in the Populus genome and 2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments.

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Dual RNA-seq of zebrafish larvae infected with Shigella sonnei

Project description:Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

2019-11-19 | GSE140544 | GEO

Shigella flexneri 2a str. 301

Project description:Human-specific pathogen that causes endemic dysentery

| PRJNA310 | ENA

Small protein enrichment improves proteomics detection of sORF encoded peptides.

Project description:With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in facilitating the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model organism Salmonella Typhimurium. An optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 relying on the differential peptide versus protein solubility is suggested and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions the SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

2021-10-07 | PXD025297 | Pride

Exploring the hidden world of Methanosarcina mazei: Unravelling the secrets of its small proteome and its nitrogen-dependent regulation

Project description:The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1, is crucial for climate and environmental research due to its ability to produce methane from H2 plus CO2, acetate, and methylamines. Here, we established the first Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency (+N) versus nitrogen limitation (-N)). Translation of 93 previously annotated and 311 novel small open reading frames (sORFs), coding for proteins ≤ 70 amino acids in length was predicted with high confidence based on Ribo-seq data. Epitope tagging followed by immunoblotting analysis confirmed the translation of 12 out of 15 selected novel sORFs, the remaining three were validated by LC-MS-MS analysis. In total, LC-MS-MS analysis validated translation for 60 annotated sORFs and 26 novel sORFs. A comprehensive differential expression analysis revealed that 29 of 311 novel sORFs were differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. Several reported sRNAs are now emerging as dual-functional sRNAs, including sRNA154, the central regulatory sRNA in regulation of nitrogen metabolism. Numerous of the novel sORFs identified in this study are conserved in Methanosarcina species, some of which showed a phenotype when overproduced, pinpointing important physiological functions. Overall, the comprehensive analysis opens a new avenue to start elucidating the function(s) of small proteins in M. mazei.

2024-08-02 | PXD045039 | Pride

Small RNA Sibling ryfA1 Influences Shigella dysenteriae Pathogenesis

Project description:Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the over production of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread and the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1; the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.

2017-04-17 | GSE87727 | GEO

ARA-PEPs : a repository of putative sORF-encoded peptides in Arabidopsis thaliana

Project description:Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). There is increasing evidence for the translation of these sORFs into bioactive peptides. Yet only a few small peptides are annotated in the model organism Arabidopsis thaliana. To aid the functional annotation of small peptides, we have developed ARA-PEPs, a repository and webserver of putative peptides encoded by sORFs in the Arabidopsis genome from in house Tiling arrays, RNA sequencing and from publicly available datasets. In order to identify novel oxidative stress-induced peptides in Arabidopsis thaliana a tiling array analysis (GeneChip® Arabidopsis Tiling 1.0R Arrays ) was performed on mRNA extracted from leaves inoculated with Botrytis cinerea (BC). Normalized log signals were obtained using the Affymetrix Tiling Analysis Software - Version 1.1, Build 2. ON and OFF probes were selected using a threshold, based on positive controls. Next, groups of 4-13 successive ON probes were combined into short TARs and a selection was made of TARs having an average signal intensity at least 2.6-fold higher after BC treatment compared to the control treatment, resulting in 195 BC induced TARs.

2016-12-23 | GSE84002 | GEO

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