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• W2944954518 abstract "The food-borne pathogen Escherichia coli O157:H7 strain Sakai (EHEC) is the causative agent of hemorrhagic colitis and hemolytic-uremic syndrome. The genome of EHEC Sakai was sequenced in 2001 and it contains 5,358 annotated genes. However, intergenic regions might harbor additional (small) protein-coding genes. Due to the DNA triplet code, it is also possible that the sequences of two genes overlap at a given locus. This study focuses on the detection of non-trivial, antiparallel overlapping genes (OLGs). The transcriptome and the translatome of EHEC were determined using the high-throughput next generation sequencing methods RNAseq and RIBOseq. Three different growth conditions were tested, representing two optimal conditions (growth in LB and BHI medium), and one severe stress condition combining long-term cold and osmotic stress. The sequencing results show excellent reproducibility. The RIBOseq data of the annotated genes correlates to previously published proteome data. About one third of the annotated genes are differentially expressed at the transcriptional and/or translational level comparing either the two optimal conditions, or the stress condition to the optimal BHI condition. Riboswitches and the ncRNA DsrA were found to be involved in regulating gene expression after adaptation to cold and osmotic stress.In addition, translation of non-annotated intergenic and antiparallel overlapping open reading frames (ORFs) was investigated. Amazingly, 465 intergenic ORFs and 380 OLGs show evidence of translation. The translatability was found to be similar to annotated genes, which supports the hypothesis these translated ORFs represent novel protein-coding genes. Further evidence for this claim includes the discovery of annotated homologs, differential regulation between growth conditions, presence of a reading frame in the sum signal of RIBOseq reads, and predicted regulatory elements (such as σ70 promoter, ρ-independent terminator, and a Shine-Dalgarno sequence).Three of the novel OLG pairs discovered, namely anoG/ECs2385, laoB/ECs5115, and slyC/slyA, were functionally characterized. The transcription initiation sites were determined, and promoter activity of sequences upstream was detected. Presence of a protein was confirmed by expressing a C-terminally EGFP-fusion. Most importantly, a phenotype was observed in competitive growth experiments using EHEC wild type against a strand-specific, translationally arrested mutant of the respective gene. Conditions with high promoter activity or those causing a phenotype provide evidence for potential functions of the novel OLGs. Phylostratigraphic analyses of the annotated mother genes and the overlapping embedded genes indicate that the OLG originated de novo by overprinting in all three cases. Characterization of another four OLG candidates provides some evidence that they might be novel functional protein-coding genes as well. Interestingly, the first potential antiparallel overlapping operon consisting of three ORFs was discovered, which is encoded antisense to ECs0535.All-in-all, this study shows that the genome of EHEC Sakai, and probably also other bacterial genomes, are under-annotated due to the systematical omission of small genes. Furthermore, genes encoded antiparallel to annotated genes seem to occur more frequently than previously presumed. The origin, evolution, and functions of these OLGs are interesting topics for future research." @default.
• W2944954518 created "2019-05-29" @default.
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• W2944954518 date "2018-01-01" @default.
• W2944954518 modified "2023-09-27" @default.
• W2944954518 title "RIBOseq-based discovery of non-annotated genes in Escherichia coli O157:H7 Sakai and their functional characterization" @default.
• W2944954518 hasPublicationYear "2018" @default.
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