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The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated. To date, only a few examples of such genes are fully established. Using RNA sequencing and ribosome profiling, we found expression of novel overlapping open reading frames in Escherichia coli O157:H7 EDL933 (EHEC). Indeed, the overlapping candidate genes are equipped with typical structural elements required for transcription and translation, i.e., promoters, transcription start sites, as well as terminators, all of which were experimentally verified. Translationally arrested mutants, unable to produce the overlapping encoded protein, were found to have a growth disadvantage when grown competitively against the wild type. Thus, the phenotypes found imply biological functionality of the genes at the level of proteins produced. The addition of 3 more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes emphasizes the underestimated coding capacity of bacterial genomes. IMPORTANCE The abundance of long overlapping genes in prokaryotic genomes is likely to be significantly underestimated, since such genes are not allowed in genome annotations. However, ribosome profiling catches mRNA in the moment of being template for protein production. Using this technique and subsequent experiments, we verified 3 novel overlapping genes encoded in antisense of known genes. This adds more examples of prokaryotic overlapping genes to the currently limited, yet constantly growing pool of such genes.

Significance: Fast and reliable detection of infectious SARS-CoV-2 virus loads is an important issue. Fluorescence spectroscopy is a sensitive tool to do so in clean environments. This presumes a comprehensive knowledge of fluorescence data., Aim: We aim at providing fully featured information on wavelength and time-dependent data of the fluorescence of the SARS-CoV-2 spike protein S1 subunit, its receptor-binding domain (RBD), and the human angiotensin-converting enzyme 2, especially with respect to possible optical detection schemes., Approach: Spectrally resolved excitation-emission maps of the involved proteins and measurements of fluorescence lifetimes were recorded for excitations from 220 to 295 nm. The fluorescence decay times were extracted by using a biexponential kinetic approach. The binding process in the SARS-CoV-2 RBD was likewise examined for spectroscopic changes., Results: Distinct spectral features for each protein are pointed out in relevant spectra extracted from the excitation-emission maps. We also identify minor spectroscopic changes under the binding process. The decay times in the biexponential model are found to be   (  2.0  ±  0.1  )   ns and   (  8.6  ±  1.4  )    ns., Conclusions: Specific material data serve as an important background information for the design of optical detection and testing methods for SARS-CoV-2 loaded media.

Laser-induced fluorescence (LIF) is a well-established technique for monitoring chemical processes and for the standoff detection of biological substances because of its simple technical implementation and high sensitivity. Frequently, standoff LIF spectra from large molecules and bio-agents are only slightly structured and a gain of deeper information, such as classification, let alone identification, might become challenging. Improving the LIF technology by recording spectral and additionally time-resolved fluorescence emission, a significant gain of information can be achieved. This work presents results from a LIF based detection system and an analysis of the influence of time-resolved data on the classification accuracy. A multi-wavelength sub-nanosecond laser source is used to acquire spectral and time-resolved data from a standoff distance of 3.5 m. The data set contains data from seven different bacterial species and six types of oil. Classification is performed with a decision tree algorithm separately for spectral data, time-resolved data and the combination of both. The first findings show a valuable contribution of time-resolved fluorescence data to the classification of the investigated chemical and biological agents to their species level. Temporal and spectral data have been proven as partly complementary. The classification accuracy is increased from 86% for spectral data only to more than 92%.

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before., (Copyright © 2016 Fellner et al.)

Background: Genomes of E. coli, including that of the human pathogen Escherichia coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which, apparently, have escaped annotation due to their small size and non-essential function. To find such genes, global gene expression of EHEC EDL933 was examined, using strand-specific RNAseq (transcriptome), ribosomal footprinting (translatome) and mass spectrometry (proteome)., Results: Using the above methods, 72 short, non-annotated protein-coding genes were detected. All of these showed signals in the ribosomal footprinting assay indicating mRNA translation. Seven were verified by mass spectrometry. Fifty-seven genes are annotated in other enterobacteriaceae, mainly as hypothetical genes; the remaining 15 genes constitute novel discoveries. In addition, protein structure and function were predicted computationally and compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. Based on this comparison, 61 of the 72 novel proteins exhibit predicted structural and functional features similar to those of annotated proteins. Many of the novel genes show differential transcription when grown under eleven diverse growth conditions suggesting environmental regulation. Three genes were found to confer a phenotype in previous studies, e.g., decreased cattle colonization., Conclusions: These findings demonstrate that ribosomal footprinting can be used to detect novel protein coding genes, contributing to the growing body of evidence that hypothetical genes are not annotation artifacts and opening an additional way to study their functionality. All 72 genes are taxonomically restricted and, therefore, appear to have evolved relatively recently de novo.

Background: Gene duplication is believed to be the classical way to form novel genes, but overprinting may be an important alternative. Overprinting allows entirely novel proteins to evolve de novo, i.e., formerly non-coding open reading frames within functional genes become expressed. Only three cases have been described for Escherichia coli. Here, a fourth example is presented., Results: RNA sequencing revealed an open reading frame weakly transcribed in cow dung, coding for 101 residues and embedded completely in the -2 reading frame of citC in enterohemorrhagic E. coli. This gene is designated novel overlapping gene, nog1. The promoter region fused to gfp exhibits specific activities and 5' rapid amplification of cDNA ends indicated the transcriptional start 40-bp upstream of the start codon. nog1 was strand-specifically arrested in translation by a nonsense mutation silent in citC. This Nog1-mutant showed a phenotype in competitive growth against wild type in the presence of MgCl2. Small differences in metabolite concentrations were also found. Bioinformatic analyses propose Nog1 to be inner membrane-bound and to possess at least one membrane-spanning domain. A phylogenetic analysis suggests that the orphan gene nog1 arose by overprinting after Escherichia/Shigella separated from the other γ-proteobacteria., Conclusions: Since nog1 is of recent origin, non-essential, short, weakly expressed and only marginally involved in E. coli's central metabolism, we propose that this gene is in an initial stage of evolution. While we present specific experimental evidence for the existence of a fourth overlapping gene in enterohemorrhagic E. coli, we believe that this may be an initial finding only and overlapping genes in bacteria may be more common than is currently assumed by microbiologists.

Overlapping embedded genes, such as htgA/yaaW, are assumed to be rare in prokaryotes. In Escherichia coli O157:H7, gfp fusions of both promoter regions revealed activity and transcription start sites could be determined for both genes. Both htgA and yaaW were inactivated strand specifically by introducing a stop codon. Both mutants exhibited differential phenotypes in biofilm formation and metabolite levels in a nontargeted analysis, suggesting that both are functional despite YaaW but not HtgA could be expressed. While yaaW is distributed all over the Gammaproteobacteria, an overlapping htgA-like sequence is restricted to the Escherichia-Klebsiella clade. Full-length htgA is only present in Escherichia and Shigella, and htgA showed evidence for purifying selection. Thus, htgA is an interesting case of a lineage-specific, nonessential and young orphan gene., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)