A novel short L-arginine responsive protein-coding gene (laoB) antiparallel overlapping to a CadC-like transcriptional regulator in Escherichia coli O157:H7 Sakai originated by overprinting.

BACKGROUND: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail. RESULTS: A small open reading frame of Escherichia coli O157:H7 strain Sakai (EHEC), designated laoB (L-arginine responsive overlapping gene), is embedded in reading frame -2 in the antisense strand of ECs5115, encoding a CadC-like transcriptional regulator. This overlapping gene shows evidence of transcription and translation in Luria-Bertani (LB) and brain-heart infusion (BHI) medium based on RNA sequencing (RNAseq) and ribosomal-footprint sequencing (RIBOseq). The transcriptional start site is 289 base pairs (bp) upstream of the start codon and transcription termination is 155 bp downstream of the stop codon. Overexpression of LaoB fused to an enhanced green fluorescent protein (EGFP) reporter was possible. The sequence upstream of the transcriptional start site displayed strong promoter activity under different conditions, whereas promoter activity was significantly decreased in the presence of L-arginine. A strand-specific translationally arrested mutant of laoB provided a significant growth advantage in competitive growth experiments in the presence of L-arginine compared to the wild type, which returned to wild type level after complementation of laoB in trans. A phylostratigraphic analysis indicated that the novel gene is restricted to the Escherichia/Shigella clade and might have originated recently by overprinting leading to the expression of part of the antisense strand of ECs5115. CONCLUSIONS: Here, we present evidence of a novel small protein-coding gene laoB encoded in the antisense frame -2 of the annotated gene ECs5115. Clearly, laoB is evolutionarily young and it originated in the Escherichia/Shigella clade by overprinting, a process which may cause the de novo evolution of bacterial genes like laoB.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933 (EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and a peptide, RyhP.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

Transcriptional and translational regulation by RNA thermometers, riboswitches and the sRNA DsrA in Escherichia coli O157:H7 Sakai under combined cold and osmotic stress adaptation.

The enteric pathogen Escherichia coli O157:H7 Sakai (EHEC) is able to grow at lower temperatures compared to commensal E. coli Growth at environmental conditions displays complex challenges different to those in a host. EHEC was grown at 37°C and at 14°C with 4% NaCl, a combination of cold and osmotic stress as present in the food chain. Comparison of RNAseq and RIBOseq data provided a snap shot of ongoing transcription and translation, differentiating transcriptional and post-transcriptional gene regulation, respectively. Indeed, cold and osmotic stress related genes are simultaneously regulated at both levels, but translational regulation clearly dominates. Special emphasis was given to genes regulated by RNA secondary structures in their 5'UTRs, such as RNA thermometers and riboswitches, or genes controlled by small RNAs encoded in trans The results reveal large differences in gene expression between short-time shock compared to adaptation in combined cold and osmotic stress. Whereas the majority of cold shock proteins, such as CspA, are translationally downregulated after adaptation, many osmotic stress genes are still significantly upregulated mainly translationally, but several also transcriptionally.

Probiotic Enterococcus faecalis Symbioflor® down regulates virulence genes of EHEC in vitro and decrease pathogenicity in a Caenorhabditis elegans model.

Enterohemorrhagic E. coli O157:H7 (EHEC) shorten the lifespan of Caenorhabditis elegans compared to avirulent bacteria. Co-feeding EHEC with Enterococcus faecalis Symbioflor® significantly increased the worms' lifespan. The transcriptome of EHEC grown in vitro with or without Symbioflor® was analyzed using RNA-seq. The analysis revealed downregulation of several virulence-associated genes in the presence of Symbioflor®, including virulence key genes (e.g., LEE, flagellum, quorum-sensing). The downregulation of the LEE genes was corroborated by lux-transposon mutants. Upregulated genes included acid response genes, due to a decrease in pH exerted by Symbioflor®. Further genes indicate cellular stress in EHEC (e.g. prophage/mobile elements involved in excision, cell lysis, and cell division inhibition). Thus, the observed protection of C. elegans during an EHEC infection by the probiotic Symbioflor® is suggested to be caused by triggering concomitant transcriptomic changes. To verify the biological relevance of this modulation, exemplary genes found to be influenced by Symbioflor® were knocked out (fliD, espB, Z3136, Z3917, and L7052). The lifespan of nematodes changed when using knock-outs as food source and the effect could be complemented in trans. In summary, Symbioflor® appears to be a protective probiotic in the nematode model.

Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids.

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.

Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis.

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Translatomics combined with transcriptomics and proteomics reveals novel functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).

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.

Evidence for the recent origin of a bacterial protein-coding, overlapping orphan gene by evolutionary overprinting.

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.

Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 (EHEC) under eleven different environmental conditions including radish sprouts and cattle feces.

BACKGROUND: Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables. The ecology of this pathogen outside its human host is largely unknown and one third of its annotated genes are still hypothetical. To identify genetic determinants expressed under a variety of environmental factors, we applied strand-specific RNA-sequencing, comparing the SOLiD and Illumina systems. RESULTS: Transcriptomes of EHEC were sequenced under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes in strain EDL933 (genome and plasmid), a surprising minority of only 144 had null sequencing reads under all conditions. We therefore developed a statistical method to distinguish weakly transcribed genes from background transcription. We find that 96% of all genes and 91.5% of the hypothetical genes exhibit a significant transcriptional signal under at least one condition. Comparing SOLiD and Illumina systems, we find a high correlation between both approaches for fold-changes of the induced or repressed genes. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. Unique sets of genes, including many hypothetical genes, are highly up-regulated on radish sprouts, cattle feces, or in the presence of antibiotics. Furthermore, we observed induction of the shiga-toxin carrying phages by antibiotics and confirmed active biofilm related genes on radish sprouts, in cattle feces, and on agar plates. CONCLUSIONS: Since only a minority of genes (2.7%) were not active under any condition tested (null reads), we suggest that the assumption of significant genome over-annotations is wrong. Environmental transcriptomics uncovered hitherto unknown gene functions and unique regulatory patterns in EHEC. For instance, the environmental function of azoR had been elusive, but this gene is highly active on radish sprouts. Thus, NGS-transcriptomics is an appropriate technique to propose new roles of hypothetical genes and to guide future research.

Mining rare associations between biological ontologies.

The constantly increasing volume and complexity of available biological data requires new methods for their management and analysis. An important challenge is the integration of information from different sources in order to discover possible hidden relations between already known data. In this paper we introduce a data mining approach which relates biological ontologies by mining cross and intra-ontology pairwise generalized association rules. Its advantage is sensitivity to rare associations, for these are important for biologists. We propose a new class of interestingness measures designed for hierarchically organized rules. These measures allow one to select the most important rules and to take into account rare cases. They favor rules with an actual interestingness value that exceeds the expected value. The latter is calculated taking into account the parent rule. We demonstrate this approach by applying it to the analysis of data from Gene Ontology and GPCR databases. Our objective is to discover interesting relations between two different ontologies or parts of a single ontology. The association rules that are thus discovered can provide the user with new knowledge about underlying biological processes or help improve annotation consistency. The obtained results show that produced rules represent meaningful and quite reliable associations.

Phenotype of htgA (mbiA), a recently evolved orphan gene of Escherichia coli and Shigella, completely overlapping in antisense to yaaW.

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.

Multivariate analysis of flow cytometric data using decision trees.

Characterization of the response of the host immune system is important in understanding the bidirectional interactions between the host and microbial pathogens. For research on the host site, flow cytometry has become one of the major tools in immunology. Advances in technology and reagents allow now the simultaneous assessment of multiple markers on a single cell level generating multidimensional data sets that require multivariate statistical analysis. We explored the explanatory power of the supervised machine learning method called "induction of decision trees" in flow cytometric data. In order to examine whether the production of a certain cytokine is depended on other cytokines, datasets from intracellular staining for six cytokines with complex patterns of co-expression were analyzed by induction of decision trees. After weighting the data according to their class probabilities, we created a total of 13,392 different decision trees for each given cytokine with different parameter settings. For a more realistic estimation of the decision trees' quality, we used stratified fivefold cross validation and chose the "best" tree according to a combination of different quality criteria. While some of the decision trees reflected previously known co-expression patterns, we found that the expression of some cytokines was not only dependent on the co-expression of others per se, but was also dependent on the intensity of expression. Thus, for the first time we successfully used induction of decision trees for the analysis of high dimensional flow cytometric data and demonstrated the feasibility of this method to reveal structural patterns in such data sets.