Abstract
BackgroundMultiple 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.ResultsTranscriptomes 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.ConclusionsSince 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-353) contains supplementary material, which is available to authorized users.
Highlights
Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables
Treatment of an EHEC infection with antibiotics is under debate since this can increase the risk for the hemolytic uremic syndrome [3]
Transcriptional activity of hypothetical genes We examined the transcriptional regulation of the 5379 protein-coding genes (GenBank and RefSeq) for the genome and plasmid in EHEC (Additional file 3: Table S4)
Summary
Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables. Most of such reads apparently do not form transcriptional units, i.e. they do not originate from nonannotated genes for most cases It is unclear whether the reads occur due to background noise introduced during deep sequencing experiments or whether they are caused by the low information content of bacterial promoters, resulting in “sloppy” transcription [34,35]. To see whether such reads mapped by chance to the EHEC genome, the RNA-seq data of LB medium in this study was mapped to the mouse Y-chromosome (95 Mbp). Out of 7 million reads, only one matched to the mouse genome sequence, all reads appear to be specific
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