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

Landstorfer R, Simon S, Schober S, Keim D, Scherer S, Neuhaus K - BMC Genomics (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. neuhaus@wzw.tum.de.

ABSTRACT

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 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 ( 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.

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Related in: MedlinePlus

Percentage of regulated hypothetical and known genes. The category of hypothetical genes includes all genes that are annotated as hypothetical protein (1771 genes). All other genes are included in the category of known genes (3608 genes). We consider a gene as regulated if its logFC is ≥ 3 or ≤ −1 in at least one condition. A gene is silent if the RPKMs in all conditions are below the threshold for random transcription. Consequently, silent genes are also not regulated.
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Fig2: Percentage of regulated hypothetical and known genes. The category of hypothetical genes includes all genes that are annotated as hypothetical protein (1771 genes). All other genes are included in the category of known genes (3608 genes). We consider a gene as regulated if its logFC is ≥ 3 or ≤ −1 in at least one condition. A gene is silent if the RPKMs in all conditions are below the threshold for random transcription. Consequently, silent genes are also not regulated.

Mentions: Filtering for transcriptionally inactive genes at any of the conditions studied, we found only 144 inactive genes which is about 2.7% of the annotated genes (Additional file 2: Table S3). These genes are covered by no read under any of the conditions investigated. 69.4% of the silent genes are hypothetical genes, indicating a potential over-annotation. On the other hand, some hypothetical genes might only be active at conditions not yet probed.We considered a gene as being regulated if its logFC was ≥ 3 or ≤ −1 under at least one condition. Accordingly, the number of regulated genes is about 4% higher for the known genes compared to the hypothetical genes (Figure 2).Figure 2


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

Landstorfer R, Simon S, Schober S, Keim D, Scherer S, Neuhaus K - BMC Genomics (2014)

Percentage of regulated hypothetical and known genes. The category of hypothetical genes includes all genes that are annotated as hypothetical protein (1771 genes). All other genes are included in the category of known genes (3608 genes). We consider a gene as regulated if its logFC is ≥ 3 or ≤ −1 in at least one condition. A gene is silent if the RPKMs in all conditions are below the threshold for random transcription. Consequently, silent genes are also not regulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048457&req=5

Fig2: Percentage of regulated hypothetical and known genes. The category of hypothetical genes includes all genes that are annotated as hypothetical protein (1771 genes). All other genes are included in the category of known genes (3608 genes). We consider a gene as regulated if its logFC is ≥ 3 or ≤ −1 in at least one condition. A gene is silent if the RPKMs in all conditions are below the threshold for random transcription. Consequently, silent genes are also not regulated.
Mentions: Filtering for transcriptionally inactive genes at any of the conditions studied, we found only 144 inactive genes which is about 2.7% of the annotated genes (Additional file 2: Table S3). These genes are covered by no read under any of the conditions investigated. 69.4% of the silent genes are hypothetical genes, indicating a potential over-annotation. On the other hand, some hypothetical genes might only be active at conditions not yet probed.We considered a gene as being regulated if its logFC was ≥ 3 or ≤ −1 under at least one condition. Accordingly, the number of regulated genes is about 4% higher for the known genes compared to the hypothetical genes (Figure 2).Figure 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. neuhaus@wzw.tum.de.

ABSTRACT

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 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 ( 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.

Show MeSH
Related in: MedlinePlus