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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

Visualization of the sequencing reads (= transcription) using the NGS overlap searcher [93]. The tool shows a plot of the read coverage in the middle. Forward strand reads are plotted above, reverse strand reads below the center line. The read starts are indicated in yellow. The bars above (forward strand) and below (reverse strand) the middle bar show all ORFs ≥ 90 nt in the six different reading frames. Annotated ORFs are in red. The tool shows the coverage also in the ORF bars according to the scaling in the lower right corner. A: selection of an “empty” region of the genome on the forward strand (4144776 – 4149762). The coverage shown is a sum signal of all eleven conditions sequenced on the SOLiD system. Only eight reads are found on the forward strand of this region. B1-B3: example for a regulated hypothetical gene, Z1023 (see Table 2), in LB medium (B1), minimal medium (B2), and on radish sprouts (B3). C1-C3: regulated hypothetical gene Z1027 (see Table 2), in LB medium (C1), minimal medium (C2), and on radish sprouts (C3). D1-D3: regulated hypothetical gene Z4396 (Table 2), in LB medium (D1), minimal medium (D2), and on radish sprouts (D3). E1-E3: regulated hypothetical gene Z5808 (see Table 2), in LB medium (E1), minimal medium (E2), and on radish sprouts (E3). The color map values range from 0.1 to 3 × 105, the exact expression values for each gene are listed in Table 2.
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Fig6: Visualization of the sequencing reads (= transcription) using the NGS overlap searcher [93]. The tool shows a plot of the read coverage in the middle. Forward strand reads are plotted above, reverse strand reads below the center line. The read starts are indicated in yellow. The bars above (forward strand) and below (reverse strand) the middle bar show all ORFs ≥ 90 nt in the six different reading frames. Annotated ORFs are in red. The tool shows the coverage also in the ORF bars according to the scaling in the lower right corner. A: selection of an “empty” region of the genome on the forward strand (4144776 – 4149762). The coverage shown is a sum signal of all eleven conditions sequenced on the SOLiD system. Only eight reads are found on the forward strand of this region. B1-B3: example for a regulated hypothetical gene, Z1023 (see Table 2), in LB medium (B1), minimal medium (B2), and on radish sprouts (B3). C1-C3: regulated hypothetical gene Z1027 (see Table 2), in LB medium (C1), minimal medium (C2), and on radish sprouts (C3). D1-D3: regulated hypothetical gene Z4396 (Table 2), in LB medium (D1), minimal medium (D2), and on radish sprouts (D3). E1-E3: regulated hypothetical gene Z5808 (see Table 2), in LB medium (E1), minimal medium (E2), and on radish sprouts (E3). The color map values range from 0.1 to 3 × 105, the exact expression values for each gene are listed in Table 2.

Mentions: Growth curve of EHEC on radish sprouts and expression ofazoR. A: growth on radish sprouts within 8 days. The sprouts were inoculated with 4 × 102 cfu/g plant and harvested after 5 days during late exponential/early stationary phase (marked with arrow). B: expression of azoR (Z2315) in LB-pH9 (RPKM = 6, color map maximum value of 1 × 106); for legend, see Figure 6. C: expression of azoR in radish sprouts (shown is the SOLiD replicate), azoR is highly covered with reads (RPKM = 190, color map maximum value of 3 × 105); for legend, see Figure 6.


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)

Visualization of the sequencing reads (= transcription) using the NGS overlap searcher [93]. The tool shows a plot of the read coverage in the middle. Forward strand reads are plotted above, reverse strand reads below the center line. The read starts are indicated in yellow. The bars above (forward strand) and below (reverse strand) the middle bar show all ORFs ≥ 90 nt in the six different reading frames. Annotated ORFs are in red. The tool shows the coverage also in the ORF bars according to the scaling in the lower right corner. A: selection of an “empty” region of the genome on the forward strand (4144776 – 4149762). The coverage shown is a sum signal of all eleven conditions sequenced on the SOLiD system. Only eight reads are found on the forward strand of this region. B1-B3: example for a regulated hypothetical gene, Z1023 (see Table 2), in LB medium (B1), minimal medium (B2), and on radish sprouts (B3). C1-C3: regulated hypothetical gene Z1027 (see Table 2), in LB medium (C1), minimal medium (C2), and on radish sprouts (C3). D1-D3: regulated hypothetical gene Z4396 (Table 2), in LB medium (D1), minimal medium (D2), and on radish sprouts (D3). E1-E3: regulated hypothetical gene Z5808 (see Table 2), in LB medium (E1), minimal medium (E2), and on radish sprouts (E3). The color map values range from 0.1 to 3 × 105, the exact expression values for each gene are listed in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Visualization of the sequencing reads (= transcription) using the NGS overlap searcher [93]. The tool shows a plot of the read coverage in the middle. Forward strand reads are plotted above, reverse strand reads below the center line. The read starts are indicated in yellow. The bars above (forward strand) and below (reverse strand) the middle bar show all ORFs ≥ 90 nt in the six different reading frames. Annotated ORFs are in red. The tool shows the coverage also in the ORF bars according to the scaling in the lower right corner. A: selection of an “empty” region of the genome on the forward strand (4144776 – 4149762). The coverage shown is a sum signal of all eleven conditions sequenced on the SOLiD system. Only eight reads are found on the forward strand of this region. B1-B3: example for a regulated hypothetical gene, Z1023 (see Table 2), in LB medium (B1), minimal medium (B2), and on radish sprouts (B3). C1-C3: regulated hypothetical gene Z1027 (see Table 2), in LB medium (C1), minimal medium (C2), and on radish sprouts (C3). D1-D3: regulated hypothetical gene Z4396 (Table 2), in LB medium (D1), minimal medium (D2), and on radish sprouts (D3). E1-E3: regulated hypothetical gene Z5808 (see Table 2), in LB medium (E1), minimal medium (E2), and on radish sprouts (E3). The color map values range from 0.1 to 3 × 105, the exact expression values for each gene are listed in Table 2.
Mentions: Growth curve of EHEC on radish sprouts and expression ofazoR. A: growth on radish sprouts within 8 days. The sprouts were inoculated with 4 × 102 cfu/g plant and harvested after 5 days during late exponential/early stationary phase (marked with arrow). B: expression of azoR (Z2315) in LB-pH9 (RPKM = 6, color map maximum value of 1 × 106); for legend, see Figure 6. C: expression of azoR in radish sprouts (shown is the SOLiD replicate), azoR is highly covered with reads (RPKM = 190, color map maximum value of 3 × 105); for legend, see Figure 6.

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