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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

Heat map representing 2026 regulated genes. Only those genes are displayed that are covered by reads in all of the conditions sequenced on the SOLiD system. Genes are clustered for similar regulation patterns among the conditions (vertical columns). Each horizontal column represents a different condition. On the right a similarity tree for the conditions is shown. The heat map was calculated on the relative differences (logFCs) in transcription levels to the reference condition LB. Transcription values higher than in LB are shown in shades of blue, transcription values lower are shown in shades of red. LB, lysogeny broth; pH9, LB-pH9; nitrite, LB-nitrite; spinach, spinach medium; radish, radish shoots; MM, minimal medium; antib, LB-antibiotics; solid, LB-solid; pH4, LB-pH4; faeces, cattle feces; 15°C, LB-15°C.
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Fig4: Heat map representing 2026 regulated genes. Only those genes are displayed that are covered by reads in all of the conditions sequenced on the SOLiD system. Genes are clustered for similar regulation patterns among the conditions (vertical columns). Each horizontal column represents a different condition. On the right a similarity tree for the conditions is shown. The heat map was calculated on the relative differences (logFCs) in transcription levels to the reference condition LB. Transcription values higher than in LB are shown in shades of blue, transcription values lower are shown in shades of red. LB, lysogeny broth; pH9, LB-pH9; nitrite, LB-nitrite; spinach, spinach medium; radish, radish shoots; MM, minimal medium; antib, LB-antibiotics; solid, LB-solid; pH4, LB-pH4; faeces, cattle feces; 15°C, LB-15°C.

Mentions: With only 2892 active genes, LB-antibiotics has the lowest number of active genes of all. In comparison, the reference condition LB displays around 4500 active genes (Table 1). Admittedly, LB-antibiotics has the lowest sequencing depth of all. However, as can be seen from Figure 3, the number of active genes is disproportionately low. After antibiotic treatment the cells elongate several times their original cell length. The indirect block of DNA synthesis influences their regulational pattern. Genes of many different pathways are turned off. We visualized this transcriptional pattern of LB-antibiotics in the heat map distance tree (Figure 4). The up-regulated genes (colored in blue) and down-regulated genes (colored in red) do not form the regulatory clusters observed in the other ten conditions: LB-antibiotics forms an outer group (antib in Figure 4). The extreme stress leads to most severe transcriptomic differences. Interestingly, it is the only LB-condition not clustering together with the other LB-based experiments. The four conditions that do not originate from LB medium, i.e. spinach medium, minimal medium, and feces, show a more related regulational pattern. Radish sprouts are closer to the conditions which originated from LB medium. We assume the high similarity of minimal medium and spinach medium as being due to a low nutrient content in both conditions. LB-pH9 and LB-nitrite have the most similar transcriptomic pattern, despite LB-nitrite being slightly acidic (Figure 4).Figure 4


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)

Heat map representing 2026 regulated genes. Only those genes are displayed that are covered by reads in all of the conditions sequenced on the SOLiD system. Genes are clustered for similar regulation patterns among the conditions (vertical columns). Each horizontal column represents a different condition. On the right a similarity tree for the conditions is shown. The heat map was calculated on the relative differences (logFCs) in transcription levels to the reference condition LB. Transcription values higher than in LB are shown in shades of blue, transcription values lower are shown in shades of red. LB, lysogeny broth; pH9, LB-pH9; nitrite, LB-nitrite; spinach, spinach medium; radish, radish shoots; MM, minimal medium; antib, LB-antibiotics; solid, LB-solid; pH4, LB-pH4; faeces, cattle feces; 15°C, LB-15°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Heat map representing 2026 regulated genes. Only those genes are displayed that are covered by reads in all of the conditions sequenced on the SOLiD system. Genes are clustered for similar regulation patterns among the conditions (vertical columns). Each horizontal column represents a different condition. On the right a similarity tree for the conditions is shown. The heat map was calculated on the relative differences (logFCs) in transcription levels to the reference condition LB. Transcription values higher than in LB are shown in shades of blue, transcription values lower are shown in shades of red. LB, lysogeny broth; pH9, LB-pH9; nitrite, LB-nitrite; spinach, spinach medium; radish, radish shoots; MM, minimal medium; antib, LB-antibiotics; solid, LB-solid; pH4, LB-pH4; faeces, cattle feces; 15°C, LB-15°C.
Mentions: With only 2892 active genes, LB-antibiotics has the lowest number of active genes of all. In comparison, the reference condition LB displays around 4500 active genes (Table 1). Admittedly, LB-antibiotics has the lowest sequencing depth of all. However, as can be seen from Figure 3, the number of active genes is disproportionately low. After antibiotic treatment the cells elongate several times their original cell length. The indirect block of DNA synthesis influences their regulational pattern. Genes of many different pathways are turned off. We visualized this transcriptional pattern of LB-antibiotics in the heat map distance tree (Figure 4). The up-regulated genes (colored in blue) and down-regulated genes (colored in red) do not form the regulatory clusters observed in the other ten conditions: LB-antibiotics forms an outer group (antib in Figure 4). The extreme stress leads to most severe transcriptomic differences. Interestingly, it is the only LB-condition not clustering together with the other LB-based experiments. The four conditions that do not originate from LB medium, i.e. spinach medium, minimal medium, and feces, show a more related regulational pattern. Radish sprouts are closer to the conditions which originated from LB medium. We assume the high similarity of minimal medium and spinach medium as being due to a low nutrient content in both conditions. LB-pH9 and LB-nitrite have the most similar transcriptomic pattern, despite LB-nitrite being slightly acidic (Figure 4).Figure 4

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