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Whole-Transcriptome Analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

Crozier L, Hedley PE, Morris J, Wagstaff C, Andrews SC, Toth I, Jackson RW, Holden NJ - Front Microbiol (2016)

Bottom Line: Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity.Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling.A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Sciences, The James Hutton Institute Dundee, UK.

ABSTRACT
Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonization. As such, we compared the ability of the VTEC strain, E. coli O157:H7 'Sakai,' to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analyzed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonization. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 h) transcriptional response to extracts as well as longer-term (10 days) plant colonization or persistence. We show that propagation temperature (37 vs. 18°C) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilization. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling. A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

No MeSH data available.


Related in: MedlinePlus

Growth analysis for E. coli O157:H7 (Sakai) in spinach plant extracts. Growth was quantified from cell density of bacteria inoculated into M9 medium supplemented with plant extracts and/or glycerol. Measurements were discontinued for the leaf lysate condition after 48 h, as growth was complete. Data represents the average of nine replicates and was analyzed by one-way ANOVA at selected time points.
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Figure 6: Growth analysis for E. coli O157:H7 (Sakai) in spinach plant extracts. Growth was quantified from cell density of bacteria inoculated into M9 medium supplemented with plant extracts and/or glycerol. Measurements were discontinued for the leaf lysate condition after 48 h, as growth was complete. Data represents the average of nine replicates and was analyzed by one-way ANOVA at selected time points.

Mentions: To determine the extent to which the global-gene-expression changes reflect the colonization potential of the bacteria in different plant tissue extracts, the ability of the plant tissue extracts to support in vitro growth was assessed. For these assays, minimal M9 medium was used as a basal medium (without carbon source) supplemented with spinach leaf lysate or root exudates (normalized on the basis of protein content), or with 0.2% glycerol as a ‘no-plant’ control. Bacterial growth could not be assessed in medium containing the (insoluble) CWPS extract and as such, is not considered here. E. coli O157:H7 (Sakai) grew well in medium supplemented with spinach leaf lysate at 18°C, reaching an OD600 of 0.7 at 48 h, which was just-under 50% of that (1.7) achieved in M9 medium plus 0.2% glycerol (Figure 6). In addition, growth with the leaf lysate exhibited a very short lag phase, unlike that with glycerol where a ∼24 h delay in rapid growth was observed. This suggests that the bacteria acclimatized more rapidly to the medium with leaf extract than that with glycerol. In contrast, no growth was evident with spinach root exudate suggesting that carbon was at least one of the limiting energy sources. Indeed, when the root exudate and glycerol were used in combination strong growth was obtained that was similar to that with glycerol alone, suggesting that the weak growth in spinach root exudates was not due to the presence of factors that supress growth (Figure 6). No significant difference was found between the growth of E. coli O157:H7 (Sakai) in the glycerol only media compared to the glycerol plus root exudates media. To test whether the root exudate was deficient in suitable carbon sources, the composition of mono- and disaccharides in the extracts was examined by HPLC. The analysis showed ∼200-fold less glucose, fructose, and sucrose in the root exudate compared to the leaf lysate, supporting the suggestion that the root exudate provides limited levels of carbohydrate (Table 2). Although root exudates were collected from plants grown under aseptic hydroponics conditions, and germinated from surface-sterilized seeds, it was apparent that there were native bacteria associated with the spinach plants. Cultivable bacteria were tentatively identified as Pseudomonas azotoformans (with 99.90% nt identity) and Pantoea agglomerans (99.95% nt identity). In our hands, the contaminating bacteria were repeatedly associated with spinach grown under these conditions indicating that they were seed-borne.


Whole-Transcriptome Analysis of Verocytotoxigenic Escherichia coli O157:H7 (Sakai) Suggests Plant-Species-Specific Metabolic Responses on Exposure to Spinach and Lettuce Extracts.

Crozier L, Hedley PE, Morris J, Wagstaff C, Andrews SC, Toth I, Jackson RW, Holden NJ - Front Microbiol (2016)

Growth analysis for E. coli O157:H7 (Sakai) in spinach plant extracts. Growth was quantified from cell density of bacteria inoculated into M9 medium supplemented with plant extracts and/or glycerol. Measurements were discontinued for the leaf lysate condition after 48 h, as growth was complete. Data represents the average of nine replicates and was analyzed by one-way ANOVA at selected time points.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940412&req=5

Figure 6: Growth analysis for E. coli O157:H7 (Sakai) in spinach plant extracts. Growth was quantified from cell density of bacteria inoculated into M9 medium supplemented with plant extracts and/or glycerol. Measurements were discontinued for the leaf lysate condition after 48 h, as growth was complete. Data represents the average of nine replicates and was analyzed by one-way ANOVA at selected time points.
Mentions: To determine the extent to which the global-gene-expression changes reflect the colonization potential of the bacteria in different plant tissue extracts, the ability of the plant tissue extracts to support in vitro growth was assessed. For these assays, minimal M9 medium was used as a basal medium (without carbon source) supplemented with spinach leaf lysate or root exudates (normalized on the basis of protein content), or with 0.2% glycerol as a ‘no-plant’ control. Bacterial growth could not be assessed in medium containing the (insoluble) CWPS extract and as such, is not considered here. E. coli O157:H7 (Sakai) grew well in medium supplemented with spinach leaf lysate at 18°C, reaching an OD600 of 0.7 at 48 h, which was just-under 50% of that (1.7) achieved in M9 medium plus 0.2% glycerol (Figure 6). In addition, growth with the leaf lysate exhibited a very short lag phase, unlike that with glycerol where a ∼24 h delay in rapid growth was observed. This suggests that the bacteria acclimatized more rapidly to the medium with leaf extract than that with glycerol. In contrast, no growth was evident with spinach root exudate suggesting that carbon was at least one of the limiting energy sources. Indeed, when the root exudate and glycerol were used in combination strong growth was obtained that was similar to that with glycerol alone, suggesting that the weak growth in spinach root exudates was not due to the presence of factors that supress growth (Figure 6). No significant difference was found between the growth of E. coli O157:H7 (Sakai) in the glycerol only media compared to the glycerol plus root exudates media. To test whether the root exudate was deficient in suitable carbon sources, the composition of mono- and disaccharides in the extracts was examined by HPLC. The analysis showed ∼200-fold less glucose, fructose, and sucrose in the root exudate compared to the leaf lysate, supporting the suggestion that the root exudate provides limited levels of carbohydrate (Table 2). Although root exudates were collected from plants grown under aseptic hydroponics conditions, and germinated from surface-sterilized seeds, it was apparent that there were native bacteria associated with the spinach plants. Cultivable bacteria were tentatively identified as Pseudomonas azotoformans (with 99.90% nt identity) and Pantoea agglomerans (99.95% nt identity). In our hands, the contaminating bacteria were repeatedly associated with spinach grown under these conditions indicating that they were seed-borne.

Bottom Line: Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity.Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling.A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Sciences, The James Hutton Institute Dundee, UK.

ABSTRACT
Verocytotoxigenic Escherichia coli (VTEC) can contaminate crop plants, potentially using them as secondary hosts, which can lead to food-borne infection. Currently, little is known about the influence of the specific plant species on the success of bacterial colonization. As such, we compared the ability of the VTEC strain, E. coli O157:H7 'Sakai,' to colonize the roots and leaves of four leafy vegetables: spinach (Spinacia oleracea), lettuce (Lactuca sativa), vining green pea (Pisum sativum), and prickly lettuce (Lactuca serriola), a wild relative of domesticated lettuce. Also, to determine the drivers of the initial response on interaction with plant tissue, the whole transcriptome of E. coli O157:H7 Sakai was analyzed following exposure to plant extracts of varying complexity (spinach leaf lysates or root exudates, and leaf cell wall polysaccharides from spinach or lettuce). Plant extracts were used to reduce heterogeneity inherent in plant-microbe interactions and remove the effect of plant immunity. This dual approach provided information on the initial adaptive response of E. coli O157:H7 Sakai to the plant environment together with the influence of the living plant during bacterial establishment and colonization. Results showed that both the plant tissue type and the plant species strongly influence the short-term (1 h) transcriptional response to extracts as well as longer-term (10 days) plant colonization or persistence. We show that propagation temperature (37 vs. 18°C) has a major impact on the expression profile and therefore pre-adaptation of bacteria to a plant-relevant temperature is necessary to avoid misleading temperature-dependent wholescale gene-expression changes in response to plant material. For each of the plant extracts tested, the largest group of (annotated) differentially regulated genes were associated with metabolism. However, large-scale differences in the metabolic and biosynthetic pathways between treatment types indicate specificity in substrate utilization. Induction of stress-response genes reflected the apparent physiological status of the bacterial genes in each extract, as a result of glutamate-dependent acid resistance, nutrient stress, or translational stalling. A large proportion of differentially regulated genes are uncharacterized (annotated as hypothetical), which could indicate yet to be described functional roles associated with plant interaction for E. coli O157:H7 Sakai.

No MeSH data available.


Related in: MedlinePlus