Project description:Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies a combination of microarray analyses and microbial genetics were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli K12 on lettuce roots using a hydroponic assay system. Here we report that after three days of interaction with lettuce roots, 193 and 131 genes were significantly up-regulated and down-regulated at least 1.5 fold, respectively. Forty-five out of the 193 up-regulated genes (23%) were involved in protein synthesis and were highly induced. Genes involved in stress response, attachment and biofilm formation were up-regulated in E. coli when they interacted with lettuce roots under conditions of hydroponic growth. In particular crl, a gene regulating the cryptic csgA gene for curli production, was significantly up regulated. The crl, csgA and fliN mutants had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy. Our microarray data showed that E. coli K12 increased the synthesis of proteins indicated that a dramatic change was induced in the physiology of the microorganism. This study indicates that E. coli K12 can efficiently colonize lettuce roots by using attachment and biofilm modulation genes and can readily adapt to the rhizosphere of lettuce plants. Further studies are needed to better characterize this interaction in pathogenic strains of this species. Escherichia coli MG1655 strains were grown in the lettuce rhizosphere for three days. Transcriptional profiling of E. coli was compared between cells grown with and without rhizosphere . Three biological replicates of each treatment were prepared, and six microarray slides were used.

Project description:Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies, a combination of microarray analyses and surface enhanced Raman spectroscopy (SERS) were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots and compared to E. coli K12 using a hydroponic system (HS) which we have reported in the previous studies. Using microarray, after three days of interaction with lettuce roots, 94 and 109 genes of E. coli O157:H7 were significantly up-regulated and down-regulated at least 1.5 fold, respectively. Only 8 genes were also found in the E. coli K12 up-regulated genes. No genes were found in the down-regulated genes clusters between those two strains. For E. coli O157:H7, forty out of the 94 up-regulated genes (43%) were involved in protein synthesis and were highly repressed compared to 40 out of 193 (23%) E. coli K12 up-regulated genes associated with protein synthesis. The wildtype of E.coli O157:H7 colonized two log CFU per root less compared to E. coli K12. Genes involved in biofilm modulation (bhsA and ybiM) were significantly up-regulated in E. coli O157:H7 and curli production (crl and csgA) were found important for E. coli K12 to attach to lettuce roots in the previous studies. BhsA mutant of E. coli O157:H7 was impaired in the colonization of lettuce roots. The SERS spectra of E. coli K12 and O157 controls (cells without interacting with roots) were very similar. The spectra of E. coli K12 and O157 exposed to the hydroponic system (HS) showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited significant differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells were also found much higher than those of E. coli O157 HS cells. These findings were in agreement to our Microarray data. Our microarray and SERS data showed that E. coli K12 and O157:H7 behavior dramatically differently in colonizing on lettuce roots. Compared to K12, E. coli O157:H7 colonized less efficiently on lettuce roots. Escherichia coli O157:H7 strains were grown in the lettuce rhizosphere for three days. Transcriptional profiling of E. coli was compared between cells grown with and without rhizosphere . Three biological replicates of each treatment were prepared, and six microarray slides were used.

Project description:affy_root-dvt-nitrogen_medicago. The biological question is the study of root development adaptation to external nitrogen availability. One of the typical responses to low N provision consists of an increased root branching which promotes soil exploration. In order to differentiate between genes specifically involved in nitrate response and genes of root development, we had performed experiments on both a wild type and a mutant affected in root architecture, both supplied with two different levels of nitrate. Global gene expression profiling of root cells using microarray analysis will be conducted to identify genes expressed during root branching. First, analysing the differential gene expression between the wild type and the mutant both at low and high level of nitrate, we will identify genes of root development but also genes specifically involved in nitrate response. Then, additional comparisons will concern the modifications of wild type and mutant gene expression between the two levels of nitrate. These genes will be compared to those found to be expressed in the wild type roots under submitted to various nitrogen sources (Ruffel et al., 2008). All together these results will allow us to determine which genes, among the firstly underlined, are specifically involved in root development. The kinetics of expression in different tissues of the most relevant of these genes will be further analysed by qRT-PCR. Three successive experiments in growth chamber were performed on two different genotypes of Medicago truncatula. Each experiment constituted a biological replicate. For each of them, after 4 days of seed cold-treatment followed by 4 days of germination, individual plantlets were transferred onto hydroponic culture tanks containing vigorously aerated nutrient solution. Each tank contained 6 plantlets of J5 and 6 plantlets of the mutant. On one shelf of the growth chamber, nutrient solution in the tanks was supplemented by 1mM of KNO3; on the other shelf, the level of KNO3 in the solution was of 10mM. Nutrient solution was renewed every week. Roots of the two genotypes were harvested separately but at the same time, 10 days after the plantlets transfer onto hydroponic culture tanks. Experiment Overall Design: 12 arrays - Medicago. Experiment Overall Design: WT and mutant (tr185) under 1mM or 10mM KNO3. Three biological replicates each genotype/condition.

Project description:affy_root-dvt-nitrogen_medicago. The biological question is the study of root development adaptation to external nitrogen availability. One of the typical responses to low N provision consists of an increased root branching which promotes soil exploration. In order to differentiate between genes specifically involved in nitrate response and genes of root development, we had performed experiments on both a wild type and a mutant affected in root architecture, both supplied with two different levels of nitrate. Global gene expression profiling of root cells using microarray analysis will be conducted to identify genes expressed during root branching. First, analysing the differential gene expression between the wild type and the mutant both at low and high level of nitrate, we will identify genes of root development but also genes specifically involved in nitrate response. Then, additional comparisons will concern the modifications of wild type and mutant gene expression between the two levels of nitrate. These genes will be compared to those found to be expressed in the wild type roots under submitted to various nitrogen sources (Ruffel et al., 2008). All together these results will allow us to determine which genes, among the firstly underlined, are specifically involved in root development. The kinetics of expression in different tissues of the most relevant of these genes will be further analysed by qRT-PCR. Three successive experiments in growth chamber were performed on two different genotypes of Medicago truncatula. Each experiment constituted a biological replicate. For each of them, after 4 days of seed cold-treatment followed by 4 days of germination, individual plantlets were transferred onto hydroponic culture tanks containing vigorously aerated nutrient solution. Each tank contained 6 plantlets of J5 and 6 plantlets of the mutant. On one shelf of the growth chamber, nutrient solution in the tanks was supplemented by 1mM of KNO3; on the other shelf, the level of KNO3 in the solution was of 10mM. Nutrient solution was renewed every week. Roots of the two genotypes were harvested separately but at the same time, 10 days after the plantlets transfer onto hydroponic culture tanks. Keywords: dose response, wt vs mutant comparison

			Action	DRS
	SRR29428917_1.fastq.gz	Fastqsanger.gz
	SRR29428917_2.fastq.gz	Fastqsanger.gz
	SRR29428918_1.fastq.gz	Fastqsanger.gz
	SRR29428918_2.fastq.gz	Fastqsanger.gz
	SRR29428919_1.fastq.gz	Fastqsanger.gz

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