Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

<p>Abstract</p> <p>Background:</p> <p>Global patterns of gene expression of <it>Escherichia coli </it>K-12 during growth transitions have been deeply investigated, however, comparable studies of <it>E. coli </it>O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR.</p> <p>Results:</p> <p>Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (<it>n </it>= 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as <it>tccP </it>and <it>espJ</it>, also decreased in expression from exponential to stationary phase. Three of the clusters (<it>n </it>= 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (<it>n </it>= 923) which includes genes involved in stress responses (e.g. <it>gadAB</it>, <it>osmBC</it>, and <it>dps</it>). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (<it>stx</it>1AB and <it>stx</it>2B) were significantly induced after transition into stationary phase.</p> <p>Conclusion:</p> <p>Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions.</p

Increased Adherence and Expression of Virulence Genes in a Lineage of Escherichia coli O157:H7 Commonly Associated with Human Infections

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a food and waterborne pathogen, can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping. One lineage (clade 8) was found to be associated with hemolytic uremic syndrome (HUS), which can lead to kidney failure and death in some cases, particularly young children. Another lineage (clade 2) differs considerably in gene content and is phylogenetically distinct from clade 8, but caused significantly fewer cases of HUS in a prior study. Little is known, however, about how these two lineages vary with regard to phenotypic traits important for disease pathogenesis and in the expression of shared virulence genes.Here, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157:H7 strains with varying Shiga toxin profiles from two common lineages. Adherence to epithelial cells was >2-fold higher for EHEC O157:H7 strains belonging to clade 8 versus clade 2, while no difference in invasiveness was observed between the two lineages. Whole-genome 70-mer oligo microarrays, which probe for 6088 genes from O157:H7 Sakai, O157:H7 EDL 933, pO157, and K12 MG1655, detected significant differential expression between clades in 604 genes following co-incubation with epithelial cells for 30 min; 186 of the 604 genes had a >1.5 fold change difference. Relative to clade 2, clade 8 strains showed upregulation of major virulence genes, including 29 of the 41 locus of enterocyte effacement (LEE) pathogenicity island genes, which are critical for adherence, as well as Shiga toxin genes and pO157 plasmid-encoded virulence genes. Differences in expression of 16 genes that encode colonization factors, toxins, and regulators were confirmed by qRT-PCR, which revealed a greater magnitude of change than microarrays.These findings demonstrate that the EHEC O157:H7 lineage associated with HUS expresses higher levels of virulence genes and has an enhanced ability to attach to epithelial cells relative to another common lineage

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-3

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p>ited MOPS minimal medium and plotted with the error bars indicating the standard deviation

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-4

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p> dark red representing expression = 1.8 and dark green representing expression = -1.8 on a log2 scale. ORFs marked with an asterisk denotes that a statistically significant change in expression levels of that ORF was detected for at least one time point

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-5

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p>island as determined by Q-PCR. . Expression differences of 3 genes encoding effectors (Esp) molecules transported through the LEE Type III Secretion System (TTSS) as determined by Q-PCR results. Average changes in expression for 2 independent replicate cultures were determined relative to the transcript level of each ORF at the 3 h mid-exponential phase time point

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-7

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p>e changes in expression for 2 independent replicate cultures were determined relative to the transcript level of each ORF at the 3 h mid-exponential phase time point

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-0

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p> phase. Error bars represent the standard deviation of four culture replicates. Samples were taken from the culture at the time points plotted and RNA was extracted. The 5 growth phases, separated by dotted lines, are defined as exponential growth, late exponential growth, transition to stationary phase, early stationary phase, and stationary phase

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-6

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p> dark red representing expression = 3 and dark green representing expression = -4 on a log2 scale. Over half of the AFI genes (7/12) had a significant increase in expression from mid- to late- exponential phas

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-1

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p>ns, representing four biological replicates: . Average signal intensity for exponential phase (3 h) vs late exponential phase (4 h). . Average signal intensity for exponential phase (3 h) vs late exponential phase (4.6 h). . Average signal intensity for exponential phase (3 h) vs transition to stationary phase (5 h). . Average signal intensity for exponential phase (3 h) vs. stationary phase (6 h. The Lowess line is plotted for each graph in red

Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium-2

<p><b>Copyright information:</b></p><p>Taken from "Global transcriptional response of O157:H7 to growth transitions in glucose minimal medium"</p><p>http://www.biomedcentral.com/1471-2180/7/97</p><p>BMC Microbiology 2007;7():97-97.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2241611.</p><p></p>ofiles were determined for the ORFs in each cluster and plotted with the standard deviation for each time point