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

Abstract

Background: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli 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.

Results: 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 (n = 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 approximately 4-fold. Most ORFs of the LEE pathogenicity island also decreased approximately 2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n = 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 (n = 923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (stx1AB and stx2B) were significantly induced after transition into stationary phase.

Conclusion: 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.

Figure 1

Average growth of E. coli O157:H7 Sakai in MOPS minimal medium. Increase in cell density is measured at OD₆₀₀ at 10 time periods (hr) of growth into stationary 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.

Figure 2

Average signal intensity plots for four of the time points of growth. The normalized signals of 4,886 ORFs for E. coli O157:H7 Sakai were averaged from four hybridizations, representing four biological replicates: A. Average signal intensity for exponential phase (3 h) vs late exponential phase (4 h). B. Average signal intensity for exponential phase (3 h) vs late exponential phase (4.6 h). C. Average signal intensity for exponential phase (3 h) vs transition to stationary phase (5 h). D. Average signal intensity for exponential phase (3 h) vs. stationary phase (6 h. The Lowess line is plotted for each graph in red.

Figure 3

Quality Threshold (QT) clusters of the 2,552 ORFs with significant changes in gene expression. The number of ORFs in each cluster is listed at the top of each plot. Average expression profiles were determined for the ORFs in each cluster and plotted with the standard deviation for each time point.

Figure 4

Residual dissolved oxygen (O₂) in MOPS minimal medium. The mean rDOT was determined for three independent cultures of E. coli O157:H7 Sakai in glucose limited MOPS minimal medium and plotted with the error bars indicating the standard deviation.

Figure 5

Heatmap of gene expression over time for the ORFs of the LEE Pathogenecity Island. Expression values determined from the ANOVA analysis are represented colorimetrically, with 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.

Figure 6

Relative changes in expression for virulence-associated genes from the LEE Pathogenecity Island. A. Expression differences of 5 genes transported through or adherence components of the LEE island as determined by Q-PCR. B. 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.

Figure 7

Expression heat map of 12 genes from the acid fitness region (AFI). Expression values determined from the ANOVA analysis are represented colorimetrically, with 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 phase

Figure 8

Relative changes in expression of Shiga toxin genes. Changes in expression of the phage-encoded toxin genes stx1A, stx2A, and stx2B as determined by Q-PCR. 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.

Figure 9

Expression profiles of 12 ORFs encoded on the tellurite resistance island (TAI). Expression values determined from the ANOVA analysis are plotted over time for the A. tellurite resistance and B. the urease-encoding ORFs. C. Average relative expression of terZ and ureD was determined by Q-PCR of two independent replicate cultures.