This purpose of this investigation was to use DNA microarrays to study gene expression in E. coli O157:H7 under refrigerated and acidic conditions. Total RNA from E. coli O157:H7 grown to 7 log CFU/mL under control (37 °C, pH 7), refrigerated (10°C, pH 7), acid adapted (37°C, pH 5.5, then lowered to 3.5), and acid shocked (37°C, pH 7, then lowered to 3.5) conditions was extracted using an optimized Qiagen RNeasy procedure. Total RNA was converted to cDNA, labeled, and hybridized onto an Affymetrix GeneChip® E. coli Genome 2.0 Array. Results were analyzed using Analysis of Variance (ANOVA) and a t-test with significance set at p<0.01 and an expression cut-off of 2-fold difference in gene expression. Expression of selected genes, including an internal control (hemX) was confirmed using real time reverse transcriptase PCR.
Microarray results revealed 293 down-regulated and 375 up-regulated genes. Cold shock genes cspE, cspA, cspG, and cspH were down-regulated; recA and SOS DNA repair genes uvrB, yebG, ruvA and B, lexA, and dinl were up-regulated. Expression of fhuA (outer membrane protein) and napG (electron transport) genes was 115- and 70-fold greater under refrigeration. Microarray analysis revealed that genes involved in DNA binding, biosynthesis, iron, transport and cellular metabolism were significantly up-or down-regulated in acid adapted cells. Genes related to iron uptake and transport (cyo, ent, fhu, fep, and feo operons) were up-regulated under acid shocked conditions. For acid adapted conditions, expression of cfa (cyclopropane fatty acid biosynthesis; decreases membrane fluidity and increases acid resistance) and genes involved in the glutamate decarboxylase acid resistant systems were significantly up-regulated. The virulence genes eae, stx1, and stx2 were not differentially expressed under either condition.
| Identifer | oai:union.ndltd.org:UTENN/oai:trace.tennessee.edu:utk_graddiss-1180 |
| Date | 01 December 2007 |
| Creators | Carter, Kristina K. |
| Publisher | Trace: Tennessee Research and Creative Exchange |
| Source Sets | University of Tennessee Libraries |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations |
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