Date of Award
Doctor of Philosophy
Food Science and Technology
David A. Golden
Michael Davidson, Doris D’Souza, Todd Reynolds, Arnold Saxton
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.
Carter, Kristina K., "Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays. " PhD diss., University of Tennessee, 2007.