TABLE 2.

Validation of microarray data by qRT-PCR of randomly selected genes relative to the housekeeping gene rpoDa

LocusbNamePrimer sequencecFragment (bp)Serovar Typhimurium gene functiondRatio of fnr mutant/WTLog2 ratio
qRT-PCReMicroarrayfqRT-PCRgMicroarrayh
STM3217aer5′-CGTACAACATCTTAATCGTAGC-3′163Aerotaxis sensor receptor; senses cellular redox state or proton motive force0.1900.210−2.4−2.3
5′-TTCGTTCAGATCATTATTACCC-3′
STM1919cheM5′-GCCAATTTCAAAAATATGACG-3′114Methyl-accepting chemotaxis protein II; aspartate sensor-receptor0.0360.120−4.8−3.1
5′-GTCCAGAAACTGAATAAGTTCG-3′
STM0441cyoC5′-TATTTAGCTCCATTACCTACGG-3′134Cytochrome o ubiquinol oxidase subunit III153.9677.0967.32.8
5′-GGAATTCATAGAGTTCCATCC-3′
STM1803dadA5′-TAACCTTTCGCTTTAATACTCC-3′155d-Amino acid dehydrogenase subunit2.8353.1691.51.7
5′-GATATCAACAATGCCTTTAAGC-3′
STM0964dmsA5′-AGCGTCTTATCAAAGAGTATGG-3′154Anaerobic dimethyl sulfoxide reductase, subunit A0.0010.005−9.8−7.6
5′-TCACCGTAGTGATTAAGATAACC-3′
STM2892invJ5′-TTGCTATCGTCTAAAAATAGGC-3′128Surface presentation of antigens; secretory proteins0.2460.182−2.0−2.5
5′-TTGATATTATCGTCAGAGATTCC-3′
STM2324nuoF5′-GGATATCGAGACACTTGAGC-3′163NADH-dehydrogenase I, chain F2.8942.6001.51.4
5′-GATTAAATGGGTATTACTGAA CG-3′
STM0650STM06505′-CAACAGCTTATTGATTTAGTGG-3′130Putative hydrolase, C terminus0.4760.219−1.1−2.2
5′-CTAACGATTTTTCTTCAATGG-3′
STM2787STM27875′-AAGCGAATACAGCTATGAACC-3′144Tricarboxylic transport28.2416.8924.82.8
5′-ATTAGCTTTTGCAGAACATGG-3′
STM4463STM44635′-AAGGTATCAGCCAGTCTACG-3′142Putative arginine repressor0.3250.181−1.6−2.5
5′-CGTATGGATAAGGATAAATTCG-3′
STM4535STM45355′-TAAGCCAGCAGGTAGATACG-3′139Putative PTS permease6.0538.2172.63.0
5′-CGACATAAAGAGATCGATAACC-3′
STM2464eutN5′-AGGACAAATCGTATGTACCG-3′153Putative detox protein in ethanolamine utilization0.0620.125−4.0−3.0
5′-ACCAGCAGTACCCACTCTCC-3′
STM2454eutR5′-GGTAAAAGAGCAGCATAAAGC-3′118Putative regulator; ethanolamine operon (AraC/XylS family)0.0430.195−4.6−2.4
5′-ATTATCACTCAAGACCTTACGC-3′
STM2470eutS5′-AATAAAGAACGCATTATTCAGG-3′137Putative carboxysome structural protein; ethanol utilization0.0490.073−4.3−3.8
5′-GTTAAAGTCATAATGCCAATCG-3′
STM1172flgM5′-AGCGACATTAATATGGAACG-3′126Anti-FliA (anti-sigma) factor; also known as RflB protein0.0500.174−4.3−2.5
5′-TTTACTCTGTAAGTAGCTCTGC-3′
STM3692lldP5′-TGATTAAACTCAAGCTGAAAGG-3′189LctP transporter; l-lactate permease76.49216.0036.34.0
5′-CCGAAATTTTATAGACAAAGACC-3′
STM3693lldR5′-GAACAGAATATCGTGCAACC-3′153Putative transcriptional regulator for lct operon (GntR family)68.37830.5976.14.9
5′-GAGTCTGATTTTCTCTTTGTCG-3′
STM1923motA5′-GGTTATCGGTACAGTTTTCG-3′194Proton conductor component of motor; torque generator0.0480.092−4.4−3.4
5′-TAGATTTTGTGTATTTCGAACG-3′
STM4277nrfa5′-GACTAACTCTCTGTCGAAAACC-3′159Nitrite reductase; periplasmic cytochrome c5520.0510.324−4.3−1.6
5′-ATTTTATGGTCGGTGTAGAGC-3′
  • a STM3211 (rpoD) was used as the reference gene where no significant change in expression level was observed. The primer sequences used for rpoD were as follows: 5′-CGATGTCTCTGAAGAAGTGC-3′ (forward) and 5′-TTCAACCATCTCTTTCTTCG-3′ (reverse). The size of the fragment generated was 150 bp.

  • b Location of the ORF in the serovar Typhimurium LT2 genome.

  • c For each set, the first sequence is the forward primer, and the second sequence is the reverse primer.

  • d Functional classification according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.

  • e Expression levels based on qRT-PCR. Values are ratios between the fnr mutant and the WT, where values of <1 indicate that FNR acts as an activator and values of >1 indicate that FNR acts as a repressor.

  • f Expression levels based on microarray data. Values are ratios between the fnr mutant and the WT, where values of <1 indicate that FNR acts as an activator and values of >1 indicate that FNR acts as a repressor.

  • g Expression levels based on qRT-PCR, comparing the fnr mutant with the WT. Values are signal-to-log2 ratios.

  • h Expression levels based on microarray data, comparing the fnr mutant with the WT. Values are signal-to-log2 ratios.