Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress

J Microbiol Methods. 2009 Feb;76(2):196-200. doi: 10.1016/j.mimet.2008.10.014. Epub 2008 Nov 5.

Abstract

Campylobacter jejuni represents one of the major causes of bacterial enteritis caused by food in humans. There are still mechanisms to be deciphered to better understand better its physiology and pathogenesis. Study of gene expression levels by RT-qPCR could be used, but to be accurate and reproducible, a good internal control has to be chosen. The aim of this study was to identify a highly stable housekeeping gene in Campylobacter jejuni that could constitute a good internal control to study gene expression variations between different growth phases or stress conditions. Expression levels of six different housekeeping genes (gyrA, ilvC, rpoA, slyD, thiC and rrs) were measured by RT-qPCR under different conditions (exponential phase, stationary phase, cold shock, cold shock+oxidative stress, oxidative stress). The rpoA gene was chosen as the best internal control. In a previous study, 9 proteins were identified as involved in oxidative stress response, among which 3 virulence factors. Expression levels of genes coding for these proteins was evaluated by RT-qPCR using rpoA as an internal control. The results obtained were concordant with what had been observed at the proteomic level, validating the methods used and confirming the hypothesis of a potential link between oxidative stress and virulence factors expression.

MeSH terms

  • Adaptation, Physiological
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Campylobacter jejuni / genetics*
  • Campylobacter jejuni / metabolism*
  • DNA Gyrase / genetics
  • DNA Gyrase / metabolism
  • DNA-Directed RNA Polymerases / genetics*
  • DNA-Directed RNA Polymerases / metabolism*
  • Gene Expression Regulation, Bacterial*
  • Ketol-Acid Reductoisomerase / genetics
  • Ketol-Acid Reductoisomerase / metabolism
  • Oxidative Stress*
  • Peptidylprolyl Isomerase / genetics
  • Peptidylprolyl Isomerase / metabolism
  • RNA Stability
  • RNA, Bacterial / genetics*
  • RNA, Bacterial / metabolism*
  • RNA, Ribosomal, 16S / genetics*
  • RNA, Ribosomal, 16S / metabolism*
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Stress, Physiological / genetics*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • RNA, Ribosomal, 16S
  • ThiC protein, Bacteria
  • Ketol-Acid Reductoisomerase
  • DNA-Directed RNA Polymerases
  • RNA polymerase alpha subunit
  • Peptidylprolyl Isomerase
  • DNA Gyrase