Semiquantitative real-time RT-PCR analysis.

WT C. jejuni and isogenic mutant strains were grown from freezer stocks on MH agar containing trimethoprim at 37°C under microaerobic conditions for 48 h. Strains were then restreaked on MH agar and grown for an additional 16 h. C. jejuni growth was suspended from the plates in MH broth and diluted into 25 ml of MH broth to an OD₆₀₀ of approximately 0.1. Strains were then grown statically at 37°C under microaerobic conditions for 8 h to achieve mid-log-phase growth. Total RNA was extracted with RiboZol (Amresco), and RNA was treated with DNase I (Invitrogen). RNA was diluted to a concentration of 50 ng/μl before analysis. Semiquantitative real-time PCR (qRT-PCR) was performed using a 7500 real-time PCR system (Applied Biosystems) with secD mRNA detection as an endogenous control. mRNA transcript levels in strains DRH212 and DRH461 served as WT controls to determine relative gene expression in isogenic mutants.

To examine the effects of different short-chain fatty acids on gene expression in C. jejuni, WT and isogeneic mutant strains were grown from freezer stocks on MH agar as detailed above. Following the additional 16 h of growth, the C. jejuni strains were suspended from plates in CDM, which is a medium containing nutrients at specific concentrations to support growth, and diluted into 25 ml of CDM broth to an OD₆₀₀ of approximately 0.1 (34). Strains were grown in CDM alone, CDM containing 0, 10, 25, 50, or 100 mM potassium acetate, sodium propionate, or sodium l-lactate (Sigma), CDM containing 0, 12.5, or 25 mM sodium butyrate (Acros Organics), or CDM containing 12.5 mM sodium butyrate and 25 mM sodium l-lactate. For analysis of how a mixture of SCFAs impacts gene expression, strains were grown in CDM alone or CDM containing 30 mM potassium acetate, 5 mM sodium butyrate, and 7.5 mM sodium propionate. All media were adjusted to pH 7.0 prior to inoculation of bacteria. Bacterial growth occurred statically at 37°C under microaerobic conditions for 8 h to achieve mid-log-phase growth. Total RNA was extracted with RiboZol (Amresco), and RNA was treated with DNase I (Invitrogen). RNA was diluted to a concentration of 50 ng/μl before analysis. qRT-PCR was performed using a 7500 real-time PCR system (Applied Biosystems) with secD mRNA detection as an endogenous control. mRNA transcript levels in the DRH461 strain grown in CDM alone served as the WT control to determine relative gene expression in the wild type and isogenic mutant at the different concentrations of SCFAs.

To examine the effect of overexpression in trans of pta and ackA on gene expression in C. jejuni, strains PML1102, PML1125, and PML1140 were grown in MH and CDM broth containing kanamycin and trimethoprim. Total RNA was extracted and purified as detailed above. mRNA transcript levels in strain PML1140 were used as the WT control, and secD mRNA detection was used as the endogenous control to determine relative gene expression.

To examine the levels of expression of colonization and virulence genes throughout the chick intestinal tract, 1-day-old chicks were orally inoculated with 100 μl of MH broth containing approximately 10² CFU of WT C. jejuni as described above. At 7 days postinfection, the chicks were sacrificed, the small intestine, cecal, or large intestine contents were removed, and total RNA was extracted with RiboZol (Amresco). RNA was then purified as described above. mRNA transcript levels in the ceca served as the organ load control, and secD mRNA detection was used as the endogenous control to determine relative gene expression.