Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

Characterization of C. jejuni pstS::kan strain in different in vitro conditions. (A) Alkaline phosphatase activity was determined in pyruvate-minimal media with 1.6 mM or 0.08 mM P_i (n = 3). Statistical analysis was done using two way ANOVA with Sidak’s multiple-comparison test: *, P < 0.05; **, P < 0.005; ns, not significant. (B) Transcription analysis of PhoRS-regulated genes phoX, peb2, and CJJ07298 at 3 h after growth of WT DRH212 and pstS::kan in pyruvate-minimal media with 1.6 mM P_i by quantitative RT-PCR. Changes in gene expression in the pstS::kan mutant and complemented strain compared with wild-type DRH212 were determined by the 2^−ΔΔCT method. Data are represented as the mean value from three independent experiments ± standard deviation (SD). (C) C. jejuni strains were grown in Mueller-Hinton (MH) broth in microaerobic conditions for 48 h and CFU/ml were determined after specified time intervals. (D) C. jejuni DRH212 was grown in MH broth for 48 h. After 48 h, sterile spent media were collected by filter sterilization. Mid-log-phase cultures of DRH212, pstS::kan, pstS::kan/C strains were inoculated (10⁸ CFU/ml) in spent media and survival was measured at different time intervals by CFU count. (E) In a nutrient downshift assay, DRH212, pstS::kan, pstS::kan/C strains grown to mid-log phase were collected and used to inoculate in minimal medium lacking carbon and phosphate sources. The survival assay was measured by CFU count at specified time intervals. (F and G) Osmotic stress tolerance was assessed by inoculating strains into MH broth containing 1% NaCl (F) or 1.5% NaCl (G) and determining growth by CFU counts. Results represent the mean value from three independent experiments. Statistical significance was assessed by Student's t test: **, P < 0.005.

Comparative analysis of polyP metabolism regulatory genes and polyP level between WT DRH212 and the pstS::kan mutant. (A and B) Transcription analysis by quantitative RT-PCR of stress regulatory genes ppk1, ppk2, spoT, and ppA at 24 h (A) and 48 h (B) after growth of DRH212 and the pstS::kan mutant. Changes in gene expression in the pstS::kan mutant compared with wild type DRH212 were determined by the 2^−ΔΔCT method. Data are represented as the mean value from three independent experiments ± standard deviation. (C and D) Levels of intracellular polyphosphate (polyP) of three strains (DRH212, pstS::kan, and pstS::kan/C strains) were measured at 24 h (CD) and 48 h (D). Statistical analysis used an unpaired t test and one-way ANOVA with Holm-Sidak’s multiple test (n = 8, ±SD): *, P < 0.05; **, P < 0.005.

Colonization ability of the pstS::kan mutant in chickens. (A to D) Day-of-hatch white Leghorn chicks were infected with either WT DRH212, pstS::kan, or pstS::kan/C C. jejuni (at two different doses of 10³ and 10⁶ CFU/ml) and cecal loads were measured at day 3 and day 7. Shown is the comparative colonization ability between DRH212 and pstS::kan strains at days 3 and 7 after oral inoculation with 10³ CFU/ml (A and B) and 10⁶ CFU/ml (C and D). Statistical analysis used one-way ANOVA with Tukey’s multiple-comparison test (n = 5, ±SD). (E) In competition analysis between DRH212 and the pstS::kan mutant, day-of-hatch chicks were infected with equal ratios of DRH212 and pstS::kan mutant strains. At day 7, the ratio of pstS::kan mutant to DRH212 was determined and is presented as a competitive index. Statistical analysis was performed using a one-simple t test against a hypothetical value of 1 (n = 5 for each group). *, P < 0.05; **, P < 0.005.

PstSCAB transporter of C. jejuni is critical for growth in lactate. C. jejuni strains were grown in minimal medium containing both d- and l-lactate (each 5 mM) with two concentrations of inorganic phosphate. Growth of WT DRH212 and pstS::kan mutant strain were measured by CFU count in minimal medium (d- and l-lactate) with 1.6 mM P_i (A) and 0.08 mM P_i (B) at 24 h and 48 h. n = 3; error bars indicate SD. Statistical analysis was done by two-way analysis of variation (ANOVA) with Sidak’s multiple comparision test: *, P < 0.05; **, P < 0.005; ns, not significant.

Comparative growth analysis of C. jejuni strains in minimal medium with d- or l-lactate. C. jejuni strains were grown in minimal medium with d-lactate or l-lactate as a carbon source and a high or low concentration of inorganic phosphate. (A and B) Growth of C. jejuni strains was measured in d-lactate minimal medium with 1.6 mM (A) and 0.08 mM (B) phosphate by CFU counts at 24 h and 48 h. (C and D) Growth of these three strains was also determined in l-lactate minimal medium with 1.6 mM (C) and 0.08 mM (D) phosphate. n = 3; error bars indicate SD. Statistical analysis was done by two-way analysis of variation (ANOVA) with Sidak’s multiple-comparison test: *, P < 0.05; **, P < 0.005; ns, not significant.

Transcript analysis of acetogenesis regulatory and acetogenesis-dependent gene expression in wild type, pstS::kan, and complemented strains. Bacterial strains were grown in minimal medium containing l-lactate with 1.6 mM P_i up to 18 h, after which cells were harvested for RNA isolation. Acetogenesis regulatory genes (ackA and pta) and acetogenesis-dependent genes (ggt, peb1C, and CJJ0683) were measured in the pstS::kan mutant and pstS::kan/C strains relative to WT DRH212 by real-time PCR. n = 3; error bars indicate SD.