Phenotypic and genotypic evidence for L-fucose utilization by Campylobacter jejuni

Abstract

Campylobacter jejuni remains among the leading causes of bacterial food-borne illness. The current understanding of Campylobacter physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for l-fucose utilization by C. jejuni. The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in fucP (encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the fucP mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10⁵ CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10⁸ CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of C. jejuni and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of C. jejuni and the role of metabolic diversity in Campylobacter pathobiology.

FIG. 1.

Identification of the genetic locus for fucose utilization. (A) Schematic representation of the chromosomal region associated with the Fuc⁺ phenotype in NCTC 11168. Solid arrows depict open reading frames. Vertical bars indicate locations of frameshift mutations resulting in premature translational termination. The location of the ΔfucP::cat mutation is indicated by the arrow above fucP. ORFs used to complement the fucP mutant are indicated by braces below each ORF. The strain designation for each complementing construct is labeled under each respective brace. The diagonal hatch marks indicate that the entire length of the ORF is not shown. (B) RT-PCR analysis of gene transcription in the wild-type strain and the ΔfucP::cat strain complemented with fucP (CjWM226a). RT+, reverse transcribed with SuperScript RT; RT−, no-SuperScript RT control. Polar effects on transcription of the genes downstream of fucP are evident by the absence of PCR bands in the RT+ lanes for cj0487 and aldA in CjWM226a.

FIG. 2.

Results of Phenotypic MicroArray carbon utilization plates (PM1) after incubation for 48 h with strain NCTC 11168, 81116, or 81-176. Wells indicating differential carbon utilization are circled. B4, l-fucose; G1, glycyl-l-glutamic acid; H1, glycyl-l-proline.

FIG. 3.

Growth of wild-type C. jejuni strains in defined medium supplemented with different carbon sources. (A) Basal medium was supplemented with d-glucose (50 mM), l-fucose (50 mM), sodium pyruvate (20 mM), or a combination of l-aspartate, l-glutamate, l-proline, and l-serine (DEPS; 390 mM, 360 mM, 560 mM, and 1.3 mM, respectively). White columns, NCTC 11168; gray columns, 81116; black columns, 81-176. Bars represent the mean viable cell counts ± standard errors from three separate experiments. (B) Passage of wild-type NCTC 11168 in defined medium containing l-fucose (50 mM). Points represent mean viable cell counts ± standard errors from three separate experiments. Arrows indicate passage after 24 h of growth to fresh medium at a 1:100 dilution.

FIG. 4.

Growth comparison of various strains in defined medium supplemented with different carbon sources. White columns, NCTC 11168 wild type; gray columns, fucP derivative of NCTC 11168 (CjWM114a); black columns, ΔfucP::cat strain complemented with fucP (CjWM226a); dotted columns, ΔfucP::cat strain complemented with fucP-cj0487 (CjWM230a); diagonally hatched columns, ΔfucP::cat strain complemented with fucP-cj0488 (CjWM231a); horizontally hatched columns, ΔfucP::cat strain complemented with fucP-aldA (CjWM227a). Bars represent the mean viable cell numbers ± standard errors from three independent experiments. Growth of the wild-type strain is shown in Fig. 3 and is included here for comparison.

FIG. 5.

RT-PCR analysis of fucose induction of gene expression in C. jejuni NCTC 11168. (A) Expression of dapA, fucP, and aldA in wild-type NCTC 11168 in defined medium supplemented with fucose (50 mM), pyruvate (20 mM), or a mixture of aspartate, glutamate, proline, and serine (DEPS). RT+, RT-PCR with SuperScript RT; RT−, no-SuperScript RT control. (B) Dose-dependent induction of fucP by fucose. R_induced represents the relative transcript abundance in cultures induced with fucose compared to that in uninduced cohorts. Error bars indicate standard errors from four independent experiments. Values above error bars indicate P values determined by a one-sample t test.

FIG. 6.

Colonization of chicken ceca by wild-type C. jejuni NCTC 11168 (circles) and its fucP mutant (squares) in mono- and coinoculated groups. In two separate experiments, young chicks were inoculated with a high dose (10⁸ CFU per chicken) (A) or a low dose (10⁵ CFU per chicken) (B). In each experiment, birds were inoculated with either a single strain (open symbols) or a 1:1 mixture of the wild type and the mutant (filled symbols) and sacrificed after 5, 10, or 20 days postinoculation (DPI). Each point represents data from a single bird. A horizontal bar indicates the mean for each group. P values above paired groups reflect significance as determined by a two-sample t test.