Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots

Abstract

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.

FIG 1

Fitness of the kdgR mutant in soft rots. S. enterica serovar Typhimurium 14028 and the isogenic kdgR mutant were coinoculated at a 1:1 ratio into intact green tomatoes or into tomatoes with P. carotovorum SR38 or Xanthomonas species. The ratio of the wild-type Salmonella to the mutant was assessed by plating and patching onto selective media after a 3-day incubation. Box plots indicate 25 to 75% quantiles, and lines within box plots are medians. Whiskers indicate the first or third quantile plus 1.5 multiplied by the inner quantile range. The zero line indicates no difference in competitive fitness. Competitive indices were compared using Student's t test, and different letters indicate statistically significant differences (P < 0.05).

FIG 2

Sequence logo for KdgR binding sites in Salmonella enterica. The position-specific scoring matrix (PSSM) was derived from the KdgR binding sequences experimentally validated in other gammaproteobacteria. A PSSM was generated from the targets identified in the first scan based on 211 sites found in the upstream regions of the Salmonella genes that are orthologous to the previously validated KdgR regulon members and 40 experimentally validated targets of KdgR. The PSSM kdgR logo was created using the Weblogo interface.

FIG 3

Overlap in utilization of carbon sources by S. enterica serovar Typhimurium, S. enterica serovar Typhimurium kdgR, P. carotovorum SR38, and Xanthomonas species. The ability of the bacteria to respire on substrates in Biolog plates PM1 and PM2 was documented (see Fig. S2 in the supplemental material).

FIG 4

In planta expression of KdgR-regulated genes. RIVET reporters in kdgM, kduID, and kdgK were constructed in the wild-type and ΔkdgR backgrounds. The resolution of the reporters was tested in soft rots and in intact green tomatoes (tom) following a 3-day incubation. Soft rots were initiated by coinoculating P. carotovorum SR38 with S. enterica RIVET reporters.

FIG 5

Fitness of the mutants in kdgR-regulated genes in soft rots and intact tomatoes. Mutants were coinoculated with the isogenic wild type in a 1:1 ratio in green tomatoes (empty boxes) or in tomatoes that also were seeded with P. carotovorum SR38 (shaded boxes). Ratios of the mutant to the wild type were assessed by patching onto selective media, and log competitive indices were determined. Box plots indicate 25 to 75% quantiles, and lines within box plots are medians. Whiskers indicate the first or third quantile plus 1.5 multiplied by the inner quantile range. The fitness of mutants in intact tomatoes and in soft rots was compared using Student's t test, and none were significant.

FIG 6

In planta expression of eda. Expression of eda::tnpR-lacZY reporters in S. enterica serovar Typhimurium 14028 was determined in wild-type and kdgR backgrounds. Reporters were inoculated into tomatoes with or without Pectobacterium carotovorum SR38. Samples were plated following 3 days of incubation and scored for the loss of the tetracycline marker.

FIG 7

Fitness of the eda and edd mutants in tomatoes. The mutants were coinoculated with the isogenic wild type in a 1:1 ratio in green tomatoes or into tomatoes that also were seeded with P. carotovorum SR38. Ratios of the mutants to the wild type were assessed by patching onto selective media, and log competitive indices were determined. Box plots indicate 25 to 75% quantiles, and lines within box plots are medians. Whiskers indicate the first or third quantile plus 1.5 multiplied by the inner quantile range.