Experimental evidence for negative selection in the evolution of a Yersinia pestis pseudogene

Abstract

Yersinia pestis, the agent of bubonic plague, evolved from the enteric pathogen Yersinia pseudotuberculosis within the past 20,000 years. Because ancestor and descendant both exist, it is possible to infer steps in molecular evolution by direct experimental approaches. The Y. pestis life cycle includes establishment of a biofilm within its vector, the flea. Although Y. pseudotuberculosis makes biofilms in other environments, it fails to do so in the insect. We show that rcsA, a negative regulator of biofilms that is functional in Y. pseudotuberculosis, is a pseudogene in Y. pestis. Replacement of the pseudogene with the functional Y. pseudotuberculosis rcsA allele strongly represses biofilm formation and essentially abolishes flea biofilms. The conversion of rcsA to a pseudogene during Y. pestis evolution, therefore, was a case of negative selection rather than neutral genetic drift.

Fig. 1.

Model of Rcs phosphorelay signaling. The histidine kinase domain (HK) of RcsC autophosphorylates, after which phosphate is transferred intramolecularly to aspartate in a receiver domain (D1). Next, phosphate is transferred intermolecularly from D1 to the histidine phosphotransfer domain (HPT) of RcsD. Last, phosphate is transferred from HPT to the RcsB receiver domain (D2). Biofilms are repressed by RcsB, and repression is increased by functional RcsA, as described in Results. Solid arrows show phosphate transfers that are well established experimentally (13, 14); dashed arrow indicates hypothesized phosphatase activity by RcsC, RcsD, or both (13, 16, 17). The histidine kinase-like ATPase (HAT) domain of RcsD does not participate in phosphotransfer. Filled arrowhead, site of frameshift in Y. pestis RcsD. Open arrowhead, site of inactivating insertion in Y. pestis RcsA.

Fig. 2.

Colony size and morphology of mutants. ΔrcsB overproduces biofilms and makes small, rugose (wrinkled) colonies. ΔhmsS is defective for biofilm formation and makes colonies slightly larger than those of the wild type. Typical colonies of each strain were photographed at identical magnifications.

Fig. 3.

Phenotypes of rcs mutants and substitutions. “Δ” indicates deletion; “-pe” and “-pstb” denote Y. pestis and Y. pseudotuberculosis alleles. ΔrcsD-pe experiments used the empty plasmid vector pET-32a(+); p-rcsD-pstb denotes the Y. pseudotuberculosis allele cloned into this plasmid under its native promoter. Congo red (CR) colony color phenotypes: −, white; +/−, pink; +, red; ++, dark red. Colony size and morphology are described in the Fig. 2 legend. In vitro biofilms grown on polystyrene culture dishes were quantified by staining with crystal violet (Methods); data are mean ± SD of two to four trials. Biofilms on C. elegans were photographed after overnight incubation on Y. pestis lawns; rare animals on rcsA-pstb acquired traces of biofilm (data not shown). Numbers are percentage of worms that grew to L4 stage in 2 d; data are aggregate of two to four trials in which a minimum of 1,100 worms were scored for each bacterial genotype.

Fig. 4.

rcsB represses biofilms. In vitro biofilms were grown in the ΔrcsB strain transformed with vector control (white) or rcsB (black) under an arabinose-inducible promoter. Inducer was added at indicated concentrations. Data are mean ± SD of two (vector) or three (rcsB) trials. The difference between control and rcsB samples in the absence of arabinose is apparently due to basal rcsB expression.

Fig. 5.

Flea blockage and colonization by rcsA-pstb. Data are mean ± SEM of three trials. (A) Cumulative blockage frequency 4 weeks after infection with Y. pestis. At least 80 fleas were analyzed for each sample. (B) Percentage of fleas infected initially and after 4 weeks. (C) Colony-forming units (CFU) in fleas sampled immediately after infection (n = 20 per strain per trial) and in fleas that remained colonized after 4 weeks (n = 4–19).

Fig. 6.

RcsA negatively regulates Y. pseudotuberculosis biofilms. No biofilm formed on adult C. elegans placed on lawns of wild-type strain IP32953 and incubated overnight. Replacement of the wild-type allele rcsA-pstb with nonfunctional rcsA-pe resulted in the production of substantial biofilms.