Transcriptome analysis of acyl-homoserine lactone-based quorum sensing regulation in Yersinia pestis [corrected]

Abstract

The etiologic agent of bubonic plague, Yersinia pestis, senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.

Figure 1. Identification of the Y. pestis quorum-sensing molecules.

(A) HPLC fractionation profiles of ¹⁴C-labeled AHL produced by R88 Y. pestis (solid circles) compared to R115 QS^- Y. pestis (empty circles). The peaks absent from organic extracts of R115 Y. pestis supernatants correspond to C8-, C6-, and oxo-C6-AHL. (B) AI-2 production during the growth of R88 Y. pestis (solid circles) and R115 (empty circles) was monitored by adding the cell-free supernatants at the indicated time points to a V. harveyii reporter strain that is bioluminescent (RLU) in response to AI-2. Data are representative of at least three independent studies. (C and D) The production of AI-2 (C) and AHL (D) signals as a function of growth.

Figure 2. AHL-based QS regulates secondary metabolism.

RNA was isolated from the indicated strains of Y. pestis, and levels of mRNA were measured by qRT-PCR as outlined in Material and Methods. Data represents the mean of triplicate measurements of the transcript differences between each mutant and that of strain R88 normalized to the 16 S rRNA of each sample, and are representative of at least three independent experiments. AHL^– = strain R114; QS^– = strain R115; AI-2^– = strain ISM1980.

Figure 3. AHLs upregulate the maltose operon and enhance growth on maltose.

(A) Growth of wild-type (WT) (black), and AHL mutant (dashed) Y. pestis in minimal maltose medium (light grey) was monitored in Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with pypeIR (black, dashed) or pyspIR (grey, dashed) restores growth on maltose, whereas control plasmid (black, solid) does not.

Figure 4. Quorum-sensing regulates fermentation of sugars.

(A) Colony phenotype after 96 hrs growth on LB plates containing Congo red and 0.2% maltose and incubated at the temperature indicated. Y. pestis ferments maltose to acid, converting the Congo red to black, whereas a yspIR ypeIR mutant does not. This fermentation does not occur at temperatures above 30°C, or on other sugars tested (data not shown.) (B) Duplicate growth stabs of Y. pestis grown on solid medium under anaerobic conditions for 96 h at 28°C. An indicator dye, Congo Red, turns dark upon production of fermentative end products. Deletion of yspIR and ypeIR in R114 and R115 results in a lag in fermentation of maltose under anaerobic conditions. 1 = Y. pseudotuberculosis; 2 = R88; 3 = R114; 4 = R115.

Figure 5. AHL quorum sensing upregulates glyoxylate bypass and enhances growth on acetate.

(A) Growth of R88 (black), and an R115 AHL null mutant strain (dashed) of Y. pestis in minimal acetate medium (light grey is medium only control) was monitored in a Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with pypeIR (black, dashed) or pyspIR (grey, dashed) restores growth on acetate, whereas control plasmid (black, solid) does not.