Transcriptome analysis of genes controlled by luxS/autoinducer-2 in Salmonella enterica serovar Typhimurium

Abstract

The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.

FIG. 1.

Growth and autoinducer-2 (AI-2) activity of Salmonella Typhimurium wild type (WT) and its isogenic ΔluxS mutant (PJ002). (A) Luria–Bertani (LB) medium supplemented with glucose 0.5%; (B) LB medium without glucose. The assays were carried out in triplicate. Bars indicate AI-2 activity, and lines indicate growth. Each bar represents mean ± standard error of the mean of all the measurements. Arrows point to the time points at which samples were collected for RNA extraction.

FIG. 2.

Effect of luxS deletion on Salmonella Typhimurium motility. Swimming and swarming zones were measured 7 h after inoculating 0.3% and 0.6% LB agar plates, respectively, in both absence and presence of glucose (0.5%). Zone diameters (mean ± standard deviations; n = 5) are listed for each strain. The zone diameters for each strain in swimming motility (A) WT (30.5 ± 6.5), (B) ΔluxS (15.0 ± 1.6), (C) WT (13.2 ± 2.3), and (D) ΔluxS (10.7 ± 1.6); swarming motility (A) WT (22.7 ± 3.5), (B) ΔluxS (16.2 ± 1.5), (C) WT (0), and (D) ΔluxS (0) are shown. Mutant was deficient in motility both in the presence and absence of glucose.

FIG. 3.

Effects of luxS mutation and glucose on biofilm (mean OD ± SE; p < 0.0001, n = 3) formation in Salmonella Typhimurium. Biofilm assays were performed in 96-well plates, and eight wells were assigned for each strain and condition. Biofilms of WT and ΔluxS mutant were determined using crystal violet binding assay after 36 h of growth at 37°C in the presence and absence of glucose. Each bar represents mean ± standard error of the mean of all the measurements. Presence of glucose inhibited biofilm formation in WT. Mutant was deficient in biofilm formation both in the presence and absence of glucose.

FIG. 4.

Categories of genes regulated by luxS/AI-2. luxS/AI-2-regulated genes are grouped into functional categories based on the Salmonella Typhimurium genome annotation (http://cmr.jcvi.org/cgi-bin/CMR/shared/MakeFrontPages.cgi?page=geneattribute). The histograms represent the number of genes in each category promoted or repressed by luxS mutation in the mid-log phase in LB without glucose.

FIG. 5.

Validation of microarray data of selected luxS/AI-2-regulated genes using real-time reverse transcriptase–polymerase chain reaction (PCR). The differences in expression of seven genes that are regulated by luxS/AI-2 in the mid-log phase in the absence of glucose were log₂ transformed and plotted against each other (microarray against reverse transcriptase–polymerase chain reaction).