Microarray-based characterization of the Listeria monocytogenes cold regulon in log- and stationary-phase cells

Abstract

Whole-genome microarray experiments were performed to define the Listeria monocytogenes cold growth regulon and to identify genes differentially expressed during growth at 4 and 37 degrees C. Microarray analysis using a stringent cutoff (adjusted P < 0.001; >/=2.0-fold change) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4 degrees C than in cells grown at 37 degrees C. A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells, respectively, grown at 4 degrees C. Genes with higher transcript levels at 4 degrees C in both stationary- and log-phase cells included genes encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas a number of genes encoding virulence factors and heat shock proteins showed lower transcript levels at 4 degrees C. Selected genes that showed higher transcript levels at 4 degrees C during both stationary and log phases were confirmed by quantitative reverse transcriptase PCR. Our data show that (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37 degrees C, with more genes showing higher transcript levels than lower transcript levels at 4 degrees C, (ii) L. monocytogenes genes with higher transcript levels at 4 degrees C include a number of genes and operons with previously reported or plausible roles in cold adaptation, and (iii) L. monocytogenes genes with lower transcript levels at 4 degrees C include a number of virulence and virulence-associated genes as well as some heat shock genes.

FIG. 1.

Growth of L. monocytogenes strain 10403S at 4 and 37°C in BHI broth. The OD₆₀₀ of L. monocytogenes 10403S was measured once a day (for 12 days) for cells grown at 4°C (•) and once an hour (for 8 h) for cells grown at 37°C (▪). The data shown represent the average OD₆₀₀ values for three independent experiments, and error bars indicate standard deviations. Cell counts were performed once a day (for 12 days) for cells grown at 4°C (○) and once an hour (for 5 h) for cells grown at 37°C (□); the cell count data shown represent the log CFU/ml determined for one growth experiment (average from duplicate platings). Numbers on the x axis represent days (for growth at 4°C) and hours (for growth at 37°C). White arrows indicate the time points used for the collection of log-phase cells (OD₆₀₀ of 0.4 ± 0.05 for both temperatures) for microarray experiments; gray arrows indicate the time points used for the collection of stationary-phase cells (OD₆₀₀ of 1.0 ± 0.25; reached 9 days and 4 h after inoculation for cells grown at 4 and 37°C, respectively).

FIG. 2.

Venn diagram of L. monocytogenes 10403S genes differentially transcribed at 4 and 37°C in log- and stationary-phase cells. Only genes that are differentially transcribed based on stringent cutoff criteria (≥2-fold change; adjusted P < 0.001) are shown (Fig. S1 in the supplemental material represents a similar Venn diagram but shows all genes that showed evidence of differential transcription based on less stringent criteria [≥2-fold change; adjusted P < 0.05]). In each circle, the total number of genes upregulated (↑) at 4°C or downregulated (↓) at 4°C under a given condition (log or stationary phase) is shown. Selected genes with relevant known or putative functions are also indicated.

FIG. 3.

Illustration of selected putative L. monocytogenes operons identified by microarray analysis to be upregulated at 4°C in log and stationary phases (A), log phase only (B), and stationary phase only (C). Operons were included even if some genes did not meet the stringent cutoff criteria (i.e., a ≥2-fold difference in transcript levels and an adjusted P value of <0.001). Transcript level ratios are given under each gene. Positive values indicate higher transcript levels at 4°C than at 37°C; while most genes have positive values, some genes have negative values for transcript level ratios, indicating higher transcript levels at 37°C (e.g., a value of −2.1 indicates a 2.1-fold-higher transcript level at 37°C). Transcript level ratios of ≥2 (indicating ≥2-fold differences in transcript levels) are in bold; adjusted P values for significance of differential expression are indicated by ** (P < 0.001), * (P < 0.05), and NS (not significant) (P > 0.05). Known promoters (GenBank accession no. AF0322444 and Y09161) (16a, 81) are marked with small arrows. Rho-independent terminators were predicted for L. monocytogenes as described by de Hoon et al. (19) and are shown as hairpins; the previously determined Rho-dependent terminator downstream of lmaA (81) is shown as a hairpin with a diamond. Genes are not drawn to scale.

FIG. 4.

Illustration of selected L. monocytogenes virulence genes identified by microarray analysis to be downregulated at 4°C. Transcript level ratios are given under each gene. Negative values indicate lower transcript levels at 4°C than at 37°C (e.g., a value of −3.3 indicates a 3.3-fold-lower transcript level at 4°C). Transcript level ratios of ≥2 (indicating ≥2-fold differences in transcript levels) are in bold; adjusted P values for significance of differential expression are indicated by ** (P < 0.001), * (P < 0.05), and NS (not significant) (P > 0.05). Previously reported promoters and terminators (79) are indicated by small arrows and hairpins, respectively. Genes are not drawn to scale.

FIG. 5.

Transcript levels for putative cold stress genes as determined by qRT-PCR. Transcript levels for the housekeeping genes rpoB and gap and for four putative cold stress genes were determined by qRT-PCR for L. monocytogenes 10403S cells grown in BHI broth to log or stationary phase at 4°C or 37°C. Transcript levels for the putative cold stress genes lmo0287, lmo0866, cspL, and lmo1722 were normalized to the geometric means for rpoB and gap. While rpoB and gap transcript levels were significantly affected (P < 0.0001 and P < 0.01, respectively) by growth phase, with lower absolute transcript levels (per 10 μg RNA) observed in stationary-phase cells, consistent with previous qRT-PCR data (16a), normalization of the transcript levels of the four target genes to the geometric means for rpoB and gap transcript levels was performed to adjust transcript levels to account for physiological differences between bacterial cells in different growth phases. The fact that temperature did not significantly (P > 0.1) affect rpoB and gap transcript levels supports that analyses of target gene transcript levels normalized to rpoB and gap transcript levels provide robust data for evaluating the effect of temperature on target gene transcript levels. Data represent the averages for three biological replicates; error bars indicate standard deviations. For a given gene, different letters above the bars indicate significantly different transcript levels.