Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Abstract

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.

FIG 1

Relative expression levels of histidine kinase-encoding genes in Listeria monocytogenes EGD-e during the logarithmic growth phase at 3°C, on a log scale, each calibrated to the expression levels during logarithmic growth at 37°C. Gene expression was normalized to the 16S rRNA gene (rrn). Error bars represent the minimum and maximum ratios for three replicate cultures. Significantly increased relative expression levels (paired t test) are indicated by asterisks (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

FIG 2

Relative expression levels of histidine kinase-encoding genes in Listeria monocytogenes EGD-e 30 min (A), 3 h (B), and 7 h (C) after cold shock from 37°C to 5°C, each calibrated to t₀ at 37°C. Gene expression was normalized to the 16S rRNA gene (rrn). Error bars represent the minimum and maximum ratios for three replicate cultures. Significantly increased relative expression levels (paired t test) are indicated by asterisks (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

FIG 3

(A) Growth curves for Listeria monocytogenes Δlmo0288 (ΔyycG), Δlmo1378 (ΔlisK), and Δlmo1947 (ΔresE) histidine kinase deletion mutant strains and the wild-type strain EGD-e in BHI broth at 3°C. All the strains were grown for 23 days, and the OD₆₀₀ was measured every hour. The other histidine kinase mutant strains showed no significantly impaired growth compared to the wild type. (B) Growth curves for L. monocytogenes ΔyycG, ΔlisK, and ΔresE histidine kinase deletion mutant strains and the wild-type strain EGD-e in BHI broth at 37°C. All the strains were grown for 24 h, and the OD₆₀₀ values were measured every 15 min. The data shown represent the mean OD₆₀₀ values and standard deviations for five replicate cultures. The correspondence between the OD₆₀₀ values and viable cell numbers was verified by plate counts.

FIG 4

Growth curves for Listeria monocytogenes wild-type EGD-e, the ΔyycGc and ΔlisKc complemented strains, and the vector controls EGD-epPL2, ΔyycGpPL2, and ΔlisKpPL2 at 3°C. All the strains were grown for 40 days, and the OD₆₀₀ was measured every hour. The data shown represent the mean OD₆₀₀ values and standard deviations for five replicate cultures.