Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes

Abstract

lisRK encodes a two-component regulatory system in the food pathogen Listeria monocytogenes LO28. Following identification of the operon in an acid-tolerant Tn917 mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.

FIG. 1

Graphic illustration of the lisRK locus. (A) Line drawing of the orientation of ORFs flanking the Tn917 insertion site. The striped regions within lisK indicate the potential membrane-spanning regions, while the lollipops indicate putative rho-independent terminators. The 498-bp region deleted in the mutant LO28ΔlisK is also indicated. (B) Alignment of the predicted receiver domains of both lisR and csrR and the consensus sequence motif for the receiver element of response regulators (27). (C) Alignment of the predicted transmitter domain of both lisR and csrR and the consensus sequence motif for the transmitter element of histidine kinase sensor proteins (27). aa, amino acids.

FIG. 2

(A) Percent survival of cultures of LO28 and LO28ΔlisK in TSB-YE (pH 3.5) after 45 min (● and ■, respectively) and 120 min (○ and □, respectively), at different points during growth ( and ░⃞, respectively). Points below the dotted line indicate that there were no survivors. (B) Growth of LO28 (○) and LO28ΔlisK (■) in TSB-YE supplemented with 5% ethanol at 37°C. Error bars represent the standard deviations of triplicate experiments. OD₆₀₀, optical density at 600 nm.

FIG. 3

Growth of LO28 (○) and LO28ΔlisK (■) in the spleens of BALB/c mice. Mice were inoculated with 1 × 10⁵ (A) and 2 × 10⁶ (B) Listeria cells by the intraperitoneal route. The arrowheads indicate the numbers inoculated into the peritoneal cavity on day 0. The cross indicates that one mouse died at that time point. Each datum point represents the mean log₁₀ number of Listeria cells per spleen for four mice. Error bars represent the standard deviations of triplicate experiments. (A) Differences were significant on days 1 and 3 (0.1 < P < 0.5). (B) Differences were significant on days 1, 3, and 4 (0.01 < P < 0.02). In all cases, significance was calculated by using the Student t test.