Identification of components of the sigma B regulon in Listeria monocytogenes that contribute to acid and salt tolerance

Abstract

Sigma B (sigma(B)) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the DeltasigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The DeltasigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that sigma(B) contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the DeltasigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of sigma(B) in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the DeltasigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of sigma(B). It also demonstrated clear roles for sigma(B) in both osmotic and low-pH stress tolerance and identified specific components of the sigma(B) regulon that contribute to the responses observed.

FIG. 1.

Rapid-growth phenotype of an L. monocytogenes ΔsigB mutant in the presence of 0.5 M NaCl. L. monocytogenes wild-type strain 10403S (squares) and the ΔsigB mutant (triangles) were grown in defined media supplemented with 0.4% (wt/vol) glucose at 37°C with shaking in the absence (DM) (filled symbols) and in the presence (DMS) (open symbols) of 0.5 M NaCl. Representative growth curves for the conditions investigated are shown; all experiments were performed in triplicate. The inset shows the specific growth rates (SGR) during exponential growth derived from the curves for the wild type (open bars) and the ΔsigB mutant (shaded bars) in DM and DMS. The error bars indicate the standard deviations from the means of triplicate measurements.

FIG. 2.

ΔsigB mutant exhibits greater specific growth rates than the wild-type strain in DM supplemented with different carbon sources in the presence of 0.5 M NaCl. Specific growth rates (SGR) of L. monocytogenes 10403S (open bars) and the corresponding sigB deletion mutant (shaded bars) in defined media supplemented with 0.04 or 0.4% (wt/vol) glucose (glu), 0.4% (wt/vol) mannose (man), 0.4% (wt/vol) fructose (fru), 0.4% (wt/vol) trehalose (tre), or 0.4% (wt/vol) cellobiose (cel) were determined. Cells were grown at 37°C in 96-well plates in the absence (A) and presence (B) of 0.5 M NaCl. The errors bars indicate the standard deviations from the means of triplicate measurements.

FIG. 3.

Eleven proteins were found to be expressed in a σ^B-dependent manner. The gel images show representative sections of 2-DGE profiles for proteins extracted from either L. monocytogenes wild-type strain 10403S or ΔsigB cells grown to exponential phase or stationary phase in DM. Proteins were extracted from cells growing either in the presence (+NaCl) or absence (−NaCl) of 0.5 M NaCl. The arrows indicate the locations of the proteins showing altered expression patterns in the ΔsigB background. (A) Proteins showing σ^B-dependent expression in stationary phase (the same proteins showed σ^B-dependent expression in exponential phase [data not shown]). The asterisk indicates the Lmo0913 protein referred to as Lmo0913a. (B) Proteins showing σ^B-dependent expression under specific conditions. Extracts of exponential-phase cells were used for Lmo2425, while extracts of stationary-phase cells were used for Lmo2829 and LmaA.

FIG. 4.

Δlmo2748 and ΔsigB mutants have a slow-growth phenotype in BHI medium supplemented with 1.75 M NaCl. L. monocytogenes wild-type strain 10403S (black squares), Δlmo0796 mutant (open squares), Δlmo0913 mutant (gray triangles), Δlmo2391 mutant (open circles), Δlmo2748 mutant (black triangles), and ΔsigB mutant (asterisks) cells were grown in BHI medium supplemented with 1.75 M NaCl at 37°C with shaking. Representative growth curves for the conditions investigated are shown; all experiments were performed in triplicate. The inset shows the specific growth rates (SGR) derived from the curves. The error bars indicate the standard deviations from the means of triplicate measurements. wt, wild type.

FIG. 5.

Δlmo0796, Δlmo0913, Δlmo2391, and ΔsigB mutants have a survival defect in BHIS at pH 2.5. L. monocytogenes wild-type strain 10403S (black squares), Δlmo0796 mutant (open squares), Δlmo0913 mutant (gray triangles), Δlmo2391 mutant (open circles), Δlmo2748 mutant (black triangles), and ΔsigB mutant (asterisks) cells were grown to stationary phase in BHIS at 37°C with shaking and were subsequently subjected to pH 2.5 in fresh BHIS at room temperature. Survival under the acid conditions was monitored in triplicate, and the errors bars indicate the standard deviations from the means.

FIG. 6.

Unusual pattern of Gram staining of the Δlmo2748 and ΔsigB mutants. L. monocytogenes wild-type strain 10403S (wt), Δlmo0796 mutant, Δlmo0913 mutant, Δlmo2391 mutant, Δlmo2748 mutant, and ΔsigB mutant cells were observed with the microscope after Gram staining.