Identification of Sigma Factor σB-Controlled Genes and Their Impact on Acid Stress, High Hydrostatic Pressure, and Freeze Survival inListeria monocytogenesEGD-e

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Abstract
The gene encoding the alternative sigma factor σBinListeria monocytogenesis induced upon exposure of cells to several stresses. In this study, we investigated the impact of asigBnull mutation on the survival ofL. monocytogenesEGD-e at low pH, during high-hydrostatic-pressure treatment, and during freezing. The survival of ΔsigBmutant exponential-phase cells at pH 2.5 was 10,000-fold lower than the survival of EGD-e wild-type cells. Moreover, the ΔsigBmutant failed to show an acid tolerance response. Upon preexposure for 1 h to pH 4.5, the survival at pH 2.5 was 100,000-fold lower for the ΔsigBmutant than for the wild type. The glutamate decarboxylase (GAD) acid resistance system is important in survival and adaptation ofL. monocytogenesin acidic conditions. The σBdependence of thegadgenes (gadA,gadB,gadC,gadD, andgadE) was analyzed in silico. Putative σB-dependent promoter sites were found upstream of thegadCBoperon (encoding a glutamate/γ-aminobutyrate antiporter and a glutamate decarboxylase, respectively) and thelmo2434gene (gadD, encoding a putative glutamate decarboxylase). Reverse transcriptase PCR revealed that expression of thegadCBoperon and expression ofgadDare indeed σBdependent. In addition, a proteomics approach was used to analyze the protein expression profiles upon acid exposure. Although the GAD proteins were not recovered, nine proteins accumulated in the wild type but not in the ΔsigBstrain. These proteins included Pfk, GalE, ClpP, and Lmo1580. Exposure to pH 4.5, in order to preload cells with active σBand consequently with σB-dependent general stress proteins, also provided considerable protection against high-hydrostatic-pressure treatment and freezing. The combined data argue that the expression of σB-dependent genes providesL. monocytogeneswith nonspecific multiple-stress resistance that may be relevant for survival in the natural environment as well as during food processing.