Proteomic Analyses of a Listeria monocytogenes Mutant Lacking σ B Identify New Components of the σ B Regulon and Highlight a Role for σ B in the Utilization of Glycerol

Abstract

ABSTRACT In Listeria monocytogenes the alternative sigma factor σ B plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σ B regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic Δ sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σ B dependent; 17 of these proteins were found to require the presence of σ B for full expression, while 21 were expressed at a higher level in the Δ sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σ B in the general stress response and highlighted a probable role for σ B in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σ B in glycerol metabolism. Five newly identified members of the σ B regulon were shown to be under direct regulation of σ B using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σ B -dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σ B -dependent genes, as well as proteomic analyses, σ B was shown to play a major role in the stationary phase of growth in complex media.