ABSTRACTInListeria monocytogenesthe alternative sigma factor σBplays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σBregulon inL. monocytogenes10403S (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 ΔsigBdeletion strain were compared. Overall, this study identified 38 proteins whose expression was σBdependent; 17 of these proteins were found to require the presence of σBfor full expression, while 21 were expressed at a higher level in the ΔsigBmutant 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 σBin the general stress response and highlighted a probable role for σBin metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σBin glycerol metabolism. Five newly identified members of the σBregulon were shown to be under direct regulation of σBusing reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σBwas shown to play a major role in the stationary phase of growth in complex media.