Proteomic analysis of a Listeria monocytogenes mutant lacking sigma B (σ^B) identify new components of the σ^B regulon and highlight a role for sigma B in the utilization of glycerol

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