Proteomic analyses of a Listeria monocytogenes mutant lacking sigmaB identify new components of the sigmaB regulon and highlight a role for sigmaB in the utilization of glycerol

Abstract

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

FIG. 1.

Transcription of bsh and lmo2085 in BHI is σ^B dependent. mRNA was extracted from stationary-phase (A) and exponential-phase (OD₆₀₀, ∼0.4) (B) L. monocytogenes 10403S wild-type (sigB +) and ΔsigB (sigB −) cells in the presence (NaCl +) or in the absence (NaCl −) of 0.5 M NaCl. The numbers in parentheses indicate the numbers of PCR cycles to which the cDNA templates were subjected. The results are representative results of at least three replicates.

FIG. 2.

Seven proteins (indicated by arrows) are expressed in a σ^B-dependent manner in stationary phase. The images are representative sections of 2-DGE profiles of proteins extracted from stationary-phase cells of L. monocytogenes wild-type strain 10403S and the ΔsigB mutant grown in BHI in the presence (+NaCl) or in the absence (−NaCl) of 0.5 M NaCl. Proteins showed σ^B-dependent expression in stationary phase in BHI either regardless of the presence of NaCl (A) or only in the presence of NaCl (B). The asterisk indicates the Lmo0913 protein referred to as Lmo0913a.

FIG. 3.

Ten proteins (indicated by arrows) found to be expressed in a growth phase-dependent manner. The images are representative sections of 2-DGE profiles of proteins extracted from L. monocytogenes wild-type 10403S or ΔsigB cells grown to exponential phase (Exp) or stationary phase (Stat) in BHI in the presence or absence of 0.5 M NaCl. (A) σ^B-dependent proteins in the wild type showing growth phase-dependent expression in BHI. A similar pattern was observed for these proteins in BHIS (not shown). (B) σ^B-dependent proteins in the wild type showing growth phase-dependent expression in BHIS. (C) Proteins not affected by σ^B that were expressed in a growth phase-dependent manner in BHI. A similar pattern was observed for these proteins in BHIS (not shown). The asterisk indicates the Lmo0913 protein referred to as Lmo0913a. Lmo2101 migrated as three spots, referred to as Lmo2101a, Lmo2101b, and Lmo2101c from left to right.

FIG. 4.

lmo0796, lmo0913, lmo1830, lmo2391, and lmo2748 are transcribed in a σ^B-dependent manner. mRNA was extracted from L. monocytogenes wild-type strain 10403S (sigB +) and ΔsigB (sigB −) cells grown in BHI to stationary phase (A) and to exponential phase (OD₆₀₀, ∼0.4) (B) in the presence and absence of 0.5 M NaCl. The numbers in parentheses indicate the numbers of cycles to which the cDNA templates were subjected. The results are representative results of at least three replicates.

FIG. 5.

ΔsigB mutant utilizes glycerol inefficiently. L. monocytogenes wild-type strain 10403S (squares) and ΔsigB (triangles) cells were grown in defined medium supplemented with 0.4% (wt/vol) glycerol at 37°C. The curves are representative growth curves for the conditions investigated; all growth curve experiments were performed in triplicate. The inset shows the specific growth rates (SGR) derived from the curves. The errors bars indicate the standard deviations from the means of triplicate measurements. wt, wild type.

FIG. 6.

Sequence logo for sequenced σ^B promoters resulting from alignments of the sequenced σ^B promoters presented in Table 3 with known L. monocytogenes σ^B promoters (upstream from lmo0596, prfA, fri, gbuA, lmo1421, lmo0699, lmo1433, lmo2230, opuCA, inlA, lmo2434, bsh, and rsbV) (4, 9, 24, 30, 32). The logo is composed of stacks of letters for the positions in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), while the height of each letter reflects the relative frequency of the corresponding nucleic acid at that position.