Protein level identification of the Listeria monocytogenes sigma H, sigma L, and sigma C regulons

Abstract

Background: Transcriptional regulation by alternative sigma (σ) factors represents an important mechanism that allows bacteria to rapidly regulate transcript and protein levels in response to changing environmental conditions. While the role of the alternative σ factor σB has been comparatively well characterized in L. monocytogenes, our understanding of the roles of the three other L. monocytogenes alternative σ factors is still limited. In this study, we employed a quantitative proteomics approach using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to characterize the L. monocytogenes σL, σH, and σC protein regulons. Proteomic comparisons used a quadruple alternative σ factor mutant strain (ΔBCHL) and strains expressing a single alternative σ factor (i.e., σL, σH, and σC; strains ΔBCH, ΔBCL, and ΔBHL) to eliminate potential redundancies between σ factors.

Results: Among the three alternative σ factors studied here, σH provides positive regulation for the largest number of proteins, consistent with previous transcriptomic studies, while σL appears to contribute to negative regulation of a number of proteins. σC was found to regulate a small number of proteins in L. monocytogenes grown to stationary phase at 37°C. Proteins identified as being regulated by multiple alternative σ factors include MptA, which is a component of a PTS system with a potential role in regulation of PrfA activity.

Conclusions: This study provides initial insights into global regulation of protein production by the L. monocytogenes alternative σ factors σL, σH, and σC. While, among these σ factors, σH appears to positively regulate the largest number of proteins, we also identified PTS systems that appear to be co-regulated by multiple alternative σ factors. Future studies should not only explore potential roles of alternative σ factors in activating a "cascade" of PTS systems that potentially regulate PrfA, but also may want to explore the σL and σC regulons under different environmental conditions to identify conditions where these σ factors may regulate larger numbers of proteins or genes.

Figure 1

Functional role category classification of alternative σ factor dependent proteins. Functional role category classification of σ^H positively-regulated (blue), σ^H negatively-regulated (red), σ^C positively-regulated (green), σ^C negatively-regulated (purple), σ^L positively-regulated (turquoise), and σ^L negatively-regulated (gray) proteins; and proteins with higher levels in L. monocytogenes parent strain 10403S (PAR.) compared to ΔBCHL (yellow) and lower levels in PAR. compared to ΔBCHL (orange). Role category numbers correspond to: (1) Amino acid biosynthesis; (2) Biosynthesis of cofactors, prosthetic groups, and carriers; (3) Cell envelope; (4) Cellular processes; (5) Central intermediary metabolism; (6) Energy metabolism; (7) Fatty acid and phospholipid metabolism; (8) Hypothetical proteins; (9) Protein fate; (10) Protein synthesis; (11) Purines, pyrimidines, nucleosides, and nucleotides; (12) Regulatory functions; (13) Transcription; (14) Transport and binding proteins; (15) Unclassified; (16) Unknown function; (17) Viral functions. One protein may be classified into more than one role category. Statistical analysis of contingency tables for regulons with > 10 proteins (i.e., proteins positively regulated by σ^H; proteins negatively regulated by σ^L; proteins with higher or lower levels in the parent strain) found that role categories were not randomly distributed among proteins negatively regulated by σ^L and proteins with lower levels in the parent strain.

Figure 2

Venn diagram of proteins identified as showing higher protein levels in comparisons of (i) L. monocytogenes parent strain 10403S (PAR.) and Δ BCHL ; (ii) ΔBCH and ΔBCHL (identifying genes positively regulated by σ^L); ΔBCL and ΔBCHL (identifying genes positively regulated by σ^H); and ΔBHL and ΔBCHL (identifying genes positively regulated by σ^C). Twelve of the 29 proteins that were found to be positively regulated in the parent strain were also found to be positively regulated by σ^B in a recent proteomics study, which compared L. monocytogenes parent strain 10403S and a ΔsigB mutant [23]; these proteins include Lmo2748, Lmo2213, Lmo2158, Lmo2047, Lmo1830, Lmo0913, Lmo0796, Lmo0794, Lmo0722, Lmo0654, Lmo0539, and Lmo0265. The 17 proteins that show higher levels in the parent strain as compared to the ΔBCHL strain, but were not identified as positively regulated by any of the alternative σ factors include Lmo1540, Lmo2610, Lmo1422, Lmo1421, Lmo1602, Lmo1426, Lmo1428, Lmo2205, Lmo2398, Lmo1601, Lmo0554, Lmo1634, Lmo0110, Lmo2558, Lmo0783, Lmo0134, and Lmo0098.