Methionine Sulfoxide Reductases Contribute to Anaerobic Fermentative Metabolism in Bacillus cereus

Abstract

Reversible oxidation of methionine to methionine sulfoxide (Met(O)) is a common posttranslational modification occurring on proteins in all organisms under oxic conditions. Protein-bound Met(O) is reduced by methionine sulfoxide reductases, which thus play a significant antioxidant role. The facultative anaerobe Bacillus cereus produces two methionine sulfoxide reductases: MsrA and MsrAB. MsrAB has been shown to play a crucial physiological role under oxic conditions, but little is known about the role of MsrA. Here, we examined the antioxidant role of both MsrAB and MrsA under fermentative anoxic conditions, which are generally reported to elicit little endogenous oxidant stress. We created single- and double-mutant Δmsr strains. Compared to the wild-type and ΔmsrAB mutant, single- (ΔmsrA) and double- (ΔmsrAΔmsrAB) mutants accumulated higher levels of Met(O) proteins, and their cellular and extracellular Met(O) proteomes were altered. The growth capacity and motility of mutant strains was limited, and their energy metabolism was altered. MsrA therefore appears to play a major physiological role compared to MsrAB, placing methionine sulfoxides at the center of the B. cereus antioxidant system under anoxic fermentative conditions.

Keywords: Bacillus cereus; anaerobiosis; methionine oxidation; methionine sulfoxide reductase.

Figure 1

Analysis of msrA and msrAB gene expression in B. cereus ATCC 14579 cells grown under anaerobiosis. mRNA levels of msrA (green) and msrAB (blue) were determined at the early exponential (EE), late exponential (LE) and stationary (S) growth phases by quantitative real-time RT-PCR and normalized to ssu mRNA levels. Samples were harvested as indicated in Figure 2. Data correspond to the mean ± SD of six samples (two technical replicates x three biological replicates). Data denoted by a common letter are not significantly different. Data denoted by different letters indicated a significant difference (two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, p ≤ 0.05).

Figure 2

Growth curves for B. cereus wild-type and msr mutant strains under anaerobic fermentative conditions. Wild-type strain ATCC 14579 (black), ΔmsrA (green), ΔmsrAB (blue), and ΔmsrAΔmsrAB mutants (red) were grown in MOD medium supplemented with 30 mM glucose. Data correspond to the mean ± SD of three biological replicates.

Figure 3

Met(O) content of B. cereus wild-type and msr mutant proteomes under anaerobic fermentative conditions. Wild-type strain ATCC 14579 (black), ΔmsrA (green), ΔmsrAB (blue), and ΔmsrAΔmsrAB mutants (red), were grown in MOD medium supplemented with 30 mM glucose. Samples were collected at early exponential (EE), late exponential (LE), and stationary (S) growth phases, as indicated in Figure 2. The relative Met(O) content in the cellular proteome (a) and exoproteome (b) was calculated as a percentage of oxidized methionine-containing peptides with respect to the total number of methionine-containing peptides identified in samples for each growth phase. Data correspond to the mean ± SD of three biological replicates. Within each panel, data denoted by a common letter are not significantly different. Data designed by different letters indicated a significant difference (two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, p ≤ 0.05).

Figure 4

Swimming motility. Representative colonies of wild-type (WT) and ΔmsrA, ΔmsrAB and ΔmsrAΔmsrAB mutants following growth on swimming TrA plates for 72 h under anaerobiosis.

Figure 5

Schematic view of the roles of MsrA and MsrAB on B. cereus physiology under anaerobic fermentative conditions. MsrA has a more important role in fermentative metabolism, compared to MsrAB (thick green vs. thin blue solid arrows). The role of MsrA could be as important as the role of MsrAB in maintaining Met(O) levels of virulence factors, such as flagella and enterotoxins (green and blue dotted arrows). PTS, phosphotransferase system; PP, pentose phosphate pathway. Succinate is formed via the reductive TCA cycle.