Time dynamics of the Bacillus cereus exoproteome are shaped by cellular oxidation

Abstract

At low density, Bacillus cereus cells release a large variety of proteins into the extracellular medium when cultivated in pH-regulated, glucose-containing minimal medium, either in the presence or absence of oxygen. The majority of these exoproteins are putative virulence factors, including toxin-related proteins. Here, B. cereus exoproteome time courses were monitored by nanoLC-MS/MS under low-oxidoreduction potential (ORP) anaerobiosis, high-ORP anaerobiosis, and aerobiosis, with a specific focus on oxidative-induced post-translational modifications of methionine residues. Principal component analysis (PCA) of the exoproteome dynamics indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their methionine sulfoxide (Met(O)) content. PCA also revealed an interesting interconnection between toxin-, metabolism-, and oxidative stress-related proteins, suggesting that the abundance level of toxin-related proteins, and their Met(O) content in the B. cereus exoproteome, reflected the cellular oxidation under both aerobiosis and anaerobiosis.

Keywords: Bacillus cereus; exoproteome; methionine oxidation; shotgun proteomics; toxins.

Figure 1

Growth curves of B. cereus in pH-regulated batch culture under aerobiosis, high-ORP anaerobiosis, and low-ORP anaerobiosis. The results from biological triplicate curves are indicated. Optical densities (OD_{600 nm}) and ORP values are shown in blue and red, respectively. Samples for exoproteomic analyses were taken during the exponential growth phase (EE), late growth phase (LE), and stationary growth phase (S), as indicated by black arrows.

Figure 2

Venn diagram–based comparison of the exoproteomes identified in the three growth conditions. (A) Number of identified proteins in the exoproteomes obtained from aerobically, and high-ORP and low-ORP anaerobically grown cells. (B) Distribution of proteins specifically detected in one growth stage (EE, LE, S) by function of the growth conditions.

Figure 3

Relative abundance of MS/MS-detected polypeptides under aerobiosis, high-ORP anaerobiosis, and low-ORP anaerobiosis. Ratio expressed in percentage of the total numbers of polypeptides (CDS), spectral counts (SC), and protein abundance normalized by the corresponding molecular weight (NSAF) per functional category are represented graphically by stacked bars for each condition.

Figure 4

Principal component analysis of the B. cereus exoproteome. (A) Fractions of the variances borne by axes 1–8. (B) Growth phase contributions to the first two principal components (PC1 and PC2), under low-ORP anaerobiosis, high-ORP anaerobiosis, and aerobiosis. Protein clusters assigned to growth phases were indicated by (i) the same capital letter (A) when they did not show abundance level change in these growth phases, or (ii) different capital letters (A,B) when the proteins showed negative correlation with abundance level changes. (C) Relative number of proteins assigned to toxins, degradation/adhesion, motility, metabolism, stress/chaperone, and “others” functional groups in protein clusters determined by PCA. Each functional group is represented by a color.

Figure 5

Dynamics of exoproteome Met(O) content under low-ORP anaerobiosis, high-ORP anaerobiosis, and aerobiosis. (A) The Met(O) content was calculated as the percentage of the number of all detected Met(O) peptides vs. the total number of MS/MS spectra. (B) Only the peptides assigned to proteins that co-clustered in CLM1 (Table S6) were considered. Data are the means of triplicate measures obtained from three independent cultures in each growth condition at the EE, LE, and S growth phases. Significant differences (p < 0.05 in Student's t-test) between two growth phases are indicated with asterisks.

Figure 6

Characteristics of the protein cluster CLM1 determined by PCA. Number of proteins assigned to toxins, degradation/adhesion, motility, metabolism, stress/chaperone, and “others” functional groups that co-clustered in CLM1 under low-ORP anaerobiosis, high-ORP anaerobiosis, and aerobiosis. The number of proteins with correlated (c) and uncorrelated (nc) abundance levels and Met(O) content changes is indicated for each growth condition.

Figure 7

Amino acid sequence of NheA. Peptides detected by LC-MS/MS are shown in red and are underlined. Met residues are shown in bold.