Growth Phases and PHB Intracellular Mobilization of B. cereus “tsu1’. Bacillus cereus tsu1 were cultured using RCS medium and cells were stained with Sudan Black to observe PHB accumulation status (Fig. 1). In 6h-culture, PHB granules were observable but smaller in size. In 9h-culture, the granules grew and formed clusters, and before 12h-culture reached the highest accumulation. Cell samples were collected at 12 h, 24h, 48h and stored at –20 °C for protein extraction. The reasons for selecting these three time points were 1) bacterial cells in 12h-culture, when examined using the Sudan black staining method were loaded with PHB; 2) in 24h-culture, most cells were still filled with PHB but some cells were sporulating with fore-spore, and spore structure clearly observable under the microscope; 3) significant degradation of PHB was observed after 48-h culture, and even though some mature endospores were released, most cells were still in vegetative state.
Quantitative Proteomic Profile and Significantly Changed Proteins Identification. Proteins were extracted from bacterial cells grown in RCS medium and harvested by centrifugation at three time-points 12, 24 and 48 hours, with three biological replicates included for each time point. After trypsin digestion, samples were labeled with nine tags from a 10-plex tandem mass tags (TMT) kit. The nano-LC-MS/MS identified 3,215 proteins, from where 2,952 proteins got quantified. Proteins identified with at least two unique peptides were subjected to a quantification study using t-test followed by false discovery rate (FDR). A protein with ≥ 1.5 SD-fold difference and a FDR adjusted p-value ≤ 0.05 was regarded as being differentially expressed in our data. When comparing 24h and 12h samples (24h–12h), 244 significantly changed proteins (SCP) passed the thresholds [FDR<0.05, and fold values (24h/12h) <0.76 or >1.31]. When comparing 48h and 12h samples, 325 proteins passed the thresholds [FDR<0.05, and fold values (48h/12h) <0.67 or >1.50]. In the 24h–12h pair, 56 proteins were up-regulated, and 188 proteins down-regulated; in 48h–12h pair, 145 proteins got up-regulated, and 180 proteins got down-regulated (Fig. 2A, Additional file 1: Table S1–1). Results of t-test and FDR analyses performed using SAS were listed in the Additional file 2.
Gene functional classification of the SCPs was identified using the PANTHER classification system (v.14.1). Fold enrichment of functional categories of 24 up-regulated SCPs (blue), 48 up-regulated SCPs (green), and 24 dn-regulated SCPs (red), 48 dn-regulated SCPs (orange) are displayed in Fig. 2B. The enriched biological processes only found in 24h up-regulated SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress; in 24h dn-regulated SCPs include carboxylic acid catabolism, cellular amino acid catabolism, peptidoglycan biosynthetic process, RNA process. For comparison between 12h and 48h sample, the biological processes enriched in the SCPs were carbohydrate metabolism, protein metabolism, oxidative phosphorylation, formation of translation ternary structure.
Enzymes for PHB Biosynthesis and Intracellular Degradation. Maxima of PHB accumulation in B. cereus ‘tsu1’ was observed reaching within 12 h. According to a previous study [20], the B. cereus ‘tsu1’ genome was predicted with three-pathway genes to produce monomers for PHB polymerization (Fig. 3). The primary pathway starts with acetyl-CoA, and through enzymes encoding by a pha locus which consists the phaR-phaB-phaC operon and phaP-phaQ-phaJ operon in the opposite direction to for PHB polymer. In the second pathway, intermediates of fatty acid β-oxidation could flow into the PHB synthesis pathway catalyzed by acyl-CoA dehydrogenase (AcdA_1 and AcdA_2) and 3-hydroxybutyryl-CoA dehydratase (PhaJ). PhaJ is the key enzyme to provide (R)–3HB-CoA monomer for PHB synthesis. Another pathway involves using succinyl-CoA from TCA cycle to produce PHB [21]. Succinyl-CoA is first converted to SSA by SSA dehydrogenase (GabD, KGT45610) followed by reduction of SSA into GABA by 4-hydroxybutyrate dehydrogenase (GabT, KGT45608). GABA is activated to R-hydroxybutyryl-CoA by a Succinyl-CoA-coenzyme A transferase enzyme (ScoT, KGT44257). The R-hydroxybutyryl-CoA could then be polymerized into PHB by PhaC [7]. STRING database (version 11.0) of B. cereus was used for protein-protein interaction network construction. In the 48h-sample, PHB was observed to have undergone significant degradation. For PHB degradation pathway, the PHB depolymerase PhaZ was not annotated on ‘tsu1’ genome. Enzyme 3-oxoadipate enol-lactonase was previously confirmed with PHB intracellular degradation activity in B. thuringiensis ATCC35646 [22]; its homologous protein (KGT42842) was annotated on the B. cereus tsu1 genome, and got quantified in this study as well.
PHB biosynthesis and intracellular degradation enzymes and their average abundance were compared among three time-point samples (Fig. 3). Poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44864) had the highest abundance level at early stage of bacterial growth, while the synthase subunit PhaR (KGT44863) showed an opposite change. PhaR protein was reported as a global regulation factor, not just influencing PHB biosynthesis [23, 24]. Both 3-oxoacyl-ACP synthase (PhaB, KGT44864) and phasin protein (PhaP, KGT44861) showed the highest abundance at 48h. PhaQ (KGT44862), previously identified as a new class of PHB synthesis transcription regulator, was not identified in this proteome profile. Both AcdA_2 (KGT41138) and PhaJ (KGT44860) involving in PHB biosynthesis using fatty acid β-oxidation intermediate had a higher abundance level at 12h [25]. Most of the enzymes converting glutamate and GABA to PHB showed higher abundance level at 12h. ScoT (KGT44257) is the enzyme involved both in PHB synthesis and consumption; its abundance reached the highest level in 48h sample. Even significant PHB degradation was observed at 48h, the abundance of 3-oxoadipate enol-lactonase (KGT42842) was higher at 12h and slightly reduced over time.
PHB mobilization and other metabolism pathways. In Bacillus spp., PHB formation and mobilization are important metabolism processes interacting with other major pathways. As Fig. 4A displays, PHB biosynthesis starts with acetyl-CoA, which is a molecule that participates in many biochemical reactions including glycolysis, lipid and protein metabolism, TCA. PHB mobilization and reuse provide carbon and energy resource for other metabolism pathway like pyruvate fermentation and butanoate metabolism [26]. Also, bacteria undergo incomplete PHB mobilization were observed with deficient sporulation and lower stress tolerance.
In this study, most enzymes involved in the EMP pathway, PP pathway, and the TCA cycle were not observed with significant change among the three time-points (Additional file 1: Table S1–3). In EMP, glucose–6-phosphate isomerase (KGT41362) was significantly down-regulated (0.7 fold) in 24h and (0.58 fold) in 48h; In PP, 6-phosphogluconate dehydrogenase (KGT42918) was down-regulated (0.66 fold) in 48h; In glyoxylate shunt bypass of TCA, malate synthase (KGT44986) was down-regulated (0.61 fold) in 48h; isocitrate lyase (KGT44987) was down-regulated (0.66 fold) in 48h. (All fold changes mentioned in the results are the abundance ratio between either 24h to 12h, or 48h to 12h samples.)
Enzymes in Butanoate Metabolism Butanoyl-CoA synthesized from acetyl-CoA is another major carbon metabolism product, through this pathway, bacteria can produce butanoate when grown at neutral pH on glucose [27]. The first step in this pathway is identical with PHB biosynthesis, afterward, acetoacetyl-CoA is converted to (S)–3-hydroxybutanoyl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (KGT41139). The final two-step conversion of butanoyl-CoA to butanoate will provide energy source for cells, as ATP is generated. This two-step conversion is catalyzed by phosphate butyryltransferase (KGT41693) and butyrate kinase (KGT41691). Phosphate butyryltransferase was up-regulated (1.86 fold) at 48h, and butyrate kinase also had a higher abundance at 48h compared to 12h and 24h (Additional file 1: Table S1–4, Fig. 4B).
Enzymes in Pyruvate Fermentation. Bacillus spp. can grow by substrate-level phosphorylation/ fermentation under anoxic condition [28]. In B. cereus ‘tsu1’, formate acetyltransferase (KGT45740) and pyruvate formate lyase-activating protein (KGT45741), which catalyze the reversible conversion of pyruvate into Acetyl-CoA using radical non-redox mechanism [29, 30], were up-regulated 2.11 fold and 2.2 fold in 48h-culture (Fig. 4B, Additional file 1: Table S1–4). Lactate dehydrogenase (KGT41354) catalyzing the interconversion of pyruvate to lactate was up-regulated 1.81 fold at 48h. Lactate utilization protein C (KGT44853) and L-lactate dehydrogenase complex protein LldF (KGT44852) were up-regulated 1.84 fold and 1.51 fold respectively in 48h-culture. Formate dehydrogenase (KGT45530) was up-regulated 1.5 fold at 48h. For alcohol fermentation, acetyl-coA was first converted to acetaldehyde by acetaldehyde dehydrogenase (KGT41893), and then to alcohol by ethanol-active dehydrogenase (KGT44011), the latter protein was up-regulated 1.68 fold at 48h. The acetyl-CoA hydrolase (KGT44257) catalyzing the reaction producing acetate from acetyl-coA also had the highest abundance at 48h sample.
Enzymes in Acetoin and 2,3-Butanediol synthesis. Acetoin or 3-hydroxybutanoate is another form of carbon and energy storage produced and excreted by bacteria when the pyruvate level is high [31]. At exponential phase, acetoin production and secretion can prevent cytoplasm and extracellular environment from over-acidification caused by acetic acid and citric acid [32]. Then at stationary phase, it can be used to provide energy for other metabolic pathways [33]. In ‘tsu1’, acetolactate synthase catalytic subunit (KGT44244) and regulatory subunit (KGT44245), acetolactate synthase (KGT44547) and catalytic subunit (KGT44546), and acetolactate synthase (KGT45211) were observed with higher abundance level at 12h (Fig. 4B, Additional file 1: Table S1–4). Acetolactate decarboxylase (KGT45212) was not identified in the proteome profile. Even though, the operon encoding for acetoin-reuptake enzymes- acetoin dehydrogenase (KGT42181), acetoin utilization protein (KGT42182), histone deacetylase (KGT42183) were not observed with significant abundance change; enzymes in the operon to convert acetoin into acetaldehyde and acetyl-CoA were all up-regulated at 48h, which include dihydrolipoamide dehydrogenase (KGT43462, 1.72 fold), branch-chain alpha-keta acid dehydrogenase subunit E2 (KGT43463), acetoin dehydrogenase E1β component (KGT43464, 1.59 fold), acetoin dehydrogenase E1α component (KGT43465, 2.59 fold). R,R-butanediol dehydrogenase (KGT45433) catalyzing the reversible oxidation of 2,3-butanediol to acetoin and the practically irreversible reduction of diacetyl to acetoin was up-regulated 1.64 fold at 48h [34].
Sporulation and stress-induced enzymes. In batch-culture process, bacteria are facing constant stresses such as nutrition depletion and suboptimal pH levels. For gram-positive bacterium like Bacillus, self-rescuing mechanisms under nutrient limitation and environmental stress include induction of chemotaxis protein [35], production of antibiotics [36], secretion of hydrolytic enzymes [37], and finally sporulation. In 24h-sample, pre-spore and spore structures were observed; in 48h-sample, mature endospores were released, meanwhile significant PHB degradation occurred.
In the quantitative proteomic analysis of ‘tsu1’, stress related proteins were identified with significant changes (Fig. 5A, Additional file 1: Table S1–5). Glyoxalase/ lactoylglutathione lyase (KGT43173, KGT42737, KGT42638, KGT44383) [38], chemotaxis protein (KGT45443, KGT41216) [39], activator of Hsp90 ATPase (KGT43768) were significantly higher at 12h (late exponential phase) compared to 24h (stationary phase). Molecular chaperone Hsp20 (KGT44005), chaperonin (KGT4577) [7], copper resistance protein CopZ (KGT42404), and RNA-binding protein Hfq (KGT42386) [40] were significantly higher at 24h compared to 12h culture. Flagellar hook protein FlgL (KGT44525), and flagellin (KGT44484), molecular chaperone DnaJ (KGT45678), disulfide bond formation protein DsbD (KGT45484), anti-terminator HutP (KGT42538) [41], general stress protein (KGT41365), PhoP family transcriptional regulator (KGT42051) [42], sigma–54 modulation protein (KGT40985) and stress protein (KGT43053) had the highest abundance level in 48h culture.
Thirty-eight proteins related to sporulation were identified with significant change over time (Fig. 5A, Additional file 1: Table S1–5). Proteins with interaction network are displayed in Fig. 5B, chemotaxis protein CheY/Spo0A (KGT41699), sporulation sigma factor SigF (σF, KGT41601), anti-sigma F factor (spoIIAB, KGT41602); anti-sigma F factor antagonist (spoIIAA, KGT41603), stage II sporulation protein E (KGT45993) are key enzymes involved in sporulation [43, 44]. Sporulation sigma factor SigF (KGT41601) is the essential enzyme for Bacillus sporulation induction. Anti-sigma F factor (KGT41602) is the antagonist of SigF, whose activity can be diminished by SpoII E (KGT45993) under the regulation of Spo0A [45]. From our result, σF was up-regulated 1.43 fold at 24h, SpoII E was up-regulated 1.5 fold at 24h, anti-sigma F factor was down-regulated 0.65 fold at 24h. The transition state regulator Abh (KGT44175) acts as a transcriptional regulator during the transition state from vegetative growth to stationary phase and sporulation [46], this protein was up-regulated 1.75 fold at 24h and 2.75 fold at 48h.
Aerobic respiration and anaerobic respiration. In aerobic bacteria, oxidative phosphorylation is the major metabolic pathway using carbohydrate oxidation to generate ATP. Most ATP molecules are synthesized by five membrane-bound enzyme complexes (electron transport chain system), which include complex I—NADH: ubiquinone oxidoreductase/ NADH dehydrogenase [47], complex II—succinate-Q oxidoreductase/ succinate dehydrogenase [48], complex III—menaquinol-cytochrome c oxidoreductase, complex IV—quinol/cytochrome c oxidase, and complex V—F0F1-ATPase (ATP synthase) [43, 49]. Most atp operon proteins had higher abundance at early stage (12h), and ATP synthase F0 subunits B (KGT41105) was significantly higher in 12h sample compared to the other two time points (Additional file 1: Table S1–6). In 48h-culture, complex III Menaquinol-cytochrome C reductase (KGT44670), complex IV—Quinol oxidase subunit 2 (KGT45463, QoxA), cytochrome D ubiquinol oxidase subunit I (KGT 42309, CydA) were significantly up-regulated by 1.77 fold, 1.57 fold, 2.33 fold, respectively.
For Bacillus spp., the final electron acceptors can also be nitrate, nitrite, nitrous oxide other than O2 when respiration happens under anaerobic condition [50, 51]. In our quantitative analysis, nitrate reductase NarG, NarH, NarJ, (KGT44113, 44114, 44115) were significantly up-regulated by 2.47, 1.92, 2.75 fold; nitrite reductase NirD, NirB (KGT44130, 44131) were up-regulated by 1.92, 2.48 fold in 48h-culture. This result indicated that, at this time point, the cellular metabolism pathways are changing towards nitrate respiration and fermentation.