Growth Phases and PHB Intracellular Mobilization of B. cereus tsu1
Bacillus cereus tsu1 was 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 aggregated and formed clusters, and reached the highest accumulation before 12 h. Bacterial cells were collected at 12 h, 24 h, 48 h and stored at -20 °C for protein extraction. The reasons for selecting these three time points were 1) bacterial cells in 12h-culture were loaded with PHB when examined using the Sudan black staining method; 2) in 24h-culture, most cells were still filled with PHB, but some cells were sporulating with fore-spore, and spore structure visible under the microscope; 3) significant degradation of PHB was observed in 48h-culture, and even though some mature endospores were released, most cells were still in vegetative state.
Quantitative Proteomic Profile and Identification of Significantly Changed Proteins
SDS phenol based method was used for proteins extraction from bacterial cells cultured in RCS medium for 12, 24 and 48 h. Three biological replicates were 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 were quantified each with two or more unique peptides (Additional file 3: Table S3). The log2-transformed abundance of all constituent peptides were subjected to a t-test followed by false discovery rate (FDR) correction analysis. A protein with ≥ 1.5 standard deviations from normal distribution curve of each quantified protein and a FDR adjusted p-value ≤ 0.05 was regarded as being significantly changed proteins (SCPs) for each pair of sampling time-points (24h/12h, or 48h/12h). Protein fold changes were obtained from anti-log conversion of log2 ratios. When comparing 24h- and 12h-samples, there were 244 significantly changed proteins (SCPs) which passed the thresholds [FDR < 0.05, and fold changes (24h/12h) < 0.76 or > 1.31], including 56 up-regulated and 188 down-regulated proteins; in 48h-12h pair of samples, 325 proteins passed the thresholds [FDR < 0.05, and fold changes (48h/12h) < 0.67 or > 1.50], with 145 proteins up-regulated and 180 down-regulated proteins (Fig. 2A, Additional file 1: Table S1-1). Results of t-test and FDR analyses using SAS were listed in the Additional file 2: Table S2-1, Table S2-2.
The identified SCPs were analyzed for functional classification using the PANTHER classification system (v.14.1). The biological processes enriched only in 24h:12h up-regulated SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress; the 24h down-regulated SCPs are classified into processes of carboxylic acid catabolism, cellular amino acid catabolism, peptidoglycan biosynthetic process, RNA process. The 48h:12h SCPs were enriched into biological processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, formation of translation ternary structure (Fig. 2B).
Enzymes for PHB Biosynthesis and Intracellular Degradation
The maximum of PHB accumulation in B. cereus tsu1 was observed within 12 h. According to a previous study [20], the B. cereus tsu1 was annotated with genes in three different pathways for PHB polymerization (Fig. 3). The primary pathway starts with acetyl-CoA, uses enzymes encoded by a pha locus which consists of phaR-phaB-phaC operon and phaP-phaQ-phaJ operon in the opposite direction. The second pathway is using intermediates of fatty acid β-oxidation and catalyzed by acyl-CoA dehydrogenase (AcdA_1 and AcdA_2) and 3-hydroxybutyryl-CoA dehydratase/ enoyl-CoA hydratase (PhaJ). The third pathway involves succinyl-CoA from TCA cycle to produce PHB [21]. And this pathway is catalyzed by SSA dehydrogenase (GabD, KGT45610), 4-hydroxybutyrate dehydrogenase (GabT, KGT45608), and succinyl-CoA-coenzyme A transferase enzyme (ScoT) [7]. STRING database (version 10.5) of B. cereus was used for protein-protein interaction network construction of all enzymes in the three PHB synthesis pathways (Fig. 3). In the 48h-sample, PHB was observed to have undergone significant degradation. For PHB degradation, the enzyme 3-oxoadipate enol-lactonase which previously confirmed with PHB intracellular degradation activity in B. thuringiensis ATCC35646 [22] was annotated on the B. cereus tsu1 genome, and this protein was quantified in this study.
Enzymes for PHB biosynthesis and intracellular degradation and their abundance were compared among the three time-point samples (Fig. 3). Poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) had the highest abundance level at early stage of bacterial growth, while the synthase subunit PhaR (KGT44863) displayed an opposite change. PhaR protein was reported as a global regulation factor, with an impact on PHB biosynthesis [23, 24]. Both 3-oxoacyl-ACP synthase (PhaB, KGT44864) and phasin protein (PhaP, KGT44861) accumulated to the highest abundance level at 48h. PhaQ (KGT44862), which previously identified as a new class of PHB synthesis transcription regulator, was not identified in the proteome analysis. Both AcdA_2 (KGT41138) and PhaJ (KGT44860) involved in PHB biosynthesis using fatty acid β-oxidation intermediate had a higher abundance level at 12 h [25]. Additionally, a majority of enzymes converting glutamate and GABA to PHB were observed with a higher abundance level at 12 h. ScoT (KGT44257) is an enzyme associated with both PHB synthesis and consumption; its abundance reached the highest level in 48h-sample. Despite of significant PHB degradation observed at 48 h, the abundance of 3-oxoadipate enol-lactonase (KGT42842) for PHB depolymerization was at highest level at 12 h and slightly reduced over time.
PHB Mobilization and Related Metabolic Pathways
In Bacillus spp., PHB formation and mobilization are important metabolic processes interacting with other major pathways. As shown in Fig. 4A, PHB biosynthesis starts with acetyl-CoA, which is a molecule that participates in several essential biochemical reactions including glycolysis, lipid and protein metabolism, TCA. PHB mobilization and recycling provide carbon and energy resource for other metabolic pathways such as pyruvate fermentation and butanoate metabolism [26].
In this study, most enzymes in Embden-Meyerhof-Parnas (EMP) pathway, pentose phosphate (PP) pathway, and TCA cycle did not show significant changes among the three time points (Additional file 1: Table S1-2). In EMP, glucose-6-phosphate isomerase (KGT41362) was significantly down-regulated at 0.7 and 0.58 fold in 24h- and 48h-samples. In PP, 6-phosphogluconate dehydrogenase (KGT42918) was down-regulated by 0.66 fold at 48 h. In glyoxylate shunt bypass of TCA, malate synthase (KGT44986), isocitrate lyase (KGT44987) was down-regulated at 0.61 and 0.66 fold respectively at 48 h.
Butanoyl-CoA converted from acetyl-CoA is another major carbon metabolic product. Using 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 provides energy source for cells, as ATP is generated. This two-step conversion process is catalyzed by phosphate butyryltransferase (KGT41693) and butyrate kinase (KGT41691). Phosphate butyryltransferase was up-regulated at 1.86 fold at 48 h, and butyrate kinase had a higher abundance at 48 h compared to the other two time points (Fig. 4B, Table 1).
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 at 2.11 and 2.2 fold in 48h-culture (Fig. 4B, Table 1). Lactate dehydrogenase (KGT41354) catalyzing the interconversion of pyruvate to lactate was up-regulated at 1.81 fold; lactate utilization protein C (KGT44853), L-lactate dehydrogenase complex protein LldF (KGT44852) and formate dehydrogenase (KGT45530) were up-regulated at 1.84, 1.51 and 1.5 fold respectively in the same culture. For alcohol fermentation, acetyl-CoA is first converted to acetaldehyde by acetaldehyde dehydrogenase (KGT41893), and then to alcohol by ethanol-active dehydrogenase (KGT44011), the latter protein was up-regulated at 1.68 fold. The acetyl-CoA hydrolase (KGT44257) catalyzing the reaction producing acetate from acetyl-CoA also had the highest abundance in 48h-sample.
Acetoin or 3-hydroxybutanoate is another form of carbon and energy storage produced and excreted by bacteria when the pyruvate level is high [31, 32]. It can be used to provide energy for other metabolic pathways at stationary phase [33]. In B. cereus tsu1, acetolactate synthase catalytic subunit (KGT44244) and regulatory subunit (KGT44245), acetolactate synthase (KGT44547) and catalytic subunit (KGT44546), acetolactate synthase (KGT45211) were observed with higher abundance level in 12h-culture (Fig. 4B, Additional file 1: Table S1-3). Acetolactate decarboxylase (KGT45212) was not identified in this proteomics analysis. The acu operon comprising of acetoin-reuptake enzymes- acetoin dehydrogenase (KGT42181), acetoin utilization protein (KGT42182), histone deacetylase (KGT42183) were not observed with significant changes in abundance. Whereas enzymes in the aco operon converting acetoin into acetaldehyde and acetyl-CoA were all up-regulated at 48 h (Table 1), which include dihydrolipoamide dehydrogenase (KGT43462, 1.72 fold), and 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 at 1.64 fold in 48h-culture [34].
Sporulation and Stress-induced Enzymes
In batch-culture process, bacteria are facing constant stresses such as nutrient depletion and suboptimal pH levels. For gram-positive bacteria like Bacillus spp., self-rescue 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 B. cereus tsu1, stress related proteins were identified with significant changes (Fig. 5A, Table 1). Glyoxalase/ lactoylglutathione lyase (KGT43173, KGT42737, KGT42638, KGT44383) [38], chemotaxis protein (KGT45443, KGT41216) [39], activator of Hsp90 ATPase (KGT43768) were significantly higher at 12 h (late exponential phase) compared to 24 h (stationary phase). Molecular chaperone Hsp20 (KGT44005), chaperonin (KGT45779) [7], copper resistance protein CopZ (KGT42404), and RNA-binding protein Hfq (KGT42386) [40] were significantly higher at 24 h. Flagellar hook protein FlgL (KGT44525), 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 S1-4). As the proteins interaction network displayed in Fig. 5B, chemotaxis protein CheY/Spo0A (KGT41699), sporulation sigma factor SigF (KGT41601), anti-sigma F factor (SpoIIAB, KGT41602); anti-sigma F factor antagonist (SpoIIAA, KGT41603), stage II sporulation protein E (SpoII E, KGT45993) are key enzymes involved in sporulation [43, 44]. SigF is the essential enzyme for Bacillus spp. sporulation induction. Anti-sigma F factor is the antagonist of SigF, whose activity can be diminished by SpoII E under the regulation of Spo0A [45]. From our results, SigF and SpoII E were up-regulated at 1.43 fold and 1.5 fold, whereas, anti-sigma F factor was down-regulated at 0.65 fold in 24h-culture. 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 at 1.75 fold and 2.75 fold in 24h- and 48h-cutlures, respectively.
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 (Additional file 1: Table S1-5) [43, 49]. Most atp operon proteins had higher abundance at early stage (12 h), and ATP synthase F0 subunits B (KGT41105) was significantly higher in 12h-sample compared to the other two time points (Table 1). In 48h-culture, complex III menaquinol-cytochrome C reductase (KGT44670), and complex IV--quinol oxidase subunit 2 (KGT45463, QoxA), cytochrome D ubiquinol oxidase subunit I (KGT42309, CydA) were significantly up-regulated at 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 proteomics analysis, nitrate reductase NarG, NarH, NarJ, (KGT44113, 44114, 44115) were significantly up-regulated at 2.47, 1.92, 2.75 fold; nitrite reductase NirD, NirB (KGT44130, 44131) were up-regulated at 1.92, 2.48 fold in 48h-culture (Table 1). These results indicate that, at this time point, the cellular metabolism pathways were changing towards nitrate respiration and fermentation.