Comparative proteomic analysis of antagonistic Bacillus amyloliquefaciens Q-426 cultivated under different pH conditions
Jing Zhao1,2 Pengchao Zhao3 ∗ Chunshan Quan1,2 1,2 Liming Jin Wei Zheng1,2 Shengdi Fan1,2
1 Key
Laboratory of Biochemical Engineering (State Ethnic Affairs Commission-Ministry of Education), Dalian Nationalities University, Dalian, People’s Republic of China
2 College
of Life Science, Dalian Nationalities University, Dalian, People’s Republic of China
3 School
of Medical Technology and Engineering, Henan University of Science and Technology, Luoyang, People’s Republic of China
Abstract Bacillus amyloliquefaciens Q-426 produces lipopeptide compounds with antifungal activities. Initial pH value has a significant influence on the production of lipopeptide compounds. The correlation between pH and intrinsic mechanism of lipopeptide production was rarely discussed. In this research, comparative proteomics, using two-dimensional gel electrophoresis and mass spectrometry, was applied to identify B. amyloliquefaciens Q-426 intracellular proteins differentially expressed under initial pH 5.0 and 7.3. A total of 24 differential spots (eight downregulated and 16 upregulated) under pH 5.0 were identified. Certain proteins were involved in
the regulation of bacillomycin and fengycin production by B. amyloliquefaciens Q-426. These proteins include four induced proteins related to stress response: Thiamine pyrophosphate-dependent acetoin dehydrogenase, butanediol dehydrogenase, two ABC-type oligopeptide transport system proteins, and two-component response regulator DegU and chorismate mutase PheB. These results indicate intrinsic differences of antagonistic B. amyloliquefaciens Q-426 under C 2014 International Union of Biochemistry different pH conditions. and Molecular Biology, Inc. Volume 00, Number 00, Pages 1–8, 2014
Keywords: Bacillus amyloliquefaciens, lipopeptide, mass spectrometry, pH, proteomic analysis, two-dimensional gel electrophoresis
1. Introduction Bacillus amyloliquefaciens belongs to Bacillus genus, and it is a Gram-positive bacteria having high similarity with Bacillus subtilis. In recent years, researchers discovered that B. amyloliquefaciens could produce mainly two types of antibacterial compounds: bacteriocins and bacteriocins-like inhibitory substances synthesized by ribosomal ways, as well as lipopep-
Abbreviations: HPLC-MS, high performance liquid chromatography-mass spectrometry; IEF, isoelectric focusing; PMF, peptide mass fingerprinting; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry . ∗ Address for correspondence: Professor Chunshan Quan, College of Life Science, Dalian Nationalities University, Dalian 116600, People’s Republic of China. Tel.: +86 411 8765 6219; Fax: +86 411 8765 6219; e-mail: [email protected]. Received 28 June 2014; accepted 5 September 2014 DOI: 10.1002/bab.1293
Published online in Wiley Online Library (wileyonlinelibrary.com)
tides and polyketides synthesized by nonribosomal ways [1–9]. Lipopeptide compounds produced by Bacillus sp. is divided into three types: surfactin family, iturin family, and fengycin family. Lipopeptide compounds, with stable antibacterial activity, broad antimicrobial spectrum, and environmental benefits, have become more attractive recently and have the potential to replace conventional antibiotics [10]. However, a relatively low yield of lipopeptides and difficulties in the separation of lipopeptide mixture greatly restrict their application in agricultural and pharmaceutical industry. Thus, screening regulation factors of lipopeptide production is of great significance. Composition and yield of lipopeptides were determined by complicated cultivation conditions [11–13]. Our previous studies showed that initial pH value, which varied with medium contents, could obviously influence lipopeptide production of B. amyloliquefaciens Q-426. The yield of lipopeptides was relatively stable under neutral and alkaline conditions; however, it declined under acidic conditions. There was no lipopeptide production below pH 5.0. This conclusion was obtained by the measurement of inhibitory activities of fermentation
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rials Vibra Cell VCX 130). The homogenate was centrifuged at 20,000g and 4 ◦ C for 20 Min. The supernatant was mixed with cold acetone at the ratio of 1:4 (v/v) and kept overnight at –20 ◦ C, and then it was centrifuged again at 20,000g and 4 ◦ C for 20 Min. After centrifugation, the supernatant was discarded. The precipitation was suspended with cold acetone solution (90% of acetone) containing 10 mM DTT and placed for 1 H at 4 ◦ C. The sample was centrifuged for at 12,000g and 4 ◦ C 10 Min. The precipitation was air-dried and solubilized in hydration liquid (8 M urea, 2% CHAPS, 0.5% IPG buffer, pH 3–10, and 10 mM DTT without bromophenol blue). The solution was centrifuged at 20,000g and 4 ◦ C for 20 Min. The supernatant was stored at –80 ◦ C. The protein samples were purified using 2D Clean Up kit (GE Healthcare, Stockholm, Sweden) and quantitatively measured using Modified BCA Protein Assay kit (Sangon Biotech Co., Shanghai, People’s Republic of China).
2.3. Two-dimensional gel electrophoresis
2. Materials and Methods 2.1. Strains and culture conditions B. amyloliquefaciens Q-426 (GenBank accession no. HM130462) was separated from compost samples and stored in LuriaBertani (LB) broth at –80 ◦ C as 30% glycerol stocks in our laboratory. The strain was cultivated in 250-mL Erlenmeyer flasks containing 50 mL of LB medium at 37 ◦ C and 180 rpm for 12–16 H. The above culture was inoculated in the fermentation medium with the inoculum concentraion of 1% for propagation. The fermentation medium composition was as follows (g/L): typsin (Oxoid), 12.40; glucose, 20.00; NaCl, 5.00; K2 HPO4 ·3H2 O, 1.50; MnSO4 ·H2 O, 0.04; FeSO4 ·7H2 O, 1.67; MgCl2 ·6H2 O, 1.22. The cultures were incubated at 30 ◦ C and 180 rpm for lipopeptide production.
2.2. Extraction and purification of intracellular proteins B. amyloliquefaciens Q-426 was cultivated in the fermentation medium with initial pH 7.3 and 5.0 separately at 30 ◦ C for 40 H. The fermentation broth was then centrifuged at 5,000g and 4 ◦ C for 10 Min. The precipitated cells were washed in the following order: initially they were washed twice with ultrapure water to remove medium components and other contaminants, then they were washed three times with TE buffer (10 mM Tris–HCl, 1 mM disodium ethylenediamine tetraacetate, pH 8.0) to remove Fe2+ in the fermentation mdium, and finally they were washed twice with cell washing solution (250 mM mannitol, 20 mM Tris–HCl, pH 7.5). The harvested cells were used for the extraction of intracellular protein. The cells were resuspended in a precooled homogenization buffer (7 M urea, 2 M thiourea, 2% 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% immobilized pH gradient (IPG) buffer, pH 3–10, 10 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride) at the ratio of 1:4 (w/v). The samples were lysed by sonication for 10 Min (2 Sec with 3 Sec intervals on an ice bath; Sonics & Mate-
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The first-dimensional protein isoelectric focusing (IEF) was performed using IPG strips (13 cm) with a linear pH range [3–10] in an Ettan IPGphor Isoelectric Focusing system (GE Healthcare). Eight hundred micrograms of protein sample was mixed with 300 µL hydration liquid (8 M urea, 2% CHAPS, 0.5% IPG buffer, pH 3–10, and 10 mM DTT). The IEF programs were performed at 30 V for 12 H, 100 V for 1 H, 200 V for 2 H, 500 V for 2 H, 1,000 V for 1 H, and 2,000 V for 1 H, and then the voltage was gradually increased to 8,000 V in 30 Min and maintained at 8,000 V for 6 H. After IEF, the strips were washed with ddH2 O and equilibrated for 15 Min in DTT equilibrium buffer (75 mM Tris–HCl, pH 7.5, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 1% DTT). Then, the strips were washed with ddH2 O and equilibrated for 15 Min in iodoacetamide (IAA) equilibrium buffer (75 mM Tris–HCl, pH 7.5, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, 2.5% IAA). The seconddimensional electrophoresis (SDS-PAGE) was performed in 12% polyacrylamide gels at 80 V for 1 H and 250 V for 4–5 H. After the electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB) G-250.
2.4. Image scanning and data analysis The CBB-stained gel images were captured using the ImageScanner III (GE Healthcare) at a resolution of 300 dots per inch. The image analysis was performed with Image Master 2D Platinum 7.0 software (GE Healthcare). Two-dimensional gel electrophoresis (2-DE) was repeated three times for correct analysis of expressed protein spots. The detected spots were normalized according to their relative volumes. Differentially expressed proteins were identified both by software and manual analysis. Only protein spots showing a statistically significant change of twofold or higher in spot volume were analyzed by MS.
2.5. Protein identification The CBB-stained protein spots were manually and selectively excised from the gels. Protein digestion,
Proteomics of B. amyloliquefaciens with pH Changes
FIG. 1
2-DE profiles of intracellular proteins from strain Q-426 cultured under different pH conditions: (a) pH 5.0 and (b) neutral pH.
peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ionization–MS (MALDI–MS) analysis, and database search were performed by Beijing Protein Innovation (Beijing, People’s Republic of China). Mass spectra were obtained on a MALDI Ultraflex II mass spectrometer (Bruker Daltonics, Bremen, Germany). Protein identification was performed using the MASCOT software and the National Center for Biotechnology Information (NCBI) database. The NCBI database was searched for the PMF data by MASCOT program. Carbamidomethylation of trypsin was set as fixed modifications, and oxidation/Gln → pyro–Glu (N-term Q) was set as variable modifications. Peptide mass tolerance was 100 ppm. Only peptides and proteins with scores higher than the Mascot threshold scores (P < 0.05) were considered significant.
3. Results 3.1. Comparative analysis of B. amyloliquefaciens Q426 intracellular protein patterns by 2-DE In our previous research, B. amyloliquefaciens Q-426 has been proved to have significant inhibitory activities against a wide spectrum of fungi, for example, Curvularia lunata (Wakker) Boed, Penicillium chrysogenum Thom, and Exserohilum turcicum (Pass.) Leonard and Suggs. This phenomenon indicated that B. amyloliquefaciens Q-426 might be a promising biological preparation for prevention and control of plant diseases. The antimicrobial substances produced by B. amyloliquefaciens Q-426 have been proved to be lipopeptides. Interestingly, our previous studies showed that production of lipopeptides varied with initial pH value of culture medium. Lipopeptides were scarcely produced under pH 5.0 [14]; however, cell density, which reflected growth, had no obvious difference between the culture medium with initial pH 5.0 and that with neutral pH
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(data not shown). To comprehend the intrinsic mechanisms of Q-426 that regulated antagonistic activities under different pH conditions, comparative analysis of intracellular proteins from strain Q-426 cultured under two pH conditions was performed. The 2-DE profiles of strain Q-426 cultivated under initial pH 7.3 and 5.0 are shown in Fig. 1. Proteins observed mainly had pI values between 4 and 7 and molecular mass less than 116 kDa. Most proteins were located near the center of the gels. Analyses using Imagemaster (GE Healthcare) showed a total of 98 protein spots differentially expressed under pH 5.0 and 7.3, and 56 proteins were upregulated and 42 were downregulated in the fermentation broth at initial pH 5.0 (data not shown). Among all spots, 27 of which represented differentially expressed proteins (twofold difference, P < 0.05) were selected for MS analysis.
3.2. PMF using MALDI–MS analysis MALDI-MS analysis results of the above 27 proteins were searched for the acquired PMF data using the MASCOT software and compared with NCBI database. However, only 24 proteins were obtained after functional analysis (Fig. 1). Information of the 24 proteins including the spot ID, encoded genes, functional analysis, NCBI accession number, Mascot scores, and theoretical pI/Mr is presented in Table 1. The differential expression levels of identified proteins based on spot volume percentages calculated from master 2D gels are shown in Fig. 2. PMF spectra were provided for several differentially expressed proteins with significant changes under different pH conditions (Fig. 3). Spot IDs 0 and 16 had 70- and 17-fold change, which indicated increased protein spot volume in cells grown under neutral pH. Spot IDs 68 and 73 had –8.6- and –28fold change, which indicated decreased protein spot volume in cells grown under neutral pH. The fold change was calculated from normalized spot volumes in Fig. 2. Proteins with spot IDs 0, 20, and 21 were identified as the same protein, encoded by acoB; proteins with spot IDs 68 and 70 were also identified as the same protein, encoded by pdhA. This phenomenon may
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The differential proteins from strain Q-426 identified by MALDI-MS
TABLE 1 Spot-Match ID
Encoded gene
Score
Sequence coverage (%)
Theoretical pI/Mr (kDa)
0
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
215
63
4.54/36.8
16
degU↑
Two-component response regulator DegU
gi|16080602
104
63
5.66/25.9
20
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
118
47
4.54/36.8
21
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
111
43
4.54/36.8
23
pheB↑
Chorismate mutase PheB
gi|497881777
87
43
6.30/16.4
24
yuaE↓
Description
NCBI accession number
DinB superfamily
gi|384266631
153
66
5.35/19.1
25
↑
mglA
ABC-type sugar transport system, ATPase component
gi|494146638
67
19
6.56/55. 4
30
mrsA↓
Methionine sulfoxide reductase A
gi|154686418
117
70
5.38/20.5
34
oppA↑
ABC-type oligopeptide transport system
gi|387897642
102
38
7.03/59.8
39
yvyD↓
Ribosome-associated σ 54 modulation protein
gi|308175263
102
46
5.62/21.9
52
folD↓
Methylenetetrahydrofolate dehydrogenase
gi|384266047
91
58
6.18/28.6
60
sucD↓
Succinyl-CoA synthetase, α subunit
gi|384265193
69
43
5.63/31.4
61
etfA↑
Electron transfer flavoprotein, α subunit
gi|375363264
151
70
4.64/34.0
66
yjlD↓
NADH dehydrogenase
gi|154685670
155
57
6.68/42.0
68
pdhA↓
Pyruvate dehydrogenase E1 component, α subunit
gi|154685875
105
54
6.16/41.4
69
appA↑
Peptide ABC transporter oligopeptide-binding protein
gi|375361789
142
38
6.75/63.4
70
pdhA↓
Pyruvate dehydrogenase E1 component, α subunit
gi|154685875
100
50
6.16/41.4
73
glyA↓
Glycine/serine hydroxymethyltransferase
gi|384267225
131
49
5.76/45.6
74
asd↓
Aspartate-semialdehyde dehydrogenase
gi|375362320
148
57
5.49/37.9
80
guaB↓
Inosine-monophosphate dehydrogenase
gi|308171900
219
71
6.11/52.9
86
groEL↓
Molecular chaperone GroEL
gi|407956284
155
51
4.71/57.4
Molecular chaperone DnaK
gi|154686807
173
39
4.73/66.1
89
dnaK
↓
(Continued)
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Proteomics of B. amyloliquefaciens with pH Changes
Continued
TABLE 1 Spot-Match ID
Encoded gene
Score
Sequence coverage (%)
Theoretical pI/Mr (kDa)
91
fusA↓
Elongation factor G
gi|311070759
124
38
4.77/76.6
92
bdhA↑
Acetoin reductase/2,3 butanediol dehydrogenase
gi|375361326
136
47
5.04/37.4
93
clpC↓
Clp protease ATP-binding subunit
gi|154684604
169
38
5.79/90.0
95
thiG↓
Thiazole synthase
gi|154685604
90
51
4.86/27.0
97
iolS↓
Aldo-keto reductases
gi|154688079
199
82
5.17/34.8
Description
NCBI accession number
↑ Represented
proteins with upregulated expression levels under neutral pH compared with pH 5.0.
↓ Represented
proteins with downregulated expression levels under neutral pH.
FIG. 2
Differential proteome of B. amyloliquefaciens Q-426. Bar plot of spot volume percentages calculated from master 2D gels at different pH conditions.
be due to posttranslational cleavage or chemical modification (Gln → pyro–Glu/oxidation) of these proteins. In the 24 differentially expressed proteins, 16 proteins were upregulated in the fermentation broth with initial pH 5.0. Protein ID 24 (belonging to DinB superfamiliy) was identified as a hypothetical protein. IolS (ID 97) was identified as an aldo-keto reductase. Elongation factor G FusA (ID 91) is a protein involved in the protein synthesis for polypeptide chain elongation and ribosome recycling. Inosine monophosphate dehydrogenase GuaB (ID 80) is involved in anabolism. The other 12 proteins were divided into four groups according to their functions: 1) four induced proteins related to stress response of B. amyloliquefaciens, including ribosome-associated σ 54 regulatory protein YvyD (ID 39), molecular chaperone DnaK
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(ID 89), and GroEL (ID 86), as well as Clp protease ClpC (ID 93); 2) three enzymes involved in metabolism of amino acids, including methionine sulfoxide reductase A MrsA (ID 30), glycine/serine hydroxymethyltransferase GlyA (ID 73), and aspartate-semialdehyde dehydrogenase Asd (ID 74); 3) three enzymes involved in coenzyme metabolism, including methylenetetrahydrofolate dehydrogenase FolD (ID 52), NADH dehydrogenase YjlD (ID 66), and thiazole synthase ThiG (ID 95); 4) two enzymes involved in TCA cycles, including succinyl-CoA synthase SucD (ID 60) and pyruvate dehydrogenase E1 PdhA (ID 70). Among the total 24 identified proteins, eight proteins were downregulated in the fermentation broth with initial pH 5.0. The sequence coverage of protein spot ID 25 was relatively low, only 19%. The analysis results of another seven differentially expressed proteins are highly reliable. Thiamine pyrophosphate (TPP)-dependent acetoin dehydrogenase AcoB (ID 0) and butanediol dehydrogenase BdhA (ID 92) are enzymes involved in glucose metabolism. ABC-type oligopeptide transport system proteins OppA (ID 34) and AppA (ID 69) are membrane proteins essential for transport and metabolism of amino acids. DegU (ID 16) is a response regulator protein belonging to DegS/DegU two-component system. Chorismate mutase PheB (ID 23) is the key enzyme in tyrosine and phenylalanine production. EtfA (ID 61) is an electron transfer flavoprotein.
4. Discussion By means of a MALDI-MS analysis and database queries, a total of 24 differentially expressed proteins were successfully identified in the intracellular proteome of B. amyloliquefaciens Q-426. Among these protein spots, the four induced proteins YvyD, DnaK, GroEL, and ClpC were upregulated in the fermentation broth with intial pH 5.0. However, antifungal lipopeptides were scarcely produced under this condition. It could be speculated that the strain Q-426 would make a stress response under acidic conditions. The stress response activated
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FIG. 3
PMF spectra of several proteins with significant changes of expression level under different pH conditions.
expression of certain regulatory proteins, including the four induced proteins that might negatively regulate the expression of nonribosomal peptide synthetases in the lipopeptide biosynthetic pathway. The four induced proteins might also regulate expression of other genes that directly involved in lipopeptide production. Three enzymes involved in metabolism of amino acids including Asd, GlyA, and MrsA were also upregulated in the fermentation broth with initial pH 5.0. Asd (ID 74) functioned as aspartate-semialdehyde dehydrogenase and it could facilitate conversion of aspartate to lysine, methionine, threonine, and isoleucine. Aspartate and threonine are precursors of bacillomycin D, whereas threonine and isoleucine are precursors of fengycin A and fengycin B [16–21]. Change in Asd expression levels might correlate with the variation of component proportions of lipopeptides. However, the relationship needed to be further discussed because the component pro-
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portions of lipopeptides were also influenced by other enzymes involved in metabolism of amino acids. GlyA (ID 73) has the physiological function of catalyzing the reversible interconversion of serine and glycine. Serine is a precursor of bacillomycin D, which belongs to the iturin family [16–18]; thus, an increase in serine would lead to the increase in lipopeptide production. However, lipopeptide production actually decreased under initial pH 5.0, accompanied by the upregulation of GlyA. Therefore, we concluded that GlyA in the strain B. amyloliquefaciens Q-426 probably had the ability to convert serine to glycine under acidic conditions. MrsA (ID 30) could repair proteins oxidized on their methionine residues. Upregulation of MrsA under initial pH 5.0 could probably increase resistance to oxidative stresses and improve cell growth. The influence of MrsA on lipopeptide production remains to be investigated. IolS (ID 97) belongs to the aldo-keto reductase superfamily, which functions in the reduction of aldehydes and ketones and played important roles in widespread cell metabolism pathways. IolS has been identified in B. subtilis [22]. However, the connection between physiological function of IolS and lipopeptide production is not clear. The other upregulated proteins under
Proteomics of B. amyloliquefaciens with pH Changes
FIG. 4
Metabolic pathway of acetoin from glucose (adapted from reference 23). AcoB and BdhA were upregulated under neutral pH, indicated by bold upward arrows.
pH 5.0 were enzymes involved in basic metabolism pathways and played important roles in cell growth, including enzymes involved in metabolism of coenzymes (ID 52, ID 66, and ID 95), enzymes ID 60 and ID 70 involved in TCA cycles, elongation factor G FusA (ID 91) involved in the protein synthesis, and GuaB (ID 80) involved in anabolism. Among the proteins downregulated in the fermentation broth with initial pH 5.0, several were found to be related with lipopeptide production. AcoB and BdhA are enzymes involved in glucose metabolism (Fig. 4) [23] and their expression were positively correlated with lipopeptide production under pH changes. Regulatory roles of AcoB in lipopoeptide production could be explained in the following aspects: First, as shown in Fig. 4, α-acetolactic acid is converted from pyruvate by catalyst AcoB and serves as a principle precursor for valine and leucine. A decrease in AcoB under pH 5.0 would consequently decrease the yield of the above amino acids. Correspondingly, production of antifungal lipopeptides as secondary metabolites would be decreased. From another aspect, a decrease in the AcoB expression level under pH 5.0 would also decrease acetoin production. Acetoin is an important secondary metabolite, which accumulates when carbon sources are abundant and can be utilized when there is a shortage of carbon sources. Thus, shortage of acetoin might indicate shortage of carbon and energy sources of the strain during stationary phase, further decreasing lipopeptide production. Third, a decrease in the AcoB expression level under pH 5.0 would accumulate pyruvate and result in feedback inhibition toward Embden-Meyerhof-Parnas (EMP) pathway, further influencing lipopeptide production. BdhA could catalyze the reversible conversion between butanediol and acetoin. A decrease in BdhA expression level under pH 5.0 probably decreased 2,3-butanediol production in the stationary phase [24]. The accumulation of acetoin might exert feedback inhibitory effects toward EMP pathway and further decrease lipopeptide production. AppA and OppA are membrane proteins responsible for transportation and metabolism of amino
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acids [25]. Thus, a decrease in their expression level under pH 5.0 implied that activities of AppA and OppA would suffer a loss under acidic conditions; consequently, lipopeptide production would be decreased. DegU was another protein downregulated under pH 5.0. As reported, DegS/DegU two-component system plays an important role in the stationary phase of Bacillus genus and regulates expression of many genes [26–30]. It was found recently that DegU could combine with two sites in the upstream sequence of bmy operon and enhance bacillomycin D production [31]. This coincided with our conclusion that DegU expression was positively related with lipopeptide production. PheB downregulated under pH 5.0 was the key enzyme in the synthesis of tyrosine and phenylalanine. It indicated that a decrease in PheB expression level would lead to a decrease in lipopeptide production because tyrosine served as a precursor of bacillomycin D, fengycin A, and fengycin B, which were separated and identified in fermentation broth of B. amyloliquefaciens Q-426 [14–21]. On the basis of positive correlation between PheB expression and lipopeptide production, it was speculated that phenylalanine could also facilitate lipopeptide production; yet, the working mechanism of phenylalanine remained unveiled. Expression levels of both MglA and EtfA were downregulated under pH 5.0. MglA was an ATPase component that participated in ATP hydrolysis to ADP and Pi. EtfA was a electron transfer vector. Their impacts on lipopeptides production still need to be investigated.
5. Conclusions Several regulatory proteins that correlate with bacillomycin D and fengycins production have been screened by comparatively proteomic analysis of B. amyloliquefaciens Q-426 intracellular proteins expression under acidic and neutral conditions. First, four induced proteins related to stress response (YvyD, DnaK, GroEL, and ClpC) were upregulated under pH 5.0, indicating that the strain Q-426 was more stress tolerant under acidic pH. This might explain why cell growth either under acidic or neutral conditions had no obvious difference as previous research has shown. However, lipopeptide production was curbed under initial pH 5.0, indicating stress response was unfavorable for lipopeptide biosynthesis. Second, upregulation of AcoB and BdhA participating in glucose metabolism under neutral pH might provide positive regulation of lipopeptide production. Third, the activities of AppA and OppA were decreased under acidic conditions; correspondingly, lipopeptide production decreased probably because of low efficiency of transportation and metabolism of amino acids. Fourth, DegU expression level was positively correlated with lipopeptide production. Finally, PheB might facilitate lipopeptide biosynthesis under neutral pH by providing tyrosine as a precursor of lipopeptides. This report provided useful information regarding mechanisms of antifungal lipopeptide production in B. amyloliquefaciens. The conclusions would provide guidance for the regulation of lipopeptide production by genetic modifications of the strain.
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6. Acknowledgement The authors are grateful for support from the Key Laboratory Project of the Department of Education of Liaoning Province (LS2010049) and the Science Research General Project of the Department of Education of Liaoning Province (L2013508).
7. References [1] Yoshida, S., Hiradate, S., Tsukamoto, T., Hatakeda, K., and Shirata, A. (2001) Phytopathology 91, 181–187. [2] Sutyak, K. E., Wirawan, R. E., Aroutcheva, A. A., and Chikindas, M. L. (2008) J. Appl. Microbiol. 104, 1067–1074. ´ [3] Abriouel, H., Franz, C. M., Ben, O. N., and Galvez, A. (2011) FEMS Microbiol. Rev. 35, 201–232. [4] Chen, X. H., Vater, J., Piel, J., Franke, P., Scholz, R., Schneider, K., Koumoutsi, ¨ A., Hitzeroth, G., Grammel, N., Strittmatter, A. W., Gottschalk, G., Sussmuth, R. D., Borriss, R. (2006) J. Bacteriol. 188, 4024–4036. ¨ [5] Chen, X. H., Scholz, R., Borriss, M., Junge, H., Mogel, G., Kunz, S., Borriss, R. (2009) J. Biotechnol. 140, 38–44. [6] Koumoutsi, A., Chen, X. H., Henne, A., Liesegang, H., Hitzeroth, G., Franke, P., Vater, J., Borriss, R. (2004) J. Bacteriol. 186, 1084–1096. [7] Nihorimbere, V., Cawoy, H., Seyer, A., Brunelle, A., Thonart, P., and Ongena, M. (2012) FEMS Microbiol. Ecol. 79, 176–191. [8] Lee, S. C., Kim, S. H., Park, I. H., Chung, S. Y., Chandra, M. S., and Choi, Y. L. (2010) Biotechnol. Bioproc. Eng. 15, 246–253. [9] Chen, L., Wang, N., Wang, X., Hu, J., and Wang, S. (2010) Bioresour. Technol. 101, 8822–8827. [10] Stein, T. (2005) Mol. Microbiol. 56, 845–857. [11] Jacques, P., Hbid, C., Destain, J., Razafindralambo, H., Paquot, M., DePauw, E., and Thonart, P. (1999) Appl. Biochem. Biotechnol. 77, 223–233. [12] Akpa, E., Jacques, P., Wathelet, B., Paquot, M., Fuchs, R., Budzikiewicz, H., and Thonart, P. (2001) Appl. Biochem. Biotechnol. 93, 551–561.
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[13] Mizumoto, S., and Shoda, M. (2007) Appl. Biochem. Biotechnol. 76, 101– 108. [14] Zhao, P. C., Quan, C. S., Jin, L. M., Wang, L. N., Wang, J. H., and Fan, S. D. (2013) World J. Microbiol. Biotechnol. 29, 401–409. [15] Zhao, P. C., Quan, C. S., Jin, L. M., Wang, L. N., and Fan, S. D. (2012) China Biotechnol. 32, 50–56. [16] Maget-Dana, R., and Peypoux, F. (1994) Toxicology 87, 151–174. [17] Stein, T. (2005) Mol. Microbiol. 56, 845–857. [18] Liu, X., Yang, S., and Mou, B. (2005) Biotechnol. Bull. 4, 18–26. [19] Nihorimbere, V., Cawoy, H., Seyer, A., Brunelle, A., Thonart, P., and Ongena, M. (2012) FEMS Microbiol. Ecol. 79, 176–191. [20] Vanittanakom, N., Loeffler, W., Koch, U., and Jung, G. (1986) J. Antibiot. 39, 888–901. [21] Pathak, K. V., Keharia, H., Gupta, K., Thakur, S. S., and Balaram, P. (2012) J. Am. Soc. Mass Spectrom. 23, 1716–1728. [22] Ellis, E. M. (2002) FEMS Microbiol. Lett. 216, 123–131. [23] Xu, H., Jia, S. R., and Liu, J. J. (2010) Food Ferment. Ind. 36, 137–144. [24] Nicholson, W. L. (2008) Appl. Environ. Microbiol. 74, 6832–6838. [25] Chen, X. H., Koumoutsi, A., Scholz, R., Eisenreich, A., Schneider, K., Heinemeyer, I., Morgenstern, B., Voss, B., Hess, W. R., Reva, O., Junge, H., ¨ Voigt, B., Jungblut, P. R., Vater, J., Sussmuth, R., Liesegang, H., Strittmatter, A., Gottschalk, G., and Borriss, R. (2007) Nat. Biotechnol. 25, 1007– 1014. [26] Kamp, H. D., and Higgins, D. E. (2009) Mol. Microbiol. 74, 421–435. [27] Dubnau, D., Hahn, J., Roggiani, M., Piazza, F., and Weinrauch, Y. (1994) Res. Microbiol. 145, 403–411. [28] Ogura, M., and Tanaka, T (1996) FEBS Lett. 397, 173–176. [29] Kobayashi, K. (2007) Mol. Microbiol. 66, 395–409. [30] Verhamme, D. T., Kiley, T., and Stanley-Wall, N. R. (2007) Mol. Microbiol. 65, 554–568. [31] Koumoutsi, A., Chen, X. H., Vater, J., and Borriss, R. (2007) Appl. Environ. Microbiol. 73, 6953–6964.
Proteomics of B. amyloliquefaciens with pH Changes
Jing Zhao1,2 Pengchao Zhao3 ∗ Chunshan Quan1,2 1,2 Liming Jin Wei Zheng1,2 Shengdi Fan1,2
1 Key
Laboratory of Biochemical Engineering (State Ethnic Affairs Commission-Ministry of Education), Dalian Nationalities University, Dalian, People’s Republic of China
2 College
of Life Science, Dalian Nationalities University, Dalian, People’s Republic of China
3 School
of Medical Technology and Engineering, Henan University of Science and Technology, Luoyang, People’s Republic of China
Abstract Bacillus amyloliquefaciens Q-426 produces lipopeptide compounds with antifungal activities. Initial pH value has a significant influence on the production of lipopeptide compounds. The correlation between pH and intrinsic mechanism of lipopeptide production was rarely discussed. In this research, comparative proteomics, using two-dimensional gel electrophoresis and mass spectrometry, was applied to identify B. amyloliquefaciens Q-426 intracellular proteins differentially expressed under initial pH 5.0 and 7.3. A total of 24 differential spots (eight downregulated and 16 upregulated) under pH 5.0 were identified. Certain proteins were involved in
the regulation of bacillomycin and fengycin production by B. amyloliquefaciens Q-426. These proteins include four induced proteins related to stress response: Thiamine pyrophosphate-dependent acetoin dehydrogenase, butanediol dehydrogenase, two ABC-type oligopeptide transport system proteins, and two-component response regulator DegU and chorismate mutase PheB. These results indicate intrinsic differences of antagonistic B. amyloliquefaciens Q-426 under C 2014 International Union of Biochemistry different pH conditions. and Molecular Biology, Inc. Volume 00, Number 00, Pages 1–8, 2014
Keywords: Bacillus amyloliquefaciens, lipopeptide, mass spectrometry, pH, proteomic analysis, two-dimensional gel electrophoresis
1. Introduction Bacillus amyloliquefaciens belongs to Bacillus genus, and it is a Gram-positive bacteria having high similarity with Bacillus subtilis. In recent years, researchers discovered that B. amyloliquefaciens could produce mainly two types of antibacterial compounds: bacteriocins and bacteriocins-like inhibitory substances synthesized by ribosomal ways, as well as lipopep-
Abbreviations: HPLC-MS, high performance liquid chromatography-mass spectrometry; IEF, isoelectric focusing; PMF, peptide mass fingerprinting; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry . ∗ Address for correspondence: Professor Chunshan Quan, College of Life Science, Dalian Nationalities University, Dalian 116600, People’s Republic of China. Tel.: +86 411 8765 6219; Fax: +86 411 8765 6219; e-mail: [email protected]. Received 28 June 2014; accepted 5 September 2014 DOI: 10.1002/bab.1293
Published online in Wiley Online Library (wileyonlinelibrary.com)
tides and polyketides synthesized by nonribosomal ways [1–9]. Lipopeptide compounds produced by Bacillus sp. is divided into three types: surfactin family, iturin family, and fengycin family. Lipopeptide compounds, with stable antibacterial activity, broad antimicrobial spectrum, and environmental benefits, have become more attractive recently and have the potential to replace conventional antibiotics [10]. However, a relatively low yield of lipopeptides and difficulties in the separation of lipopeptide mixture greatly restrict their application in agricultural and pharmaceutical industry. Thus, screening regulation factors of lipopeptide production is of great significance. Composition and yield of lipopeptides were determined by complicated cultivation conditions [11–13]. Our previous studies showed that initial pH value, which varied with medium contents, could obviously influence lipopeptide production of B. amyloliquefaciens Q-426. The yield of lipopeptides was relatively stable under neutral and alkaline conditions; however, it declined under acidic conditions. There was no lipopeptide production below pH 5.0. This conclusion was obtained by the measurement of inhibitory activities of fermentation
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Biotechnology and Applied Biochemistry broth and by the measurement of lipopeptide production using reversed-phase high-performance liquid chromatography (HPLC), HPLC–mass spectrometry (HPLC–MS), and tandem MS techniques [14, 15]. This phenomenon would be explained intrinsically if the metabolic mechanisms are comprehensively understood. Proteomics is a tool for providing overall protein expression patterns and facilitates understanding of the metabolism of bacteria. On the basis of previous studies, this article aimed at using proteomics for studying protein expression patterns of B. amyloliquefaciens Q-426 cultivated under two different pH conditions. By analysis of differentiated proteomics and identification of proteins by MS, genes related to lipopeptide production of B. amyloliquefaciens Q-426 could be screened; furthermore, regulation mechanism of lipopeptide production and metabolism pathways of B. amyloliquefaciens could be illustrated.
rials Vibra Cell VCX 130). The homogenate was centrifuged at 20,000g and 4 ◦ C for 20 Min. The supernatant was mixed with cold acetone at the ratio of 1:4 (v/v) and kept overnight at –20 ◦ C, and then it was centrifuged again at 20,000g and 4 ◦ C for 20 Min. After centrifugation, the supernatant was discarded. The precipitation was suspended with cold acetone solution (90% of acetone) containing 10 mM DTT and placed for 1 H at 4 ◦ C. The sample was centrifuged for at 12,000g and 4 ◦ C 10 Min. The precipitation was air-dried and solubilized in hydration liquid (8 M urea, 2% CHAPS, 0.5% IPG buffer, pH 3–10, and 10 mM DTT without bromophenol blue). The solution was centrifuged at 20,000g and 4 ◦ C for 20 Min. The supernatant was stored at –80 ◦ C. The protein samples were purified using 2D Clean Up kit (GE Healthcare, Stockholm, Sweden) and quantitatively measured using Modified BCA Protein Assay kit (Sangon Biotech Co., Shanghai, People’s Republic of China).
2.3. Two-dimensional gel electrophoresis
2. Materials and Methods 2.1. Strains and culture conditions B. amyloliquefaciens Q-426 (GenBank accession no. HM130462) was separated from compost samples and stored in LuriaBertani (LB) broth at –80 ◦ C as 30% glycerol stocks in our laboratory. The strain was cultivated in 250-mL Erlenmeyer flasks containing 50 mL of LB medium at 37 ◦ C and 180 rpm for 12–16 H. The above culture was inoculated in the fermentation medium with the inoculum concentraion of 1% for propagation. The fermentation medium composition was as follows (g/L): typsin (Oxoid), 12.40; glucose, 20.00; NaCl, 5.00; K2 HPO4 ·3H2 O, 1.50; MnSO4 ·H2 O, 0.04; FeSO4 ·7H2 O, 1.67; MgCl2 ·6H2 O, 1.22. The cultures were incubated at 30 ◦ C and 180 rpm for lipopeptide production.
2.2. Extraction and purification of intracellular proteins B. amyloliquefaciens Q-426 was cultivated in the fermentation medium with initial pH 7.3 and 5.0 separately at 30 ◦ C for 40 H. The fermentation broth was then centrifuged at 5,000g and 4 ◦ C for 10 Min. The precipitated cells were washed in the following order: initially they were washed twice with ultrapure water to remove medium components and other contaminants, then they were washed three times with TE buffer (10 mM Tris–HCl, 1 mM disodium ethylenediamine tetraacetate, pH 8.0) to remove Fe2+ in the fermentation mdium, and finally they were washed twice with cell washing solution (250 mM mannitol, 20 mM Tris–HCl, pH 7.5). The harvested cells were used for the extraction of intracellular protein. The cells were resuspended in a precooled homogenization buffer (7 M urea, 2 M thiourea, 2% 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% immobilized pH gradient (IPG) buffer, pH 3–10, 10 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride) at the ratio of 1:4 (w/v). The samples were lysed by sonication for 10 Min (2 Sec with 3 Sec intervals on an ice bath; Sonics & Mate-
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The first-dimensional protein isoelectric focusing (IEF) was performed using IPG strips (13 cm) with a linear pH range [3–10] in an Ettan IPGphor Isoelectric Focusing system (GE Healthcare). Eight hundred micrograms of protein sample was mixed with 300 µL hydration liquid (8 M urea, 2% CHAPS, 0.5% IPG buffer, pH 3–10, and 10 mM DTT). The IEF programs were performed at 30 V for 12 H, 100 V for 1 H, 200 V for 2 H, 500 V for 2 H, 1,000 V for 1 H, and 2,000 V for 1 H, and then the voltage was gradually increased to 8,000 V in 30 Min and maintained at 8,000 V for 6 H. After IEF, the strips were washed with ddH2 O and equilibrated for 15 Min in DTT equilibrium buffer (75 mM Tris–HCl, pH 7.5, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 1% DTT). Then, the strips were washed with ddH2 O and equilibrated for 15 Min in iodoacetamide (IAA) equilibrium buffer (75 mM Tris–HCl, pH 7.5, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, 2.5% IAA). The seconddimensional electrophoresis (SDS-PAGE) was performed in 12% polyacrylamide gels at 80 V for 1 H and 250 V for 4–5 H. After the electrophoresis, the gels were stained with Coomassie Brilliant Blue (CBB) G-250.
2.4. Image scanning and data analysis The CBB-stained gel images were captured using the ImageScanner III (GE Healthcare) at a resolution of 300 dots per inch. The image analysis was performed with Image Master 2D Platinum 7.0 software (GE Healthcare). Two-dimensional gel electrophoresis (2-DE) was repeated three times for correct analysis of expressed protein spots. The detected spots were normalized according to their relative volumes. Differentially expressed proteins were identified both by software and manual analysis. Only protein spots showing a statistically significant change of twofold or higher in spot volume were analyzed by MS.
2.5. Protein identification The CBB-stained protein spots were manually and selectively excised from the gels. Protein digestion,
Proteomics of B. amyloliquefaciens with pH Changes
FIG. 1
2-DE profiles of intracellular proteins from strain Q-426 cultured under different pH conditions: (a) pH 5.0 and (b) neutral pH.
peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ionization–MS (MALDI–MS) analysis, and database search were performed by Beijing Protein Innovation (Beijing, People’s Republic of China). Mass spectra were obtained on a MALDI Ultraflex II mass spectrometer (Bruker Daltonics, Bremen, Germany). Protein identification was performed using the MASCOT software and the National Center for Biotechnology Information (NCBI) database. The NCBI database was searched for the PMF data by MASCOT program. Carbamidomethylation of trypsin was set as fixed modifications, and oxidation/Gln → pyro–Glu (N-term Q) was set as variable modifications. Peptide mass tolerance was 100 ppm. Only peptides and proteins with scores higher than the Mascot threshold scores (P < 0.05) were considered significant.
3. Results 3.1. Comparative analysis of B. amyloliquefaciens Q426 intracellular protein patterns by 2-DE In our previous research, B. amyloliquefaciens Q-426 has been proved to have significant inhibitory activities against a wide spectrum of fungi, for example, Curvularia lunata (Wakker) Boed, Penicillium chrysogenum Thom, and Exserohilum turcicum (Pass.) Leonard and Suggs. This phenomenon indicated that B. amyloliquefaciens Q-426 might be a promising biological preparation for prevention and control of plant diseases. The antimicrobial substances produced by B. amyloliquefaciens Q-426 have been proved to be lipopeptides. Interestingly, our previous studies showed that production of lipopeptides varied with initial pH value of culture medium. Lipopeptides were scarcely produced under pH 5.0 [14]; however, cell density, which reflected growth, had no obvious difference between the culture medium with initial pH 5.0 and that with neutral pH
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(data not shown). To comprehend the intrinsic mechanisms of Q-426 that regulated antagonistic activities under different pH conditions, comparative analysis of intracellular proteins from strain Q-426 cultured under two pH conditions was performed. The 2-DE profiles of strain Q-426 cultivated under initial pH 7.3 and 5.0 are shown in Fig. 1. Proteins observed mainly had pI values between 4 and 7 and molecular mass less than 116 kDa. Most proteins were located near the center of the gels. Analyses using Imagemaster (GE Healthcare) showed a total of 98 protein spots differentially expressed under pH 5.0 and 7.3, and 56 proteins were upregulated and 42 were downregulated in the fermentation broth at initial pH 5.0 (data not shown). Among all spots, 27 of which represented differentially expressed proteins (twofold difference, P < 0.05) were selected for MS analysis.
3.2. PMF using MALDI–MS analysis MALDI-MS analysis results of the above 27 proteins were searched for the acquired PMF data using the MASCOT software and compared with NCBI database. However, only 24 proteins were obtained after functional analysis (Fig. 1). Information of the 24 proteins including the spot ID, encoded genes, functional analysis, NCBI accession number, Mascot scores, and theoretical pI/Mr is presented in Table 1. The differential expression levels of identified proteins based on spot volume percentages calculated from master 2D gels are shown in Fig. 2. PMF spectra were provided for several differentially expressed proteins with significant changes under different pH conditions (Fig. 3). Spot IDs 0 and 16 had 70- and 17-fold change, which indicated increased protein spot volume in cells grown under neutral pH. Spot IDs 68 and 73 had –8.6- and –28fold change, which indicated decreased protein spot volume in cells grown under neutral pH. The fold change was calculated from normalized spot volumes in Fig. 2. Proteins with spot IDs 0, 20, and 21 were identified as the same protein, encoded by acoB; proteins with spot IDs 68 and 70 were also identified as the same protein, encoded by pdhA. This phenomenon may
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The differential proteins from strain Q-426 identified by MALDI-MS
TABLE 1 Spot-Match ID
Encoded gene
Score
Sequence coverage (%)
Theoretical pI/Mr (kDa)
0
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
215
63
4.54/36.8
16
degU↑
Two-component response regulator DegU
gi|16080602
104
63
5.66/25.9
20
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
118
47
4.54/36.8
21
acoB↑
TPP-dependent acetoin dehydrogenase E1
gi|154685285
111
43
4.54/36.8
23
pheB↑
Chorismate mutase PheB
gi|497881777
87
43
6.30/16.4
24
yuaE↓
Description
NCBI accession number
DinB superfamily
gi|384266631
153
66
5.35/19.1
25
↑
mglA
ABC-type sugar transport system, ATPase component
gi|494146638
67
19
6.56/55. 4
30
mrsA↓
Methionine sulfoxide reductase A
gi|154686418
117
70
5.38/20.5
34
oppA↑
ABC-type oligopeptide transport system
gi|387897642
102
38
7.03/59.8
39
yvyD↓
Ribosome-associated σ 54 modulation protein
gi|308175263
102
46
5.62/21.9
52
folD↓
Methylenetetrahydrofolate dehydrogenase
gi|384266047
91
58
6.18/28.6
60
sucD↓
Succinyl-CoA synthetase, α subunit
gi|384265193
69
43
5.63/31.4
61
etfA↑
Electron transfer flavoprotein, α subunit
gi|375363264
151
70
4.64/34.0
66
yjlD↓
NADH dehydrogenase
gi|154685670
155
57
6.68/42.0
68
pdhA↓
Pyruvate dehydrogenase E1 component, α subunit
gi|154685875
105
54
6.16/41.4
69
appA↑
Peptide ABC transporter oligopeptide-binding protein
gi|375361789
142
38
6.75/63.4
70
pdhA↓
Pyruvate dehydrogenase E1 component, α subunit
gi|154685875
100
50
6.16/41.4
73
glyA↓
Glycine/serine hydroxymethyltransferase
gi|384267225
131
49
5.76/45.6
74
asd↓
Aspartate-semialdehyde dehydrogenase
gi|375362320
148
57
5.49/37.9
80
guaB↓
Inosine-monophosphate dehydrogenase
gi|308171900
219
71
6.11/52.9
86
groEL↓
Molecular chaperone GroEL
gi|407956284
155
51
4.71/57.4
Molecular chaperone DnaK
gi|154686807
173
39
4.73/66.1
89
dnaK
↓
(Continued)
4
Proteomics of B. amyloliquefaciens with pH Changes
Continued
TABLE 1 Spot-Match ID
Encoded gene
Score
Sequence coverage (%)
Theoretical pI/Mr (kDa)
91
fusA↓
Elongation factor G
gi|311070759
124
38
4.77/76.6
92
bdhA↑
Acetoin reductase/2,3 butanediol dehydrogenase
gi|375361326
136
47
5.04/37.4
93
clpC↓
Clp protease ATP-binding subunit
gi|154684604
169
38
5.79/90.0
95
thiG↓
Thiazole synthase
gi|154685604
90
51
4.86/27.0
97
iolS↓
Aldo-keto reductases
gi|154688079
199
82
5.17/34.8
Description
NCBI accession number
↑ Represented
proteins with upregulated expression levels under neutral pH compared with pH 5.0.
↓ Represented
proteins with downregulated expression levels under neutral pH.
FIG. 2
Differential proteome of B. amyloliquefaciens Q-426. Bar plot of spot volume percentages calculated from master 2D gels at different pH conditions.
be due to posttranslational cleavage or chemical modification (Gln → pyro–Glu/oxidation) of these proteins. In the 24 differentially expressed proteins, 16 proteins were upregulated in the fermentation broth with initial pH 5.0. Protein ID 24 (belonging to DinB superfamiliy) was identified as a hypothetical protein. IolS (ID 97) was identified as an aldo-keto reductase. Elongation factor G FusA (ID 91) is a protein involved in the protein synthesis for polypeptide chain elongation and ribosome recycling. Inosine monophosphate dehydrogenase GuaB (ID 80) is involved in anabolism. The other 12 proteins were divided into four groups according to their functions: 1) four induced proteins related to stress response of B. amyloliquefaciens, including ribosome-associated σ 54 regulatory protein YvyD (ID 39), molecular chaperone DnaK
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(ID 89), and GroEL (ID 86), as well as Clp protease ClpC (ID 93); 2) three enzymes involved in metabolism of amino acids, including methionine sulfoxide reductase A MrsA (ID 30), glycine/serine hydroxymethyltransferase GlyA (ID 73), and aspartate-semialdehyde dehydrogenase Asd (ID 74); 3) three enzymes involved in coenzyme metabolism, including methylenetetrahydrofolate dehydrogenase FolD (ID 52), NADH dehydrogenase YjlD (ID 66), and thiazole synthase ThiG (ID 95); 4) two enzymes involved in TCA cycles, including succinyl-CoA synthase SucD (ID 60) and pyruvate dehydrogenase E1 PdhA (ID 70). Among the total 24 identified proteins, eight proteins were downregulated in the fermentation broth with initial pH 5.0. The sequence coverage of protein spot ID 25 was relatively low, only 19%. The analysis results of another seven differentially expressed proteins are highly reliable. Thiamine pyrophosphate (TPP)-dependent acetoin dehydrogenase AcoB (ID 0) and butanediol dehydrogenase BdhA (ID 92) are enzymes involved in glucose metabolism. ABC-type oligopeptide transport system proteins OppA (ID 34) and AppA (ID 69) are membrane proteins essential for transport and metabolism of amino acids. DegU (ID 16) is a response regulator protein belonging to DegS/DegU two-component system. Chorismate mutase PheB (ID 23) is the key enzyme in tyrosine and phenylalanine production. EtfA (ID 61) is an electron transfer flavoprotein.
4. Discussion By means of a MALDI-MS analysis and database queries, a total of 24 differentially expressed proteins were successfully identified in the intracellular proteome of B. amyloliquefaciens Q-426. Among these protein spots, the four induced proteins YvyD, DnaK, GroEL, and ClpC were upregulated in the fermentation broth with intial pH 5.0. However, antifungal lipopeptides were scarcely produced under this condition. It could be speculated that the strain Q-426 would make a stress response under acidic conditions. The stress response activated
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FIG. 3
PMF spectra of several proteins with significant changes of expression level under different pH conditions.
expression of certain regulatory proteins, including the four induced proteins that might negatively regulate the expression of nonribosomal peptide synthetases in the lipopeptide biosynthetic pathway. The four induced proteins might also regulate expression of other genes that directly involved in lipopeptide production. Three enzymes involved in metabolism of amino acids including Asd, GlyA, and MrsA were also upregulated in the fermentation broth with initial pH 5.0. Asd (ID 74) functioned as aspartate-semialdehyde dehydrogenase and it could facilitate conversion of aspartate to lysine, methionine, threonine, and isoleucine. Aspartate and threonine are precursors of bacillomycin D, whereas threonine and isoleucine are precursors of fengycin A and fengycin B [16–21]. Change in Asd expression levels might correlate with the variation of component proportions of lipopeptides. However, the relationship needed to be further discussed because the component pro-
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portions of lipopeptides were also influenced by other enzymes involved in metabolism of amino acids. GlyA (ID 73) has the physiological function of catalyzing the reversible interconversion of serine and glycine. Serine is a precursor of bacillomycin D, which belongs to the iturin family [16–18]; thus, an increase in serine would lead to the increase in lipopeptide production. However, lipopeptide production actually decreased under initial pH 5.0, accompanied by the upregulation of GlyA. Therefore, we concluded that GlyA in the strain B. amyloliquefaciens Q-426 probably had the ability to convert serine to glycine under acidic conditions. MrsA (ID 30) could repair proteins oxidized on their methionine residues. Upregulation of MrsA under initial pH 5.0 could probably increase resistance to oxidative stresses and improve cell growth. The influence of MrsA on lipopeptide production remains to be investigated. IolS (ID 97) belongs to the aldo-keto reductase superfamily, which functions in the reduction of aldehydes and ketones and played important roles in widespread cell metabolism pathways. IolS has been identified in B. subtilis [22]. However, the connection between physiological function of IolS and lipopeptide production is not clear. The other upregulated proteins under
Proteomics of B. amyloliquefaciens with pH Changes
FIG. 4
Metabolic pathway of acetoin from glucose (adapted from reference 23). AcoB and BdhA were upregulated under neutral pH, indicated by bold upward arrows.
pH 5.0 were enzymes involved in basic metabolism pathways and played important roles in cell growth, including enzymes involved in metabolism of coenzymes (ID 52, ID 66, and ID 95), enzymes ID 60 and ID 70 involved in TCA cycles, elongation factor G FusA (ID 91) involved in the protein synthesis, and GuaB (ID 80) involved in anabolism. Among the proteins downregulated in the fermentation broth with initial pH 5.0, several were found to be related with lipopeptide production. AcoB and BdhA are enzymes involved in glucose metabolism (Fig. 4) [23] and their expression were positively correlated with lipopeptide production under pH changes. Regulatory roles of AcoB in lipopoeptide production could be explained in the following aspects: First, as shown in Fig. 4, α-acetolactic acid is converted from pyruvate by catalyst AcoB and serves as a principle precursor for valine and leucine. A decrease in AcoB under pH 5.0 would consequently decrease the yield of the above amino acids. Correspondingly, production of antifungal lipopeptides as secondary metabolites would be decreased. From another aspect, a decrease in the AcoB expression level under pH 5.0 would also decrease acetoin production. Acetoin is an important secondary metabolite, which accumulates when carbon sources are abundant and can be utilized when there is a shortage of carbon sources. Thus, shortage of acetoin might indicate shortage of carbon and energy sources of the strain during stationary phase, further decreasing lipopeptide production. Third, a decrease in the AcoB expression level under pH 5.0 would accumulate pyruvate and result in feedback inhibition toward Embden-Meyerhof-Parnas (EMP) pathway, further influencing lipopeptide production. BdhA could catalyze the reversible conversion between butanediol and acetoin. A decrease in BdhA expression level under pH 5.0 probably decreased 2,3-butanediol production in the stationary phase [24]. The accumulation of acetoin might exert feedback inhibitory effects toward EMP pathway and further decrease lipopeptide production. AppA and OppA are membrane proteins responsible for transportation and metabolism of amino
Biotechnology and Applied Biochemistry
acids [25]. Thus, a decrease in their expression level under pH 5.0 implied that activities of AppA and OppA would suffer a loss under acidic conditions; consequently, lipopeptide production would be decreased. DegU was another protein downregulated under pH 5.0. As reported, DegS/DegU two-component system plays an important role in the stationary phase of Bacillus genus and regulates expression of many genes [26–30]. It was found recently that DegU could combine with two sites in the upstream sequence of bmy operon and enhance bacillomycin D production [31]. This coincided with our conclusion that DegU expression was positively related with lipopeptide production. PheB downregulated under pH 5.0 was the key enzyme in the synthesis of tyrosine and phenylalanine. It indicated that a decrease in PheB expression level would lead to a decrease in lipopeptide production because tyrosine served as a precursor of bacillomycin D, fengycin A, and fengycin B, which were separated and identified in fermentation broth of B. amyloliquefaciens Q-426 [14–21]. On the basis of positive correlation between PheB expression and lipopeptide production, it was speculated that phenylalanine could also facilitate lipopeptide production; yet, the working mechanism of phenylalanine remained unveiled. Expression levels of both MglA and EtfA were downregulated under pH 5.0. MglA was an ATPase component that participated in ATP hydrolysis to ADP and Pi. EtfA was a electron transfer vector. Their impacts on lipopeptides production still need to be investigated.
5. Conclusions Several regulatory proteins that correlate with bacillomycin D and fengycins production have been screened by comparatively proteomic analysis of B. amyloliquefaciens Q-426 intracellular proteins expression under acidic and neutral conditions. First, four induced proteins related to stress response (YvyD, DnaK, GroEL, and ClpC) were upregulated under pH 5.0, indicating that the strain Q-426 was more stress tolerant under acidic pH. This might explain why cell growth either under acidic or neutral conditions had no obvious difference as previous research has shown. However, lipopeptide production was curbed under initial pH 5.0, indicating stress response was unfavorable for lipopeptide biosynthesis. Second, upregulation of AcoB and BdhA participating in glucose metabolism under neutral pH might provide positive regulation of lipopeptide production. Third, the activities of AppA and OppA were decreased under acidic conditions; correspondingly, lipopeptide production decreased probably because of low efficiency of transportation and metabolism of amino acids. Fourth, DegU expression level was positively correlated with lipopeptide production. Finally, PheB might facilitate lipopeptide biosynthesis under neutral pH by providing tyrosine as a precursor of lipopeptides. This report provided useful information regarding mechanisms of antifungal lipopeptide production in B. amyloliquefaciens. The conclusions would provide guidance for the regulation of lipopeptide production by genetic modifications of the strain.
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Biotechnology and Applied Biochemistry
6. Acknowledgement The authors are grateful for support from the Key Laboratory Project of the Department of Education of Liaoning Province (LS2010049) and the Science Research General Project of the Department of Education of Liaoning Province (L2013508).
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Proteomics of B. amyloliquefaciens with pH Changes
