Differential proteomics research of Bacillus amyloliquefaciens and its genome-shuffled saltant for improving fengycin production

Abstract

In the previous study, we used genome shuffling to improve fengycin production of the original strain Bacillus amyloliquefaciens ES-2–4. After two rounds of genome shuffling, a high-yield recombinant FMB72 strain that exhibited 8.30-fold increase in fengycin production was obtained. In this study, comparative proteomic analysis of the parental ES-2–4 and genome-shuffled FMB72 strains was conducted to examine the differentially expressed proteins. In the shuffled strain FMB72, 50 differently expressed spots (p < 0.05) were selected to be excised and analyzed using Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry, and finally 44 protein spots were confidently identified according to NCBI database. According to clusters of orthologous groups (COG) functional category analysis and related references, the differentially expressed proteins could be classified into several functional categories, including proteins involved in metabolism, energy generation and conversion, DNA replication, transcription, translation, ribosomal structure and biogenesis, cell motility and secretion, signal transduction mechanisms, general function prediction. Of the 44 identified proteins, signaling proteins ComA and Spo0A may positively regulate fengycin synthesis at transcriptional level. Taken together, the present study will be informative for exploring the exact roles of ComA and Spo0A in fengycin synthesis and explaining the molecular mechanism of fengycin synthesis.

Introduction

Bacillus strains can produce many kinds of bioactive peptides synthesized non-ribosomally by a large multifunctional enzyme complex. Of these, fengycin specifically acting against filamentous fungi¹ is biosynthesized by fengycin synthetase encompassing the five non-ribosomal peptide synthetases (NRPSs) Fen1-Fen5, respectively is coded by the gene fen A-E.² Fengycin consists of a β-hydroxy fatty acid connected to the N-terminus of a decapeptide including four d-amino acid residues and the rare amino acid l-ornithine. The C-terminal residue of the peptide moiety is linked to the tyrosine residue at position 3, forming the branching point of the acylpeptide and the eight-membered cyclic lactone.³ Fengycin has potential applications in plant disease biocontrol,⁴ biomedicine, food⁵ and cosmetics⁶ industries. Therefore, it is particularly significant to improve fengycin production by industrial Bacillus strains.

Genome shuffling is an efficient approach for the rapid improvement of microbial phenotypes.⁷ We previously described the generation of a high-yield recombinant Bacillus amyloliquefaciens FMB72 strain that exhibited 8.30-fold increases in fengycin production, following two rounds of genome shuffling. Comparative research of synthetase gene expression was conducted between the parent strain and mutant strain using FQ (fluorescent quantitation) RT-PCR. Delta CT (threshold cycle) relative quantitation analysis indicated that fengycin synthetase gene (fenA) expression in the FMB72 strain was 12.77-fold greater than in the parent strain ES-2–4 at the transcriptional level.

However, the results only indirectly identified differences in fengycin synthetase gene at the transcriptional level. Because proteins execute molecular functions and are in charge of almost all the biochemical activities of the cell, a deep-dyed comprehension of biological systems requires the direct research of proteins. The proteomics technology based on two-dimensional electrophoresis, identification by MALDI-TOF/MS, and bioinformatics provides a good approach for large-scale proteomic analyses. In this research, the molecular mechanism of high-yield fengycin will be explored by comparative proteomics analysis of differentially expressed proteins between the parental and genome-shuffled strains.

Materials and methods

Strains and culture conditions

B. amyloliquefaciens ES-2–4 was the initial strain.^8,9B. amyloliquefaciens FMB72 was the genome-shuffled mutant strain of B. amyloliquefaciens ES-2–4.¹⁰ The yield of fengycin increased by 8.30-fold compared to ES-2–4. These strains are preserved by the Key Laboratory of Food Processing and Quality Control of the Food Science and Technology College at Nanjing Agricultural University, Nanjing, China. B. amyloliquefaciens ES-2–4 was cultured in PDA (potato dextrose agar) media at 37 °C. All microorganisms were conserved in BPY supplemented with 20% (v/v) glycerol and stored at −70 °C. Seed medium (BPY) (beef extract 5.0 g/L, peptone 10.0 g/L, yeast extract paste 5.0 g/L, NaCl 5.0 g/L, glucose 10.0 g/L) and fermentation medium (modified Landy) (l-sodium glutamate 4.0 g/L, glucose 42.0 g/L, KCl 0.5 g/L, MgSO₄ 0.5 g/L, CuSO₄ 0.16 mg/L, KH₂PO₄ 1.0 g/L, MnSO₄ 5.0 mg/L, FeSO₄ 0.15 mg/L) were adjusted to pH 7.0.

Protein sample preparation

The strains were cultured at 30 °C, 180 rpm for 36 h. Cells were harvested by centrifugation at 6000 rpm for 5 min at 4 °C, and then washed 3 times with 20 mmol/L Tris–HCl (pH 6.8). Cells were subsequently resuspended in lysis buffer containing 2 mol/L thiourea, 7 mol/L urea, 40 mmol/L DTT, 4% (w/v) CHAPS, and 2% (v/v) pH 3–10 IPG buffer.^11,12 Cells were cracked by sonication in an ultrasonic cell pulverizer (Ningbo Xin-zhi Biotechnology Co., China), equipped with a cup horn, for 45 min on ice. Following ultrasonication, Nuclease Mix (GE Healthcare, Little Chalfont, United Kingdom) was added to a final concentration of 1% (v/v). The mixture was incubated for 1 h at room temperature and then centrifuged for 30 min at 13,000 × g at 4 °C. 2-D Quant kit (GE Healthcare) was used to assay the protein concentration, with bovine serum albumin as the standard.¹³ The samples were stored at −80 °C until 2-DE.

2-DE analysis and staining

In the first dimension, total whole-cell protein (250 μg) was loaded onto the IPG strips (24 cm, pH 4–7, GE Healthcare) which had been rehydrated 14 h with 120 mL rehydration solution (7 mol/L urea, 2 mol/L thiourea, 18 mmol/L DTT, 2% Bio-Lyte, 2% (w/v) CHAPS, and 0.002% (w/v) bromophenol blue). Isoelectric focusing was performed on an EttanIPGphor 3 IEF system (GE Healthcare) for a total of 80 kVh at 20 °C. The voltage was set at 50 V for 10 h, 250 V for 3 h, 500 V for 3 h, 1000 V for 1 h, and 8000 V for 1 h, followed by 8000 V until final volt-hours were reached. Subsequently, the strips were equilibrated for 15 min in 2% (w/v) DTT in equilibration buffer (6 mol/L urea, 75 mmol/L Tris–HCl (pH 8.8), 30% (v/v) glycerol and 2% (w/v) SDS) followed by 15 min in 2.5% (w/v) IAA in equilibration buffer. The strips were then transferred to 12.5% (w/v) SDS-polyacrylamide gels. The second dimension electrophoresis was carried out in an Ettan DALTII system (GE Healthcare) with a constant power of 5 W per gel for the first 30 min, followed by 12 W per gel for 6.5–7.5 h until the bromophenol blue front reached the bottom of the gels. The gels were placed into fixative solutions (10% acetic acid,40% methanol) overnight and then stained with 0.25% (w/v) silver nitrate.¹⁴ The biological replicates were performed for each treatment at least three times.

Image acquisition and data analysis

The silver-stained 2-DE gels were imaged by an ImageScanner (GE Healthcare), and analyzed on the Image Master 2D Elite software Version 2.00 (GE Healthcare). Images were properly cropped and optimized, and then gel-to-gel matching of the standard protein maps was performed. The spot detection parameters were optimized by checking different protein spots in certain regions of the gel and then automatically detected, followed by visual inspection for removal or addition of undetected spots. Spot detection was refined by manual spot edition when needed. The percentage volumes were used to designate the significant differentially expressed spots (at least two-fold increase/decrease and statistically significant as calculated by one-way ANOVA, p < 0.05). Triplicate gels were used for each sample and the SD was calculated. Finally, only those protein spots that showed reproducible and changed more than 2-fold were considered to be differentially expressed proteins.

Protein in-gel digestion

Spots showing changes statistically significant (p < 0.05) and above a 2-fold threshold were excised from the gels and washed with double-distilled water and then transferred to sterilized Eppendorf tubes. Then, the protein spots were washed at room temperature with 25 mmol/L NH₄HCO₃, followed by dehydration with 50% (v/v) acetonitrile (ACN) in 25 mmol/L NH₄HCO₃. The proteins were then reduced with 10 mmol/L DTT in 25 mmol/L NH₄HCO₃ at 56 °C for 1 h, and then alkylated in 55 mmol/L iodoacetamide in 25 mmol/L NH₄HCO₃ for 45 min at room temperature in darkness. The liquid was discarded and gel pieces were washed three times in 25 mmol/L NH₄HCO₃, dehydrated in CAN and dried in a vacuum centrifuge. Gel pieces were then rehydrated in 25 mmol/L NH₄HCO₃ containing 40 ng trypsin, and incubated at 4 °C for 1 h. Excess liquid was discarded and gel plugs were incubated at 37 °C overnight, with tubes inverted to keep gel pieces wet for sufficient enzymatic cleavage. Then, 5% (v/v) trifluoroacetic acid (TFA) was added and samples were incubated at 37 °C for 1 h. Supernatants were collected and the proteins were extracted twice by incubating the gel pieces in 8 μL of 2.5% TFA in 50% ACN at 37 °C for 1 h. The resulting peptides were gathered and stored a −20 °C until analysis.

Protein identification by MALDI-TOF/TOF and database search

Samples were air-dried and analyzed by a Biflex IV MALDI-TOF-MS (Bruker, Billerica, MA, USA). The N₂ laser was operated at an accelerating voltage of 19 kV with a wavelength of 337 nm (3 ns pulse length).

Data analysis was performed with the National Center for Biotechnology Information (NCBI) nr database using the MASCOT search program (Matrix Science, Boston, MA, USA). The following parameters were allowed: taxonomy restrictions to other firmicutes, 120 ppm mass tolerance in MS, one missed cleavage, oxidation (M) as a variable modification and carbamidomethyl (C) as a fixed modification. The confidence in the peptide mass fingerprinting (PMF) matches (p < 0.05) was based on the MOWSE score and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum. Only the best matches with high confidence levels were chosen when the software gave more than one eligible result.

qRT-PCR verification

The total RNA were isolated from B. amyloliquefaciens cultures using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and then treated with RNase-free DNase. First-strand cDNA was conversed from total RNA using an RT-PCR kit (Fermentas, Vilnius, Lithuania¹⁵). Real-time PCR was performed as described in the paper.¹⁶ Band intensities were normalized to the 16S rDNA transcript band for 2^−ΔΔCT relative quantification. The B. amyloliquefaciens nucleotide sequences for these genes were obtained from the NCBI GenBank database. Primer pairs were designed from these sequences with Primer Premier 5.0 software (Applied Biosystems), the 16S rDNA primers used were F(5-CCTACGGGAGGCAGCAG-3) and R (5-ATTACCGCGGCT GCTGG-3), the comA primers were F (5-TCAAAGTGAGCAGGATCGGTTAA-3) and R (5-CTTCTGTACGGGAGCCGACAT-3) and spo0A primers were F (5-TTGCGGCG ATGAAGTGAATG-3) and R (5-CGATGGAAAGCTGCGGTGTA-3).

Results

Identification of differentially expressed proteins

Two-DE profiles of soluble proteins were analyzed from parental (ES-2–4) and mutant (FMB72) strains. We found 50 protein spots that differed between the strains (Fig. 1). These 50 protein spots were identified by MALDI-TOF/MS analysis and their complete peptide fingerprints were gained. A search through the NCBI nr database using Mascot revealed that protein spots 87 and 433, 115 and 132, 443 and 446, 461 and 493, 473 and 501, 508 and 697 were the same proteins, meaning that a total of 44 proteins were successfully identified. In B. amyloliquefaciens FMB72, 37 proteins had increased expression, 4 proteins had decreased expression, 5 proteins appeared only in ES-2–4 and 4 proteins appeared only in FMB72 (Table 1).

Fig. 1.

2-DE maps of the differentially regulated cellular proteins (>2-fold change in expression) of B. amyloliquefaciens FMB72. (A) ES-2–4; (B) FMB72.

Table 1.

Identification of differentially regulated cellular proteins (>2-fold change in expression) of B. amyloliquefaciens FMB72.

Spot no.^a	Protein name^b	Accession no.^c	Locus^d	Gene^e	Theor.^f Mr/pI	Exper.^g Mr/pI	Protein score^h	Sequence coverage (%)^i	Fold change^j (p < 0.05)
55	Hypothetical protein RBAM_013590	gi|154685792	YP_001420953	ykvT	23302/9.85	23189/9.76	362	84	+2.3452
61	Hypothetical protein RBAM_008300	gi|154685284	YP_001420445	acoA	36400/5.03	35338/4.97	186	48	+2.8632
63	MtnD	gi|154685773	YP_001420934	mtnD	20933/4.61	18754/4.75	149	79	+3.2361
70	Recombination protein	gi|387898274	YP_006328570	recA	36946/5.04	35092/5.28	229	71	+2.9971
72	Elongation factor Ts	gi|154686067	YP_001421228	tsf	32401/5.24	31429/5.11	191	62	+3.3894
86	Hypothetical protein RBAM_020150	gi|154686447	YP_001421608	ypcP	33480/5.51	31884/5.39	243	55	+2.5722
87	Translaldolase	gi|154687826	YP_001422987	tal	23055/5.23	21291/5.19	92	49	−2.6738
93	Flagellar motor protein MotS	gi|154687112	YP_001422273	ytxE	25773/9.61	23782/9.93	285	60	+2.6744
97	Hypothetical protein RBAM_031300	gi|154687531	YP_001422692	gapA	35875/5.36	34849/5.19	197	44	+2.4325
99	Spo0A	gi|154686686	YP_001421847	spo0A	29725/5.84	27893/5.54	162	41	+8.2345
101	Hypothetical protein RBAM_022210	gi|154686652	YP_001421813	yqjE	39599/5.07	38211/5.15	79	36	No
102	Alanine dehydrogenase	gi|387899785	YP_006330081	ald	39881/5.26	38327/5.13	205	68	No
103	Heat-inducible transcription repressor	gi|154686809	YP_001421970	hrcA	38878/5.79	37432/5.62	260	58	No
107	Aspartate-semialdehyde dehydrogenase	gi|154686092	YP_001421253	asd	38025/5.39	36711/5.17	197	54	+3.2421
108	Chemotaxis-specific methylesterase	gi|154686059	YP_001421220	cheB	38860/7.03	37262/6.83	278	54	No
110	DNA-directed RNA polymerase subunit alpha	gi|154684661	YP_001419822	rpoA	34849/4.80	32156/4.52	239	50	+2.3821
115	Hypothetical protein RBAM_031300	gi|154687531	YP_001422692	gapA	35875/5.36	34177/5.12	196	42	−2.6374
118	Bifunctional pyrimidine regulatory protein PyrR uracil phosphoribosyltransferase	gi|154685963	YP_001421124	pyrR	20270/5.22	19746/4.97	183	64	+2.7889
132	Hypothetical protein RBAM_031300	gi|154687531	YP_001422692	gapA	35875/5.36	34928/5.22	198	44	+2.3672
416	Transcription elongation factor GreA	gi|154686872	YP_001422033	greA	17348/4.75	16738/4.63	348	77	Have
431	6,7-Dimethyl-8-ribityllumazine synthase	gi|308174115	YP_003920820	ribH	16322/5.43	15829/5.34	105	68	Have
433	Translaldolase	gi|154687826	YP_001422987	tal	23055/5.23	22238/5.19	233	83	+3.6733
434	SdaAB	gi|154686001	YP_001421162	sdaAB	23931/5.19	22378/5.06	225	67	+3.3303
435	Hypothetical protein RBAM_008040	gi|154685258	YP_001420419	yfkM	18877/4.83	17357/4.59	121	61	−12.2543
443	Hypothetical protein RBAM_035480	gi|154687947	YP_001423108	ywcC	24036/8.73	23743/8.53	300	54	−2.6378
446	Hypothetical protein RBAM_035480	gi|154687947	YP_001423108	ywcC	24036/8.73	23283/8.45	255	54	+3.6738
461	tcyK gene product	gi|384266899	YP_005422606	tcyK	29775/7.70	28466/7.64	182	55	+3.5367
473	Thiol peroxidase	gi|154687070	YP_001422231	tpx	18262/4.99	17398/4.64	122	73	+28.4183
476	Hypothetical protein RBAM_015250	gi|154685958	YP_001421119	divIVA	19303/5.06	17647/4.96	183	76	+2.5732
483	Pyridoxal biosynthesis lyase PdxS	gi|16077079	NP_387892	yaaD	31705/5.26	30738/5.17	84	25	+2.5267
493	tcyK gene product	gi|384266899	YP_005422606	tcyK	29775/7.70	27643/6.97	189	57	+2.4253
501	Thiol peroxidase	gi|154687070	YP_001422231	tpx	18262/4.99	17382/4.73	164	59	+2.5626
505	Pyridoxine biosynthesis protein	gi|308171902	YP_003918607	pdxS	31706/5.34	30567/5.32	95	32	+2.4266
506	Hypothetical protein HMPREF0984_00182	gi|373451213	ZP_09543140	–	50547/5.69	49657/5.53	87	24	Have
507	Hypothetical protein RBAM_035310	gi|154687930	YP_001423091	ywcI	11993/11.42	10374/10.78	190	81	Have
508	Phosphomethylpyrimidine kinase	gi|154685606	YP_001420767	yjbV	29037/5.93	28637/5.78	219	69	+2.5672
513	ATP-dependent Clp protease proteolytic subunit	gi|154687585	YP_001422746	clpP	21874/4.96	20374/4.87	112	31	+3.0213
516	Hypothetical protein RBAM_028130	gi|154687215	YP_001422376	yuaE	19112/5.46	18291/5.34	101	54	+4.168332
522	Hypothetical protein RBAM_008300	gi|154685284	YP_001420445	acoA	36400/5.03	35666/4.98	196	42	+2.5671
533	NfrA	gi|154687935	YP_001423096	nfrA	28297/5.93	27536/5.87	191	38	+2.6782
546	ComA	gi|154687277	YP_001422438	comA	24371/5.19	22878/5.13	108	48	+27.5012
550	S-ribosylhomocysteinase	gi|154687196	YP_001422357	luxS	17913/5.27	16562/5.12	97	59	+67.8196
572	Hypothetical protein RBAM_014930	gi|154685926	YP_001421087	ylbN	20048/4.60	19672/4.46	165	70	+2.6738
577	Hypothetical protein RBAM_028130	gi|154687215	YP_001422376	yuaE	19112/5.46	18233/5.34	134	61	+4.2202
581	50S ribosomal protein L10	gi|308171995	YP_003918700	rplJ	17993/5.24	16738/4.97	101	61	+63.1951
633	Two-component response regulator	gi|16080602	NP_391429	degU	25907/5.66	24562/5.53	98	53	+2.5319
638	YraA	gi|363723843	EHM03981	–	18714/4.94	17436/4.75	100	59	+20.1731
685	Hypothetical protein RBAM_003150	gi|154684784	YP_001419945	yceC	21763/5.10	20637/4.68	337	65	+2.5326
697	Phosphomethylpyrimidine kinase	gi|154685606	YP_001420767	yjbV	29037/5.93	28436/5.53	211	52	+2.5372
705	Hypothetical protein RBAM_020380	gi|154686470	YP_001421631	ypqE	17961/5.22	16367/5.13	80	22	+65.1469

a

Spot numbers assigned by the software refer to the proteins labeled in Fig. 1.

b

Protein name in the National Center for Biotechnology Information (NCBI) database for B. amyloliquefaciens.

c

Accession number in the NCBI database for B. amyloliquefaciens.

d

The specific location of a gene or DNA sequence on of the B. amyloliquefaciens chromosome.

e

Gene designation in the NCBI database for B. amyloliquefaciens.

f

Theoretical molecular mass (Mr) and isoelectric point (pI) were obtained from the protein database in the NCBI database for B. amyloliquefaciens.

g

Experimental molecular mass (Mr) and isoelectric point (pI) were obtained from the 2-DE gels.

h

MASCOT protein score from MS.

i

Percentage of amino acids in reference proteins covered by matching peptides from MS.

j

Fold change: positive values represent over-expressed proteins, negative values represent under-expressed proteins, “have” indicates that the protein appeared only in high-yield strain FMB72, “no” indicates that the protein appeared only in strain ES-2–4.

Cellular localization analysis of experimentally identified proteins

PSORTb tool version 3.0.2 (http://www.psort.org/psortb/index.html) was used to predict the cellular localization of the 44 identified proteins (Table 2). Thirty-nine proteins were found to be located at cytoplasm, one protein was in cytoplasmic membrane, one protein was extracellular, and four proteins had an unknown cellular location (Fig. 2).

Table 2.

Cellular localization and function of differentially regulated cellular proteins (>2-fold change in expression) of B. amyloliquefaciens FMB72.

Spot no.^a	Protein name^b	COG^c	Celluarlocalization^d	Biological process^e	Molecular functional annotation^f
Energy production and conversion
63	MtnD	C	Cytoplasmic	Cellular metabolic process; mitochondrial electron transport, NADH to ubiquinone; respiratory electron transport chain	NADH dehydrogenase (ubiquinone) activity
Amino acid transport and metabolism
102	Alanine dehydrogenase	E	Cytoplasmic	l-Alanine catabolic process; alanine catabolic process; sporulation resulting in formation of a cellular spore	Alanine dehydrogenase activity; metal ion binding; nucleotide binding
107	Aspartate-semialdehyde dehydrogenase	E	Cytoplasmic	’De novo’ l-methionine biosynthetic process; diaminopimelate biosynthetic process; isoleucine biosynthetic process; lysine biosynthetic process via diaminopimelate; threonine biosynthetic process	Aspartate-semialdehyde dehydrogenase activity; N-acetyl-gamma-glutamyl-phosphate reductase activity; NAD binding; NADP binding
461, 493	tcyK gene product	E	Unknown	Amino acid transport	Transporter activity
Carbohydrate transport and metabolism
87, 433	Translaldolase	G	Cytoplasmic	Phosphate shunt	Transferase activity
434	SdaAB	G	Cytoplasmic	Gluconeogenesis	4 iron, 4 sulfur cluster binding; amino acid binding; l-serine ammonia-lyase activity
Coenzyme metabolism
431	6,7-Dimethyl-8-ribityllumazine synthase	H	Cytoplasmic	Riboflavin biosynthetic process	6,7-Dimethyl-8-ribityllumazine synthase activity; transferase activity
483	Pyridoxal biosynthesis lyasePdxS	H	Cytoplasmic	Pyridoxal phosphate biosynthetic process	Lyase activity
505	Pyridoxine biosynthesis protein	C	Cytoplasmic	Thiamine biosynthetic process	ATP binding; metal ion; binding; phosphomethylpyrimidine kinase activity; pyridoxal kinase activity
508, 697	Phosphomethylpyrimidine kinase	H	Cytoplasmic	Thiamine biosynthetic process; thiamine diphosphate biosynthetic process	ATP binding; hydroxymethylpyrimidine kinase activity; phosphomethylpyrimidine kinase activity
533	NfrA	H	Unknown	Aromatic compound catabolic process; response to toxic substance	FMN reductase (NADH) activity; FMN reductase (NADPH) activity
Lipid metabolism
108	Chemotaxis-specific methylesterase	I	Cytoplasmic	Chemotaxis	Phospho relay response regulator activity
473, 501	Thiol peroxidase		Cytoplasmic	Cell redox homeostasis	Thioredoxin peroxidase activity
Translation, ribosomal structure, and biogenesis
72	Elongation factor Ts	J	Cytoplasmic	Positive regulation of GTPase activity	Guanyl-nucleotide exchange factor activity; translation elongation factor activity; zinc ion binding
581	50S ribosomal protein L10	J	Cytoplasmic	Response to stress; translation	Large ribosomal subunit rRNA binding
Transcription
103	Heat-inducible transcription repressor	K	Cytoplasmic	Regulation of transcription, DNA-dependent; response to stress; transcription, DNA-dependent	DNA binding
110	DNA-directed RNA polymerase subunit alpha	K	Cytoplasmic	Transcription, DNA-templated	DNA binding; DNA-directed RNA polymerase activity; zinc ion binding
118	Bifunctional pyrimidine regulatory protein PyrR uracil phosphoribosyltransferase	K	Cytoplasmic	DNA-templated transcription, termination; nucleoside metabolic process; regulation of transcription, DNA-templated	RNA binding; uracil phosphoribosyltransferase activity
416	Transcription elongation factor GreA	K	Cytoplasmic	Regulation of DNA-dependent transcription, elongation; response to stress; transcription, DNA-dependent	DNA binding
DNA replication
70	Recombination protein	L	Cytoplasmic	ATP catabolic process; DNA duplex unwinding; DNA geometric change; DNA recombination;DNA repair; heteroduplex formation; meiotic sister chromatid segregation; reciprocal meiotic recombination	ATP binding; DNA-dependent ATPase activity; DNA topoisomerase activity; DNA translocase activity; double-stranded DNA binding; helicase activity
Cell motility and secretion
93	Flagellar motor protein MotS	N	Cytoplasmic membrane	Rotation of the flagellar motor	The flagellar motor switch
513	ATP-dependent Clp protease proteolytic subunit	NO	Cytoplasmic	Misfolded or incompletely synthesized proteincatabolic;process; response to heat; response to temperature stimulus	Identical protein binding; serine-type endopeptidase activity; serine-type peptidase activity
General function prediction
638	YraA	R	Cytoplasmic	Proteolysis; response to stress	Hydrolase activity, acting on glycosyl bonds; peptidase activity
Signal transduction mechanisms
99	Spo0A	T	Cytoplasmic	Intracellular signal transduction; regulation of sporulation resulting in formation of a cellular spore	DNA binding; calcium ion binding; phosphorelay response regulator activity; sequence-specific DNA binding transcription factor activity
546	ComA	T	Cytoplasmic	Intracellular signal transduction; transcription, DNA-dependent	DNA binding; phosphorelay response regulator activity; sequence-specific DNA binding transcription factor activity
550	S-ribosylhomocysteinase	T	Cytoplasmic	Quorum sensing	Iron ion binding
633	Two-component response regulator	TK	Cytoplasmic	Circadian rhythm; negative regulation of transcription, DNA-templated; phosphorelay signal transduction system; red or far-red light signaling pathway; regulation of transcription, DNA-templated;response to temperature stimulus; transcription, DNA-templated	DNA binding
Others
55	Hypothetical protein RBAM_013590	–	Extracellular	–	–
61	Hypothetical protein RBAM_008300	–	Cytoplasmic	–	–
86	Hypothetical protein RBAM_020150	–	Cytoplasmic	–	–
97	Hypothetical protein RBAM_031300	–	Cytoplasmic	–	–
101	Hypothetical protein RBAM_022210	–	Cytoplasmic	–	–
115, 132	Hypothetical protein RBAM_031300	–	Cytoplasmic	–	–
435	Hypothetical protein RBAM_008040	–	Cytoplasmic	–	–
443, 446	Hypothetical protein RBAM_035480	–	Unknown	–	–
476	Hypothetical protein RBAM_015250	–	Cytoplasmic	–	–
506	Hypothetical protein HMPREF0984_00182	–	Cytoplasmic	–	–
507	Hypothetical protein RBAM_035310	–	Unknown	–	–
516	Hypothetical protein RBAM_028130	–	Cytoplasmic	–	–
522	Hypothetical protein RBAM_008300	–	Cytoplasmic	–	–
572	Hypothetical protein RBAM_014930	–	Cytoplasmic	–	–
577	Hypothetical protein RBAM_028130	–	Cytoplasmic	–	–
685	Hypothetical protein RBAM_003150	–	Cytoplasmic	–	–
705	Hypothetical protein RBAM_020380	–	Cytoplasmic	–	–

a

Spot numbers assigned by the software refer to the proteins labeled in Fig. 1.

b

Protein name in the National Center for Biotechnology Information (NCBI) database for B. amyloliquefaciens.

c

Cellular localization of proteins.

d

Clusters of orthologous groups.

e

Biological process was assigned according to the protein knowledge base (www.uniprot.org) for B. amyloliquefaciens.

f

Molecular functional annotation was assigned according to the protein knowledge base (www.uniprot.org) for B. amyloliquefaciens.

Fig. 2.

Cellular localization of the differentially expressed proteins identified in B. amyloliquefaciens FMB72 predicted by the PSORTb database.

Classification and functional analysis of differential proteins

Experimentally identified proteins were functionally characterized by clusters of orthologous groups (COG) analysis (Table 2). The separated proteins were chiefly divided into the following categories: energy production and conversion (C), amino acid transport and metabolism (E), carbon transport and metabolism (G), coenzyme metabolism (H), lipid metabolism (I), translation, ribosomal structure, and biosynthesis (J), transcription (K), DNA replication (L), cell motility and secretion (N), general function prediction (R), signal transduction mechanisms (T) and not included in the COG classification (–). To determine the mechanism of increased antimicrobial peptide yield from B. amyloliquefaciens FMB72, biological process and molecular function data were acquired from the UniProKB (www.uniprot.org) database.

Gene expression verification by qRT-PCR

Expression of the two genes encoding differentially expressed proteins related to fengycin synthesis (comA, spo0A) was analyzed by qRT-PCR analysis of mRNA from FMB72. The mRNA expression profiles of these genes are shown in Fig. 3. The mRNA levels of comA and spo0A were upregulated1 5.8 and 12.1 fold in FMB72. The upregulated expression of comA and spo0A mRNA in FMB72 agreed with their protein levels.

Fig. 3.

qRT-PCR analysis of mRNA expression of comA and spo0A genes. Asterisks indicate a statistically significant difference (p < 0.05) between the parental strain ES-2–4 and recombination strain FMB72.

Discussion

The majority of the identified proteins in this experiment are related to energy production and conversion, amino acid transport and metabolism, carbon transport and metabolism, coenzyme metabolism, lipid metabolism, translation, ribosomal structure, and biosynthesis, transcription, DNA replication, cell motility and secretion, general function prediction, signal transduction mechanisms, and not included in the COG classification. These proteins are analyzed as the following.

Proteins related to signal transduction mechanisms

There are four proteins involved in signal transduction mechanisms. The prokaryotes are mainly regulated at the transcriptional level. The activator protein bind the sequences close to promoter, the affinity enhancement of RNA polymerase with the promoter, and RNA polymerase activity augmentation. The repressor protein can hinder gene transcription by binding manipulation sequence. Spo0A is a very important regulatory protein in bacterial gene regulation system¹⁷ and the main control fact or of biofilm formation.¹⁸ It can control the opening and closing of downstream genes through phosphorylation and dephosphorylation. As previously mentioned Spo0A¹⁹ (upregulated) and ComA²⁰ (upregulated) as a transcription factor, it is speculated that the expression change and fengycin production increase in the high-yield strain are closely related (Fig. 4). Antibiotics synthesis is closely related to the biofilm formation and the growth of the spore. It is showed that lipopeptide biosynthesis, biofilm formation and the growth of spores in the same metabolic network. Bacillus sporulation and biofilm formation are governed by the regulatory protein Spo0A. The upregulation of Spo0A is possible helpful to the formation of biofilm. It is reported that antibiotic is produced in a biofilm. Thereby, it is very well understood that the synthesis of fengycin is modulated by Spo0A. ComA could also work through modulating transition state gene expression. It is assumed that ComA acts as a transcriptional regulatory protein, and can directly bind to the fen promoter, thus promoting the synthesis and secretion of fengycin. The further functional verification of transcription proteins will be carried out in subsequent experiments.

Fig. 4.

Simplified scheme showing some of the regulators of fengycin synthesis and the roles of ComA and Spo0A in positive (→) regulation. The solid lines show the regulation has been confirmed in the literature, the dash lines show the regulation speculated in this study.

S-ribosylhomocysteinase upregulated in the recombination strain, has lyase activity, which is both combined with the iron ions and related to quorum sensing.²¹

Metabolism-related proteins

We identified 12 differentially expressed proteins related to the metabolism of lipids, carbohydrates, coenzymes, and amino acids. The synthesis levels of key enzymes involved in glycolysis and the pentose phosphate pathway of glucose metabolism were higher in the genome-shuffled strain. The putative implications of these increases are described below. Transaldolase in the non-oxidative stage of the pentose phosphate pathway can catalyze the reaction between glyceraldehyde-3-phosphate and 7-sedoheptulose monophosphate to generate 4-phosphate erythrose and fructose 6-phosphate. NADP produced in the pentose phosphate pathway could provide reducing power for the biosynthesis reaction, while pentose phosphate generated in this pathway could then participate in nucleic acid metabolism. SdaAB can enhance gluconeogenesis, thus amino acids into sugar may be the main pathway of amino acid metabolism.

Alanine dehydrogenase plays an important role in amino acid transport and metabolism. Because of this function, it was widely believed that Aspartate-semialdehyde dehydrogenase played an important regulatory function in a series of pathological and physiological processes, has aspartate-semialdehyde dehydrogenase activity, and catalyzes the synthesis of aspartate. We speculate that the increased fengycin production by strain FMB72 is a result of increases in this enzyme.

Furthermore, expression levels of key enzymes in lipid metabolism and coenzyme metabolism processes were also raised. The upregulation of chemotaxis-specific methylesterase and thiol peroxidaseimprove lipid metabolism, and 6,7-dimethyl-8-ribityllumazine synthase, pyridoxal biosynthesis lyase PdxS, phosphomethylpyrimidine kinase, NfrA, pyridoxine biosynthesis protein would greatly improve the metabolic activities of coenzyme in the recombination strain. Thus, we speculate that the increase in fengycin yield accompanies the increased synthesis of key enzymes of the glycolysis and pentose phosphate pathways. Additionally, the abundance of alanine dehydrogenase and other key enzymes in amino acid metabolism likely enhances fengycin production.

Proteins related to energy generation and conversion

One of the identified protein (MtnD) was related to energy production and conversion, which is upregulated in the recombination strain. MtnD is in respiratory electron transport chain with NADH dehydrogenase (ubiquinone) activity. The enhancement of synthesis of enzymes associated with energy production and conversion may cause the increase of the fengycin yield indirectly.

Proteins related to DNA replication

The DNA replication-related protein, recombination protein is upregulated with the function of DNA duplex unwinding, DNA recombination, DNA repair and heteroduplex formation. Thus, the capability of DNA replication is improved in fengycin high-yield strain FMB72.

Proteins related to transcription

The three proteins related to transcription, DNA-directed RNA polymerase subunit alpha, bifunctional pyrimidine regulatory protein PyrR uracil phosphoribosyltransferase and transcription elongation factor GreA are all up-regulated. The heat-inducible transcription repressor is down-regulated. Therefore, the level of transcription is strengthened in the shuffled strain.

Proteins related to translation, ribosomal structure, and biogenesis

There are two proteins related to translation, ribosomal structure, and biogenesis. One is elongation factor Ts, the other is 50S ribosomal protein L10.

Translation elongation factor Ts belong to the protein elongation factor family and is related to protein synthesis. The elongation factor Ts is one of the three elongation factors in prokaryotes and necessary for prokaryotic protein synthesis.

Ribosome is the place of protein biosynthesis. Ribosome size is demonstrated by the sedimentation coefficient S. There are approximately 20,000 ribosomes in a eugenic bacteria, wherein the proteins account for 10% of the total cellular proteins, rRNA account for 80% of the total cellular RNA. In prokaryote 70S ribosome, the 30 S subunit contain 22 kinds of the ribosomal protein, the 50 S subunit contain 34 kinds of the ribosomal protein, accounting for 35% of the ribosome. Ts and methionine aminopeptidase play an important role in protein processing. For this reason, the upregulation of two proteins in the recombination strain will more effectively ensure the process of protein translation.

Proteins related to cell motility and secretion

There are two proteins involved in cell motility and secretion, flagellar motor protein MotS and ATP-dependent Clp protease proteolytic subunit. In addition, the upregulation of the two proteins can improve the cell motility and secretion in FMB72.

Proteins related to general function prediction and hypothetical proteins

There is one protein, YraA related to general function prediction. 17 spots subjected to mass spectrometry are identified as hypothetical proteins. They may play important roles directly and/or indirectly in response to fengycin synthesis. Thus, the more additional experiments are needed to gain the related protein function message in fengycin synthesis process.

Conclusions

All these indicated that the metabolic capability of mutant was improved by the genome shuffling. We obtained two metabolic proteins in the database for which we are uncertain about their specific function. They are ComA (spot 546) and Spo0A (spot 99), respectively. They may play important roles directly and/or indirectly in response to fengycin synthesis.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 31571887).