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Biotechnology and Biological Transformations

Knockout of rapC improves bacillomycin D yield based on de novo genome sequencing of Bacillus amyloliquefaciens fmbJ Jing Sun, Shiquan Qian, Jing Lu, Yanan Liu, Fengxia Lu, Xiaomei Bie, and Zhaoxin Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00418 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of Agricultural and Food Chemistry

Knockout of rapC improves bacillomycin D yield based on de novo genome sequencing of Bacillus amyloliquefaciens fmbJ Jing Sun, Shiquan Qian, Jing Lu, Yanan Liu, Fengxia Lu, Xiaomei Bie, Zhaoxin Lu*

College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing, China, 210095

*

Corresponding author, Tel.: +86-25-84396583; Fax: +86-25-84396583. E-mail address: [email protected] (Zhaoxin Lu) Present address: Weigang 1, Nanjing, Jiangsu Province 210095, P. R. China

ACS Paragon Plus Environment


1

Abstract

2

Bacillus amyloliquefaciens, a gram-positive and soil-dwelling bacterium, could

3

produce secondary metabolites that suppress plant pathogens. In this study, we

4

provided the whole genome sequence results of B. amyloliquefaciens fmbJ which had

5

one circular chromosome of 4,193,344 bp with 4,249 genes, 87 transfer RNA genes,

6

and 27 rRNA genes. In addition, fmbJ was found to contain several gene clusters of

7

antimicrobial lipopeptides (bacillomycin D, surfactin, and fengycin) and bacillomycin

8

D homologues were further comprehensively identified. To clarify the influence of

9

rapC regulating the synthesis of lipopeptide on the yield of bacillomycin D, rapC

10

gene in fmbJ was successfully deleted by marker-free method. Finally, it was found

11

that the deletion of rapC gene in fmbJ significantly improved bacillomycin D

12

production from 240.7 ± 18.9 mg/L to 360.8 ± 30.7 mg/L, attributed to the increased

13

the expression of bacillomycin D synthesis-related genes through enhancing the

14

transcriptional level of comA, comP, and phrC. These results showed that the

15

production of bacillomycin D in B. amyloliquefaciens fmbJ might be regulated by the

16

RapC-PhrC system. The findings are expected to advance further agricultural

17

application of bacillus spp. as a promising source of natural bioactive compounds.

18

Keywords：Bacillus amyloliquefaciens, genome sequence, bacillomycin D, rapC,

19

knockout

20


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1. Introduction

22

Many plant diseases caused by pathogenic microorganisms lead to decrease in

23

quality and yield of the important crops. With increasing concern about environmental

24

sustainability and the health of consumers, the application of synthetically chemical

25

fungicides in agriculture is restricted 1. As the plant growth-promoting rhizobacteria

26

(PGPR), Bacillus spp. have shown good prospects for the replacement of chemical

27

fungicides in sustainable agriculture 2. They were well known to secrete compounds

28

promoting plant growth, and produce many secondary metabolites with antimicrobial

29

activity

30

bio-control strain, showed strong antagonistic effect on some fungal pathogens, such

31

as Fusarium graminearum 5, Aspergillus ochraceus 6, A. flavus 7, and Rhizopus

32

stolonifer 8. Its inhibitory effect on these pathogens is mainly due to the produced

33

secondary metabolites. However, their practical application were restricted by poor

34

yield of these secondary metabolites. To improve their yield in B. amyloliquefaciens

35

fmbJ, the sequencing of whole genome will provide a deeper understanding on the

36

biosynthesis of secondary metabolites, and important information for the

37

biotechnological modification of the strain.

3, 4

. Bacillus amyloliquefaciens fmbJ (formerly Bacillus subtilis fmbJ), a

38

B. amyloliquefaciens can be used as a biological control agent to destroy its

39

rivals (such as fungi and bacteria). They not only need complex regulatory networks

40

to respond to the extracellular stimuli variation and control the community

41

differentiation9, but also can secrete antimicrobial active substances (such as

42

lipopeptides). There is the inseparable relationship between many two-component



43

systems and other systems and the synthesis of antimicrobial lipopeptides in Bacillus

44

strains. The system of Rap-Phr, as one of the core components of the complicated

45

regulatory network, is composed by response regulator aspartate phosphatase (Rap)

46

and its inhibitory oligopeptide (Phr) 10. Several Rap proteins can regulate the process

47

of community differentiation through reducing phosphorylation modification or

48

binding of the response regulator proteins 11. The literature showed that RapC was

49

able to suppress DNA-binding ability of ComA and the phosphorylation modification

50

of ComA (ComA~P), which combines with target DNA and increases the expression

51

of surfactin biosynthetic genes

52

adjust several subpopulations differentiation in B. subtilis 14. In addition, the study of

53

Yang et al.

54

sporulation, and competent cells in Bacillus sp. However, few studies have explored

55

the regulation of antimicrobial lipopeptides except for surfactin by the Rap-Phr

56

system.

15

12, 13

. Recently, a system of RapP-PhrP was used to

revealed that RapQ-PhrQ system could regulate surfactin production,

57

Here, the whole genome sequence of B. amyloliquefaciens fmbJ was reported.

58

The strain has remarkable variations in colonial feature with other Bacillus strains

59

isolated in our laboratory. Moreover, we found that there are gene clusters of

60

bacillomycin D in fmbJ genome which are different from B. subtilis 168.

61

Bacillomycin D as a part of the iturin family’s lipopeptide is composed by one

62

β-amino fatty acid and seven α-amino acids

63

antimicrobial bioactivity

64

agriculture. However, the production of bacillomycin D in wild strain is too low to

16

. It was reported to have high

5-7

. Therefore, it has a promising potential application in


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65

satisfy the production and application. To improve the bacillomycin D yield, we

66

successfully deleted the rapC gene in fmbJ. Furthermore, we present several evidence

67

that the RapC-PhrC can regulate production of bacillomycin D in B.

68

amyloliquefaciens.

69

2. Materials and methods

70

2.1 Strain and cultivation

71

B. amyloliquefaciens fmbJ strain (used name B. subtilis fmbJ) was isolated and

72

characterized in our laboratory. The strain was stored in the China General

73

Microbiological Culture Collection Center as Bacillus sp. (CGMCC 0943). For DNA

74

analysis, the bacteria were cultivated in Luria-Bertani (LB) medium containing 0.5

75

g/L yeast extract, 1 g/L tryptone, and 1 g/L NaCl (pH 7.0) at 37 °C with 180 rpm for

76

24 h. The strain was inoculated in beef extract medium (3% beef extract, 10% peptone,

77

and 5% NaCl (pH 7.2)) and cultivated at 37 °C with 180 rpm as a pre-culture. The

78

Landy medium (20 g/L glucose, 5 g/L L-glutamic acid, 1 g/L yeast extract, 1.0 g/L

79

KH2PO4, 0.5 g/L MgSO4·7H2O; 0.5 g/L KCl, 5.0 mg/L MnSO4, 0.16 mg/L

80

CuSO4·5H2O, 0.15 mg/L FeSO4·7H2O, pH 7.0) was used as a fermentation medium

81

for the preparation of bacillomycin D. The fermentation conditions were 33 °C, 180

82

rpm, and for 72 h. Escherichia coli DH5α was severed as a host for plasmid

83

replication and E. coli JM110 (dam-/dcm-) was utilized for demethylation. They were

84

cultivated in LB medium at 37 °C. If necessary, 100 µg/mL ampicillin and 5 µg/mL

85

erythromycin were added for E. coli and B. amyloliquefaciens, respectively. In the

86

study, all the strains and plasmids are summarized in Table 1. All the chemicals and



87

culture medium compositions were bought from Sinopharm Chemical Reagent Co.,

88

Ltd, Nanjing, China.

89

2.2 Whole genome sequencing and assembly

90

The complete genomic sequencing of the fmbJ was carried out by the Beijing

91

Genomic Institute (BGI, Shenzhen, China) by a union of randomly sheared libraries.

92

Illumina Hiseq 4000 sequencing platform (Illumina; CA, USA) and PacBio RSII

93

sequencing platform (Pacific Biosciences, USA) were applied to perform the genomic

94

DNA sequencing, and the evaluations of all generated reads were qualitative 17, 18. De

95

novo assembly was carried out with the Short Oligonucleotides Alignment Program

96

(SOAP) denovo_v2.04 using the clean data

97

was used to estimate the size of genome, the degree of heterozygosis and the degree

98

of duplication. The result exhibited that the genome size of fmbJ was 4.28 Mb (Fig.

99

S1). After genome assembling, the GC distribution of fmbJ was obtained by

100

GC-Depth analysis (Fig. S2).

101

2.3 Genome analysis and annotation

19, 20

. Before assembling, k-mer analysis

102

Putative genes were accomplished through Glimmer_v3.02 to identification 21, 22.

103

All putative genes were checked with databases to acquire their consistent annotations.

104

In order to make sure the biological significance, the best alignment result was

105

selected as annotation. Functional annotation was finished via BLAST with different

106

public databases. Repeating DNA sequences were identified using Tandem Repeat

107

Finder (version 4.04, http://tandem.bu.edu/trf/trf.html)

108

minisatellite DNA and microsatellite DNA were determined by the length and number


23

. The selection of the

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109

of repeat units. The tRNA genes and the tRNA secondary structure were predicted

110

using tRNAscan-SE 24, and the genes of rRNA were identified via BLAST according

111

to the current rRNA database. The result of rRNA database blasting is precise but not

112

complete, and rRNAmmer was used to predict rRNA when there was no homology

113

reference 25. The sRNA genes were identified using Rfam database 26. The structural

114

variation in fmbJ genome was determined according to that of the reference bacterium

115

based on Mummer

116

information is summarized in Fig. S3, S4. Meanwhile, the Core-Pan genes in fmbJ

117

and four other Bacillus. strains (B. amylpliquefaciens FZB42, B. amyloliquefaciens

118

NAU-B3, B. amyloliquefaciens Y2, and B. subtilis 168) were confirmed using

119

BLAST based on the method of Qin et al. 28.

120

2.4 Lipopeptide extraction and HPLC/ESI/CID-MS analysis

27

, including amino acid level and nucleic acid level. Detailed

121

After centrifugation, the fermentation broth supernatant was collected, and its pH

122

was adjusted to 2.0 by 4 M HCl. Then, the solution was stored at 4 °C until further

123

treatment. Subsequently, the precipitation was collected using centrifugation and

124

discarding the supernatant. The right amount of methanol was added to the

125

precipitation to extraction of the substance, and the pH was adjusted to 7.0. After

126

10,000 g × 10 min centrifugation, the crude products of bacillomycin D were obtained.

127

Bacillomycin D crude products were separated by Sephadex LH-20 column.

128

Preparation HPLC (Waters 600, USA) with a C18 column (Eclipse XDB, 5µm

129

4.6×250 mm, Agilent, USA) was used for further purification. Water with 0.1%

130

trifluoroacetic acid (TFA) and acetonitrile with 0.1% TFA were utilized for moving



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131

phase. The structure of bacillomycin D was identified by high performance liquid

132

chromatography/electrospray

133

spectrometry (HPLC/ESI/CID-MS) and Surveyor-LCQ DECA XP Plus of Thermo

134

Finnegan (Thermo Electron Corporation, San Jose, CA, USA). Specific HPLC and

135

MS conditions were carried out according to Gong et al. 7 and Qian et al. 29.

136

2.5 Strain construction

ionization/collision-induced

dissociation-mass

137

The temperature-sensitive vector pCBS was used to delete the rapC gene of

138

strain fmbJ. The upstream/ downstream regions of rapC were amplified with two

139

pairs of primers rapC-P1: 5′-gtcgacGAAGAAACGAAGCGGATG (SalI restriction

140

site

141

5′-CAAATAACAAACCATTCCTTCACCCTCCCCATCCA,

142

GGGGAGGGTGAAGGAATGGTTTGTTATTTGTTTAG,

143

agatctGCAGGAACTTCAAGCAGA (BglII restriction site underlined). The two

144

fragments were spliced via splicing by overlap extension (SOE) PCR, followed by

145

insertion of the spliced fragments (1407 bp) into pCBS using the SalI/ BamHI

146

restriction sites (because BamHI and BglII are the same tailed enzymes), resulting in

147

pCBS△rapC. The recombined plasmid pCBS△rapC was transferred into fmbJ

148

successfully through electro transformation technology

149

the rapC gene in B. amyloliquefaciens fmbJ was conducted according to the

150

previously procedure reported

151

selected. Then the rapC-deleted mutants were validated by PCR amplification and

152

sorting by the primer pair rapC-P1 and rapC-P4.

underlined),

rapC-P2: rapC-P3: and

rapC-P4:

5′5′-

30

. A marker-free deletion of

30

. Colonies without erythromycin resistance were


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153

2.6 Reverse transcription quantitative real-time PCR (RT-qPCR)

154

Total RNA was extracted with Trizol Reagent (TranGen Biotech, Beijing, China)

155

according to the protocol of manufacturer. RNA quality was examined by

156

electrophoresis on a 2.0% agarose gel, and its amount was analyzed by a

157

spectrophotometer (NanoDrop 2000, Thermo Scientific, USA). For RT-qPCR analysis,

158

1 µg RNA sample was used for cDNA synthesis with 5X All-In-One RT MasterMix

159

(AccuRT Genomic DNA Removal Kit; Applied Biological Materials Inc., Canada)

160

according to the protocol of manufacturer. Real-time PCR was carried out with a

161

mixture containing 1 µL cDNA, 0.2 µM forward primer, 0.2 µM reverse primer, 10 µL

162

Hieff™ qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen, Shanghai, China),

163

and ddH2O in 20 µL of total volume. DNA was amplified with Real-Time PCR

164

System (StepOnePlus™,Applied Biosystems, USA) to analyze the expression of target

165

genes (bymA, bymB, bymC, bymD, TE, comA, comP, and phrC) under the PCR

166

procedure below: denaturation 5 min at 95 °C and 40 cycles of 95 °C for 10 s, 60 °C

167

for 30 s. The primers used for amplification of the reference gene 16sRNA and target

168

genes were presented in Table S1. The relative fold change of the target genes

169

expression was evaluated by the calculation of the 2-△△Ct. The threshold cycle (Ct)

170

values were obtained by the Real-Time PCR System software (StepOnePlusTM,

171

Applied Biosystems,USA)) 31.

172

2.7 Statistical analysis

173

All statistical analyses were carried out with one-way analysis of variance

174

(ANOVA) by SPSS (SPSS version 17.0, IBM, USA). After checking analysis results



175

of ANOVA, the p-value was given. Duncan's test was used to examine significance

176

below 0.05. Means ± standard deviation (SD) and triplicate of assay were used to

177

express all results.

178

3. Results

179

3.1. General genomic features of B. amyloliquefaciens fmbJ

180

The complete genomic sequencing of B. amyloliquefaciens fmbJ was carried out

181

by Illumina Hiseq 4000 sequencing platform to produce 605 Mb clean data and

182

Pacbio RSII sequencing platform to produce 594 Mb clean data. Based on the

183

assemble result of sample fmbJ, we found that the genome contains a 4,193,344 bp

184

circular chromosome, with 45.98% G+C (Fig. 1). With 134 tandem repeats (including

185

103 minisatellite DNA and 10 microsatellite DNA), 11 small RNA (sRNA), 87

186

transfer RNA genes, and 27 rRNA genes (Table S2), the whole genome of fmbJ was

187

predicted to contain 4,249 genes. Compared with B. amylpliquefaciens FZB42, fmbJ

188

may have a much more complicated gene regulation, because it has more number of

189

genes than FZB42 (3892 genes) (Table 2). Although there was no obvious difference

190

in the size of genome, to find the functional differences and similarities among five

191

strains, the core and pan genes were analyzed by taking the genome of B.

192

amyloliquefaciens FZB42 as a reference, B. amyloliquefaciens NAU-B3, B.

193

amyloliquefaciens Y2, B. subtilis 168, and fmbJ as query

194

fmbJ genome, the core genes organized by the five Bacillus strains consisted of 3,342

195

genes were sharing more than 50% identity to each other (Fig. 2a). They are necessary

196

for growth. Some genes are special genes when they are contained only by one of the


28

. Out of 4,249 genes in

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bacteria. 5,143 pan genes were detected in five strains (Fig. 2b). Furthermore, we

198

analyzed the differences in five strains after removing the core genes (Fig. 3). The

199

results indicated that the difference between fmbJ and B. subtilis 168 was the largest,

200

with little difference among fmbJ, B. amyloliquefaciens NAU-B3 and B.

201

amyloliquefaciens Y2. The same results were found in fmbJ genomic structural

202

variation including nucleic acid level (Fig. S3) and amino acid level (Fig. S4).

203

After annotation, 2,956 CDSs (coding sequences) were assigned to putative

204

biological function, while 1,293 CDSs were considered as unknown function proteins.

205

For all CDSs without allocation functions, 1,238 CDSs were consistent with

206

conserved hypothetical proteins, whereas 55 CDSs were not homologous with any

207

previously reported sequences. By the analysis for the Cluster of Orthologous Groups

208

(COG), 3,424 CDSs were allocated to one or more COG functional groups. In

209

addition, 64 genes were related to controlling the bacterial mobility, 213 genes were

210

connected with the biosynthesis of membrane and cell wall, and 121 genes were

211

associated with transport, catabolism and the biosynthesis of secondary metabolites

212

(Fig. 4). The genome encodes a large number of pathways, including the production

213

of large amounts of antimicrobial substances. These genes may be favorable to

214

promote the growth of fmbJ strain and to protect its plant host from pathogens.

215

3.2. Cluster of non-ribosomal biosynthesized bacillomycin D in fmbJ

216

Three gene clusters (including bacillomycin D, fengycin and surfactin) for the

217

non-ribosomal biosynthesis of lipopeptides were found in fmbJ genome. The 37,250

218

bp bmy gene cluster was inserted in the fmbJ genome, which comprised four genes



219

(bmyD, bmyA, bmyB, bmyC). By compared the genome sequence of B.

220

amyloliquefaciens FZB42 (a strain of bacillomycin D production) with fmbJ, it was

221

found that bmyD (002202), bmyA (002201), bmyB (002200), and bmyC (002199) are

222

very similar to those of FZB42. BmyD, bmyA, bmyB, and bmyC are the synthetase

223

genes encoding bacillomycin D. The first gene bmyD codes a putative malonyl

224

coenzyme A transacylase which is nearly identical to the function of FabD in fatty

225

acid synthesis

226

synthetase, which are constituted as their relatives in the operons of mycosubtilin and

227

iturin A 32. A thioesterase (TE) domain is at the C-terminal end of bmyC, which has

228

the function of bacillomycin D cyclization and release.

229

3.3. HPLC-ESI mass spectrometry identification of bacillomycin D

16

. BmyA-C code the peptide forming subunits of bacillomycin D

230

Bacillomycin D was prepared by Sephadex LH-20 chromatography combined

231

with HPLC. Its liquid chromatographic analysis showed that 11 active peaks were

232

obtained in Fig. 5. These active peaks were further analyzed by MS/MS. The

233

corresponding compounds were confirmed by match up with the MS data of

234

bacillomycin D

235

contained seven peptide structures (-Asn-Tyr-Asn-Pro-Glu-Ser-Thr-). The MS of

236

peaks 1-3 displayed their [M + H]+ ion peaks at m/z 1030.60, 1031.30, and 1032.30,

237

respectively. Through analysis and comparison, we found that these peaks could be

238

identified to bacillomycin D of C14 fatty acid chain. The MS of C15 bacillomycin D

239

gave two [M+H]+ ion peaks at m/z 1045.30 (peaks 4) and 1046.31(peaks 5). The MS

240

of peaks 6-9 were confirmed as C16 bacillomycin D, which showed [M+H]+ ion peaks

7, 33, 34

. The results (Fig. S5) showed that the isolated bacillomycin D


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241

at m/z 1058.76, 1059.18, 1060.20, and 1061.50. In addition, the MS of C17

242

bacillomycin D gave two [M+H]+ ion peaks at m/z 1072.91 (peak 10) and

243

1073.61(peak 11). These molecular weight intervals of 14 (-CH2-) were homologues

244

of bacillomycin D 7.

245

3.4. The influence of rapC on bacillomycin D biosynthesis

246

In Bacillus, the phosphatase RapC was found to involve in the biosynthesis of

247

lipopeptide surfactin 35. Bacillomycin D and surfactin are lipopeptides, and does rapC

248

gene also regulate bacillomycin D biosynthesis? Furthermore, we found that there is

249

rapC (003893) gene in fmbJ from the results of genome sequencing. Subsequently,

250

the rapC gene in fmbJ was successfully knocked out using marker-free biological

251

technique. In this study, the lipopeptides collected from fmbJ strain and its mutant

252

fmbJ△rapC strain were detected by HPLC. Fig. 6 showed the production of

253

bacillomycin D between in fmbJ strain and its mutant fmbJ△rapC in 24-120 h. As

254

shown in Fig. 6, there was no obvious difference between the bacillomycin D

255

production of the deletion of rapC mutant and that of wild strain during 48 h.

256

However, the bacillomycin D production of the mutant was remarkably higher than

257

that of fmbJ after 72 h (Fig. 6). Its maximum yield was 360.8 mg/L, which was 1.5

258

times higher than the original yield of 240.7 mg/L. These results indicated that the

259

rapC gene could negatively regulate the synthesis of bacillomycin D.

260

3.5. The relationship between rapC and bacillomycin D genes and related signal

261

factors

262

To further validate the regulation of bacillomycin D production by rapC,



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263

RT-qPCR was used to detect the expression of bacillomycin D synthetase genes

264

(bmyA, bmyB, bmyC, bmyD and TE) and relative regulatory signal genes (comA,

265

comP and phrC) in fmbJ and its mutant. The results were displayed in Fig. 7A-D,

266

which presented that genes expression had a certain time-space properties. As shown

267

in Fig. 7A, compared with fmbJ strain, the expression of bacillomycin D synthetase

268

genes was decreased in fmbJ△rapC, except for bmyC and TE at 12h, whereas the

269

signal gene comP increased significantly at this time, 1.8 times as much as fmbJ strain.

270

As can be seen from Fig. 7B, TE and signal genes have increased significantly,

271

especially that the expression of the comA gene was 18.9 times as high as that of fmbJ

272

at 24 h. All the bacillomycin D synthetase genes and signal genes were significantly

273

increased at 36 h and 48 h (Fig. 7 C, D). Bacillomycin D was a kind of secondary

274

metabolites which was produced after the bacterial growth to a certain stage. It was

275

also explained that rapC knockout improved bacillomycin D synthase genes

276

expression in the late stage of strain growth, thus increasing bacillomycin D yield.

277

This confirmed that the rapC gene negatively regulated the expression of

278

bacillomycin D synthase genes in strain fmbJ.

279

4. Discussion

280

The genomic sequencing of fmbJ provides the theoretical basis for

281

biotechnological means to improve the bacillomycin D production. Bacillomycin D

282

that composed of 14–17 carbon atoms of β-amino fatty acid chain and a seven amino

283

acids of cyclic peptide, had high antifungal and antitumor activities

284

the signal proteins (such as ComA, ComP, PhrC and RapC) were also closely related


5, 7, 29, 36

. Some of

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16, 29, 37

285

to the biosynthesis of lipopeptides

. The fmbJ genome showed that as a major

286

transcriptional regulator, comA (001020) was probably to be related to histidine

287

kinases comP (001019) through its phosphorylated form comA~P 9. PhrC (003892),

288

as a regulator of phosphatase rapC (003893), plays an inhibitory role 11. As shown in

289

Fig. 7, the deletion of rapC gene led to an increase in the expression level of three

290

genes (comA, comP and phrC) in varying degrees, compared to the original strain.

291

The systems of Rap-Phr have been found to be take part in biosynthesis of

292

lipopeptides through the mutual effect with major regulatory factor (ComA)15. When

293

the density of cell population reaches a very high level, Phr peptides are re-entered

294

inside the cell by the oligopeptide permease (Opp), then they inhibit cognate Rap

295

proteins activities

296

involved in regulation of bacillomycin D production in B. amyloliquefaciens fmbJ.

297

The finding was in close agreement to Yang’s researchresult that RapQ-PhrQ system

298

could regulate the production of surfactin in B. subtilis OKB105

299

transcriptional regulatory factor was related to the regulation of lipopeptide

300

production and sporulation in Bacillus

301

ComA enable to initiate the srfA operon expression by binding with promotor, so the

302

synthesis of surfactin was promoted

303

with ComA restrained the ability of response regulator to bind its target DNA

304

promoter. It was found in the work that the knockout of rapC gene increased the

305

expression of comA and comP genes, which would be beneficial for starting the

306

transcription and for improving the transcriptional ability of bacillomycin D

38

. Our results revealed that the RapC-PhrC system might be

13

15

. ComA, a

15, 37

. ComA~P, the product phosphorylated

. Core et al.

12

reported that RapC interaction



307

synthetase genes, indicated the bacillomycin D synthesis was of similar regulation

308

mode to surfactin production.

309

In summary, whole genome sequencing shows that B. amyloliquefaciens fmbJ

310

has a good prospect of bio-control and could produce many kinds of bioactive

311

secondary metabolites, especially bacillomycin D. To improve bacillomycin D yield,

312

the rapC regulation gene in the wild strain fmbJ was successfully knocked out by

313

marker-free method. It was found by deletion of RapC gene that RapC-PhrC system

314

in B. amyloliquefaciens fmbJ are involved in the regulation of bacillomycin D and

315

regulate the expression of comA, comP, and phrC genes, as well bacillomycin D

316

production of rapC-null strain was significantly increased. Further studies using the

317

multi-omics and biotransformation techniques of B. amyloliquefaciens fmbJ on the

318

basis of genome sequencing will contribute to clarify the function of the putative

319

genes and the synthetic pathways of bioactive substances.

320 321

Acknowledgements

322

The work was supported by the National Natural Science Foundation of China

323

(No. 31571887), Agricultural Innovation Foundation of Jiangsu Province (CX

324

16-1058), Jiangsu Collaborative Innovation Center (2011 plan) of Meat Production

325

and Processing, Quality and Safety Control.

326 327 328


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Figure Captions and Tables Fig.1 The circular map of the Bacillus amyloliquefaciens fmbJ genome for several specific genomic features. Innermost circle (1st): scale (bps); 2nd circle: GC skew-(violet); 3rd circle: GC skew+ (green); 4th circle: GC content (black); 5th circle: all genes are labeled color according to their function. Fig. 2 Dilution curve of bacterial genes. A, core genes; B, pan genes. Fig. 3 Heat-map after core gene deletion. Fig. 4 COG annotation of sample bacillus. fmbJ. Fig. 5 Analysis of bacillomycin D by HPLC. Peaks 1-11: bacillomycin D. Fig. 6 The influence of rapC gene knockout in fmbJ on bacillomycin D production. The strains fmbJ and fmbJ△rapC were cultured in100 mL Landy at 33°C for 24-120 h. * and ** were significantly different from controls at 0.05 and 0.01. Fig. 7 Effect of rapC gene knockout on relative expression of bacillomycin D synthase genes and signal genes. The strains fmbJ and fmbJ△rapC were cultured in 100 mL Landy at 33°C for 12 h, 24 h, 36 h, and 48 h (A-D). * and ** were significantly different from controls at 0.05 and 0.01.

Tables Table 1 Microorganisms and plasmids presented in the study. Table 2 General features of the genomes of the fmbJ and other Bacillus species



Page 24 of 32

Tables Table 1 Microorganisms and plasmids presented in the study. Strains or plasmids

Characteristics

Source

Wild-type strain, producer of bacillomycin

Laboratory stock

B. amyloliquefaciens strain fmbJ

D fmbJ∆rapC

rapC deletion mutant, derivative of strain

Current study

fmbJ Trans 5α (E. coli DH 5α)

F-φ80 lac Z∆M15 ∆(lacZYA-arg F) U169

TransGen Biotech

endA1 recA1 hsdR17(rk-,mk+) supE44λ- thi -1 gyrA96 relA1 phoA Trans 110 (E. coli JM110)

rpsL (Str R) thr leu thi-1 lacY galK galT ara

TransGen Biotech

tonA tsx dam dcm supE44 Δ (lac-proAB) /F′[traD36 proAB lacIqlacZΔM15] Plasmids pMAD

E.

coli

and

B.

subtilis

shuttle,

Laboratory stock

temperature-sensitive vector. Apr Emr (9666 bp) pCBS

pMAD

with

minor

modification.

The

Current study

3928-6049 bases in pMAD were removed, and Pamy, SamyE and lacZ were added in that location. Apr Emr (8102 bp) pCBS∆rapC

pCBS with rapC deletion box. Apr Emr

Apr, Emr indicate resistance to ampicillin and erythromycin, respectively.


Current study

Page 25 of 32


Table 2 General features of the genomes of the fmbJ and other Bacillus species Species Genome size (bp)

B. amyloliquefaciens




B. subtilis

fmbJ

FZB42

NAU-B3

Y2

168

4,193,344

3,918,589

4,204,608

4,238,624

4,215,606

gene

4,249

3,892

4,194

4,246

4,420

tRNA

87

88

91

86

86

rRNA

27

29

30

29

30

GC%

45.98

46.50

45.99

45.90

43.50



Fig. 1 The circular map of the Bacillus amyloliquefaciens fmbJ genome for several specific genomic features. Innermost circle (1st): scale (bps); 2nd circle: GC skew-(violet); 3rd circle: GC skew+ (green); 4th circle: GC content (black); 5th circle: all genes are labeled color according to their function. 101x76mm (300 x 300 DPI)


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Fig. 2 Dilution curve of bacterial genes. A, core genes; B, pan genes. 168x84mm (300 x 300 DPI)



Fig. 3 Heat-map after core gene deletion. 82x77mm (300 x 300 DPI)


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Fig. 4 COG annotation of sample bacillus. fmbJ. 101x72mm (300 x 300 DPI)



Fig. 5 Analysis of bacillomycin D by HPLC. Peaks 1-11: bacillomycin D. 101x65mm (300 x 300 DPI)


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Fig. 6 The influence of rapC gene knockout in fmbJ on bacillomycin D production. The strains fmbJ and fmbJ△rapC were cultured in100 mL Landy at 33°C for 24-120 h. * and ** were significantly different from controls at 0.05 and 0.01. 212x174mm (300 x 300 DPI)



Fig. 7 Effect of rapC gene knockout on relative expression of bacillomycin D synthase genes and signal genes. The strains fmbJ and fmbJ△rapC were cultured in100 mL Landy at 33°C for 12 h, 24 h, 36 h, and 48 h (A-D). * and ** were significantly different from controls at 0.05 and 0.01. 229x170mm (300 x 300 DPI)


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Knockout of rapC improves bacillomycin D yield ... - ACS Publications

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