Advertisement

A comprehensive genomic and growth proteomic analysis of antitumor lipopeptide bacillomycin Lb biosynthesis in Bacillus amyloliquefaciens X030

  • Jiao Yang Lu
  • Kexuan Zhou
  • Wei Tao Huang
  • Pengji Zhou
  • Shuqing Yang
  • Xiaoli Zhao
  • Junyan Xie
  • Liqiu Xia
  • Xuezhi DingEmail author
Genomics, transcriptomics, proteomics

Abstract

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering).

Keywords

Genomics Growth proteomics Bacillomycin Lb Lipopeptide Anticancer 

Notes

Funding information

This work was financially supported by the National key Research and Development program of China (2017YFD0201201), the National Natural Science Foundation of China (31370116), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486).

Compliance with ethical standards

This study did not involve any research involving human participants or animals.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10019_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1173 kb)
253_2019_10019_MOESM2_ESM.xlsx (3 mb)
ESM 2 (XLSX 3053 kb)

References

  1. Bartolini M, Cogliati S, Vileta D, Bauman C, Ramirez W, Grau R (2019) The stress-responsive alternative sigma factor SigB plays a positive role in the antifungal proficiency of Bacillus subtilis. Appl Environ Microbiol 85.  https://doi.org/10.1128/AEM.00178-19
  2. Belbahri L, Chenari Bouket A, Rekik I, Alenezi FN, Vallat A, Luptakova L, Petrovova E, Oszako T, Cherrad S, Vacher S, Rateb ME (2017) Comparative genomics of Bacillus amyloliquefaciens strains reveals a core genome with traits for habitat adaptation and a secondary metabolites rich accessory genome. Front Microbiol 8:1438.  https://doi.org/10.3389/fmicb.2017.01438 PubMedGoogle Scholar
  3. Burgard C, Zaburannyi N, Nadmid S, Maier J, Jenke-Kodama H, Luxenburger E, Bernauer HS, Wenzel SC (2017) Genomics-guided exploitation of lipopeptide diversity in Myxobacteria. ACS Chem Biol 12(3):779–786.  https://doi.org/10.1021/acschembio.6b00953 PubMedGoogle Scholar
  4. Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J (2019) Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol 10:302.  https://doi.org/10.3389/fmicb.2019.00302 PubMedGoogle Scholar
  5. Cochrane SA, Vederas JC (2016) Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Med Res Rev 36(1):4–31.  https://doi.org/10.1002/med.21321 PubMedGoogle Scholar
  6. Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6):673–679.  https://doi.org/10.1093/bioinformatics/btm009 PubMedGoogle Scholar
  7. Demain AL (1998) Induction of microbial secondary metabolism. Int Microbiol 1(4):259–264PubMedGoogle Scholar
  8. D'Souza C, Nakano MM, Zuber P (1994) Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis. Proc Natl Acad Sci U S A 91(20):9397–9401.  https://doi.org/10.1073/pnas.91.20.9397
  9. Du L, Lou L (2010) PKS and NRPS release mechanisms. Nat Prod Rep 27(2):255–278.  https://doi.org/10.1039/b912037h PubMedGoogle Scholar
  10. Eshita SM, Roberto NH, Beale JM, Mamiya BM, Workman RF (1995) Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the congeners. J Antibiot 48(11):1240–1247PubMedGoogle Scholar
  11. Frikha-Gargouri O, Ben Abdallah D, Bhar I, Tounsi S (2017) Antibiosis and bmyB gene presence as prevalent traits for the selection of efficient Bacillus biocontrol agents against crown gall disease. Front Plant Sci 8:1363.  https://doi.org/10.3389/fpls.2017.01363 PubMedGoogle Scholar
  12. Hamon MA, Stanley NR, Britton RA, Grossman AD, Lazazzera BA (2004) Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis. Mol Microbiol 52(3):847–860.  https://doi.org/10.1111/j.1365-2958.2004.04023.x PubMedGoogle Scholar
  13. Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M (2005) The H2O2 stress-responsive regulator PerR positively regulates srfA expression in Bacillus subtilis. J Bacteriol 187(19):6659–6667.  https://doi.org/10.1128/JB.187.19.6659-6667.2005 PubMedGoogle Scholar
  14. He H, Zhu YL, Chi LQ, Zhao ZZ, Wang T, Zuo MX, Zhang T, Zhou FJ, Xia LQ, Ding XZ (2015) Screening and antibacterial function of Bacillus amyloliquefaciens X030. Wei Sheng Wu Xue Bao 55(9):1133–1143.  https://doi.org/10.13343/j.cnki.wsxb.201501 PubMedGoogle Scholar
  15. Higgins CF, Linton KJ (2004) The ATP switch model for ABC transporters. Nat Struct Mol Biol 11(10):918–926.  https://doi.org/10.1038/nsmb836 PubMedGoogle Scholar
  16. Hu Y, Nan F, Maina SW, Guo J, Wu S, Xin Z (2018) Clone of plipastatin biosynthesis gene cluster by transformation-associated recombination technique and high efficient expression in model organism Bacillus subtilis. J Biotechnol 288:1–8.  https://doi.org/10.1016/j.jbiotec.2018.10.006 PubMedGoogle Scholar
  17. Hu F, Liu Y, Li S (2019) Rational strain improvement for surfactin production: enhancing the yield and generating novel structures. Microb Cell Factories 18(1):42.  https://doi.org/10.1186/s12934-019-1089-x Google Scholar
  18. Kimmel PL, Fwu CW, Eggers PW (2013) Segregation, income disparities, and survival in hemodialysis patients. J Am Soc Nephrol 24(2):293–301.  https://doi.org/10.1681/ASN.2012070659 PubMedGoogle Scholar
  19. Koumoutsi A, Chen XH, Vater J, Borriss R (2007) DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloliquefaciens strain FZB42. Appl Environ Microbiol 73(21):6953–6964.  https://doi.org/10.1128/AEM.00565-07 PubMedGoogle Scholar
  20. Kraas FI, Giessen TW, Marahiel MA (2012) Exploring the mechanism of lipid transfer during biosynthesis of the acidic lipopeptide antibiotic CDA. FEBS Lett 586(3):283–288.  https://doi.org/10.1016/j.febslet.2012.01.003 PubMedGoogle Scholar
  21. Ladoukakis E, Pereira V, Magny EG, Eyre-Walker A, Couso JP (2011) Hundreds of putatively functional small open reading frames in Drosophila. Genome Biol 12(11):R118.  https://doi.org/10.1186/gb-2011-12-11-r118 PubMedGoogle Scholar
  22. Lim SM, Yoon M-Y, Choi GJ, Choi YH, Jang KS, Shin TS, Park HW, Yu NH, Kim YH, Kim J-C (2017) Diffusible and volatile antifungal compounds produced by an antagonistic Bacillus velezensis G341 against various phytopathogenic fungi. Plant Pathol J 33(5):488–498.  https://doi.org/10.5423/ppj.oa.04.2017.0073 PubMedGoogle Scholar
  23. Luo Y, Ding X, Xia L, Huang F, Li W, Huang S, Tang Y, Sun Y (2011) Comparative proteomic analysis of Saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield. Proteome Sci 9:40.  https://doi.org/10.1186/1477-5956-9-40 PubMedGoogle Scholar
  24. Luo C, Liu X, Zhou H, Wang X, Chen Z (2015) Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions. Appl Environ Microbiol 81(1):422–431.  https://doi.org/10.1128/AEM.02921-14 PubMedGoogle Scholar
  25. Luo S, Chen XA, Mao XM, Li YQ (2018) Regulatory and biosynthetic effects of the bkd gene clusters on the production of daptomycin and its analogs A21978C1-3. J Ind Microbiol Biotechnol 45(4):271–279.  https://doi.org/10.1007/s10295-018-2011-y PubMedGoogle Scholar
  26. Ma Z, Hu J, Wang X, Wang S (2014) NMR spectroscopic and MS/MS spectrometric characterization of a new lipopeptide antibiotic bacillopeptin B1 produced by a marine sediment-derived Bacillus amyloliquefaciens SH-B74. J Antibiot 67(2):175–178.  https://doi.org/10.1038/ja.2013.89 PubMedGoogle Scholar
  27. Medini D, Serruto D, Parkhill J, Relman DA, Donati C, Moxon R, Falkow S, Rappuoli R (2008) Microbiology in the post-genomic era. Nat Rev Microbiol 6(6):419–430.  https://doi.org/10.1038/nrmicro1901 PubMedGoogle Scholar
  28. Moussatova A, Kandt C, O'Mara ML, Tieleman DP (2008) ATP-binding cassette transporters in Escherichia coli. BBA-Biomembranes 1778(9):1757–1771.  https://doi.org/10.1016/j.bbamem.2008.06.009 PubMedGoogle Scholar
  29. Palazzotto E, Weber T (2018) Omics and multi-omics approaches to study the biosynthesis of secondary metabolites in microorganisms. Curr Opin Microbiol 45:109–116.  https://doi.org/10.1016/j.mib.2018.03.004 PubMedGoogle Scholar
  30. Podgornaia AI, Laub MT (2013) Determinants of specificity in two-component signal transduction. Curr Opin Microbiol 16(2):156–162.  https://doi.org/10.1016/j.mib.2013.01.004 PubMedGoogle Scholar
  31. Pretorius D, van Rooyen J, Clarke KG (2015) Enhanced production of antifungal lipopeptides by Bacillus amyloliquefaciens for biocontrol of postharvest disease. New Biotechnol 32(2):243–252.  https://doi.org/10.1016/j.nbt.2014.12.003 Google Scholar
  32. Qian S, Lu H, Meng P, Zhang C, Lv F, Bie X, Lu Z (2015) Effect of inulin on efficient production and regulatory biosynthesis of bacillomycin D in Bacillus subtilis fmbJ. Bioresour Technol 179:260–267.  https://doi.org/10.1016/j.biortech.2014.11.086 PubMedGoogle Scholar
  33. Ramamurthi KS, Storz G (2014) The small protein floodgates are opening; now the functional analysis begins. BMC Biol 12:96.  https://doi.org/10.1186/s12915-014-0096-y PubMedGoogle Scholar
  34. Reddy VS, Shlykov MA, Castillo R, Sun EI, Saier MH Jr (2012) The major facilitator superfamily (MFS) revisited. FEBS J 279(11):2022–2035.  https://doi.org/10.1111/j.1742-4658.2012.08588.x PubMedGoogle Scholar
  35. Salzberg LI, Botella E, Hokamp K, Antelmann H, Maass S, Becher D, Noone D, Devine KM (2015) Genome-wide analysis of phosphorylated PhoP binding to chromosomal DNA reveals several novel features of the PhoPR-mediated phosphate limitation response in Bacillus subtilis. J Bacteriol 197(8):1492–1506.  https://doi.org/10.1128/JB.02570-14 PubMedGoogle Scholar
  36. Sandrin TR, Goldstein JE, Schumaker S (2013) MALDI TOF MS profiling of bacteria at the strain level: a review. Mass Spectrom Rev 32(3):188–217.  https://doi.org/10.1002/mas.21359 PubMedGoogle Scholar
  37. Shaheen M, Li JR, Ross AC, Vederas JC, Jensen SE (2011) Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics. Chem Biol 18(12):1640–1648.  https://doi.org/10.1016/j.chembiol.2011.09.017 PubMedGoogle Scholar
  38. Singh P, Patil Y, Rale V (2019) Biosurfactant production: emerging trends and promising strategies. J Appl Microbiol 126(1):2–13.  https://doi.org/10.1111/jam.14057 PubMedGoogle Scholar
  39. Smith DDN, Williams AN, Verrett JN, Bergbusch NT, Manning V, Trippe K, Stavrinides J (2019) Resistance to two vinylglycine antibiotic analogs is conferred by inactivation of two separate amino acid transporters in Erwinia amylovora. J Bacteriol 201(9).  https://doi.org/10.1128/JB.00658-18
  40. Soussi S, Essid R, Hardouin J, Gharbi D, Elkahoui S, Tabbene O, Cosette P, Jouenne T, Limam F (2018) Utilization of grape seed flour for antimicrobial lipopeptide production by Bacillus amyloliquefaciens C5 strain. Appl Biochem Biotechnol 187(4):1460–1474.  https://doi.org/10.1007/s12010-018-2885-1 PubMedGoogle Scholar
  41. Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 56(4):845–857.  https://doi.org/10.1111/j.1365-2958.2005.04587.x PubMedGoogle Scholar
  42. Strauch MA, Bobay BG, Cavanagh J, Yao F, Wilson A, Le Breton Y (2007) Abh and AbrB control of Bacillus subtilis antimicrobial gene expression. J Bacteriol 189(21):7720–7732.  https://doi.org/10.1128/JB.01081-07 PubMedGoogle Scholar
  43. Sun J, Qian S, Lu J, Liu Y, Lu F, Bie X, Lu Z (2018) Knockout of rapC improves the bacillomycin D yield based on de novo genome sequencing of Bacillus amyloliquefaciens fmbJ. J Agric Food Chem 66(17):4422–4430.  https://doi.org/10.1021/acs.jafc.8b00418 PubMedGoogle Scholar
  44. Verhamme DT, Murray EJ, Stanley-Wall NR (2009) DegU and Spo0A jointly control transcription of two loci required for complex colony development by Bacillus subtilis. J Bacteriol 191(1):100–108.  https://doi.org/10.1128/JB.01236-08 PubMedGoogle Scholar
  45. Weinrauch Y, Penchev R, Dubnau E, Smith I, Dubnau D (1990) A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev 4(5):860–872PubMedGoogle Scholar
  46. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362.  https://doi.org/10.1038/nmeth.1322 PubMedGoogle Scholar
  47. Wüthrich K (2017) NMR with proteins and nucleic acids. Europhys News 17(1):11–13.  https://doi.org/10.1051/epn/19861701011 Google Scholar
  48. Xu B-H, Ye Z-W, Zheng Q-W, Wei T, Lin J-F, Guo L-Q (2018) Isolation and characterization of cyclic lipopeptides with broad-spectrum antimicrobial activity from Bacillus siamensis JFL15. 3 Biotech 8(10):444.  https://doi.org/10.1007/s13205-018-1443-4 PubMedGoogle Scholar
  49. Yang Q, Ding X, Liu X, Liu S, Sun Y, Yu Z, Hu S, Rang J, He H, He L, Xia L (2014) Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa. Microb Cell Factories 13(1):27.  https://doi.org/10.1186/1475-2859-13-27 Google Scholar
  50. Yaseen Y, Diop A, Gancel F, Bechet M, Jacques P, Drider D (2018) Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis. Arch Microbiol 200(5):783–791.  https://doi.org/10.1007/s00203-018-1483-5 PubMedGoogle Scholar
  51. Zhang Z, Ding ZT, Zhong J, Zhou JY, Shu D, Luo D, Yang J, Tan H (2017) Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes. Lett Appl Microbiol 64(6):452–458.  https://doi.org/10.1111/lam.12739 PubMedGoogle Scholar
  52. Zhao H, Shao D, Jiang C, Shi J, Li Q, Huang Q, Rajoka MSR, Yang H, Jin M (2017) Biological activity of lipopeptides from Bacillus. Appl Microbiol Biotechnol 101(15):5951–5960.  https://doi.org/10.1007/s00253-017-8396-0 PubMedGoogle Scholar
  53. Zhao J, Zhang C, Lu Z (2018) Differential proteomics research of Bacillus amyloliquefaciens and its genome-shuffled saltant for improving fengycin production. Braz J Microbiol 49:166–177.  https://doi.org/10.1016/j.bjm.2018.04.010 PubMedGoogle Scholar
  54. Zhi Y, Wu Q, Xu Y (2017) Genome and transcriptome analysis of surfactin biosynthesis in Bacillus amyloliquefaciens MT45. Sci Rep 7:40976.  https://doi.org/10.1038/srep40976 PubMedGoogle Scholar
  55. Zhou B, Yang Y, Chen T, Lou Y, Yang XF (2018a) The oligopeptide ABC transporter OppA4 negatively regulates the virulence factor OspC production of the Lyme disease pathogen. Ticks Tick Borne Dis 9(5):1343–1349.  https://doi.org/10.1016/j.ttbdis.2018.06.006 PubMedGoogle Scholar
  56. Zhou M, Liu F, Yang X, Jin J, Dong X, Zeng K-W, Liu D, Zhang Y, Ma M, Yang D (2018b) Bacillibactin and bacillomycin analogues with cytotoxicities against human cancer cell lines from marine Bacillus sp PKU-MA00093 and PKU-MA00092. Mar Drugs 16(1):22.  https://doi.org/10.3390/md16010022 Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory of Microbial Molecular Biology, College of Life ScienceHunan Normal UniversityChangshaPeople’s Republic of China

Personalised recommendations