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Applied Microbiology and Biotechnology

, Volume 99, Issue 10, pp 4255–4263 | Cite as

Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless derivative strains FZBREP and FZBSPA enhances antibacterial activity

  • Liming Wu
  • Huijun Wu
  • Lina Chen
  • Ling Lin
  • Rainer Borriss
  • Xuewen Gao
Biotechnologically relevant enzymes and proteins

Abstract

Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites (lox71 and lox66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6 % more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

Keywords

Bacillus amyloliquefaciens Bacilysin Promoter replacement Markerless Antibiotic overproduction Antibacterial 

Notes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31100056, 31471811), the Special Fund for the Fundamental Research Funds for the Central Universities (KYZ201404), the Agro-scientific Research in the Public Interest (20130315), the Doctoral Fund of Ministry of Education of China (20100097120011), and the National High-tech R&D Program of China (2012AA101504).

Supplementary material

253_2014_6251_MOESM1_ESM.pdf (118 kb)
ESM 1 (PDF 118 kb)

References

  1. Chen XH, Vater J, Piel J, Franke P, Scholz R, Schneider K, Koumoutsi A, Hitzeroth G, Grammel N, Strittmatter AW, Gottschalk G, Süssmuth RD, Borriss R (2006) Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42. J Bacteriol 188(11):4024–4036CrossRefPubMedCentralPubMedGoogle Scholar
  2. Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R (2007) Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25(9):1007–1014CrossRefPubMedGoogle Scholar
  3. Chen XH, Koumoutsi A, Scholz R, Schneider K, Vater J, Süssmuth R, Piel J, Borriss R (2009a) Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol 140(1–2):27–37CrossRefPubMedGoogle Scholar
  4. Chen XH, Scholz R, Borriss M, Junge H, Mögel G, Kunz S, Borriss R (2009b) Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease. J Biotechnol 140(1–2):38–44CrossRefPubMedGoogle Scholar
  5. Chmara H, Zähner H, Borowski E, Milewski S (1984) Inhibition of glucosamine-6-phosphate synthetase from bacteria by anticapsin. J Antibiot 37(6):652–658CrossRefPubMedGoogle Scholar
  6. Claessen D, Emmins R, Hamoen LW, Daniel RA, Errington J, Edwards DH (2008) Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis. Mol Microbiol 68(4):1029–1046CrossRefPubMedGoogle Scholar
  7. Foster JW, Woodruff HB (1946) Bacillin, a new antibiotic substance from a soil isolate of Bacillus subtilis. J Bacteriol 51(3):363–369PubMedCentralGoogle Scholar
  8. Hilton MD, Alaeddinoglu NG, Demain AL (1988) Bacillus subtilis mutant deficient in the ability to produce the dipeptide antibiotic bacilysin: isolation and mapping of the mutation. J Bacteriol 170(2):1018–1020PubMedCentralPubMedGoogle Scholar
  9. Hoflack L, Seurinck J, Mahillon J (1997) Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons. J Bacteriol 179(16):5000–5008PubMedCentralPubMedGoogle Scholar
  10. Inaoka T, Ochi K (2011) Scandium stimulates the production of amylase and bacilysin in Bacillus subtilis. Appl Environ Microbiol 77(22):8181–8183CrossRefPubMedCentralPubMedGoogle Scholar
  11. Kenig M, Abraham EP (1976) Antimicrobial activities and antagonists of bacilysin and anticapsin. J Gen Microbiol 94(1):37–45CrossRefPubMedGoogle Scholar
  12. Kenig M, Vandamme E, Abraham EP (1976) The mode of action of bacilysin and anticapsin and biochemical properties of bacilysin-resistant mutants. J Gen Microbiol 94(1):46–54CrossRefPubMedGoogle Scholar
  13. Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R (2004) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J Bacteriol 186(4):1084–1096CrossRefPubMedCentralPubMedGoogle Scholar
  14. Lambert JM, Bongers RS, Kleerebezem M (2007) Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol 73(4):1126–1135CrossRefPubMedCentralPubMedGoogle Scholar
  15. Leclère V, Béchet M, Adam A, Guez JS, Wathelet B, Ongena M, Thonart P, Gancel F, Chollet-Imbert M, Jacques P (2005) Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Appl Environ Microbiol 71(8):4577–4584CrossRefPubMedCentralPubMedGoogle Scholar
  16. Leibig M, Krismer B, Kolb M, Friede A, Götz F, Bertram R (2008) Marker removal in Staphylococci via Cre recombinase and different lox sites. Appl Environ Microbiol 74(5):1316–1323CrossRefPubMedCentralPubMedGoogle Scholar
  17. Ozcengiz G, Alaeddinoglu NG, Demain AL (1990) Regulation of biosynthesis of bacilysin by Bacillus subtilis. J Ind Microbiol 6(2):91–100CrossRefPubMedGoogle Scholar
  18. Parker JB, Walsh CT (2013) Action and timing of BacC and BacD in the late stages of biosynthesis of the dipeptide antibiotic bacilysin. Biochemistry 52(5):889–901CrossRefPubMedCentralPubMedGoogle Scholar
  19. Pomerantsev AP, Sitaraman R, Galloway CR, Kivovich V, Leppla SH (2006) Genome engineering in Bacillus anthracis using Cre recombinase. Infect Immun 74(1):682–693CrossRefPubMedCentralPubMedGoogle Scholar
  20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  21. Scholz R, Molohon KJ, Nachtigall J, Vater J, Markley AL, Süssmuth RD, Mitchell DA, Borriss R (2011) Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. J Bacteriol 193(1):215–224CrossRefPubMedCentralPubMedGoogle Scholar
  22. Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci U S A 44(10):1072–1078CrossRefPubMedCentralPubMedGoogle Scholar
  23. Steinborn G, Hajirezaei MR, Hofemeister J (2005) bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains. Arch Microbiol 183(2):71–79CrossRefPubMedGoogle Scholar
  24. Sun H, Bie X, Lu F, Lu Y, Wu Y, Lu Z (2009) Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the Pspac promoter. Can J Microbiol 55(8):1003–1006CrossRefPubMedGoogle Scholar
  25. Suzuki N, Okayama S, Nonaka H, Tsuge Y, Inui M, Yukawa H (2005) Large-scale engineering of the Corynebacterium glutamicum genome. Appl Environ Microbiol 71(6):3369–3372CrossRefPubMedCentralPubMedGoogle Scholar
  26. Yan X, Yu HJ, Hong Q, Li SP (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol 74(17):5556–5562CrossRefPubMedCentralPubMedGoogle Scholar
  27. Yang YS, Hughes TE (2001) Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells. Biotechniques 31(5):1036–1040PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Liming Wu
    • 1
    • 2
  • Huijun Wu
    • 1
    • 2
  • Lina Chen
    • 1
    • 2
  • Ling Lin
    • 1
    • 2
  • Rainer Borriss
    • 3
  • Xuewen Gao
    • 1
    • 2
  1. 1.Department of Plant Pathology, College of Plant ProtectionNanjing Agricultural UniversityNanjingChina
  2. 2.Key Laboratory of Monitoring and Management of Crop Diseases and Pest InsectsMinistry of EducationNanjingChina
  3. 3.ABiTEP GmbHBerlinGermany

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