Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless derivative strains FZBREP and FZBSPA enhances antibacterial activity
- 617 Downloads
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.
KeywordsBacillus amyloliquefaciens Bacilysin Promoter replacement Markerless Antibiotic overproduction Antibacterial
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).
- 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
- 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
- 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
- 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
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar