Applied Microbiology and Biotechnology

, Volume 74, Issue 3, pp 667–675 | Cite as

Fusion of the genes for AHL-lactonase and S-layer protein in Bacillus thuringiensis increases its ability to inhibit soft rot caused by Erwinia carotovora

  • Lei Zhang
  • Lifang Ruan
  • Chaohua Hu
  • Huaiguang Wu
  • Shouwen Chen
  • Ziniu Yu
  • Ming SunEmail author
Applied Microbial and Cell Physiology


Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA 4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.


N-acyl-homoserine lactonase Fusion protein AiiA4Q7 protein Bacillus thuringiensis Surface layer Soft rot disease 



Strain DH5a (pJZ365) was kindly donated by Professor Stephen C. Winans in Cornell University. This study was supported by grants from the National Basic Research Program (973) of China (2003CB114201), National Natural Science Foundation of China (30270053, and 30080013), and National High Technology Research and Development project (863) of China (2006, 2004AA214092 and 2003AA223081). We thank Donald H. Dean at the Ohio State University, Chen Guo-qiang at Tsinghua University, Louis S. Tisa at University of New Hampshire, AP James Chin at the University of Queensland for their critical reading of this manuscript.


  1. Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119CrossRefGoogle Scholar
  2. Bassler BL (1999) How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol 2:582–587CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Byers JT, Lucas C, Salmond GPC, Welch M (2002) Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule. J Bacteriol 184:1163–1171CrossRefGoogle Scholar
  5. Dong YH, Xu JL, Li XZ, Zhang LH (2000) AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci USA 97:3526–3531CrossRefGoogle Scholar
  6. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH (2001) Quenching quorum sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813–817CrossRefGoogle Scholar
  7. Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH (2002) Identification of Quorum-Quenching N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol 68:1754–1759CrossRefGoogle Scholar
  8. Espinasse S, Gohar M, Lereclus D, Sanchis V (2004) An extracytoplasmic-function sigma factor is involved in a pathway controlling beta-exotoxin I production in Bacillus thuringiensis subsp. thuringiensis strain 407-1. J Bacteriol 186:3108–3116CrossRefGoogle Scholar
  9. Fuqua C, Winans SC, Greenberg EP (1996) Census and consensus in bacterial ecosystem: the luxR-luxI family of quorum-sensing transcriptional regulaters. Annu Rev Microbiol 50:727–751CrossRefGoogle Scholar
  10. Juárez-Pérez V, Guerchicoff A, Rubinstein C, Delécluse1 A (2002) Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin. Appl Environ Microbiol 68:1228–1231CrossRefGoogle Scholar
  11. Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK (2002) Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl Environ Microbiol 68:3919–3924CrossRefGoogle Scholar
  12. Lupas A, Engelhardt H, Peters J, Santarius U, Volker S, Baumeister W (1994) Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis. J Bacteriol 176:1224–1233Google Scholar
  13. Mesnage S, Tosi-Couture E, Fouet A (1999) Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase. Mol Microbiol 31:927–936CrossRefGoogle Scholar
  14. Möllenkvist A, Nordström T, Halldén C, Christensen JJ, Forsgren A, Riesbeck K (2003) The Moraxella catarrhalis immunoglobulin D-binding protein MID has conserved sequences and is regulated by a mechanism corresponding to phase variation. J Bacteriol 185:2285–2295CrossRefGoogle Scholar
  15. Palva A, Vigren G, Simonen M, Rintala H, Laamanen P (1990) Nucleotide sequence of the tetracycline resistance gene of pBC16 from Bacillus cereus. Nucleic Acids Res 18:1635CrossRefGoogle Scholar
  16. Park HW, Ge B, Bauer LS, Federici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938Google Scholar
  17. Pérombelon MCM (1992) The genus Erwinia. In: Truper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes, 2nd edn., vol 3. SpringerBerlin Heidelberg New York, pp 2899–2921Google Scholar
  18. Pérombelon MCM, Kelman A (1980) Ecology of the soft rot Erwinias. Annu Rev Phytopathol 18:361–387CrossRefGoogle Scholar
  19. Ries W, Hotzy C, Schocher I, Sleytr UB, Sára M (1997) Biophysical characterization of SbsB. J Bacteriol 179:3892–3898Google Scholar
  20. Rünzler D, Huber C, Moll D, Köhler G, Sára M (2004) Biophysical characterization of SbsB biophysical characterization of the entire bacterial surface layer protein SbsB and its two distinct functional domains. J Biol Chem 279:5207–5215CrossRefGoogle Scholar
  21. Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  22. Sára M, Sleytr UB (2000) S-Layer proteins. J Bacteriol 182:859–868CrossRefGoogle Scholar
  23. Sára M, Dekitsch C, Mayer HF, Egelseer EM, Sleytr UB (1998) Influence of the secondary cell wall polymer on the reassembly, recrystallization, and stability properties of the S-layer protein from Bacillus stearothermophilus PV72/p2. J Bacteriol 180:4146–4153Google Scholar
  24. Silo-Suh LA, Stabb EV, Raffle SJ, Handelsman J (1998) Target range of zwittermicin A, an aminopolyol antibiotic from Bacillus cereus. Curr Microbiol 37:6–11CrossRefGoogle Scholar
  25. Sleytr UB, Beveridge TJ (1999) Bacterial S-layers. Trends Microbiol 7:253–260CrossRefGoogle Scholar
  26. Stohl EA, Milner JL, Handelsman J (1999) Zwittermicin a biosynthetic cluster. Gene 237:403–411CrossRefGoogle Scholar
  27. Sun M, Zhu C, Yu Z (2001) Cloning of parasporal body protein gene resembling to S-layer protein genes from Bacillus thuringiensis CTC strain (in Chinese). Wei Sheng Wu Xue Bao 41:141–147Google Scholar
  28. Umelo-Njaka E, Nomellini JF, Bingle WH, Glasier LG, Irvin RT, Smit J (2001) Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system. Vaccine 19:1406–1415CrossRefGoogle Scholar
  29. Zhang Q (2001) Autoinducer inactivity protein Aii in Bacillus thuringiensis and its effect on pathogenicity of plant-pathogen bacteria. Ph.D. Dissertation, The Huazhong Agricultural University, Wuhan, ChinaGoogle Scholar
  30. Zhu J, Beaber JW, More MI, Fuqua C, Eberhard A, Winans SC (1998) Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J Bacteriol 180:5398–5405Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Lei Zhang
    • 1
  • Lifang Ruan
    • 1
  • Chaohua Hu
    • 1
  • Huaiguang Wu
    • 1
  • Shouwen Chen
    • 1
  • Ziniu Yu
    • 1
  • Ming Sun
    • 1
    Email author
  1. 1.State Key Laboratory of Agricultural MicrobiologyCollege of Life Science and Technology, Huazhong Agricultural UniversityWuhanChina

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