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Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation


A Bacillus thuringiensis respiratory mutant (AB1 strain) that shows premature sporulation and insecticidal crystal protein (ICP) production was isolated. The mutant strain harbours the cryIC and cryID insecticidal genes and could be important for the production of ICP highly toxic to Spodoptera sp. The mutant was selected by its increased capacity to oxidize. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). In this strain, cytochrome aa 3 expression is not repressed during the sporulation phase, in contrast with the wild-type strain. The growth, spore production, dissolved O2, O2 consumption, CO2 evolution rate and ICP production were recorded as a function of time. The AB1 mutant strain has a similar growth yield to the wild-type strain, but begins sporulation at least 4 h earlier. The AB1 strain consumes 74.5% more O2 than the wild-type strain, during the fermentation process. The mutation on strain AB1 has an important positive effect on ICP production. This procedure shows that ICP production could be increased during fermentation by increasing the respiration capacity of Bacillus thuringiensis.

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  1. Boulter D, Gatehouse AMR, Hilder V (1989) Use of cowpea trypsin inhibitor (CpTI) to protect plants against insect predation. Biotechnol Adv 7:489–497

  2. Garcia-Horsman JA, Barquera B, Escamilla JE (1991) Two different aa 3-type cytochromes can be purified from the bacterium Bacillus cereus. Eur J Biochem 199:761–768

  3. Heierson A, Landen R, Lovgren A, Dalhammar G, Bowman HG (1987) Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA. J Bacteriol 169:1147–1152

  4. Hofmann C, Luthy P, Hutter R, Pliska V (1988) Binding of the δ-endotoxin from Bacillus thuringiensis to brush border membrane vesicles of the cabbage butterfly (Pieris brassicae). Eur J Biochem 173:85–91

  5. Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255

  6. Höfte H, Soetaert P, Jansens S, Peferoen M (1990) Nucleotide sequence and deduced amino acid sequence of a new lepidopteran specific crystal protein gene from Bacillus thuringiensis. Nucleic Acids Res 18:5545

  7. Honée G, Vander Salm T, Visser B (88) Nucleotide sequence of crystal protein gene isolated from Bacillus thuringiensis subspecies entomocidus 60.5 coding for a toxic protein highly active against Spodoptera species. Nucleic Acids Res 16:6240

  8. Johnson DE, Niezgodski DM, Twaddle GM (1980) Parasporal crystal produced by oligosporogenous mutants of Bacillus thuringiensis (SpoCr+). Can J Microbiol 26:486–491

  9. Lecadet A, Blunde S, Ribier W (1980) Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber. J Gen Microbiol 121:203–212

  10. Liu JK, Jurtchuk D (1986) N,N,N',N',-tetramethyl-p-phenylene-diamine-dependent cytochrome oxidase analysis of Bacillus species. In J Sys Bacteriol 36:38–48

  11. Markwell MAK, Haas SM, Vieber ZZ, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210

  12. Poole RK (1983) Bacterial cytochrome oxidase. A structural and functional diverse group of electron transfer proteins. Biochim Biophys Acta 726:205–243

  13. Sacchi VF, Parenti P, Hanozet GM, Giordana B, Luthy P, Wolfersberger MG (1986) Bacillus thuringiensis toxin inhibits K+ gradient-dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells. FEBS 204:213–218

  14. Soberón M, Williams HD, Poole RK, Escamilla E (1989) Isolation of a Rhizobium phasseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation. J Bacteriol 171:465–472

  15. Starzak M, Bajpai RK (1991) A structured model for vegetative growth and sporulation in Bacillus thuringiensis. Appl Biochem Biotechnol 28/29:699–718

  16. Thorne CB (1978) Transduction in Bacillus thuringiensis. Appl Environ Microbiol 35:1109–1115

  17. Vander Oost J, Wachenfeld C van, Hederstedt L, Saraste M (1991) Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa 3-type oxidase. Mol Microbiol 5:2063–2072

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Correspondence to: A. Bravo

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Bravo, A., Quintero, R., Díaz, C. et al. Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation. Appl Microbiol Biotechnol 39, 558–562 (1993).

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  • Fermentation
  • Bacillus Thuringiensis
  • Spore Production
  • Respiration Capacity
  • Terminal Oxidase