Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. 1997 Jun; 63(6): 2311–2317.
PMCID: PMC168523

Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal protein.


The nprA gene, encoding Bacillus thuringiensis neutral protease A, was cloned by the use of gene-specific oligonucleotides. The size of neutral protease A deduced from the nprA sequence was 566 amino acids (60,982 Da). The cloned nprA gene was partially deleted in vitro, and the deleted allele, designated nprA3, was used to construct an nprA3 strain (neutral protease A-deficient strain) of B. thuringiensis. Growth and sporulation of the nprA3 strain were similar to those of an isogenic nprA+ strain, although the extracellular proteolytic activity of the nprA3 strain was significantly less than that of the nprA+ strain. The nprA3 strain produced insecticidal crystal proteins that were more stable than those of the isogenic nprA+ strain after solubilization in vitro, and sporulated cultures of the nprA3 strain contained higher concentrations of full-length insecticidal crystal proteins than did those of its isogenic counterpart. The absence of neutral protease A did not affect the insecticidal activity of a lepidopteran-specific crystal protein of B. thuringiensis. These results indicate that crystal protein stability and yield may be improved by deletion of specific proteases from B. thuringiensis.

Full Text

The Full Text of this article is available as a PDF (332K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Andrews RE, Jr, Bibilos MM, Bulla LA., Jr Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki. Appl Environ Microbiol. 1985 Oct;50(4):737–742. [PMC free article] [PubMed]
  • Aronson AI. The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol Microbiol. 1993 Feb;7(4):489–496. [PubMed]
  • Brousseau R, Saint-Onge A, Préfontaine G, Masson L, Cabana J. Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl Environ Microbiol. 1993 Jan;59(1):114–119. [PMC free article] [PubMed]
  • Bulla LA, Jr, Kramer KJ, Davidson LI. Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J Bacteriol. 1977 Apr;130(1):375–383. [PMC free article] [PubMed]
  • Bulla LA, Jr, Kramer KJ, Cox DJ, Jones BL, Davidson LI, Lookhart GL. Purification and characterization of the entomocidal protoxin of Bacillus thuringiensis. J Biol Chem. 1981 Mar 25;256(6):3000–3004. [PubMed]
  • Carroll J, Li J, Ellar DJ. Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis. Biochem J. 1989 Jul 1;261(1):99–105. [PMC free article] [PubMed]
  • Chestukhina GG, Zalunin IA, Kostina LI, Kotova TS, Kattrukha SP, Stepanov VM. Crystal-forming proteins of Bacillus thuringiensis. The limited hydrolysis by endogeneous proteinases as a cause of their apparent multiplicity. Biochem J. 1980 May 1;187(2):457–465. [PMC free article] [PubMed]
  • Donovan WP, Kushner SR. Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1986 Jan;83(1):120–124. [PMC free article] [PubMed]
  • Donovan WP, Dankocsik CC, Gilbert MP, Gawron-Burke MC, Groat RG, Carlton BC. Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki. J Biol Chem. 1988 Jan 5;263(1):561–567. [PubMed]
  • González JM, Jr, Carlton BC. A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis. Plasmid. 1984 Jan;11(1):28–38. [PubMed]
  • Haider MZ, Knowles BH, Ellar DJ. Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. Eur J Biochem. 1986 May 2;156(3):531–540. [PubMed]
  • Höfte H, Whiteley HR. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. [PMC free article] [PubMed]
  • Kawamura F, Doi RH. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. [PMC free article] [PubMed]
  • Li E, Yousten AA. Metalloprotease from Bacillus thuringiensis. Appl Microbiol. 1975 Sep;30(3):354–361. [PMC free article] [PubMed]
  • Macaluso A, Mettus AM. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA. J Bacteriol. 1991 Feb;173(3):1353–1356. [PMC free article] [PubMed]
  • Matar GM, Slieman TA, Nabbut NH. Subtyping of Bacillus cereus by total cell protein patterns and arbitrary primer polymerase chain reaction. Eur J Epidemiol. 1996 Jun;12(3):309–314. [PubMed]
  • Norton NB, Orzech KA, Burke WF., Jr Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593. Plasmid. 1985 May;13(3):211–214. [PubMed]
  • Ogiwara K, Indrasith LS, Asano S, Hori H. Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae. J Invertebr Pathol. 1992 Sep;60(2):121–126. [PubMed]
  • Perlman D, Halvorson HO. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. [PubMed]
  • Sidler W, Niederer E, Suter F, Zuber H. The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase. Biol Chem Hoppe Seyler. 1986 Jul;367(7):643–657. [PubMed]
  • Sloma A, Rufo GA, Jr, Theriault KA, Dwyer M, Wilson SW, Pero J. Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis. J Bacteriol. 1991 Nov;173(21):6889–6895. [PMC free article] [PubMed]
  • Stepanov VM, Chestukhina GG, Rudenskaya GN, Epremyan AS, Osterman AL, Khodova OM, Belyanova LP. A new subfamily of microbial serine proteinase? Structural similarities of Bacillus thuringiensis and Thermoactinomyces vulgaris extracellular serine proteinases. Biochem Biophys Res Commun. 1981 Jun;100(4):1680–1687. [PubMed]
  • Thanabalu T, Porter AG. Efficient expression of a 100-kilodalton mosquitocidal toxin in protease-deficient recombinant Bacillus sphaericus. Appl Environ Microbiol. 1995 Nov;61(11):4031–4036. [PMC free article] [PubMed]
  • Tinoco I, Jr, Borer PN, Dengler B, Levin MD, Uhlenbeck OC, Crothers DM, Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. [PubMed]
  • Vasantha N, Thompson LD, Rhodes C, Banner C, Nagle J, Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. [PMC free article] [PubMed]
  • Wetmore DR, Wong SL, Roche RS. The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus. Mol Microbiol. 1992 Jun;6(12):1593–1604. [PubMed]
  • Wong HC, Chang S. Identification of a positive retroregulator that stabilizes mRNAs in bacteria. Proc Natl Acad Sci U S A. 1986 May;83(10):3233–3237. [PMC free article] [PubMed]
  • Yamamoto T, Iizuka T. Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki. Arch Biochem Biophys. 1983 Nov;227(1):233–241. [PubMed]
  • Yang MT, Gardner JF. Transcription termination directed by heteroduplex thr attenuator templates. Evidence that the transcript stem and loop structure is the termination signal. J Biol Chem. 1989 Feb 15;264(5):2634–2639. [PubMed]
  • Yang MY, Ferrari E, Henner DJ. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J Bacteriol. 1984 Oct;160(1):15–21. [PMC free article] [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene links
  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...