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

Abstract

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.

Metabolic-network-driven analysis of bacterial ecological strategies

“…For the ubiquitous B. thuringiensis we compute high environmental diversity and high competition scores (ESI, 0.09; maximal-CHS, 59; Additional data file 1). We applied the SVM classifier to characterize the growth rates of these species; in accordance with experimental data, B. thuringiensis falls into the fast-growing category and Parachlamydia UWE25 falls into the slow-growing category (corresponding to the experimentally observed doubling times of 40 minutes [ 30 ] and 48 hours, respectively [ 11 ]).…”

A cell–cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group

“…The neutral protease NprA is the major extracellular protein produced during sporulation of bacteria of the B. cereus group (Fig. S1) (Donovan et al ., 1997; Chitlaru et al ., 2006; Chung et al ., 2006). Here we show that nprA gene expression depends on a cell–cell communication system consisting of the regulatory protein NprR and the signalling peptide NprX.…”

“…The metalloprotease NprB, also known as Npr599, has been described as a potential virulence factor because of its degradative properties against human tissue proteins (Chung et al ., 2006). This protein was initially called NprA in B. thuringiensis and was characterized for its important degradative properties during sporulation (Donovan et al ., 1997).…”

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

“…Homologous recombination has been used to create null mutants in vivo. Applications of this technique have included disruptions of cry and cyt genes to assess their contribution to pesticidal activity (85,301) and inactivation of protease production genes to increase crystal production and stability (97,370). Recent progress in understanding cry gene expression has allowed the construction of asporogenous B. thuringiensis strains that nevertheless produce crystals; these crystals remain encapsulated in the mother cell compartment (48,213).…”

“…There are several other insect species in which Cry toxins with different receptor specificities are known (93,105,113,163,198,394,395). For many insect species, multiple Cry1A proteins would not be an appropriate choice, since some of these proteins share binding sites with one another (94,106,395,413) and even with other toxins of the Cry1 class (97). Yet for other insects, Cry1A proteins have been shown, at least on ligand blots, to recognize different binding proteins (211,385,386,388).…”