Molecular characterization of Bacillus thuringiensis strains from Argentina

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.     

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.     

Molecular characterization of a protease secreted by Erwinia amylovora.

A protease with a molecular mass of 48 kDa is secreted by the fire blight pathogen Erwinia amylovora in minimal medium. We characterized this activity as a metalloprotease, since the enzyme was inhibited by EDTA and o -phenanthroline. A gene cluster was determined to encode four genes connected to protease expression, including a structural gene (prtA) and three genes (prtD, prtE, prtF) for secretion of the protease, which are transcribed in the same direction. The organization of the protease gene cluster in E. amylovora is different from that in other Gram-negative bacteria, such as Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. On the basis of the conservative motif of metalloproteases, PrtA was identified to be a member of the metzincin subfamily of zinc-binding metalloproteases, and was confirmed to be the 48 kDa protease on gels by sequencing of tryptic peptide fragments derived from the protein. The protease is apparently secreted into the external medium through the type I secretion pathway via PrtD, PrtE and PrtF which share more than 90% identity with the secretion apparatus for lipase of S. marcescens. A protease mutant was created by Tn 5 -insertions, and the mutation localized in the prtD gene. The lack of protease reduced colonization of an E. amylovora secretion mutant labelled with the gene for the green fluorescent protein (gfp) in the parenchyma of apple leaves.     

Discovery and characterization of Sip1A: a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).     

Purification and characterization of a secreted protease from Tetrahymena pyriformis

A simple major protease, secreted into the medium during growth of Tetrahymena pyriformis strain W, has been purified about 4000-fold by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and affinity chromatography on organomercurial-Sepharose. The purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22 000-23 000. Amino acid analysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena protease consists of multiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5-8.0 with alpha-N-benzoyl-DL-arginine-p-nitroanilide and with azocasein, while it was in the lower pH range (4.5-5.5) for denatured hemoglobins. The purified enzyme was inhibited by compounds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.     

Molecular characterization of a novel vegetative insecticidal protein from Bacillus thuringiensis effective against sap-sucking insect pest

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on LC(50) values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.     

Molecular and insecticidal characterization of a Bacillus thuringiensis strain isolated during a natural epizootic

A new Bacillus thuringiensis strain belonging to the serovar aizawai was isolated from a dead larva of the lepidopteran Mythimna loreyi collected in a corn crop in Spain during a natural epizootic. This strain, which was named Leapi01, was compared with the kurstaki and aizawai strains isolated from Dipel(R) and Xentari(R), by electron microscopy, SDS-PAGE, plasmid pattern, PCR and insecticidal activity. This strain showed similar morphological and biochemical characteristics to the standard strains. The content in cry genes of Leapi01 was analysed with a set of general and specific primers recognizing most of the cry genes reported to date. DNA amplification was obtained with primers corresponding to six genes and, to clearly determine the identity of the genes, the amplified fragments were sequenced and corresponded to cry1Aa, cry1Ab, cry1Ca, cry1Da, cry2Ab and cry1Ia. However, the proteins encoded by two of these genes, Cry2 and Cry1I, were not detected in the SDS-PAGE of the purified parasporal bodies. The insecticidal activity of Leapi01 was determined by bioassays against two Lepidoptera species, Helicoverpa armigera and Spodoptera littoralis, that were found to be very susceptible to Leapi01 purified crystals. Since two of the cry genes identified in Leapi01 appear to be silent, other factors may be involved in the toxicity of the strain. As a result of this study, the potential of Leapi01 as biological control agent is discussed, with special emphasis on the high toxicity and relatively broad spectrum activity compared with two B. thuringiensis strains that are the active ingredients of commercial preparations commonly used as bioinsecticides.     

Neuroserpin, an axonally secreted serine protease inhibitor.

We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult.     

Molecular characterization and sequence of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI-specific phospholipase C, PI-PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38095). The NH₂-terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI-PLC consists of 299 amino acid residues with a molecular weight of 34586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA-donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingo-myelin. The cleaving specifity of PI-PLC was examined by thin layer chromatography.     

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .     

Purification and characterization of a putative virulence factor,serine protease,from Vibrio parahaemolyticus

Binding of immobilized collagen-I (Cn-I) and fibronectin (Fn) by Lactobacillus acidophilus CRL 639 depends on cell-surface proteins. Capsule formation during the stationary growth phase has a negative effect on adherence of Cn-I and Fn. However, cells from the exponential growth phase, which produce no capsule, exhibit maximal binding. Binding is sensitive to trypsin, proteinase K, pronase E, and heat. Gelatin and soluble Cn-I partially inhibit binding of Cn-I although various proteins, sugars and amino acids do not affect binding to Fn. These results indicate that protein-protein interactions mediate adhesion to extracellular matrix proteins. SDS-PAGE and Western blot analyses of surface proteins revealed that several proteins including the major 43-kDa protein of the S-layer are expressed. Monoclonal antibodies showed that Fn binds to a 15-kDa protein, while Cn-I binds to proteins of 45 and 58 kDa.     

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.     

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis.

A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.     

Purification and partial characterization of hemolysins from Bacillus thuringiensis

A strain of Bacillus thuringiensis serotype I was investigated for hemolytic activity against horse, sheep, and rabbit erythrocytes. Two distinct hemolytic proteins were found; one was similar to streptolysin 0 in its properties and behavior. Both hemolysins were purified and characterized by molecular sieve filtration and by isoelectric focusing. The specific activities of the purified hemolysins were determined.     

Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.