Parasporal Inclusions Research Articles

Diversity analysis and characterization of Coleoptera-, Hemiptera- and Nematode-active cry genes in native isolates of Bacillus thuringiensis

Applications to combat non-lepidopteran insects are not as common as applications against lepidopteran insects. The aim of the present work was to isolate and identify Bacillus thuringiensis isolates from soil samples using five approaches, viz., analysis of crystal protein production by microscopy; detection of cry gene content by PCR, SDS-PAGE profiling; cloning and sequencing; phylogenetic analysis; and toxicity testing. Two hundred soil samples were used for isolation of B. thuringiensis and a total of 69 putative isolates of B. thuringiensis that produce parasporal crystalline inclusions were isolated from 5,267 Bacillus-like colonies. A bipyramidal inclusion was predominant in 32.2 % of the B. thuringiensis isolates compared to other shapes. Crystal protein profiling of B. thuringiensis isolates by SDS-PAGE analysis showed the presence of bands of 130, 73, 34, 25 and 13 kDa, among which 50–60 kDa bands were present abundantly. PCR analysis revealed the predominance of Coleopteran-active cry genes in these isolates. Variation in nucleotide sequences, crystal morphology and mass of crystal protein(s) purified from the isolates of B. thuringiensis revealed genetic and molecular diversity. Four strains containing Coleopteran-active cry genes showed higher toxicity against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) adults when compared with B. thuringiensis subsp. morrisoni pathovar tenebrionis. These results are useful in emphasizing the distribution of cry genes and for prognostication of toxicity, and may contribute to the identification of novel candidate genes for bioengineered crop protection.

Expression and characterization of a recombinant Cry1Ac crystal protein fused with an insect-specific neurotoxin ω-ACTX-Hv1a in Bacillus thuringiensis

In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.

Isolation and sequencing of the cryIC-like delta endotoxin gene from Egyptian strains of Bacillus thuringiensis toxic against Lepidoptera

The aim of the present work was to explore the diversity of Bacillus thuringiensis (Bt) among local Egyptian isolates that produce parasporal crystalline inclusions. On the basis of bioactivities, nine isolates were collected from different ecological environments within Egypt. The Bt2K isolate was effective against Lepidoptera as compared to B. thurigiensis ssp. kurstawi that was used as a reference standard. The gene isolated from the strain was shown to be homologous to the Lepidoptera active cryIC gene from B. thurigiensis ssp. ansawai as shown by PCR and alignment of the complete nucleotide sequences (98% identical). Therefore, a cryIC insecticidal toxin coding gene was cloned from an Egyptian isolate of B. thuringiensis. Sequence analyses of the cryIC gene showed the absence of potential polyadenylation signal sequences within the coding region and a less biased codon usage than most cryIC like genes. Therefore, the gene may be a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cryIC gene into a plant expression vector and then transferring it to tobacco and potato through Agrobacteriummediated transformation. Tobacco and potato plants with a stably integrated native cryIC gene were completely protected from predation by Lepidoptera. It was concluded that novel insecticidal genes exist in nature that may not require extensive modifications for efficient expression in plants. Key words: Bacillus thuringiensis, cryIC gene, plant transformation, Bt toxins, DNA sequencing.

Bacillus thuringiensis isolates from Korean forest environments

We investigated the distribution, toxicity, morphology, and protein profiles of Bacillus thuringiensis isolates from forests in Korea to isolate naturally occurring novel B. thuringiensis. A total of 170 B. thuringiensis isolates were obtained from 832 samples producing spore and parasporal inclusion bodies. In toxicity tests for lepidopteran, dipteran, and coleopteran insects, 57.6% isolates were toxic only to Lepidoptera, 5.3% were toxic only to Diptera, and 24.1% were toxic to both Diptera and Lepidoptera. The remaining collections (13.0%) were not toxic to the tested insects. The shapes of the parasporal crystals produced in B. thuringiensis isolates were bipyramidal, spherical, ovoid, or irregular. As their toxicities varied with parasporal crystal shape, B. thuringiensis isolates possessing bipyramidal or irregular parasporal crystals were largely toxic to lepidopteran species whereas those producing spherical parasporal crystals were mainly toxic to dipteran species. B. thuringiensis toxic to both dipteran and lepidopteran insects contained 130- and 70-kDa parasporal crystals, whereas B. thuringiensis toxic to lepidopteran insects expressed 130-kDa parasporal crystals. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates.

ISOLATION AND IDENTIFICATION OF NOVEL LOCAL ISOLATES OF Bacillus thuringiensis ACTIVE AGAINST RED PALM WEEVIL (RPW)

The gram-positive bacterium B. thuringiensis is well-known as sources of insecticidal proteins, most of which accumulate in crystalline inclusions during sporulation, these parasporal inclusions contain various types of insecticidal crystal protein (ICP). The crystal proteins from B. thuringiensis are among the most successful biological control agents for the suppression of agriculturally and medically important insect pests (Roh et al., 2007), being toxic to the larvae of lepidopteron, dipteran, and coleopteran insects (Johnson et al., 1998), as well as certain hymenoptera, homoptera, and mallophaga, in addition to many nematodes, flatworms, and Sarcomastigophora (Walters and English, 1995; Horak et al., 1996). At present, more than 130 B. thuringiensis crystal proteins have been described based on their gene sequences and amino acid homologies (Crickmore et al., 1998). Recently, all crystal proteins show some relatedness is grouped into four main groups. B. thuringiensis isolates are distributed worldwide, and more than 60,000 have already been collected by various industries in an effort to obtain novel crystal proteins (Martin and Travers 1989; Li et al., 2007). In addition, B. thuringiensis isolates showing different crystal protein gene patterns from the reference strains have also been reported (Li et al., 2002). The red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier), a concealed tissue borer, is a lethal pest of palms and is reported to attack 17 palm species worldwide. Although the weevil was first reported on coconut (Cocos nucifera) in South Asia during the last two decades it has gained a foothold on date palm Phoenix dactylifera in several Middle Eastern countries from where it has moved to Africa and Europe, mainly due to the movement of infested planting material. In the Mediterranean region, RPW also severely damages Phoenix canariensis. Infested palms, if not early detected and treated, often declined and die. However, palms in the early stages of infestation usually respond to chemical insecticide treatments. RPW has been managed in several countries through an integrated pest management (IPM) program (Faleiro, 2006). The search for effective natural enemies (biological control) for RPW still continues. A novel Bacillus thuringiensis strain, NCIMB 40152, was isolated from dead Tenebrio molitor L. Larvae. The isolate is endowed with specific insecticide activity against the Colorado potato beetle (Leptinotarsa decemlineata (Say), Coleoptera, Crysomelidae), but has no effect against lepidopteran or dipteran insects (Cidaria et al., 1991). Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, with high toxicity against lepidopteran and coleopteran pests have been isolated from Northeast Mexico (Tamez-Guerra et al., 2004). Bacillus thuringiensis soil isolates A21, A51 and C17 isolated from different regions of the Cuban archipelago showed higher larvicide activity than Bactivec's isolated reference strain, against both Aedes aegypti and Culex quinquefasciatus (Gonzalez et al., 2011). Accordingly, This study describe the isolation and identification of novel isolates of B. thuringiensis, in order to study insecticidal activity of isolates produced proteins against Red Palm Weevil insect.

Two groups of S-layer proteins, SLP1s and SLP2s, in Bacillus thuringiensis co-exist in the S-layer and in parasporal inclusions

We screened four B. thuringiensis strains whose parasporal inclusions contained the S-layer protein (SLP), and cloned two slp genes from each strain. Phylogenetic analysis indicated these SLPs could be divided into two groups, SLP1s and SLP2s. To confirm whether SLPs were present in the S-layer or as a parasporal inclusion, strains CTC and BMB1152 were chosen for further study. Western blots with whole-cell associated proteins from strains CTC and BMB1152 in the vegetative phase showed that SLP1s and SLP2s were constituents of the S-layer. Immunofluorescence utilizing spore-inclusion mixtures of strains CTC and BMB1152 in the sporulation phase showed that SLP1s and SLP2s were also constituents of parasporal inclusions. When heterogeneously expressed in the crystal negative strain BMB171, four SLPs from strains CTC and BMB1152 could also form parasporal inclusions. This temporal and spatial expression is not an occasional phenomenon but ubiquitous in B. thuringiensis strains.

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.

Bacillus thuringiensis subsp. israelensis CAB199균주의 제형에 따른 모기유충방제 효과

Among 18 Bacillus thuringiensis isolates with spherical parasporal inclusion from soils, B. thuringiensis subsp. israelensis CAB199 was selected. It was showing over 90% mortality against Aedes aegypti and Culex pipiens moletus. It was confirmed that this B. thuringiensis subsp. israelensis CAB199 isolate also had a insecticidal activity against Culex inatomii that was occurred in the marsh. Because most of mosquito larva were primarily situated or shifted from under- to surface water, we need to select long floating formulations on surface water for controlling mosquito larva. It was tested the pesticidal and control effects in the laboratory and wetland with two formulation types of B. thuringiensis subsp. israelensis, for example, wettable power (WP) and suspension concentrate type (SC). Laboratory test showed that SC formulation type was relatively faster and more effective against 3 tested mosquito species, C. pipiens, Aedes aegypti, and C. inatomii. Otherwise, the control efficacy of SC formulation type was more rapidly appeared against C. inatomii in the wetland.

Three Cry Toxins in Two Types fromBacillus thuringiensisStrain M019 Preferentially Kill Human Hepatocyte Cancer and Uterus Cervix Cancer Cells

Bacillus thuringiensis strain M019, non-pathogenic to lepidopteran and dipteran insects, produces a parasporal inclusion that consists of three 84-kDa Cry proteins (CPs). CP78A and CP78B, which exhibit 83.5% amino acid identity, were new variants of the previously reported HeLa cell-killing protein (parasporin-1). CP84 was a novel CP showing low-level homology, of 21.9% (56.4% similarity), with the insecticidal Cry2 toxin. In vitro solubilization with dithiothreitol at pH 10.2 and limited hydrolysis with trypsin resulted in the removal of N-terminal portions of the CPs and their activation. The 70-kDa proteins (15- and 55-kDa fragments) from CP78A and CP78B and the 73-kDa protein (14- and 59-kDa fragments) from CP84 exhibited varying degrees of cytocidal activity preferentially toward human hepatocyte cancer HepG2 cells and uterus cervix cancer HeLa cells causing cell swelling or the formation of vacuoles in the cytoplasm. These toxins appeared to attack an identical target on human cells.

Diversity of Bacillus thuringiensis Isolated from Western Ghats of Tamil Nadu State, India

The Western Ghats of India is the one of the world's 10 "Hottest biodiversity hotspots" that runs along the western part of India through four states including Tamil Nadu. The only biodiversity reserve in the Western Ghats is the Nilgiri biosphere located in the Tamil Nadu state. In the present study, 525 soil samples were collected from all the 14 different divisions of the Western Ghats in Tamil Nadu state, India. A total of 316 new isolates of Bacillus thuringiensis (Bt) that produce parasporal crystalline inclusions were isolated from 525 soil samples. Seven different types of crystalline inclusions were observed in the 316 new isolates of Bt. Cuboidal inclusion was predominantly present in 26.9% of the Bt isolates when compared to other shapes. Further characterization of 70 of the 316 Bt isolates for crystal protein profile through SDS-PAGE revealed six different types of crystal protein profile viz., 135 and 65, 135, 95, 65, 43, and 30 kDa crystal proteins. Variation in the mass of crystal protein(s) purified from the isolates of Bt revealed molecular diversity of this bacterium prevalent in the Western Ghats of Tamil Nadu, India.

Construction of a Recombinant Bacillus velezensis Strain as an Integrated Control Agent Against Plant Diseases and Insect Pests

To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.

Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity

The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS–PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.

A novel mosquitocidal Bacillus thuringiensis strain LLP29 isolated from the phylloplane of Magnolia denudata

Eleven Bacillus thuringiensis isolates were recovered from phylloplanes of Magnolia denudata, a specific source of new strains of B. thuringiensis. Among these, a new strain, LLP29, was found to be most toxic to mosquitoes based on the results of preliminary toxicity analysis. Phase contrast microscopy, mosquitocidal activity, polymerase chain reaction (PCR) analysis and parasporal inclusion were performed to learn more about the characteristics of this novel mosquitocidal isolate. The LC(50) values of LLP29 against Aedes albopictus and Culex quinquefasciatus were 0.33 and 0.04 ng of protein/ml, respectively. The cyt1 gene, which encodes the Cyt protein that is toxic to mosquitoes, was subsequently detected, cloned, sequenced and expressed in acrystalliferous Bt HD73 Cry(-). The results indicated that it might be a member of the cyt1Aa gene group. The novel strain LLP29 appears to be a new subspecies of B. thuringiensis and should prove useful in the control of mosquitoes and mosquito-borne diseases.

모기유충에 활성 있는 Bacillus thuringiensis subsp. tohokuensis CAB167 균주의 특성

Eight Bacillus thuringiensis strains activated against mosquito larva were compared their characterization. Spherical-shaped parasporal inclusion of B. thuringiensis subsp. tohokuensis CAB167 was observed by phase-contrast microscopy and scanning electron microscopy. values of B. thuringiensis subsp. tohokuensis CAB167 against Culex pipiens molestus, Culex pipiens pallens, and Aedes aegyti were 173, 190 and 580 ng/ml, respectively. B. thuringiensis subsp. tohokuensis CAB167 had a parasporal inclusion containing 4 major protein components, for example, 135, 80, 49 and 28-kDa by SDS-PAGE. Otherwise, after trypsin digestion of parasporal inclusion, SDS-PAGE was showed new protease-resistant peptides at 72 and 63-kDa. Activated toxins of isolated CAB167 were different from other reference strains on a serological by immuno-diffusion test.

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 Å. The protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

Occurrence of Bacillus thuringiensis in Canopies of a Natural Lucidophyllous Forest in Japan

A total of 39 Bacillus thuringiensis isolates were recovered from 38 leaves collected from 5- to 10-m-high canopies of 8 micro-/meso-phanerophyte species in a lucidophyllous forest of Japan. B. thuringiensis-positive leaves accounted for 1.4% of a total of 2805 leaves from 15 tree species. The frequency of the organism was 0.8% among the Bacillus cereus/B. thuringiensis group. Of 39 isolates obtained, 27 (69.2%) were allocated to 11 H serovars, and 12 isolates remained unidentified: 11 were motile but lacked reactivity to the 55 reference antisera, and 1 isolate was not flagellated. Two H serovars, kurstaki (H3abc) and tohokuensis (H17), occurred predominantly on canopy phylloplanes. Larvicidal activities against Bombyx mori and/or Aedes aegypti were associated with 49% of the canopy isolates. Strong hemolysis was induced by parasporal inclusion proteins of the two isolates of serovar israelensis (H14). Hemagglutinating (lectin) activity was associated with parasporal proteins of nine isolates. There was little correlation between insecticidal activity and lectin activity.

The surface layer protein of Bacillus thuringiensis CTC forms unique intracellular parasporal inclusion body

Bacillus thuringiensis subsp. finitimus strain CTC forms round parasporal inclusion body. The inclusion body protein gene ctc has been cloned and characterized. Sequence homology analysis reveals that the amino acid sequence of CTC protein shows 87% identity with the surface layer (S-layer) protein Sap (GenBank Z36946) in B. anthracis. In this report, transmission electron microscope observation showed that CTC formed intracellular parasporal inclusion body and sheet structure of S-layer-like protein at the spore phase. Furthermore, the ctc gene was transformed into an acrystalliferous B. thuringiensis strain BMB171. The resulting transformant could form parasporal body which had the same shape and molecular weight of protein with that of B. thuringiensis CTC. These results, together with the sequence homology analysis of ctc gene, confirmed that the unique intracellular parasporal inclusion body of B. thuringiensis was comprised of S-layer protein.

Bacillus thuringiensis improved isolation methodology from soil samples

Bacillus thuringiensis is a sporulating bacterium, which produces parasporal inclusions toxic to insects. Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores. The results presented here suggest that a preliminary 5 h dry-heat treatment largely enhance the selectivity of these procedures.

A new group of parasporal inclusions encoded by the S-layer gene ofBacillus thuringiensis

Bacillus thuringiensis produces various groups of active proteins, such as Cyt, Vip and Parasporin, in addition to the Cry protein. In this study we show S-layer proteins to be a new group of parasporal inclusions of B. thuringiensis. The S-layer consists of a two-dimensional lattice structure and is the outermost component of many archaeobacteria and eubacteria. The parasporal inclusion of B. thuringiensis strain CTC was found to be not a typical crystal protein encoded by the cry gene, but a proteinaceous inclusion encoded by the S-layer gene. Furthermore, the CTC-like strains (with their parasporal inclusions coded by the S-layer gene) are widely distributed and accounted for 25.4% of the B. thuringiensis strains tested. These strains constitute a new group of parasporal inclusions encoded by the S-layer gene of B. thuringiensis and shed new light on B. thuringiensis nontoxic strains.

Failure to phenotypically discriminate between non-insecticidal Bacillus thuringiensis strains with anticancer parasporins (PS2, PS3, and PS4) and Bacillus thuringiensis strains that produce insecticidal Cry proteins

This study examined the microbiological features of the three reference strains of Bacillus thuringiensis whose non-insecticidal parasporal inclusions contain anticancer parasporins (PS2, PS3, and PS4). Morphometric analysis showed that the sizes and aspect ratios of the spores of these strains were in the range of those reported for the Bacillus cereus/B. thuringiensis group. The API 50CH test revealed that the carbohydrate utilization profiles of these strains were typical of the B. cereus/B. thuringiensis group. These parasporin producers proved resistant to ampicillin, and induced β-hemolysis on sheep blood agar. Overall results show that there is no significant difference in several phenotypic key characters between B. thuringiensis strains with PS2, PS3, and PS4 and those with insecticidal crystal proteins.