Seasonal distribution and characterization of Bacillus thuringiensis isolated from sericultural environments in Korea

To isolate naturally occurring novel Bacillus thuringiensis strains, we investigated the distribution and characteristics of B. thuringiensis from samples of sericultural farms in various regions of Korea in the spring and fall. Fifty-four B. thuringiensis strains out of 164 samples and 34 B. thuringiensis strains out of 135 samples were isolated in the spring and fall, respectively. Seventy percent of the isolates in the spring and 15% in the fall were toxic to lepidopteran larvae. Dipteran-active isolates were rare (7% in spring and 3% in fall isolation). Particularly, B. thuringiensis isolates, which are toxic to both Lepidoptera and Diptera, were widely distributed (19% in spring and 62% in fall isolation). Non-toxic isolates were also found (4% in spring and 20% in fall isolation). B. thuringiensis isolates in the sericultural farms represented 11 H serotypes; they were principally B. thuringiensis subsp. aizawai in the spring and kurstaki in the fall. B. thuringiensis isolates of serotypes 1, 3a, 3a3b, 4a4c, 6, 7 and 12 were toxic to Lepidoptera. Seventy isolates produced typical rhomboidal inclusions, and the remainder produced parasporal inclusions with various morphologies. PCR analysis using cryI gene type-specific primers showed that cryIAa and cryIC genes are frequently found in the spring and cryIAa gene is a predominant type in the fall. Toxicity, H serotype and the cryI gene contents of B. thuringiensis isolated from sericultural farms showed that distribution varied depending on the season.     

Microbial Ecology and Association of Bacillus thuringiensis in Chicken Feces Originating from Feed

To explain the association of Bacillus thuringiensis (Bt) with animal feces, an ecological analysis in chickens was conducted by introducing a cry ^? strain marked by production of green fluorescent protein (GFP). After feeding with the tagged Bt strains, the feces of the tested chickens were collected at different times, isolated, and the morphology of Bt was observed. It was shown that Bt strain HD-73GFP in spore form could be isolated from feces of chickens for a period of 13 d, and then it disappeared thereafter. Bt could be detected only up to day 4 (but not thereafter), when chickens were fed with vegetative cells of HD-73GFP. To confirm the source of newly isolated strains, the gfp gene was examined by polymerase chain reaction (PCR), which showed that all the isolated strains harbored the marker gene. Recent data from isolation and PCR had suggested that fecal Bt strains had originated from food. Chicken tissues were thus dissected to isolate Bt strains and to investigate whether Bt could be located in vivo. Bt was located within the duodenum in spore form. Compared to the morphology of the isolated strains at different growth times, the growth rates of all the tested Bt had little changes when passing through the digestive system to the feces. Dissection of the chickens confirmed that Bt was safe for the tested animal.     

Microbial control and biotechnology research on Bacillus thuringiensis in China

The current status of production and application of biopesticides for pest control in China is briefly reviewed, with a focus on research advances in microbial control with Bacillus thuringiensis (Bt). These have led to improvements in Bt production, exploitation of Bt gene resources, and development of engineered Bt insecticides and transgenic Bt crops that have expanded host ranges and increased efficacy against target pests. Both conventional and biotechnology approaches need to be employed to achieve further progress in discovery, production technology, formulation processing, development of quality standards and recommended use patterns.     

Metalloprotease from Bacillus thuringiensis

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...     

Biological, Immunological, and Genetic Analysis of Bacillus thuringiensis Isolated from Granary in Korea

To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types. Received: 15 January 1998 / Accepted: 18 February 1998     

Bacillus thuringiensis: from biodiversity to biotechnology

Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of δ-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection. Received 25 February 1997/ Accepted in revised form 15 August 1997     

Extracellular ribonuclease from Bacillus thuringiensis

The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.     

Bacillus thuringiensis: ecology,the significance of natural genetic modification,and regulation

Legislation and regulation in respect of genetically modified microorganisms must be based on rick-assessment principles rather than on the current European concept of pathogenicity, which is a measure of hazard. Natural genetic modification has commerical attractions where the processes can be exempted from regulation. Bacillus thuringiensis is used to illustrate this in the context of the European Directives and UK regulation.     

Characterization of eight Bacillus thuringiensis isolates originated from fecal samples of Fuzhou Zoo and Fuzhou Panda Center

In eight fecal samples of 5 herbivorous animals from Fuzhou Zoo and Fuzhou Panda Center were found eight Bacillus thuringiensis isolates. Obtained isolates were characterized by crystal microscopy, cry identification, and assay against the development of Eimeria tenella oocyst in chicken embryo. Bt WCB1, WCB2, WCB3 and WCB8, originated from horse, spotted deer, giant panda and lesser panda, respectively, all exhibited bipyramidal and cuboidal crystal and harbored the same cry genes, which were cry1Cb, cry1Db, cry1Fa, cry1Ib and cry2Ab. WCB7, a Bt isolate with bipyramidal crystal from zebra, showed a different cry pattern, cry1Cb, cry1Db, cry1Fa and cry1Ia. Bt WCB6, the third Bt isolate from giant panda, also contained bipyramidal crystal, however, harbored cry1Ib only. It was noted that Bt isolate WCB5 contained cuboidal crystal and harbored an unknown cry3 gene. It was very interesting that no cry genes were detected from Bt isolate WCB4 with dot-like crystal. Parasporal crystal proteins of WCB3, WCB4 and WCB7 obviously inhibited the development of E. tenella oocyst in chicken embryo.     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Distribution, Serological Identification, and PCR Analysis of Bacillus thuringiensis Isolated from Soils of Korea

A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea. Serological tests showed that B. thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B. thuringiensis. But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates. In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates. Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found. Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape. In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions. PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%). But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive. Several isolates had unusual PCR products and multiple insecticidal crystal protein genes. PCR results showed varied distribution of the cry-type gene. Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis. Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C. pipiens produced spherical inclusion. Received: 13 March 1998 / Accepted: 14 April 1998     

Microbial biofilms: from ecology to molecular genetics.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.     

Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries

Mulberry leaves were examined for the occurrence of Bacillus thuringiensis. This organism was recovered from both abaxial and adaxial surfaces: a total of 186 B. thuringiensis colonies were isolated from 24 (96·0%) out of 25 mulberry trees, and from 112 (11·2%) out of 1004 leaves from 25 trees. The frequency of B. thuringiensis colonies was 3·2% among 5900 colonies belonging to the Bacillus cereus/B. thuringiensis group. Single colonies were associated with 75·9% of the B. thuringiensis -positive leaves and 2–16 colonies were occasionally found on a single phylloplane. Flagellar (H) serotypying of the isolates revealed that, among the 19 H serotypes (serovars) detected, the H serotype 13 (serovar pakistani ) was the predominant, followed by the H serotypes 3abc ( kurstaki ), 6ac ( oyamensis ), 16 ( indiana ), 24 ( neoleonesis ), 4ac ( kenyae ), 7 ( aizawai ) and 10 ( darmstadiensis ). Larvicidal activity, against the silkworm ( Bombyx mori ) and/or the mosquito ( Aedes aegypti ), was exhibited by 18 isolates (9·7%) belonging to H serovars kurstaki, kenyae, canadensis and aizawai , and an unidentified H serogroup.     

Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.     

Detection and ecology of leptospirosis in Iowa wildlife

To gain additional information on the extent of leptospirosis in wildlife following a human outbreak in Iowa, wild mammals and lower forms of life were collected. Isolation, darkfield microscopic, serologic and pathologic procedures were used to identify past or present evidence of leptospiral infection. Leptospires were isolated from 7 of 75 (9%) mammals. Serotype grippotyphosa was isolated from three raccoons (procyon lotor) and one Western Harvest Mouse (Reithrodontomys megalotis). Serotype ballum was isolated from three opossums (Didelphis marsupialis). Leptospires, unidentified to date, were isolated from frog (Rana pipiens) kidneys. Other positive serologic and pathologic tests gave evidence of infection or previous infection. Utilization of Darkfield microscopic and silver staining techniques did not detect all cases of leptospiral infection. Macroscopic and microscopic serologic methods failed to identify evidence of leptospirosis in all mammals from which leptospires were isolated. Pathologic lesions could only be considered presumptive evidence for leptospirosis. These findings indicate that detection of leptospirosis in wildlife cannot be limited to a single diagnostic test. A combination of diagnostic procedures and clinical evaluation is necessary. Although serotype pomona was implicated as the predominant infecting leptospire in the human cases and domestic animals and was isolated from water at a swimming site, only serotypes grippotyphosa, ballum and ICF (frog isolate) were isolated from wild mammals and lower forms of life in the same vicinity.