Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the bacillus anthracis exosporium

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the DeltaexsFA and DeltaexsFA DeltaexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The DeltaexsFA and DeltaexsFA DeltaexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.     

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.     

苏云金芽孢杆菌杀虫活性的增效机制研究进展

为了克服苏云金芽孢杆菌（Bacillius thuringiensis, Bt）制剂在实践过程中存在的速效性差、持效期短、防效不稳定以及使害虫逐渐产生抗药性等诸多缺陷,各类添加剂已经被广泛应用于防治过程中以起到增效作用。就目前国内外已开展的各类添加剂对苏云金芽孢杆菌杀虫活性的增效机制和方式的研究进行了概述,并指出其作用方式的潜在可能与途径,以期为苏云金芽孢杆菌生物农药作用机制的研究提供参考。     

Identification and characterization of strains of Bacillus thuringiensis by genomic fingerprinting using biotinylated phage M13 DNA]

Genomic DNA of the entomopathogenic bacterium Bacillus thuringiensis was analyzed by the genomic fingerprinting technique. The biotin-labeled single-stranded DNA of the phage M13 was used as a marker of hypervariable sequences. A procedure for analyzing the differentiation among various Bacillus thuringiensis strains was developed. Characteristic patterns of fingerprints were obtained for several strains, the main representatives of subspecies that are most frequently used in the manufacture of bacterial insecticides, such as subsp. thuringiensis, subsp. kurstaki, and subsp. galleriae. Because no essential differences were revealed in band patterns upon comparing fingerprints of crystal-producing bacterial strains with those of acrystallic mutants, it was assumed that the loss of crystal-producing ability in the insect pathogen Bacillus thuringiensis is not connected with significant rearrangement of its genome.     

Ultra-violet-resistant mutants of Bacillus thuringiensis

One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B. thuringiensis toxin.     

Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.     

Comparative characteristics of spore-forming and asporogenic strains of Bacillus thuringiensis

Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".     

Presence of inducible DNA repair in Bacillus thuringiensis

Weigle reactivation of ultraviolet-irradiated luminal diameter 8 bacteriophage was observed after ultraviolet treatment of Bacillus thuringiensis cells. A slight increased frequency of clear plaque mutants was detected among the survivors. The kinetics of induction of the phage reactivation and phage mutagenesis have been determined. The presence of chloramphenicol before and after irradiation abolished the induction of repair and mutagenesis. These experiments suggest that, in spite of the relatively small mutagenic response in bacteriophage progeny, B. thuringiensis has an inducible repair system responsible to the significant Weigle reactivation of irradiated phage.     

Toxicity of Bacillus thuringiensis Spo Cr Mutants for the European Corn Borer Ostrinia nubilalis

Inclusion bodies isolated from Spo Cr mutants of Bacillus thuringiensis were toxic for larvae of the European corn borer. Probit analysis revealed comparable toxicity between wild-type crystals (isolated from B. thuringiensis subsp. kurstaki) and crystals produced from two spore-free mutants of the same subspecies. Death of the larvae was due to starvation, presumably through delta-endotoxin-induced gut paralysis. Inclusion bodies pretreated with alpha-chymotrypsinogen were equally as toxic as native crystals for the insect larvae.     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis.

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.     

Spermidine Requirement for Bacillus thuringiensis Ribosomes in Cell-Free Phenylalanine Incorporation

A cell-free system from Bacillus thuringiensis was found to actively incorporate phenylalanine into hot trichloroacetic acid-precipitable material in the presence of synthetic polynucleotide, ribosomes, S-100 supernatant, an energy-generating system, and guanosine triphosphate. Phenylalanine incorporation was absolutely dependent on the presence of spermidine in addition to magnesium ions, even when highly purified ribosomes were used. The spermidine effect could not be attributed to inhibition of nucleases. The ribosomal and supernatant fractions from Escherichia coli and B. thuringiensis could be substituted for each other in this system. The spermidine requirement was shown to be limited to the B. thuringiensis ribosome fraction.     

Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.     

Mutagenic analysis of a conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis.

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.     

Transduction ability of mutants of phage CP51, virulent for bacteria of the Bacillus cereus group

The virulent phage CP51 used usually to transfer chromosomal and plasmid markers between bacteria of the Bacillus cereus group was treated with N-methyl-N'-nitro-N-nitroguanidine. Mutants with reduced viability and ts-mutants were isolated. Some of the mutants were found to have an increased efficiency of transduction and allow to simplify the process. Transfer frequencies of the plasmid pBC16 by the phages CP51-26 and CP51-4-59 were 5 x 10(-4) per plaque-forming unit and 4-5 x 10(-3) per bacterial cell, respectively. Possibilities of further increasing the transduction efficiency of Bacillus thuringiensis genetic material using phage CP51 mutants are discussed.     

1-Tetradecanol, a new alcohol found in the cell wall of some rapidly growing chromogenic mycobacteria

Abstract Three oligosporogenic mutants of Bacillus thuringiensis serovar. israelensis which continue to produce the toxic parasporal body were isolated. The mutants were blocked late in sporulation and were approximately as toxic to mosquito larvae as were the parental strains.