Toxicity of Bacillus sphaericus LP1-G against susceptible and resistant Culex quinquefasciatus and the cloning of the mosquitocidal toxin gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.     

<Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> S-layer protein toxicity against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

The main toxicity mechanism of Lysinibacillus sphaericus, which is used in the control of mosquitoes, is its binary toxin produced during sporulation; additionally the Mtx1, Mtx2 and Mtx 3 toxins are expressed in vegetative cells. Mosquito larvicidal potency of the S-layer protein that is expressed in vegetative cells has been determined. The protein is similar to other S-layer proteins of mosquitocidal L. sphaericus strains. The LC50 values of the S-layer protein of the L. sphaericus OT4b25, OT4b26, and III(3)7 strains against third-instar larvae of Culex quinquefasciatus were 8.7, 24 and 0.68 μg/ml, respectively. To our knowledge this is the first study showing the mosquito larvicidal potency of the S-layer protein from Lysinibacillus sphaericus.     

Efficient Expression of the Mosquito Larvicidal Binary Toxin Gene from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E. coli. Low expression level was found when both proteins were expressed from a single operon. High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter. The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB). Both forms of BinB were equally stable. Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA. The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied. The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable.     

High-Level Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, Purification and Mosquito-Larvicidal Activity of the Binary Toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis>

The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin, the binA and binB genes were separately cloned in Eschericia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity of the fusion protein.     

Expression of Binary Toxin Genes in the Mosquito-Colonizable Bacteria, <Emphasis Type="Italic">Bacillus cereus</Emphasis>, Leads to High Toxicity Against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> Larvae

Two B. cereus strains, Ae10 and Cx5, isolated from mosquito larval guts, were transformed with a recombinant plasmid, pBS373, harboring binary toxin genes from Bacillus sphaericus 2297. Immunoblotting analysis clearly revealed the production and presence of the 51-kDa toxin protein in both strains. Two recombinant B. cereus strains Ae10 and Cx5 showed very high toxicity against C. quinquefasciatus larvae. Since both strains have a close relationship with the mosquito larvae in the native environment and are capable of recolonizing in the guts of mosquito larvae, these strains can be considered promising new hosts for an effective delivery of mosquito-larvicidal toxins.     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Characterization of a novel <Emphasis Type="Italic">cry8</Emphasis> gene specific to Melolonthidae pests: <Emphasis Type="Italic">Holotrichia oblita</Emphasis> and <Emphasis Type="Italic">Holotrichia parallela</Emphasis>

A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita.     

Stability,oviposition attraction,and larvicidal activity of binary toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?相似文献   

Cloning and Expression of the Insecticidal Crystal Protein Gene <Emphasis Type="Italic">Cry</Emphasis>1Ca9 of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> G10-01A from Taiwan Granaries

A new cry gene (cry1Ca9) was cloned and sequenced from a Bacillus thuringiensis isolate native to Taiwan (G10-01A). The cry1C-type gene, designated cry1Ca9, consisted of an open reading frame of 3,567 bp, encoding a protein of 1,189 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa. The gene sequence was compared against the GenBank nucleotide sequence data base. It was found that the cry1Ca9 gene coded for a 134.7-kDa protoxin which had greater than 99.8% homology with the previously reported cry1Ca1 gene, as only three mismatches were found between the two amino acid sequences. When the Cry1Ca9 toxin was expressed in a crystal-negative strain of B. thuringiensis (cryB^-), elliptical crystals were produced. Cell extracts from this recombinant strain appear to have high insecticidal activity against lepidopteran larvae (Plutella xylostella).Received: 23 September 2002 / Accepted: 6 December 2002     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> ser. <Emphasis Type="Italic">jordanica</Emphasis> (Serotype H71), a Novel Serovariety Isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3^rd instar larvae of Drosophila melanogaster and to 2^nd stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present. Received: 9 August 2002 / Accepted: 4 September 2002     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Antimicrobial activity of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains

A bacterial strain with a high level of antimicrobial activity was isolated from soil and identified as Bacillus megaterium. Production of antibiotics by nine strains of this species from the collection of the State Research Institute for Genetics and Selection of Industrial Microorganisms was investigated. In submerged cultures, nine out of ten B. megaterium strains were found to produce antibacterial antibiotics differing in their spectra of action. Physicochemical characteristics of five compounds were described. Three of them belonged to peptide antibiotics. All five compounds were active against the methicillin-resistant strain Staphylococcus aureus INA 00761. Three of them were shown to be the previously undescribed compounds. Antibiotics produced by various B. megaterium strains were also active against the Leuconostoc mesenteroides VKPM B-4177 strain resistant to glycopeptide antibiotics and against gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922.     

Susceptibility of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> strains different in oxalate production to infection by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g⁻¹ medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.