Tianpei Huang

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

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.

Aerobic Cr(VI) Reduction by an Indigenous Soil Isolate Bacillus thuringiensis BRC-ZYR2

Abstract Chromium (Cr) may cause losses in the yield of field plant, which is one of the favorite habitats of Bacillus thuringiensis (Bt). The purposes of our study were to assess the Cr(VI)-resistance and Cr(VI)-reducing abilities of an indigenous soil isolate of Bt and to determine the factors governing Cr(VI) reduction. Towards this end a novel dichromate-reducing Bt BRC-ZYR2, characterized with insecticidal crystal proteins (ICPs), was isolated from a uranium deposit. Minimum inhibitory concentrations (MICs) of Cr(VI) were determined by broth dilution method and the concentrations of Cr(VI) and total Cr in the supernatant were quantified colorimetrically using 1,5-diphenylcarbazide (DPC) reagent and a mixture of sulfuric-nitric acids, respectively. The isolate contained five ICP genes ( cry1Ba, cry1Bb, cry1Be/cry1Bf, cry9Ca and cry9Da ) and exhibited a high level of Cr(VI) resistance with MICs of 150 mg L −1 at pH 7.0 and 30 °C, and 500 mg L −1 under optimal conditions (pH 9.0 and 40 °C). The total Cr concentration was similar to initial concentration of Cr(VI) under the optimal condition, suggesting that the essential removal of the Cr(VI) was dependent on Bt reduction. Under optimal conditions, the initial Cr(VI) concentrations from 25 to 75 mg L −1 significantly decreased in 24 h after incubation. Addition of Mn 2+ , Co 2+ , Mo 2+ and Cu 2+ activated Bt-mediated Cr(VI) reduction, while Zn 2+ , Ni 2+ and glucose were found to inhibit the reduction. Our results indicated that this isolate could be a promising biopesticide with the potential for both insect biocontrol and Cr bioremediation in the field.

Effect of Chemical Additives on Bacillus thuringiensis (Bacillales: Bacillaceae) Against Plutella xylostella (Lepidoptera: Pyralidae)

ABSTRACT To examine the effect of chemical additives on Bacillus thuringiensis (Berliner) against Plutella xylostella (L.), inorganic salts, nitrogenous compounds, protein solubilizing agents, and organic acids were selected and tested. The chosen materials are low in cost and environmentally safe. Results show that many inorganic salts can increase the activity of B. thuringiensis in a range of 1.31-to 3.08-fold. These include calcium acetate, calcium chloride, calcium hydroxide, calcium sulfate, calcium carbonate, sodium carbonate, sodium acetate, potassium hydroxide, potassium carbonate, potassium acetate, magnesium chloride, magnesium sulfate, and zinc sulfate. Nitrogenous compounds, including peptone, sodium nitrate, and ammonium nitrate, can enhance the activity of B. thuringiensis 1.62-, 1.32-, and 1.37-fold, respectively. Among the protein solubilizing agents, EDTA, urea, mercaptoethanol and dipotassium hydrogen phosphate increased the activity of B. thuringiensis 1.62- to 2.34-fold. Among the organic acids, maleic and citric acids boosted the activity 1.45- and 1.55-fold, respectively. Meanwhile, sodium benzoate and resorcinol led to 1.74- and 1.44-fold activity gains, respectively. Use of appropriate additives could provide great benefit not only in reducing the costs for field applications of biological insecticides but also by boosting the efficacy of B. thuringiensis.

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.

Cry11Aa Interacts with the ATP-Binding Protein from Culex quinquefasciatus To Improve the Toxicity

Cry11Aa displays high toxicity to the larvae of several mosquito species, including Aedes, Culex, and Anopheles. To study its binding characterization against Culex quinquefasciatus, Cry11Aa was purified and western blot results showed that Cry11Aa could bind successfully to the brush border membrane vesicles. To identify Cry11Aa-binding proteins in C. quinquefasciatus, a biotin-based protein pull-down experiment was performed and seven Cry11Aa-binding proteins were isolated from the midgut of C. quinquefasciatus larvae. Analysis of liquid chromatography-tandem mass spectrometry showed that one of the Cry11Aa-binding proteins is the ATP-binding domain 1 family member B. To investigate its binding property and effect on the toxicity of Cry11Aa, western blot, far-western blot, enzyme-linked immunosorbent assay, and bioassays of Cry11Aa in the presence and absence of the recombinant ATP-binding protein were performed. Our results showed that the ATP-binding protein interacted with Cry11Aa and increased the toxicity of Cry11Aa against C. quinquefasciatus. Our study suggests that midgut proteins other than the toxin receptors may modulate the toxicity of Cry toxins against mosquitoes.

RAP-PCR fingerprinting reveals time-dependent expression of development-related genes following differentiation process of Bacillus thuringiensis

Gene expression profiles are important data to reveal the functions of genes putatively involved in crucial biological processes. RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and specifically primed reverse transcription polymerase chain reaction (RT-PCR) were combined to screen differentially expressed genes following development of a commercial Bacillus thuringiensis subsp. kurstaki strain 8010 (serotype 3a3b). Six differentially expressed transcripts (RAP1 to RAP6) were obtained. RAP1 encoded a putative triple helix repeat-containing collagen or an exosporium protein H related to spore pathogenicity. RAP2 was homologous to a ClpX protease and an ATP-dependent protease La (LonB), which likely acted as virulence factors. RAP3 was homologous to a beta subunit of propionyl-CoA carboxylase required for the development of Myxococcus xanthus. RAP4 had homology to a quinone oxidoreductase involved in electron transport and ATP formation. RAP5 showed significant homology to a uridine kinase that mediates phosphorylation of uridine and azauridine. RAP6 shared high sequence identity with 3-methyl-2-oxobutanoate-hydroxymethyltransferase (also known as ketopantoate hydroxymethyltransferase or PanB) involved in the operation of the tricarboxylic acid cycle. The findings described here would help to elucidate the molecular mechanisms underlying the differentiation process of B. thuringiensis and unravel novel pathogenic genes.

Different Toxicity of the Novel Bacillus thuringiensis (Bacillales: Bacillaceae) Strain LLP29 Against Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae)

ABSTRACT Bacillus thuringiensis (Bt) (Berliner) strain LLP29 produces a crystal protein Cyt1Aa6 toxic to mosquito vectors of human diseases. However, the susceptibility of Culex quinquefasciatus (Say) in the current study was 8.25 times higher than that of Aedes albopictus (Skuse) with this single protein Cyt1Aa6 purified from LLP29. To understand the mechanism of the novel mosquitocidal protein, the binding characteristic of brush border membrane vesicles from the two tested mosquitoes was investigated. Enzyme-linked immunosorbent assay showed that Cyt1Aa6 bound to the two mosquitoes’ brush border membrane vesicles. However, the titer of Ae. albopictus was a little higher than that of Cx. quinquefasciatus, with 3.21 and 2.91, respectively. Ligand Western blot analysis showed Cyt1Aa6 toxin specifically bound to the same three proteins (i.e., 68, 54, and 26 kDa) in the two mosquitoes, but one another protein, approximately to 37 kDa, could just be detected in Cx. quinquefasciatus. However, little difference was found in the test of immunohistochemistry. Cyt1Aa6 was detected in the midguts of both mosquitoes with histopathological changes. It would of great importance to the knowledge of the novel toxin against to Cx. quinquefasciatus and Ae. albopictus.

A Differentially Displayed mRNA Related to Resistance to Bacillus thuringiensis israelensis of Aedes albopictus Selected in Vitro-Activated CYT1AA6

Abstract We compared the differential display of Aedes albopictus cells, both resistant and susceptible to Bacillus thuringiensis israelensis (Bti), using differentially displayed reverse transcription polymerase chain reaction. We found 1 band about 200 base pairs long. After cloning and sequencing, the differentially expressed gene was similar to some partial messenger ribonucleic acid of Ae. aegypti, Culex quinquefasciatus, and Anopheles gambiae, rather than Ae. albopictus. This will be of some value for clarifying the mechanism of mosquito resistance to Bti products.

Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin

Globally, Aedes aegypti is one of the most dangerous mosquitoes that plays a crucial role as a vector for human diseases, such as yellow fever, dengue, and chikungunya. To identify (1) transcriptomic basis of midgut (2) key genes that are involved in the toxicity process by a comparative transcriptomic analysis between the control and Bacillus thuringiensis (Bt) toxin (LLP29 proteins)-treated groups. Next-generation sequencing technology was used to sequence the midgut transcriptome of A. aegypti. A total of 17130 unigenes, including 574 new unigenes, were identified containing 16358 (95.49%) unigenes that were functionally annotated. According to differentially expressed gene (DEG) analysis, 557 DEGs were annotated, including 226 upregulated and 231 downregulated unigenes in the Bt toxin-treated group. A total of 442 DEGs were functionally annotated; among these, 33 were specific to multidrug resistance, 6 were immune-system-related (Lectin, Defensin, Lysozyme), 28 were related to putative proteases, 7 were lipase-related, 8 were related to phosphatases, and 30 were related to other transporters. In addition, the relative expression of 28 DEGs was further confirmed through quantitative real time polymerase chain reaction. The results provide a transcriptomic basis for the identification and functional authentication of DEGs in A. aegypti.