Quanxin Cai

The synergistic activity between Cry1Aa and Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera

Aims:  To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor.

Occurrence of psychrotolerant Bacillus cereus group strains in ice creams

The occurrences of Bacillus cereus group strains in 40 ice cream samples were investigated. Among 109 isolated B. cereus group strains confirmed by 16S rDNA sequence analysis only 50 were identified as B. cereus and one as B. thuringiensis by using FDA (U.S. Food and Drug Administration) standard, indicating the two identification standards were highly inconsistent. Furthermore, the psychrotolerant growth properties and the occurrence of specific psychrotolerant genes of the isolates were also studied. Both psychrotolerant 16S rDNA fragments and enterotoxic genes could be detected among mesophilic and psychrotolerant strains. No relationship among psychrotolerance, presence of psychrotolerant 16S rDNA fragments and enterotoxic genes were found and the specific cspA fragment was only detected in a small fraction (9.5%) of the psychrotolerant isolates. One psychrotolerant isolate Bw2-1 was identified as B. weihenstephanensis, but no clear distinguishing characteristics between B. weihenstephanensis and psychrotolerant B. cereus were found. These results might be of importance for gaining further understanding of the growth properties of B. weihenstephanensis and psychrotolerant B. cereus as well as their contribution to food poisoning.

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptors C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.

Prevalence of Burkholderia pseudomallei in Guangxi, China.

Melioidosis, an infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei, is now recognized as an important public health problem in Southeast Asia and tropical northern Australia. Although B. pseudomallei has been detected in various water and soil samples in southeast China, the enviromental distribution of B. pseudomallei in China is unclear. In the winter months of 2007, 154 and 130 soil and water samples, respectively, were collected from several locations in Guangxi, China. The samples were screened for B. pseudomallei by bacterial culture and identification and confirmed by PCR for species-specific 16S rDNA and flagellin genes. B. pseudomallei was detected in 8.4% of the soil samples but in none of the water samples. All positive samples were confined to a single low-lying region from rice paddy fields. Counts of B. pseudomallei ranged from 23 to 521 c.f.u./g soil. This is the first geographical distribution survey of B. pseudomallei in soil in Guangxi, China, and the data are of importance for further evaluating the impact of this pathogen on melioidosis in this region.

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph

pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

Mosquitocidal Toxin from Bacillus sphaericus Induces Stronger Delayed Effects Than Binary Toxin on Culex quinquefasciatus (Diptera: Culicidae)

Abstract We studied the toxicity and delayed effects of a mosquitocidal toxin (Mtx1) and a binary toxin (Bin) produced in Escherchia coli E-TH21 and Bacillus thuringiensis B-CW1, respectively, on Culex quinquefasciatus (Diptera: Culicidae). Bioassay results showed that both E-TH21 powder and B-CW1 sporulated culture were highly toxic against susceptible Cx. quinquefasciatus, with LC50 values of 0.65 and 1.70 mg/liter against third and fourth instars at 48 h, respectively. After initial 48-h exposure of larvae to different concentrations of Mtx1 and Bin, significant continued mortality could be observed in larval, pupal, and emergence stages of Cx. quinquefasciatus. Importantly, the Mtx1 could induce higher cumulative larval and preadult mortalities than Bin toxin on the target mosquito. This finding is important for understanding the mode of action of Mtx1 and Bin toxins and for developing a new bioassay procedure for evaluation of toxicity of Bacillus sphaericus Neide, some strains of which produce Mtx1 and Bin, in the laboratory and field.

A Novel Transcriptional Activator, tubX, Is Required for the Stability of Bacillus sphaericus Mosquitocidal Plasmid pBsph

Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning (par) system that consists of a filament-forming protein, B. sphaericus TubZ (TubZ-Bs); a centromere-binding protein, TubR-Bs; and a centromere-like DNA site, tubC, composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding the TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here orf187 (encoding TubX), a gene downstream of tubZ-Bs, was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in the level of tubRZ-Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR-Bs, and a higher level of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region.

Conjugative Transfer of Insecticidal Plasmid pHT73 from Bacillus thuringiensis to B. anthracis and Compatibility of This Plasmid with pXO1 and pXO2

ABSTRACT Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1+ pXO2−) and Ba63605R (pXO1− pXO2+), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 × 10−4 and 1.6 × 10−4 CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment.

Interaction of Lysinibacillus sphaericus Cry48Aa/Cry49Aa toxin with midgut brush-border membrane fractions from Culex quinquefasciatus larvae

The Cry48Aa/Cry49Aa mosquitocidal toxin from Lysinibacillus sphaericus was uniquely composed of a three‐domain (Cry) toxin and binary (Bin) toxin‐like protein, with high toxicity against Culex spp. However, its mode of action against the target mosquitoes is still unknown. In this study, Cry48Aa, Cry49Aa and its N‐ and C‐terminal truncated proteins were expressed and purified, and the binding affinities of the purified proteins with midgut brush‐border membrane fractions (BBMFs) from Culex quin‐quefasciatus larvae were performed. The results showed that both Cry48Aa and Cry49Aa have specific and high binding affinity to BBMFs, with dissociation constants of 9.5 ± 1.8 and 25.4 ± 3.8 nM, respectively. Competition assays demonstrated that Cry49Aa C‐terminal derivatives were able to bind to the BBMFs, whereas Far‐Western dot blot analysis revealed that its N‐terminal constructs interacted with Cry48Aa. Nevertheless, larvicidal activity was almost lost when Cry49Aa truncated proteins, either individually or in pairs, combined with Cry48Aa. It is concluded that Cry49Aa is responsible for receptor binding and interaction with Cry48Aa and plays an important role in the mechanism of action of these two‐component toxins.