Dasheng Zheng

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

Bacillus sphaericus strain C3-41 is an aerobic, mesophilic, spore-forming bacterium that has been used with great success in mosquito control programs worldwide. Genome sequencing revealed that the complete genome of this entomopathogenic bacterium is composed of a chromosomal replicon of 4,639,821 bp and a plasmid replicon of 177,642 bp, containing 4,786 and 186 potential protein-coding sequences, respectively. Comparison of the genome with other published sequences indicated that the B. sphaericus C3-41 chromosome is most similar to that of Bacillus sp. strain NRRL B-14905, a marine species that, like B. sphaericus, is unable to metabolize polysaccharides. The lack of key enzymes and sugar transport systems in the two bacteria appears to be the main reason for this inability, and the abundance of proteolytic enzymes and transport systems may endow these bacteria with exclusive metabolic pathways for a wide variety of organic compounds and amino acids. The genes shared between B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, including mobile genetic elements, membrane-associated proteins, and transport systems, demonstrated that these two species are a biologically and phylogenetically divergent group. Knowledge of the genome sequence of B. sphaericus C3-41 thus increases our understanding of the bacilli and may also offer prospects for future genetic improvement of this important biological control agent.

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.

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

ABSTRACT Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had Km values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.

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.

Allelic Diversity and Population Structure of Bacillus sphaericus as Revealed by Multilocus Sequence Typing

ABSTRACT The genetic diversity of 35 Bacillus sphaericus strains was analyzed by a newly developed multilocus sequence typing (MLST) scheme, toxin gene pool survey, and mosquito bioassay. The results demonstrated that strains assigned to the same sequence type (ST) had the same occurrence of toxin genes. Further sequence analysis revealed that toxic strains presented a nearly clonal population structure, whereas nontoxic strains had a high level of heterogeneity and were significantly distinct from toxic strains.

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.

Expression of mel gene improves the UV resistance of Bacillus thuringiensis

Aims:  To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory.

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.