Mingshun Li

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.

Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.

Proteomic analysis reveals the strategies of Bacillus thuringiensis YBT-1520 for survival under long-term heat stress†

Bacillus thuringiensis (Bt) has been widely used for 50 years as a safe biopesticide for controlling agricultural and sanitary insect pests because of its insecticidal crystal proteins. In this study a proteomic approach was used to investigate the responses and survival strategies of Bt YBT‐1520 under a long‐term heat stress condition (42°C). Heat stress mainly influenced the characteristics of YBT‐1520 on four aspects: (i) the abilities to synthesise insecticidal crystal proteins and other potential pathogenic factors were almost lost, (ii) cell adhesion and motility were also lost, (iii) cell did not sporulate, (iv) cell kept accumulating poly(3‐hydroxybutyrate) (PHB). Proteomic analyses to the physiological changes of the strain revealed three strategies of YBT‐1520 for survival under long‐term heat stress. The first strategy is to up‐regulate enzymes (BDH1, GuaB and PepA) for long‐term heat stress tolerance. The second one is to down‐regulate metabolic enzymes to reduce metabolic burden. The third strategy is to increase the synthesis and accumulation of PHB. Under heat stress condition, the bacterium adjusted its metabolism by up‐/down‐regulation and continuous accumulation of PHB. These strategies would help cells to gain more tolerance to heat stress.

Comparative proteomic analysis revealed metabolic changes and the translational regulation of Cry protein synthesis in Bacillus thuringiensis.

B. thuringiensis strain YBT-1520 is highly toxic to some Lepidopteran pests and is used to control pests in southern part of China. The aim of this work is to uncover the metabolic changes associated with the ICP synthesis. A comparative proteomic analysis on the strain was performed with 2-DE and MALDI-TOF-MS methods with the bacterial culture grown in a modified defined minimal medium. Transcriptional expression of some key enzymes for carbohydrate metabolism was also investigated with qPCR. Seventy-two proteins differentially expressed at least two folds were identified. Significant changes were observed in metabolisms when comparing stationary growth phase to mid-log phase. In late-stationary phase, five major phenomena were observed: 1) the glycolysis cycle was redirected into pentose phosphate pathway and ended in serine formation; 2) the tricarboxylic acid (TCA) cycle was suppressed; 3) poly(3-hydroxybutyrate) (PHB) was considered to be the important energy and carbon supply; 4) collectively increased proteases suggested massive protein degradation to release amino acids, and 5) translation-related factors were up-regulated including EF-Tu, GroEL and GatB, while the negative regulator YfiA was down. The metabolic changes of bacterium during growth shift indicated that the cells acquired energy, amino acid precursors and translation-related factors for the ICP synthesis.

Prevalence and diversity of insertion sequences in the genome of Bacillus thuringiensis YBT-1520 and comparison with other Bacillus cereus group members

Members of the Bacillus cereus group are closely related bacteria that exhibit highly divergent pathogenic properties. Sequencing of Bacillus thuringiensis ssp. kurstaki strain YBT-1520 revealed an increased number of insertion sequences (ISs) compared with those of the published B. cereus group genomes. Although some of these ISs have been observed and summarized in B. thuringiensis previously, a genomic characterization of their content is required to reveal their distribution and evolution. The result shows that the larger number of transposase coding genes on YBT-1520 chromosome is mainly caused by the amplification of IS231C, IS232A and ISBth166. Some functional genes have been disrupted through the insertion of ISs, preferentially IS231C. By comparing the Southern hybridization profiles of different B. thuringiensis strains, the existence of ISBth166 was mainly found in serovar kurstaki and the recent expansion of IS231C between different kurstaki isolates was suggested. In addition to revealing the ISs profile in YBT-1520 as well as the comparison in the B. cereus group, this study will contribute to further comparative analyses of multiple B. thuringiensis strains aimed at understanding the IS-mediated genomic rearrangements among them.

Proteomic analysis of Bacillus thuringiensis ΔphaC mutant BMB171/PHB− 1 reveals that the PHB synthetic pathway warrants normal carbon metabolism

A phaC knockout mutant from Bacillus thuringiensis (Bt) strain BMB171, named BMB171/PHB(-1), was constructed. A physiological and metabolic investigation and a proteomic analysis were conducted for both ΔphaC mutant and its parent strain. Grown in peptone medium with 5 gram glucose per liter as sole carbon source, BMB171/PHB(-1) produced various organic acids. Here the excreted pyruvate, citrate, lactate, acetate and glutamate were quantitatively analyzed. Deletion of phaC gene from the BMB171 strain resulted in 1) growth delay; 2) higher consumption of dioxigen but lower cell yield; 3) stagnation of pH movement; 4) overproduction of organic acids; 5) rapid descent of cell density in the stationary phase; and 6) a sporulation-deficient phenotype. Our proteomic study with qPCR reconfirmation reveals that the absence of PhaC led to a metabolic turmoil which showed repressed glycolysis, and over-expressed TCA cycle, various futile pathways and amino acid synthesis during vegetative growth. It is thus thought that B. thuringiensis BMB171 effectively regulated its carbon metabolism upon the presence of the functional PHB synthetic pathway. The presence of this pathway warrants a PHB-producing bacterium better surviving under different environmental conditions.

Two novel transposon delivery vectors based on mariner transposon for random mutagenesis of Bacillus thuringiensis.

Two new mariner transposon delivery vector systems, pMarA333 and pMarB333, had been constructed to randomly mutagenize Bacillus thuringiensis in vivo. The results showed that both systems could randomly insert into the genome of B. thuringiensis YBT881 (CCAM 020673). These systems could have potential for further construction of mutant libraries of B. thuringiensis and other Bacillus strains.