Jieping Wang

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.

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Bacillus thuringiensis is a well-known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20–30% of the cells dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: (1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; (2) stored poly-β-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; (3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; (4) the tricarboxylic acid cycle was significantly modified during sporulation; (5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; (6) most F0F1-ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels.

Mapping of centromeric regions on the molecular linkage map of rice (Oryza sativa L.) using centromere-associated sequences

Abstract Genetic mapping of centromeres has been a challenge for plant geneticists. The objective of this study was to develop a new strategy for determining the locations of centromeric regions on genetic maps by mapping centromere-associated sequences, to make it possible to define the centromeric region of each chromosome as a single Mendelian locus on the molecular linkage map. Two DNA probes containing sequences specifically associated with the centromeres of grass species were used for genetic mapping. The centromere-associated sequences for all 12 rice chromosomes were mapped on the molecular map with either or both of the probes, and flanking molecular markers on one or both sides were localized 0 to 8 cM away. The map locations of the centromere-associated markers corresponded very well with the positions of centromeric regions determined previously using trisomic analyses for 11 of the 12 chromosomes. The precise mapping of the centromeric regions using these probes makes the molecular map a more complete and informative tool for genomic studies, which will facilitate studies of the structure and function of the rice centromeres. The simplicity of this technique, together with the fact that these probes are also associated with the centromeric regions in other grass species, may provide a general approach to the mapping of centromeric regions in the genomes of other cereal crops.

High-Throughput Identification of Promoters and Screening of Highly Active Promoter-5′-UTR DNA Region with Different Characteristics from Bacillus thuringiensis

In bacteria, both promoters and 5′-untranslated regions (5′-UTRs) of mRNAs play vital regulatory roles in gene expression. In this study, we identified 1203 active promoter candidates in Bacillus thuringiensis through analysis of the genome-wide TSSs based on the transcriptome data. There were 11 types of σ-factor and 34 types of transcription factor binding sites found in 723 and 1097 active promoter candidates, respectively. Moreover, within the 1203 transcriptional units (TUs), most (52%) of the 5′-UTRs were 10–50 nucleotides in length, 12.8% of the TUs had a long 5′-UTR greater than 100 nucleotides in length, and 16.3% of the TUs were leaderless. We then selected 20 active promoter candidates combined with the corresponding 5′-UTR DNA regions to screen the highly active promoter-5′-UTR DNA region complexes with different characteristics. Our results demonstrate that among the 20 selected complexes, six were able to exert their functions throughout the life cycle, six were specifically induced during the early-stationary phase, and four were specifically activated during the mid-stationary phase. We found a direct corresponding relationship between σ-factor-recognized consensus sequences and complex activity features: the great majority of complexes acting throughout the life cycle possess σA-like consensus sequences; the maximum activities of the σF-, σE-, σG-, and σK-dependent complexes appeared at 10, 14, 16, and 22 h under our experimental conditions, respectively. In particular, complex Phj3 exhibited the strongest activity. Several lines of evidence showed that complex Phj3 possessed three independent promoter regions located at −251∼−98, −113∼−31, and −54∼+14, and that the 5′-UTR +1∼+118 DNA region might be particularly beneficial to both the stability and translation of its downstream mRNA. Moreover, Phj3 successfully overexpressed the active β-galactosidase and turbo-RFP, indicating that Phj3 could be a proper regulatory element for overexpression of proteins in B. thuringiensis. Therefore, our efforts contribute to molecular biology research and the biotechnological application of B. thuringiensis.

Expression Profile and Regulation of Spore and Parasporal Crystal Formation-Associated Genes in Bacillus thuringiensis

Bacillus thuringiensis, a Gram-positive endospore-forming bacterium, is characterized by the formation of parasporal crystals consisting of insecticidal crystal proteins (ICPs) during sporulation. We reveal gene expression profiles and regulatory mechanisms associated with spore and parasporal crystal formation based on transcriptomics and proteomics data of B. thuringiensis strain CT-43. During sporulation, five ICP genes encoded by CT-43 were specifically transcribed; moreover, most of the spore structure-, assembly-, and maturation-associated genes were specifically expressed or significantly up-regulated, with significant characteristics of temporal regulation. These findings suggest that it is essential for the cell to maintain efficient operation of transcriptional and translational machinery during sporulation. Our results indicate that the RNA polymerase complex δ and ω subunits, cold shock proteins, sigma factors, and transcriptional factors as well as the E2 subunit of the pyruvate dehydrogenase complex could cooperatively participate in transcriptional regulation via different mechanisms. In particular, differences in processing and modification of ribosomal proteins, rRNA, and tRNA combined with derepression of translational inhibition could boost the rate of ribosome recycling and assembly as well as translation initiation, elongation, and termination efficiency, thereby compensating for the reduction in ribosomal levels. The efficient operation of translational machineries and powerful protein-quality controlling systems would thus ensure biosyntheses of a large quantity of proteins with normal biological functions during sporulation.

Mechanistic insights into manganese oxidation of a soil-borne Mn(II)-oxidizing Escherichia coli strain by global proteomic and genetic analyses

An iTRAQ-based comparative and quantitative proteomics analysis of a soil-borne Mn(II)-oxidizing bacterium, Escherichia coli MB266, was conducted during the exponential and stationary growth phases. A total of 1850 proteins were identified in 4 samples, of which 373 and 456 proteins were significantly up- or down-regulated in at least one pairwise comparison, respectively. The iTRAQ data indicated that several enzymes involved in fatty acid metabolism (i.e., FabA, FabD and FabZ) and pyruvate metabolism (particularly pyruvate oxidase PoxB) were significantly up-regulated, while those related to the tricarboxylic acid cycle (such as FrdB, FumB and AcnA) and methylcitrate cycle (i.e., PrpC) were inactivated in the presence of 1 mM Mn(II); the amounts of some stress response and signal transduction system-related proteins (i.e., Spy) were remarkably increased, and the cold shock protein CspD was significantly up-regulated during the exponential growth phase. However, all verified heat shock proteins remained unchanged. The reactive oxygen species response and some redox enzymes might also be involved in Mn oxidation processes. The involvement of several cellular proteins in Mn(II) oxidation, including PoxB, Spy and MCO266, was further confirmed by gene disruption and expression complementation experiments. Based on these results, a signal transduction mechanism coupled to Mn oxidation was proposed.