| Bacillus thuringiensis is a Gram-positive, spore-forming bacterium, and it is a member of the B. cereus sensu lato group bacteria, which produces parasporal crystals during sporulation if the nutrient is limited. The crystals consist of insecticidal crystal proteins (ICPs, also called Cry proteins), and have outstanding contribution to the insecticidal activity of B. thuringiensis against a variety of insects and nematodes.. As a natural insecticidal bacterium, B. thuringiensis has been globally used as a microbial pesticide with the advantages of safety and high efficiency. However, thousands of B. thuringiensis strains showed considerable variety in their insecticidal toxicity, and over the past 70 years people still didn’t known why the highly toxic strains, which were used in biopesticide production, have more effective insecticidal activity.Genomics is a key to answer this question. Based on the genomic analysis, systematic studies of multiple virulence related factors could be performed. However, it’s still a challenge that using high-throughput sequencing strategy to complete a genome of B. thuringiensis with a lot of plasmids. Up to Sep 2015, there were 26 completed genomes of B. thuringiensis in NCBI database, the plasmid content of these strains is low. And no completed genomes of biopesticide production strains or highly toxic strains are currently available.. Furthermore, there was a lack of systematic explanations on the effective insecticidal toxicity phenotype of these B. thuringiensis strains at the genomic, transcriptomic, or other omics levels.In this work, based on the complete genomes of the highly toxic B. thuringiensis strains, we performed the comparative genomic and transcriptomic analysis of B. cereus sensu lato group strains to discuss the pathogenic mechanism.1. Based on the high-throughput sequencing technologies, we established a platform for genome assembly and bioinformatics analysis. Combined with the optics mapping, BAC library strategy and so on we obtained the whole genome sequence of B. thuringiensis globally insecticide standard strain HD-1. Significantly, the BAC library strategy played a very important role for plasmids assembly. Then using HD-1 genome as a reference, we quickly finished the genome sequencing of the "MianFeng" biopesticide production strain YBT-1520 and "Entobacterin" biopesticide similar strain HD-29, which are used in China.2. We predicted and summarized the 24 insecticidal toxicity-related gene families and clusters (ITRGs) of B. thuringiensis, which were represented by Cry protein genes. And they are divided into three groups:the insecticidal and other virulence synergic genes, including the Cry and Vip protein genes; the pathogenic and virulence assistant genes, including novel pore-forming toxin, hemolysin, non-hemolysin enterotoxin, phospholipases and immune inhibitor; and the genes for saprophytic colonization, which includes the bacteriocins, enhancin proteins and various degradation enzymes. In addition, we also found that compared to the complete genome data, using the daft genome data of strians HD-1 and YBT-1520, we only obtained 88.6% and 90.3% of the total ITRGs genes, repectively. It indicated that the complete genome data is indispensable for functional gene resource mining and other researches.3. Highly toxic B. thuringiensis strains have more ITRGs. The comparative genomic analysis of the 318 B. cereus group strains showed that the B. thuringiensis strains with high insecticidal toxicity had significantly larger genome sizes and were richer in plasmid content than the other ones. And we also found that highly toxic strains have 20.0%-87.5% more ITRGs and a 22.2% higher variety than other B. cereus group bacteria.4. Highly toxic strains have two nitrogen utilization systems. We found that besides the nitrate-nitrite reduction related genes conserved in the B.cereus group strains, the highly toxic strains encode an extra system, a nine-gene urease synthesis cluster, for nitrogen source utilization. It could help Btk to survive in the complex host environment.5. A microarray strategy was used to identify the transcription modulation of the ITRGs during the different growth phases of Btk YBT-1520 under culture. It showed that almost the ITRGs in YBT-1520 were transcribed, including the urease synthesis cluster, indicating that the plentiful ITRGs in highly toxic Bt strains are active. Significantly, the comparative transcriptomic analysis and the qRT-PCR confirmatory experiment both indicated that the ITRGs and related regulator genes of highly toxic B. thuringiensis strains have 10-20 times higher transcriptional level than those in other strains. The transcriptional differences of some genes can even reach hundredfolds. It also suggested that in the Bc group and other bacteria population, the expression of the conserved virulence related genes directly related to their different phenotypes.6. We also investigated the impact of the plasmids in the insecticidal phenotype of B. thuringiensis. After exploring the plasmid patterns of all the serotype standard B. thuringiensis strains, based on the serotype and related insecticidal activity, we extracted and mixed over 76 plasmids DNA from 21 standard serotype B. thuringiensis strains for high-throughput sequencing. Then we obtained 143 novel insecticidal protein genes. It showed that it is practicable for novel insecticidal or other functional protein genes mining, and proved that the plasmids provide abundant gene resources and armed the highly toxic strains.Based on these results, we revealed the core essence for the highly insecticidal phenotype of the Bt pesticide production strains:The highly toxic strains contained significantly more ITRGs, both on the species and the quantity. Thereby they can provide additional strategies for infection, immune evasion, and cadaver utilization.And the high expression of the ITRGs and related regulator genes is a key factor in efficient entomopathogenicity. This study provides novel insights into the strategies used by B. thuringiensis for interaction with insect hosts and maximized utilization of high-throughput sequencing; it also helps further identify novel toxicity factors. |
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