BtToxin_Digger: a comprehensive and high-throughput pipeline for mining toxin protein genes from Bacillus thuringiensis.

SUMMARY: Bacillus thuringiensis (Bt) has been used as the most successful microbial pesticide for decades. Its toxin genes are used for the development of genetically modified crops against pests. We previously developed a web-based insecticidal gene mining tool BtToxin_scanner. It has been frequently used by many researchers worldwide. However, it can only handle the genome one by one online. To facilitate efficiently mining toxin genes from large-scale sequence data, we re-designed this tool with a new workflow and the novel bacterial pesticidal protein database. Here, we present BtToxin_Digger, a comprehensive and high-throughput Bt toxin mining tool. It can be used to predict Bt toxin genes from thousands of raw genome and metagenome data, and provides accurate results for downstream analysis and experiment testing. Moreover, it can also be used to mine other targeting genes from large-scale genome and metagenome data with the replacement of the database. AVAILABILITY AND IMPLEMENTATION: The BtToxin_Digger codes and web services are freely available at https://github.com/BMBGenomics/BtToxin_Digger and https://bcam.hzau.edu.cn/BtToxin_Digger, respectively. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparative genomic analysis of multidrug-resistant Streptococcus pneumoniae isolates.

INTRODUCTION: Multidrug resistance in Streptococcus pneumoniae has emerged as a serious problem to public health. A further understanding of the genetic diversity in antibiotic-resistant S. pneumoniae isolates is needed. METHODS: We conducted whole-genome resequencing for 25 pneumococcal strains isolated from children with different antimicrobial resistance profiles. Comparative analysis focus on detection of single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) was conducted. Moreover, phylogenetic analysis was applied to investigate the genetic relationship among these strains. RESULTS: The genome size of the isolates was ~2.1 Mbp, covering >90% of the total estimated size of the reference genome. The overall G+C% content was ~39.5%, and there were 2,200-2,400 open reading frames. All isolates with different drug resistance profiles harbored many indels (range 131-171) and SNPs (range 16,103-28,128). Genetic diversity analysis showed that the variation of different genes were associated with specific antibiotic resistance. Known antibiotic resistance genes (pbps, murMN, ciaH, rplD, sulA, and dpr) were identified, and new genes (regR, argH, trkH, and PTS-EII) closely related with antibiotic resistance were found, although these genes were primarily annotated with functions in virulence as well as carbohydrate and amino acid transport and metabolism. Phylogenetic analysis unambiguously indicated that isolates with different antibiotic resistance profiles harbored similar genetic backgrounds. One isolate, 14-LC.ER1025, showed a much weaker phylogenetic relationship with the other isolates, possibly caused by genomic variation. CONCLUSION: In this study, although pneumococcal isolates had similar genetic backgrounds, strains were diverse at the genomic level. These strains exhibited distinct variations in their indel and SNP compositions associated with drug resistance.

The complete mitochondrial genome of Hericium coralloides (Hericiaceae, Basidiomycota).

In this study, the complete mitochondrial genome of the medicinal mushroom Hericium coralloides (Hericiaceae, Basidiomycota) was sequenced. This mitochondrial genome is 72,961 bp in length and consisted of 14 protein-coding genes, 21 hypothetical open reading frames, 2 ribosomal RNA subunits and 27 transfer RNAs. The overall nucleotide composition of is 41.33% A, 40.71% T, 9.06% C and 8.90% G, with GC content of 17.96%. A phylogenetic tree with the complete mitochondrial genome sequences of Hericium coralloides together with 9 other affinis mushrooms was constructed. The newly achieved mitochondrial genome sequence seem to be useful for addressing taxonomic issues and studying related evolution events, which would contribute to enrich the fungal mitochondrial genome resource and promote the biological research.

Genome and metagenome analyses reveal adaptive evolution of the host and interaction with the gut microbiota in the goose.

The goose is an economically important waterfowl that exhibits unique characteristics and abilities, such as liver fat deposition and fibre digestion. Here, we report de novo whole-genome assemblies for the goose and swan goose and describe the evolutionary relationships among 7 bird species, including domestic and wild geese, which diverged approximately 3.4~6.3 million years ago (Mya). In contrast to chickens as a proximal species, the expanded and rapidly evolving genes found in the goose genome are mainly involved in metabolism, including energy, amino acid and carbohydrate metabolism. Further integrated analysis of the host genome and gut metagenome indicated that the most widely shared functional enrichment of genes occurs for functions such as glycolysis/gluconeogenesis, starch and sucrose metabolism, propanoate metabolism and the citrate cycle. We speculate that the unique physiological abilities of geese benefit from the adaptive evolution of the host genome and symbiotic interactions with gut microbes.

Donkey genome and insight into the imprinting of fast karyotype evolution.

The donkey, like the horse, is a promising model for exploring karyotypic instability. We report the de novo whole-genome assemblies of the donkey and the Asiatic wild ass. Our results reflect the distinct characteristics of donkeys, including more effective energy metabolism and better immunity than horses. The donkey shows a steady demographic trajectory. We detected abundant satellite sequences in some inactive centromere regions but not in neocentromere regions, while ribosomal RNAs frequently emerged in neocentromere regions but not in the obsolete centromere regions. Expanded miRNA families and five newly discovered miRNA target genes involved in meiosis may be associated with fast karyotype evolution. APC/C, controlling sister chromatid segregation, cytokinesis, and the establishment of the G1 cell cycle phase were identified by analysis of miRNA targets and rapidly evolving genes.

Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis.

Bacillus thuringiensis has been globally used as a microbial pesticide for over 70 years. However, information regarding its various adaptions and virulence factors and their roles in the entomopathogenic process remains limited. In this work, we present the complete genomes of two industrially patented Bacillus thuringiensis strains (HD-1 and YBT-1520). A comparative genomic analysis showed a larger and more complicated genome constitution that included novel insecticidal toxicity-related genes (ITRGs). All of the putative ITRGs were summarized according to the steps of infection. A comparative genomic analysis showed that highly toxic strains contained significantly more ITRGs, thereby providing additional strategies for infection, immune evasion, and cadaver utilization. Furthermore, a comparative transcriptomic analysis suggested that a high expression of these ITRGs was a key factor in efficient entomopathogenicity. We identified an active extra urease synthesis system in the highly toxic strains that may aid B. thuringiensis survival in insects (similar to previous results with well-known pathogens). Taken together, these results explain the efficient entomopathogenicity of B. thuringiensis. It provides novel insights into the strategies used by B. thuringiensis to resist and overcome host immune defenses and helps identify novel toxicity factors.

High-quality draft genome sequence of nematocidal Bacillus thuringiensis Sbt003.

Bacillus thuringiensis represents one of the six species of "Bacillus cereus group" in the genus Bacillus within the family Bacillaceae. Strain Sbt003 was isolated from soil and identified as B. thuringiensis. It harbors at least seven plasmids and produces three shapes of parasporal crystals including oval, bipyramidal and rice. SDS-PAGE analysis of spore-crystal suspension of this strain reveals six major protein bands, which implies the presence of multiple parasporal crystal genes. Bioassay of this strain reveals that it shows specific activity against nematodes and human cancer cells. In this study, we report the whole genomic shotgun sequences of Sbt003. The high-quality draft of the genome is 6,175,670 bp long (including chromosome and plasmids) with 6,372 protein-coding and 80 RNA genes.

Analysis of horse genomes provides insight into the diversification and adaptive evolution of karyotype.

Karyotypic diversification is more prominent in Equus species than in other mammals. Here, using next generation sequencing technology, we generated and de novo assembled quality genomes sequences for a male wild horse (Przewalski's horse) and a male domestic horse (Mongolian horse), with about 93-fold and 91-fold coverage, respectively. Portion of Y chromosome from wild horse assemblies (3 M bp) and Mongolian horse (2 M bp) were also sequenced and de novo assembled. We confirmed a Robertsonian translocation event through the wild horse's chromosomes 23 and 24, which contained sequences that were highly homologous with those on the domestic horse's chromosome 5. The four main types of rearrangement, insertion of unknown origin, inserted duplication, inversion, and relocation, are not evenly distributed on all the chromosomes, and some chromosomes, such as the X chromosome, contain more rearrangements than others, and the number of inversions is far less than the number of insertions and relocations in the horse genome. Furthermore, we discovered the percentages of LINE_L1 and LTR_ERV1 are significantly increased in rearrangement regions. The analysis results of the two representative Equus species genomes improved our knowledge of Equus chromosome rearrangement and karyotype evolution.

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline.

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.

Complete genome sequence of Bacillus thuringiensis serovar finitimus strain YBT-020.

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.

Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs) of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb) in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp) was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp) at the mid-exponential growth stage (OD(600) = 2.0) of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp) and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively). These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation.

The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding to the brush border membrane microvilli prepared from H. armigera. When the residues (1423)GVLSLNFQ(1430) were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1, which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops.

Genetic characterization of two putative toxin-antitoxin systems on cryptic plasmids from Bacillus thuringiensis strain YBT-1520.

A novel putative toxin-antitoxin segregational stability system named KyAB system was identified in a novel native plasmid pBMB8240 from Bacillus thuringiensis strain YBT-1520, based on sequences homology with other toxin-antitoxin systems, the lethal activity of the KyB putative toxin in Escherichia coli and the stabilizing effect of the kyAB system in Bacillus thuringiensis. Secondarily, the native plasmid pBMB9741 from the same strain was resequenced and the corrected plasmid was named as pBMB7635. Based on sequence homology with the tasAB system and the lethal activity of toxin protein in Escherichia coli, a tasAB-like putative toxin-antitoxin system was identified on pBMB7635.

A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis.

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.