Donghai Peng

Complete Genome Sequence of Bacillus subtilis BSn5, an Endophytic Bacterium of Amorphophallus konjac with Antimicrobial Activity for the Plant Pathogen Erwinia carotovora subsp. carotovora

Here, we present the complete genome sequence of Bacillus subtilis strain BSn5, isolated from Amorphophallus konjac calli tissue and showing strong inhibitory activity to Erwinia carotovora subsp. carotovora, which causes Amorphophallus soft rot disease and affects the industry development of this organism.

New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

ABSTRACT We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis.

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis.

Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline

ABSTRACT 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 laboratorys 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.

Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua

Aims:  The objective of this work was to enhance the insecticidal activity or widen the pesticidal spectrum of a commercial Bacillus thuringiensis strain YBT1520.

Bacillus thuringiensis Bel Protein Enhances the Toxicity of Cry1Ac Protein to Helicoverpa armigera Larvae by Degrading Insect Intestinal Mucin

ABSTRACT Bacillus thuringiensis has been used as a bioinsecticide to control agricultural insects. Bacillus cereus group genomes were found to have a Bacillus enhancin-like (bel) gene, encoding a peptide with 20 to 30% identity to viral enhancin protein, which can enhance viral infection by degradation of the peritrophic matrix (PM) of the insect midgut. In this study, the bel gene was found to have an activity similar to that of the viral enhancin gene. A bel knockout mutant was constructed by using a plasmid-free B. thuringiensis derivative, BMB171. The 50% lethal concentrations of this mutant plus the cry1Ac insecticidal protein gene were about 5.8-fold higher than those of the BMB171 strain. When purified Bel was mixed with the Cry1Ac protein and fed to Helicoverpa armigera larvae, 3 μg/ml Cry1Ac alone induced 34.2% mortality. Meanwhile, the mortality rate rose to 74.4% when the same amount of Cry1Ac was mixed with 0.8 μg/ml of Bel. Microscopic observation showed a significant disruption detected on the midgut PM of H. armigera larvae after they were fed Bel. In vitro degradation assays showed that Bel digested the intestinal mucin (IIM) of Trichoplusia ni and H. armigera larvae to various degrading products, similar to findings for viral enhancin. These results imply Bel toxicity enhancement depends on the destruction of midgut PM and IIM, similar to the case with viral enhancin. This discovery showed that Bel has the potential to enhance insecticidal activity of B. thuringiensis-based biopesticides and transgenic crops.

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 1423GVLSLNFQ1430 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.

Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis

Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli–Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBankTM and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).

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 (OD600 = 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.

Are nematodes a missing link in the confounded ecology of the entomopathogen Bacillus thuringiensis

Bacillus thuringiensis, which is well known as an entomopathogen, has been accepted by the public as a safe bioinsecticide. The natural ecology of this bacterium has never been particularly clear, with views ranging from it being an obligate pathogen to an opportunist pathogen that can otherwise exist as a soil saprophyte or a plant endophyte. This confusion has recently led to it being considered as an environmental pathogen that has evolved to occupy a diverse set of environmental niches in which it can thrive without needing a host. A significant driving force behind this classification is the fact that B. thuringiensis is found in high numbers in environments that are not occupied by the insect hosts to which it is pathogenic. It is our opinion that the ubiquitous presence of this bacterium in the environment is the result of a variety of vectoring systems, particularly those that include nematodes.