Complete genome sequence of Bacillus thuringiensis serovar galleriae strain HD-29, a typical strain of commercial biopesticide.

Bacillus thuringiensis serovar galleriae is highly toxic to Lepidoptera insect pests, and has been widely used as Bt biopesticide in many countries. Here we reported the complete genome of strain HD-29, a standard serotype strain in galleriae serovariety. More than previous work reported, it harbors ten plasmids, and three large ones carry eight insecticidal protein genes (cry1Aa, cry1Ac, cry1Ca, cry1Da, cry1Ia, cry2Ab, cry9Ea and vip3Aa) and an intact zwittermicin A biosynthetic gene cluster.

ApnI, a transmembrane protein responsible for subtilomycin immunity, unveils a novel model for lantibiotic immunity.

Subtilomycin was detected from the plant endophytic strain Bacillus subtilis BSn5 and was first reported from B. subtilis strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes apnI and apnT downstream of apnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of the apnI and/or apnT genes in the subtilomycin-sensitive strain CU1065 and inactivation of apnI and apnT in the producer strain BSn5, we showed that the single gene apnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in Bacillus and Paenibacillus. We cloned the paeI gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant B. subtilis expressing apnI interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.

Validation of the intact zwittermicin A biosynthetic gene cluster and discovery of a complementary resistance mechanism in Bacillus thuringiensis.

Zwittermicin A (ZmA) is a hybrid polyketide-nonribosomal peptide produced by certain Bacillus cereus group strains. It displays broad-spectrum antimicrobial activity. Its biosynthetic pathway in B. cereus has been proposed through analysis of the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules involved in ZmA biosynthesis. In this study, we constructed a bacterial artificial chromosome (BAC) library from Bacillus thuringiensis subsp. kurstaki strain YBT-1520 genomic DNA. The presence of known genes involved in the biosynthesis of ZmA in this BAC library was investigated by PCR techniques. Nine positive clones were identified, two of which (covering an approximately 60-kb region) could confer ZmA biosynthesis ability upon B. thuringiensis BMB171 after simultaneous transfer into this host by two compatible shuttle BAC vectors. Another previously unidentified gene cluster, named zmaWXY, was found to improve the yield of ZmA and was experimentally defined to function as a ZmA resistance transporter which expels ZmA from the cells. Putative transposase genes were detected on the flanking regions of the two gene clusters (the ZmA synthetic cluster and zmaWXY), which suggests a mobile nature of these two gene clusters. The intact ZmA gene cluster was validated, and a resistance mechanism complementary to that for zmaR (the previously identified ZmA self-resistance gene) was revealed. This study also provided a straightforward strategy to isolate and identify a huge gene cluster from Bacillus.

Protein elicitor PemG1 from Magnaporthe grisea induces systemic acquired resistance (SAR) in plants.

Elicitors can stimulate defense responses in plants and have become a popular strategy in plant disease control. Previously, we isolated a novel protein elicitor, PemG1, from Magnaporthe grisea. In the present study, PemG1 protein expressed in and purified from Escherichia coli improved resistance of rice and Arabidopsis to bacterial infection, induced transient expression of pathogenesis-related (PR) genes, and increased accumulation of hydrogen peroxide in rice. The effects of PemG1 on disease resistance and PR gene expression were mobilized systemically throughout the rice plant and persisted for more than 28 days. PemG1-induced accumulation of OsPR-1a in rice was prevented by the calcium channel blockers LaCl3, BAPTA, EGTA, W7, and TFP. Arabidopsis mutants that are insensitive to jasmonic acid (JA) and ethylene showed increased resistance to bacterial infection after PemG1 treatment but PemG1 did not affect resistance of mutants with an impaired salicylic acid (SA) transduction pathway. In rice, PemG1 induced overexpressions of the SA signal-related genes (OsEDS1, OsPAL1, and OsNH1) but not the JA pathway-related genes (OsLOX2 and OsAOS2). Our findings reveal that PemG1 protein can function as an activator of plant disease resistance, and the PemG1-mediated systemic acquired resistance is modulated by SA- and Ca(2+)-related signaling pathways.

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 GenBank(TM) 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).

[Cloning and expression of tyrosinase-encoding gene (mel) of Bacillus thuringiensis and its initial research of application].

Tyrosinase, which is encoded by Tyrosinase gene (mel), is the key enzyme in the process of melanin formation in animals, plants and microorganisms. Using the primers designed by comparing the conserved domain of tyrosinase, a DNA fragment was amplified which contain the mel gene from Bacillus thuringiensis 4D11. The recombinant E. coli EMB1179 was gained by subcloning this DNA fragment onto the vector pGEM-7zf and transformed it into E. coli DH5alpha. EMB1179 express the tyrosinase activity and produce melanin under the presence of L-tyrosin. The effect of melanin on survival of E. coli was also determined. The results showed that the melanin produced by EMB1179 effectively increased its resistance against UV light.

Determination of the fungicide validamycin A by capillary zone electrophoresis with indirect UV detection.

Validamycin A, a main component of the antibiotic validamycin complex, which is widely used to control sheath blight disease of rice plants, can be determined by capillary zone electrophoresis with indirect UV detection. The influence of various separation conditions including background electrolyte and modifier concentration, pH, and voltage was investigated. By using 10 mM aminopyrine-2 mM ethylenediaminetetraacetic acid at pH 5.2 as the carrier electrolyte, high efficiency separation of validamycin A was achieved with the number of theoretical plates up to 350000 plates/m. The linear range was across 3 orders of magnitude. The relative standard deviations for migration times and peak areas were less than 0.5 and 3.0%, respectively. The limit of detection for validamycin A was 1.0 microg/mL. The average recoveries ranged from 103.0 to 104.8%. This method has many advantages as compared with high-performance liquid chromatography and micellar electrokinetic capillary chromatography in the determination of commercial formulations.