Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis. Methods and Results:  The effect of DNA desalting, wall‐weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis. By using this improved method, the greatest efficiency was reached 2 × 1010 CFU μg−1 with pHT304, which is 104 times higher than previously reported. Four large plasmids (29·1, 44·9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully. Conclusions:  This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures. Significance and Impact of the Study:  The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis. This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis. It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.

Plant-parasitic nematodes were found in 4 of the 12 clades of phylum Nematoda. These nematodes in different clades may have originated independently from their free-living fungivorous ancestors. However, the exact evolutionary process of these parasites is unclear. Here, we sequenced the genome sequence of a migratory plant nematode, Ditylenchus destructor. We performed comparative genomics among the free-living nematode, Caenorhabditis elegans and all the plant nematodes with genome sequences available. We found that, compared with C. elegans, the core developmental control processes underwent heavy reduction, though most signal transduction pathways were conserved. We also found D. destructor contained more homologies of the key genes in the above processes than the other plant nematodes. We suggest that Ditylenchus spp. may be an intermediate evolutionary history stage from free-living nematodes that feed on fungi to obligate plantparasitic nematodes. Based on the facts that D. destructor can feed on fungi and has a relatively short life cycle, and that it has similar features to both C. elegans and sedentary plant-parasitic nematodes from clade 12, we propose it as a new model to study the biology, biocontrol of plant nematodes and the interaction between nematodes and plants.

Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs. P lant-parasitic nematodes (PPNs) are an important agricultural pest of many crops, and they cause agricultural losses amounting to an estimated $157 billion worldwide every year (1, 2). Root knot nematodes (RKNs; Meloidogyne spp.) are the most damaging crop PPNs, and they are capable of infecting almost all crops (2, 3). The management of RKNs is more difficult than that of other pests because RKNs are obligate root parasites of thousands of plant species, and most of the life cycle of RKNs is spent as parasites in the host roots (2, 4). The current means of controlling RKNs relies on chemical nematicides, which are often toxic and harmful to humans and the environment (3). In addition, chemical nematicides are generally expensive and cannot provide longterm RKN suppression (5).Biological control of RKNs is a better management strategy that avoids the potential environmental problems associated with the chemical control of RKNs. Indeed, the utilization of beneficial bacterial nematicides has been proposed as a sustainable alternative for RKN management, and bacterial nematicides are also regarded as economical and ecologically friendly nematicides for the control of RKNs (3, 6). Currently, for the bacterial control of RKNs, many problems remain, one of which is that nematicidal bacteria are relatively scarce. Only a few bacteria have been reported to possess nematicidal activity and to show potential for the biological control of RKNs. The typical nematicidal bacteria are Bacillus, Pseudomonas, and Pasteuria species (4, 7). Identifying novel nematicidal bacteria has become urgent for the bacterial control of RKNs and is of vital practical and economic significance.However, until now, it has been a challenge to isolate novel nematici...

Nattokinase is a serine protease in the subtilisin family which is produced by Bacillus subtilis subsp. natto and exhibits vigorous fibrinolytic activity that has been suggested to be able to prevent and treat thromboembolic diseases. In this study, WTC016, a spore-forming and rod-shaped bacterium with fibrinolytic activity was successfully isolated from soil, which was identified as Bacillus subtilis subsp. natto based on morphological and physiological tests, and phylogenetic analysis of 16S rRNA and gyrA. According to the growth curve of WTC016, the nattokinase production reached the highest amount in the stationary phase. To optimize the liquid fermentation condition for nattokinase yield of WTC016, further optimal tests of four factors, including the temperature, pH, inoculum size, and loading volume, followed by orthogonal test of all these factors, was performed. The optimal fermentation conditions were determined as 30 °C, 7.0 pH, 2% inoculum size, and 60 mL of loading volume in 250 mL conical flask, which indicates the highest nattokinase production of 3284 ± 58 IU/mL while fermented for 26 h. This work laid the foundation for producing nattokinase using Bacillus subtilis subsp. natto WTC016.