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.

A strain of marine bacterium Bacillus firmus YBf-10 with nematicidal activity was originally isolated by our group. In the present study, the systemic nematicidal activity and biocontrol efficacy in pot experiment of B. firmus YBf-10 were investigated. Our results showed that YBf-10 exhibits systemic nematicidal activity against Meloidogyne incognita, including lethal activity, inhibition of egg hatch and motility. Pot experiment suggested that soil drenching with YBf-10 efficiently reduced damage of M. incognita to tomato plants, such as reduction of galls, egg masses on roots, and final nematode population in soil; and moreover, YBf-10 significantly promoted host plant growth. In addition, our results also indicated that the systemic nematicidal activity is likely attributed to the secondary metabolites produced by YBf-10. The obtained results of the current study confirmed that B. firmus YBf-10 is a promising nematicidal agent, and has great potential in plant-parasitic nematicidal management.

BackgroundSaccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins.ResultsTwo-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis.ConclusionsThis study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance the production yield of spinosad in future studies.

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Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla . Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla . To monitor the entry pathway of crystal proteins into M. hapla , second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1–84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l−1.

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-P-pnp via overlap extension PCR. The vector pOJ260-P-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement.

Eight new α-pyrones 1-8 and three known α-pyrones 9-11 were isolated from three marine-derived Nocardiopsis strains SCSIO 10419, SCSIO 04583, and SCSIO KS107. The structures of compounds 1-8 were elucidated by comprehensive spectral analyses. The absolute configurations of 4-deoxyphomapyrone C (1), 4-deoxy-11-hydroxyphomapyrone C (3), 4-deoxy-7R-hydroxyphomapyrone C (5), and phomapyrone C (11) were determined by TDDFT-ECD calculations for the solution conformers, which revealed that the conformation of the side chain was decisive for the sign of the characteristic high-wavelength ECD transition. (-)-4-Deoxy-8-hydroxyphomapyrone C (4) was isolated from SCSIO 10419 and was deduced as a diastereomeric mixture containing (8S)- and (8R)-4-deoxy-8-hydroxyphomapyrone C in a ratio of 2.6:1 (8R:8S), by chiral-phase HPLC analysis and Mosher's ester analysis. Interestingly, 7-hydroxymucidone (9) was isolated from both SCSIO 04583 and SCSIO KS107, as an enantiomeric mixture containing (7S)-hydroxymucidone (major in 9 from SCSIO 04583) and (7R)-hydroxymucidone (major in 9 from SCSIO KS107). α-Pyrones 3-5 were identified as three isomers of phomapyrone C (11) with diverse hydroxy substitutions. α-Pyrones 10-hydroxymucidone (6), 4-hydroxymucidone (8), and 9, differed in the position of the hydroxy group. Several α-pyrones exhibited moderate growth inhibitory activity against Micrococcus luteus and Bacillus subtilis.