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Probiotics are alternatives to antibiotics that resist infection and benefit the host in several aspects. Here, we isolated and identified a novel strain of Bacillus methylotrophicus (XA-8) with a broad spectrum of activity against Aeromonas hydrophila AhX040 and other aquatic bacterial pathogens. The strain was non-pathogenic as it revealed no cytotoxic effects in hepatic L8824 cells or pathophysiological signs in the tissue of Ctenopharyngodon idella (grass carp). Using a co-culture approach, XA-8 (1×109 CFU mL−1) when co-cultivated with AhX040 (1×108 CFU mL−1) revealed a reduction in the cell density, cytotoxicity, and infection rate of AhX040. More interestingly, genes such as aer, act, hylA, lafA, hcp, and luxS, associated with the virulence of AhX040, were significantly down-regulated during co-cultivation (P

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

The emerging outbreak of bacterial diseases poses a major threat to the aquaculture industry, and the abuse of antibiotics leads to the emergence of drug-resistant pathogens; hence, the development of antibacterial agents from natural resources to control and manage fish bacterial diseases has become a promising strategy. In this study, seven potential Streptomyces spp. with antimicrobial activity against fish pathogenic bacteria were isolated from the soils of different regions in China. Streptomyces termitum N-15, which showed the strongest and most stable antibacterial activity, was selected for the fish disease control experiment. Then, we investigated the effect of a basal diet and N-15 supplementation (108 ​CFU/mg diet) on growth performance, blood immune parameters (ACP and AKP), and the expression of immune genes. The results demonstrated that the ACP and AKP activities of serum were significantly increased in the N-15-supplemented group. Moreover, N-15 upregulated the expression level of Toll-like receptor 4 (TLR-4) in the kidney, liver, spleen and head kidney. The challenge test showed that supplementation with N-15 could significantly improve the survival rates of grass carp infected with A. veronii. Chalcomycin A was purified from Streptomyces termitum N-15 and found to have antimicrobial activity against fish pathogenic bacteria for the first time. Furthermore, the complete genome of S. termitum N-15 was found to consist of an 8,887,222 bp circular chromosome with 72.89% GC content and a plasmid genome, including 7744 protein-coding sequences (CDSs). The genome of S. termitum N-15 contained gene clusters for the synthesis of multiple secondary metabolites, including melingomycin, sulforaphane, oromycin, chlortetracycline, isomycin, and primycin, by antiSMASH online software. Gene cluster 1.5 revealed a chalcomycin A biosynthesis gene cluster (chmGⅠ, chmGⅡ, chmGⅢ, chmGⅣ, chmGⅤ). In summary, S. termitum N-15 and its secondary metabolites are expected to be developed into inoculants for promoting fish health in aquacultural systems.

Abstract Background Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involve in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently understanding its regulatory mechanism, and improving its production by metabolic engineering. Results Here, we identified a TetR family transcriptional regulator, SP_2854, that can positively regulate butenyl-spinosyn biosynthesis and affect strain growth, glucose consumption, and mycelial morphology in S. pogona. Using targeted metabolomic analyses, we found that SP_2854 overexpression enhanced glucose metabolism, while SP_2854 deletion had the opposite effect. To decipher the overproduction mechanism in detail, comparative proteomic analysis was carried out in the SP-2854 overexpressing mutant and the original strain, and we found that SP_2854 overexpression promoted the expression of proteins involved in glucose metabolism. Conclusion Our findings suggest that SP_2854 can affect strain growth and development and butenyl-spinosyn biosynthesis in S. pogona by controlling glucose metabolism. The strategy reported here will be valuable in paving the way for genetic engineering of regulatory elements in actinomycetes to improve important natural products production.

Summary Butenyl‐spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild‐type S. pogona strain is too low to meet the industrial production requirements. Deletion of non‐target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl‐spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS‐T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona‐Δura4‐Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl‐spinosyn in S. pogona‐Δura4‐Δc14 was increased by 4.72‐fold compared with that in the wild‐type strain. qRT‐PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl‐spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl‐spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl‐spinosyn by deleting other secondary metabolite synthetic gene clusters.

Abstract Background Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. Results Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. Conclusion Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Abstract Background Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. Results The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. Conclusions This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.

Butenyl-spinosyn, a highly effective biological insecticide, is produced by Saccharopolyspora pogona. However, its application has been severely hampered by its low yield. Recent studies have shown that PhoU plays a pivotal role in regulating cell growth, secondary metabolite biosynthesis and intracellular phosphate levels. Nevertheless, the function of PhoU remains ambiguous in S. pogona. In this study, we investigated the effects of PhoU on the growth and the butenyl-spinosyn biosynthesis of S. pogona by constructing the mutants. Overexpression of phoU increased the production of butenyl-spinosyn to 2.2-fold that of the wild-type strain. However, the phoU deletion resulted in a severe imbalance of intracellular phosphate levels, and suppression of the growth and butenyl-spinosyn biosynthesis. Quantitative Real-time PCR (qRT-PCR) analysis, distinctive protein detection and mass spectrometry revealed that PhoU widely regulated primary metabolism, energy metabolism and DNA repair, which implied that PhoU influences the growth and butenyl-spinosyn biosynthesis of S. pogona as a global regulator.

Abstract Background A new Bacillus thuringiensis X023 (BtX023) with high insecticidal activity was isolated in Hunan Province, China. The addition of metals (Cu, Fe, Mg and Mn) to the medium could influence the formation of spores and/or insecticidal crystal proteins (ICPs). In previous studies, Cu ions considerably increased the synthesis of ICPs by enhancing the synthesis of poly-β-hydroxy butyrate. However, the present study could provide new insights into the function of Cu ions in ICPs. Results Bioassay results showed that wild strain BtX023 exhibited high insecticidal activity against Plutella xylostella. The addition of 1 × 10−5 M Cu2+ could considerably increase the expression of cry1Ac and vip3Aa, and the insecticidal activity was enhanced. Quantitative real-time polymerase chain reaction (qRT-PCR) and proteomic analyses revealed that the upregulated proteins included amino acid synthesis, the glyoxylate pathway, oxidative phosphorylation, and poly-β-hydroxy butyrate synthesis. The Cu ions enhanced energy metabolism and primary amino acid synthesis, will providing abundant raw material accumulation for ICP synthesis. Conclusion The new strain BtX023 exerted a strong insecticidal effect on P. xylostella by producing ICPs. The addition of 1 × 10−5 M Cu2+ in the medium could considerably enhance the expression of the cry 1Ac and vip 3Aa genes, thereby further increasing the toxicity of BtX023 to Helicoverpa armigera and P. xylostella by enhancing energy synthesis, the glyoxylate cycle, and branched-chain amino acids synthesis, but not poly-β-hydroxy butyrate synthesis.

Abstract Background Filamentous fungi have long been used as hosts for the production of proteins, enzymes and valuable products in various biotechnological applications. However, recombinant proteins are expressed with highly secreted host proteins when stronger promoters are used under inducing conditions. In addition, the efficiency of target protein expression can be limited by the application of constitutive promoters in recently developed filamentous fungal expression systems. Results In this study, a novel expression system was constructed by using a Penicillium oxalium strain that has powerful protein secretion capability. The secretory background of the host was reduced by knocking out the Amy13A protein and utilizing the starch as a carbon source. The strong promoter amy15A(p) was further improved by overexpressing the transcription activator AmyR and deleting of putative repressor CreA. By using the native amylase Amy15A as a reporter, the efficiency of expression from the amy15A promoter was dramatically and specifically enhanced after redesigning the regulatory network of amylase expression. Conclusions Our researches clearly indicated that the triple-gene recombinant strain Δ13A-OamyR-ΔCreA, with the amy15A(p) promoter could be used as a suitable expression system especially for high-level and high-purity protein production.

Abstract Background Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. Results In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. Conclusion This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.

Beneficial microorganisms to control bacterial diseases has been widely used in aquaculture, Bacillus amyloliquefaciens (BaX030) as a probiotic feed additive was a commonly biological control method. Added sucrose promoted the growth of BaX030, and the yield of its antibacterial substance macrolactin A was enhanced by 1.46-fold. A total of 2055 proteins were screened through proteomics, with 143 upregulated and 307 downregulated. Differential protein expression analysis and qRT-PCR verification showed that the pentose phosphate pathway and the fatty acid synthesis pathway were upregulated, thereby providing sufficient energy and precursors for the synthesis of macrolactin A. The influence of some potential regulatory factors (SecG, LiaI, MecG and ComG) on macrolactin A was discovered. After grass carp were fed with BaX030, the abundance of probiotics (Fusobacterium, Proteobacteria, Gemmobacter) were higher than the control group, and the abundance of potential pathogenic bacteria (Planctomycetes, Aeromonas) were significantly lower than the control group. The cell and challenge experiments showed that BaX030 can significantly increase the expression of C3 and IL8 in the liver and kidney, which decreases the risk of immune organ disease. Moreover, BaX030 effectively reduced the mortality of grass carp. The results revealed that BaX030 can significantly improve the structure of the intestinal flora, enhance immunity and it is beneficial to the control of grass carp Aeromonas.

Abstract Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions. In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase LuxCDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins.

Summary: The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTEPsy) from Pseudomonas syringae pv. syringae B728a. Compared with RecTEPsy, the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas. Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P. aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P. syringae. Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E. coli. The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas. : Biological Sciences; Molecular Biology; Bioengineering; Biotechnology; Cell Biology Subject Areas: Biological Sciences, Molecular Biology, Bioengineering, Biotechnology, Cell Biology

Summary: Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

The presence of healthy gut microbiota in the gastrointestinal tract of fish is important for the optimal function of gut, which plays a significant role in the host growth and health. The aim of the present study was to investigate the effect of dietary stress worry free (SWF®) on growth, feed utilization and disease resistance of hybrid sturgeon (Acipenser baerii x Acipenser schrenckii). Sturgeon were fed for three weeks with SWF® supplemented or basal diet. The weight gain and FCR of sturgeon fed on the diet supplemented with SWF® were significantly improved (P

One of the common shortcomings with Bacillus thuringiensis (Bt) biopesticides in field application is their instability under UV irradiation. In Bt, the leuB gene encodes the 3-isopropylmalate dehydrogenase. In addition to its role in leucine biosynthesis, LeuB would be likely recruited to catalyze the dehydrogenation of malate in the final step of tricarboxylic acid cycle during sporulation. In this study, we constructed a Bt recombinant strain in which the gene leuB was deleted by using the markerless gene deletion system. The ΔleuB mutant strain showed a conditionally asporogenous phenotype while overproducing insecticidal crystal proteins and retaining its insecticidal activity well in both fermentation and LB media. Furthermore, the metabolic regulation mechanisms of LeuB was elucidated by iTRAQ-based quantitative proteomics approach. Evidences from proteomics data suggested that the inhibited supply of pyruvate (carbon source) was an important factor related to the conditionally asporogenous feature of the mutant. Consistently, the mutant regained its ability to sporulate in LB medium by adding 1% glucose or 1% sodium pyruvate. Taken together, our study demonstrated that deletion of the leuB gene resulted in delayed or completely blocked mother cell lysis, allowing the crystals encapsulated within cells, which makes this recombinant strain a good candidate for developing Bt preparations with better UV-stability.

Abstract Gut microbiota are known to play an important role in health and nutrition of the host and have been attracting an increasing attention. Farming of new lineages of grass carp and crucian carp has been developed rapidly as these species were found to outperform indigenous ones in terms of growth rate and susceptibility to diseases. Despite this rapid development, no studies have addressed the characteristics of their gut microbiota as a potential factor responsible for the improved characteristics. To reveal whether microbiomes of the new lineages are different from indigenous ones, and therefore could be responsible for improved growth features, intestinal microbiota from the new lineages were subjected to high‐throughput sequencing. While the phyla Firmicutes, Fusobacteria and Proteobacteria were representing the core bacterial communities that comprised more than 75% in all fish intestinal samples, significant differences were found in the microbial community composition of the new linages versus indigenous fish populations, suggesting the possibility that results in the advantages of enhanced disease resistance and rapid growth for the new fish lineages. Bacterial composition was similar between herbivorous and omnivorous fish. The relative abundance of Bacteroidetes and Actinobacteria was significantly higher in omnivores compared to that of herbivores, whereas Cetobacterium_sp. was abundant in herbivores. We also found that the gut microbiota of freshwater fish in the Dongting lake area was distinct from those of other areas. Network graphs showed the reduced overall connectivity of gut bacteria in indigenous fish, whereas the bacteria of the new fish lineage groups showed hubs with more node degree. A phylogenetic investigation of communities by reconstruction of unobserved states inferred function profile showed several metabolic processes were more active in the new lineages compared to indigenous fish. Our findings suggest that differences in gut bacterial community composition may be an important factor contributing to the rapid growth and high disease resistance of the new fish lineages.

Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. pogona limit its broad application and metabolic engineering. Here, we showed that S. pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69- and 3.03-fold, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

Abstract Background Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts. Results A 22 kb genomic fragment containing the sylA–sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC–MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects. Conclusions This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.

Abstract Cry6A represents a novel family of nematicidal crystal proteins from Bacillus thuringiensis. It has distinctive architecture as well as mechanism of action from Cry5B, a highly focused family of nematicidal crystal proteins, and even from other insecticidal crystal proteins containing the conserved three-domain. However, how nematode defends against Cry6A toxin remains obscure. In this study, the global defense pattern of Caenorhabditis elegans against Cry6Aa2 toxin was investigated by proteomic analysis. In response to Cry6Aa2, 12 proteins with significantly altered abundances were observed from worms, participating in innate immune defense, insulin-like receptor (ILR) signaling pathway, energy metabolism, and muscle assembly. The differentially expressed proteins (DEPs) functioning in diverse biological processes suggest that a variety of defense responses participate in the stress responses of C. elegans to Cry6Aa2. The functional verifications of DEPs suggest that ILR signaling pathway, DIM-1, galectin LEC-6 all are the factors of defense responses to Cry6Aa2. Moreover, Cry6Aa2 also involves in accelerating the metabolic energy production which fulfills the energy demand for the immune responses. In brief, our findings illustrate the global pattern of defense responses of nematode against Cry6A for the first time, and provide a novel insight into the mechanism through which worms respond to Cry6A.

The small heat shock protein plays an important role in response to stresses. We wanted to investigate how Hsp20 affects sporulation and production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis (Bt) at the stationary growth phase when cells are starved. The hsp20 gene was knocked out in Bt4.0718 (wide type), which is a B. thuringiensis strain screened in our laboratory, using endonuclease I-SceI mediated unmarked gene replacement method. Deletion of Hsp20 resulted in a decrease in both sporulation and ICPs production. Bt4-Δhsp20 cells and its ICP did not have a significant difference in shape and size but entered the decline phase 2 h earlier than the Bt4.0718. In order to find the mechanism that underlies these phenotypes, we completed a proteomic study of differentially expressed proteins (DEPs). In Bt4-Δhsp20 cells, 11 DEPs were upregulated and 184 DEPs downregulated. These affected DEPs are involved in multiple metabolic pathways: (1) six DEPs (two upregulated and four downregulated) are directly related to the sporulation and ICPs synthesis; (2) supply of amino acids including amino acid synthesis and protein recycling; (3) the energy supplementation (the tricarboxylic acid cycle and glycolysis); (4) purine metabolism and mRNA stability. These results suggest that hsp20 may be critical in maintaining the homeostasis of B. thuringiensis during the production of spores and ICPs, and could provide new sight into the sporulation and ICPs formation in B. thuringiensis.

Probiotic-feeding continues to be a promising strategy to control the bacterial pathogens in aquaculture. A new Pediococcus pentosaceus strain (SL001) was isolated from 1000s of soil samples, which exhibited wide antimicrobial spectrum of against fish pathogens, involving Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Edwardsiella tarda, Lactococcus garvieae, and Plesiomonas shigelloide. The challenge test against A. hydrophila showed that the survival rate of SL001-supplemented group was significantly higher than that of control group (P < 0.05). Moreover, SL001 could stably colonize in gut of grass carp and increased mucus-secreting goblet cells and extended intestinal villi could be observed in SL001-supplemented group (P < 0.05). Feeding with SL001 supplemented diet could significantly enhance the growth rate (P < 0.05) and markedly affect gut microbiota structure of grass carps, resulting in reduced potential pathogens and increased potential probiotics. Furthermore, feeding grass carps with SL001 caused the up-regulated expression of insulin-like growth factor (IGF-1 and IGF-2) and down-regulated expression of myostatin (MSTN-1 and MSTN-2) (P < 0.05), which probably also account for the increased growth rate of SL001-fed group. Meanwhile, relative mRNA expression levels of immune-related genes in liver, spleen, and head kidney were analyzed in grass carps after feeding for 30 days with SL001 supplemented diets. In all three immune organs, the expression levels of immunoglobulin M (IgM) and complement 3 (C3) were significantly increased (P < 0.05), whereas the interleukin-8 (IL-8) was down-regulated (P < 0.05). Besides, whole genome sequencing revealed several probiotics properties of SL001, including organic acid synthesis, bacteriocin synthesis (coagulin), superoxide dismutase, and digestive enzymes. In conclusion, P. pentosaceus SL001 which could enhance immunity and promoter growth rate of grass carps, is prospective to be used as a dietary probiotic in freshwater fish aquaculture.

Our previous study showed that the srfABC operon, which was originally identified in Salmonella enterica as an SsrB-regulated operon clustered with the flagellar class 2 operon, exhibited significant cytotoxicity against insect midgut CF-203 cells and injectable insecticidal activity against Helicoverpa armigera larvae. The srfABC operon was widely distributed among bacteria, which raises the question of their biological roles in different species. In this study, we investigated the cytotoxic effect of SrfABC toxin on mammalian cell lines. When simultaneously expressed in the Escherichia coli cytoplasm, SrfABC exhibited cytotoxicity against all tested mammalian cancer cell lines (B16, 4T-1, Hep-3B, and HeLa) in a dose-dependent manner. Intracellular expression of SrfA−FLAG, SrfB−FLAG, or SrfC−FLAG also resulted in inhibition of proliferation and apoptosis on HeLa cells. When incubated with HeLa cells separately, SrfA, SrfB, and SrfC proteins alone could enter HeLa cells, then induce apoptosis and cytotoxicity. SrfC protein shifts its localization from cytoplasm to nucleus with the aid of SrfA and/or SrfB protein. Although SrfA, SrfB, and SrfC proteins alone exhibited a cytotoxic effect against HeLa cells, all three components were essential for the full cytotoxicity. Native PAGE and co-immunoprecipitation assay demonstrated that SrfA, SrfB, and SrfC proteins could interact with each other and form a heteromeric complex.

Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for promoting the application of proteogenomics in the life sciences.

The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains.

Combination of PCR and mass spectrometry is used to detect five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cryIIa) in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718 and identify three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) in the strain's parasporal crystals. The complementary method might be useful to evaluate the B. thuringiensis strains at both the gene and protein levels.

Contact-dependent growth inhibition (CDI) is a type of competitive mechanisms and has been identified in various strains including Burkholderia, Dickeya, E. coli and Yersinia. Classical CDI systems contain three genes, cdiB, cdiA and cdiI. CdiB encoded by cdiB gene is a conserved β-barrel protein and required for export of CdiA. CdiA protein encoded by cdiA gene includes a conserved N-terminal domain and variable C-terminal toxic domain (CdiA-CT). Immunity protein CdiI binds and inactivates toxin protein CdiA-CT. Here, we identified two CDI systems, an intact cdiBAI operon with a truncated CdiB due to an unexpected mutation and an 'orphan' cdiA-CT/cdiI module in the probiotic Escherichia coli Nissle 1917 (EcN) genome. Both CdiA-CTs from EcN showed auto-inhibition activity when transferring into E. coli DH5α, as well the sequential deletion of amino acid residues resulted in the generation of the most potent mutant of CdiA-CT. CdiI neutralized the toxicity activity of CdiA and was immunity protein as previous report. In conclusion, this is the first report that the functional CDI system is in probiotic EcN and might provide a potential competitive mechanism for probiotic EcN in intestinal microenvironment. [ABSTRACT FROM AUTHOR]

Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and Orf3, were modulated in the epothilone production strain Burkholderia DSM7029::Tn5-km-epo (named G32) via Red/ET engineering to increase heterologous polyketide epothilone yields. Compared with the prior G32 strain, the total production of several epothilones in the G32::orf14-orf3 mutant was meaningfully doubled according to high-performance liquid chromatography-mass spectrometer analysis. Typically for epothilone B, in simple and clear liquid medium CYMG, the overall productivity in the engineered high-yield producer G32::orf14-orf3 was improved for almost 3-fold, from 2.7 to about 8.1 μg/l. Additionally, the ratio of extracellular to intracellular accumulation of epothilone B was raised from 9.3:1 to 13.7:1 in response to expression of two putative transport genes orf14 and orf3. Hence, we strongly recommend that the Orf14 and Orf3 transporters export epothilone, thus promotes the forward reaction of biosynthesis on epothilone manufacture inside the cells. Our results afford a practical stage for yield improvement of other heterologous natural products in broad chassis cells. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) X022 is a novel strain isolated from soil in China, and showed strong insecticidal activity against several Lepidopteran pests. In this work, we performed whole genome sequencing of this Bt strain using the next-generation sequencing technology, and further conducted a comparative analysis with the proteomics data of the specific spore-release period based on LC-MS/MS approach. The Bt X022 genome consisted of one circular chromosomal DNA and seven plasmids, which were further functionally annotated using the RAST server. Comparative analysis of insecticidal substances showed that X022 contained genes coding for three Cry proteins (Cry1Ac, Cry1Ia and Cry2Ab) and a vegetative insecticidal protein (Vip3A). However, three insecticidal crystal proteins (ICPs) (Cry1Ca, Cry1Ac and Cry1Da) were detected by proteomics in the spore-release period. Moreover, a putative biosynthetic gene cluster and the metabolic pathway for poly-β-hydroxybutyrate in Bt X022 were deduced based on the comparative analysis of genomic and proteomic data, which revealed the metabolic regulation mechanism of carbon flux correlated with ncreased production of ICPs caused by Cu2+. Hence, these results provided a deeper understanding of the genetic background and protein expression profile of Bt X022. This study established a foundation for directed genetic modification and further application of this new isolated Bt strain. [ABSTRACT FROM AUTHOR]

Background: Bacillus thuringiensis X022, a novel strain isolated from soil in China, produces diamond-shaped parasporal crystals. Specific mineral nutrients, such as Mg, Cu, and Mn, influence insecticidal crystal proteins (ICP) expression and the effects of these elements vary significantly. However, the molecular mechanisms of the effects caused by mineral elements have yet to be reported. Results: The ICP are mainly composed of Cry1Ca, Cry1Ac, and Cry1Da, which have molecular weights of about 130 kDa. ICP production was most efficient when Cu2+ was added at concentrations ranging from 10-6 to 10-4 mol/L at an initial pH of 8.0. Addition of Cu2+ also evidently increased the toxicity of fermentation broth to Spodoptera exigua and Helicoverpa armigera. After analyzing changes in proteome and fermentation parameters caused by Cu2+ addition, we propose that Cu2+ increases PhaR expression and consequently changes the carbon flow. More carbon sources was used to produce intracellular poly-β-hydroxybutyrate (PHB). Increases in PHB as a storage material bring about increases of ICP production. Conclusions: Bacillus thuringiensis X022 mainly expresses Cry1Ca, Cry1Ac, and Cry1Da. Cu2+ increases the expression of Cry1Da, Cry1Ca, and also enhances the toxicity of fermentation broth to S. exigua and H. armigera. [ABSTRACT FROM AUTHOR]

A 8.7 kDa lectin with high agglutin activity was isolated by affinity chromatography and cloned from Allium chinense in this study. For the MTT assay, approximately 60 μg/ml A. chinense lectin (ACL) inhibited 50% of the human hepatoma Hep-3B cells grown after 48 h. In addition, no antiproliferative effect was observed on normal human umbilical vein endothelial cells (HUVEC) even at 100 ug/ml concentration. After treatments with ACL on Hep-3B cells, morphologic changes in the nucleus and cytoskeleton were observed under laser scanning confocal microscopy with 4',6-diamidino-2-phenylindole and tubulin Alexa Fluor 488 staining; whereas, the mitochondrial membrane potential was observed through Mito Tracker Red CMXRos staining. The results showed that ACL led to cell morphology and structure change (e.g., round cell shrinkage). Moreover, ACL resulted in significant change in the shape of the nucleus, damaged the cytoskeleton when tubulin was degraded, and reduced the mitochondrial transmembrane potential. By contrast, no changes were observed on HUVEC cells under the same treatment conditions. DNA fragmentation analysis was used to detect DNA damage. Western blot showed that ACL upregulated caspase-3 and Bax expression during apoptosis and cloned the structural gene of ACL with an open reading frame of 456 bp encoding 151 amino acid residues. The results showed that ACL is a potential anticancer drug. [ABSTRACT FROM AUTHOR]

Background Saccharopolyspora 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. Results Two-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. Conclusions This 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 [ABSTRACT FROM AUTHOR]

The calY gene, encoding metalloprotease camelysin in the Bacillus thuringiensis acrystalliferous strain XBU001, was amplified and sequenced. The camelysin from the calY sequence was 199 amino acids in size ( c. 22 000 Da). The temperature-sensitive plasmid pKESX was used to construct a metalloprotease camelysin-deficient strain of B. thuringiensis. The calY gene was replaced by an erythromycin-resistant gene in KCTF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and MS analysis showed that the metalloprotease InhA was not expressed after knocking out the gene calY. The temperature-sensitive plasmid pKPC was used to construct a metalloprotease camelysin complementation strain KCTFC. The InhA protein was found in KCTFC. Analysis of the expression of InhA in the wild-type strain KCTF12, camelysin-deficient and complementation strains indicated that inhA expression depended on camelysin. Although camelysin did not directly regulate the expression of the InhA through binding to the promoter of the inhA, the results suggest that camelysin can positively regulate the expression of the InhA protein. [ABSTRACT FROM AUTHOR]

Domain III of Bacillus thuringiensis Cry δ-endotoxins are considered to be related to the stability of the structure and avoidance of overdigestion by proteases. In this study, some residues of potential chymotrypsin and trypsin sites in Domain III of B. thuringiensis Cry1Aa were replaced individually with alanine by site-directed mutagenesis, in order to investigate their functional roles. Except F574A, all mutants F536A, R543A, F550A, F565A, R566A, F570A, F576A, F583A, and F590A were highly expressed the 130 kD protoxins at levels comparable to the wild-type tested by SDS-PAGE. In bioassays, F536A, R566A, and F590A increased toxicity against Spodoptera exigua Hüner larve by 20, 40, and 40%, respectively, as compared to the wild-type. F536A and F565A showed an increase of 6 and 10% in toxicity against Heliothis armigera Hubner than the wild-type. Toxicities of some mutants were altered greatly, and the same mutants were shown to have different toxicities against those two insects. Structural analyses showed that mutants R543A, F574A, F576A-affecting insecticidal activity might be relational to structural stability of toxin or decreased affinity for receptor binding. These results indicated that those residues were involved in the larvicidal activity of the Cry1Aa toxin. [ABSTRACT FROM AUTHOR]

The β20-β21 loop is a unique structure in the domain III of Bacillus thuringiensis Cry proteins. In this study, the role of β20-β21 loop on insecticidal activity of Cry1Ac toxin was investigated. 10 residues in β20-β21 loop were substituted with alanine using PCR-based site-directed mutagenesis. All mutants were capable of producing diamond-shaped crystal and expressing a protein sized 130 kDa. The mutants S581A and I585A enhanced toxicity against Helicoverpa armigera larvae dramatically, while most of the rest mutants possess a reduced toxicity at different degrees. Indoor bioassay result revealed that mutants S581A and I585A had a 1.72- and 1.89-fold increasing in toxicity against Helicoverpa armigera larvae compared with the wild-type strain, respectively; On the contrary, G583A experienced a significant reduced insecticidal activity. Three-dimensional analysis of Cry1Ac5 protein demonstrated that the side chain of residues T579, S580, L582, and I585 extended to the surface of the protein, and might participate in the interaction between the protein and its receptor, whereas side chain of residues N576, F578, S581, N584, and V586 preferred the inside of the protein, and which might be critical to the stability of the protein structure. Our study for the first time clarified the special properties and the functions of the β20-β21 loop in domain III of Cry1Ac5. These findings also provided the latest biological evidence for the recognition and binding mechanism of the domain III in Cry1Ac, and its role in maintaining the structure stability of Cry1Ac. [ABSTRACT FROM AUTHOR]

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis ( Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16-β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules. [ABSTRACT FROM AUTHOR]

Background: Saccharopolyspora spinosa produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type S. spinosa. Recently, a spinosadhyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type S. spinosa SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. Results: In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Conclusions: The present study is the first comprehensive and comparative proteome analysis of S. spinosa strains. Our results highlight the differentially expressed proteins between the two S. spinosa strains and provide some clues to understand the molecular and metabolic mechanisms that could lead to the increased spinosad production yield. [ABSTRACT FROM AUTHOR]

A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and p I = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc–CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc–CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry−B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry−B–pHAc–CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC50) of Cry−B–pHAc–CDEP2 strain (8.5 μl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry−B–pHAc strain (16.7 μl/ml) at 72 h. [ABSTRACT FROM AUTHOR]

We obtained and compared a new cry2Ac6 gene from Bacillus wuhanensis 140, and Bacillus thuringiensis (Bt) subsp. kurstaki 4.0718 and B.t. kurstaki XL004 that share a similar genetic background but occupy different ecological niches. Using a proteomic approach and function-based activity profiling, we systemically identified the insecticidal crystal proteins (ICPs) from the three Bt species, which were found to be mainly distributed at pH 4-7 on two-dimensional electrophoresis (2DE) gels by PDQuest software. The proteins that exhibited a significant difference in expression were excised, digested in-gel and identified by MALDI-TOF-MS. Thirty-three differently expressed proteins were identified from the three Bt strains. The Cry2Ac6, Cry1Ab16, CryIG, CryH2, CryI, CryINA67-1 and CryI+ crystal protein mixture from B.t. wuhanensis 140, Cry1Ac, Cry2Aa and endotoxin delta1 from B.t. kurstaki 4.0718 were further analyzed by bioinformatics analysis. Two common proteins were founded in three strains, the heat shock proteins (HSP60) and the translation elongation factor Tu, which help with protein refolding and prevent protein degradation. The different enzymes of metabolism, including glutamate racemase, chemotaxis protein histidine kinase and related kinases pyruvate dehydrogenase complex E1orE3 were identified. Some protein spots could not be identified. The results indicate that each Bt strain has unique ICPs as well as some common proteins related to ICPs formation, and that the virulence of Bt strains is closely related to the expression of specific ICPs. [ABSTRACT FROM AUTHOR]

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC50 (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. [ABSTRACT FROM AUTHOR]

The β18-β19 loop in domain III of Cry1Ac toxin is unique among Bacillus thuringiensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin. [ABSTRACT FROM AUTHOR]

In order to improve the insecticidal activity, the chitinase gene from tobacco ( Nicotiana tabacum) endochitinase and the cry1Ac gene from Bacillus thuringiensis were cloned into the vector pHT315 and designated as pHUAccB5 plasmid. The constructed transcriptional fusion was attempted under the control of the native cry1Ac promoter. Plasmid pHUAccB5 was introduced into B. thuringiensis acrystalliferous by electroporation. Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, the transformant XBU-HUAccB5 produced 130–kDa Cry1Ac protein and 30-kDa chitinase protein. During the chitinase active analysis, the transformant, XBU-HUAccB5 chitinase active, reached 7.5 U/mL at 72 h, and was 5 times higher than the HTX-42 and 6 times higher than the parent strains. When the insecticidal activity of the transformant was evaluated against Helicoverpa armigera Hubner, the XBU-HUAccB5 toxicity was 11.30 times higher than the transformant HTX-42 expressed single cry1Ac at 48 h and was 18.76 times higher at 72 h. [ABSTRACT FROM AUTHOR]

The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains Cry¯B and HD73, producing recombinant strains Cry¯B(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant Cry¯B(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than Cry¯B(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins. [ABSTRACT FROM AUTHOR]

An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein (CSAP). CSAP was single-chained, with an apparent molecular mass of 35×103 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inhibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Furthermore, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts. [ABSTRACT FROM AUTHOR]