Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation.

Understanding the genetic mechanisms of phenotypic variation in hybrids between domestic animals and their wild relatives may aid germplasm innovation. Here, we report the high-quality genome assemblies of a male Pamir argali (O ammon polii, 2n = 56), a female Tibetan sheep (O aries, 2n = 54), and a male hybrid of Pamir argali and domestic sheep, and the high-throughput sequencing of 425 ovine animals, including the hybrids of argali and domestic sheep. We detected genomic synteny between Chromosome 2 of sheep and two acrocentric chromosomes of argali. We revealed consistent satellite repeats around the chromosome breakpoints, which could have resulted in chromosome fusion. We observed many more hybrids with karyotype 2n = 54 than with 2n = 55, which could be explained by the selfish centromeres, the possible decreased rate of normal/balanced sperm, and the increased incidence of early pregnancy loss in the aneuploid ewes or rams. We identified genes and variants associated with important morphological and production traits (e.g., body weight, cannon circumference, hip height, and tail length) that show significant variations. We revealed a strong selective signature at the mutation (c.334C > A, p.G112W) in TBXT and confirmed its association with tail length among sheep populations of wide geographic and genetic origins. We produced an intercross population of 110 F2 offspring with varied number of vertebrae and validated the causal mutation by whole-genome association analysis. We verified its function using CRISPR-Cas9 genome editing. Our results provide insights into chromosomal speciation and phenotypic evolution and a foundation of genetic variants for the breeding of sheep and other animals.

Discovery of an antitumor compound from xenorhabdus stockiae HN_xs01.

Xenorhabdus, known for its symbiotic relationship with Entomopathogenic nematodes (EPNs), belongs to the Enterobacteriaceae family. This dual-host symbiotic nematode exhibits pathogenic traits, rendering it a promising biocontrol agent against insects. Our prior investigations revealed that Xenorhabdus stockiae HN_xs01, isolated in our laboratory, demonstrates exceptional potential in halting bacterial growth and displaying anti-tumor activity. Subsequently, we separated and purified the supernatant of the HN_xs01 strain and obtained a new compound with significant inhibitory activity on tumor cells, which we named XNAE. Through LC-MS analysis, the mass-to-nucleus ratio of XNAE was determined to be 254.24. Our findings indicated that XNAE exerts a time- and dose-dependent inhibition on B16 and HeLa cells. After 24 h, its IC50 for B16 and HeLa cells was 30.178 µg/mL and 33.015 µg/mL, respectively. Electron microscopy revealed conspicuous damage to subcellular structures, notably mitochondria and the cytoskeleton, resulting in a notable reduction in cell numbers among treated tumor cells. Interestingly, while XNAE exerted a more pronounced inhibitory effect on B16 cells compared to HeLa cells, it showed no discernible impact on HUVEC cells. Treatment of B16 cells with XNAE induced early apoptosis and led to cell cycle arrest in the G2 phase, as evidenced by flow cytometry analysis. The impressive capability of X. stockiae HN_xs01 in synthesizing bioactive secondary metabolites promises to significantly expand the reservoir of natural products. Further exploration to identify the bioactivity of these compounds holds the potential to shed light on their roles in bacteria-host interaction. Overall, these outcomes underscore the promising potential of XNAE as a bioactive compound for tumor treatment.

Lignocellulose degradation by rumen bacterial communities: New insights from metagenome analyses.

Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.

Microbial Lipopeptide Supramolecular Self-Assemblies as a Methuosis-Like Cell Death Inducer with In Vivo Antitumor Activity.

Discovering new drugs and improving action mechanisms is a promising strategy to overcome chemotherapy ineffectiveness caused by cancer cell apoptosis resistance. Natural products (like cyclic lipopeptides, CLPs) are potential sources of nonapoptotic cell death inducers and can form diverse supramolecular structures, closely relating to their bioactivities. Herein, it is found for the first time that fatty chain is the key to maintain self-assembled form and antitumor activity of microbial-derived amphiphilic CLP bacillomycin Lb (B-Lb). Compared with B-Lb analogues assemblies without antitumor activity, B-Lb supramolecular self-assemblies (including nanomicelles, nanofibers, giant micrometer rods) can be generated in a multilevel and cross-scale manner and served as a methuosis-like cell death inducer triggered by cytoplasmic vacuolation through macropinocytosis in MDA-MB-231-Luc and MCF-7 cells and in vivo tumor-bearing mice. This study will promote constructing of customized CLP micro-/nanostructures with multipurposes and functions, and boost designing of new antitumor drugs as nonapoptotic cell death modulators based on structure-activity relationship.

Age-dependent variations in rumen bacterial community of Mongolian cattle from weaning to adulthood.

BACKGROUND: Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that the rumen microbiome of Mongolian cattle has adapted to degrade the rough forage to provide sufficient energy to tolerate the harsh desert ecological conditions. However, little is known about the succession of rumen microbes in different developmental stages of post-weaning Mongolian cattle. METHODS: Here, we examined the succession of the rumen microbial composition and structure of 15 post-weaning Mongolian cattle at three developmental stages i.e., 5 months (RM05), 18 months (RM18) and, 36 months (RM36) by using the 16S rRNA gene sequencing method. RESULTS: We did not find any age-dependent variations in the ruminal concentrations of any volatile fatty acid (VFA) of Mongolian cattle. The diversity of the rumen bacterial community was significantly lower in RM05 group, which reached to stability with age. Bacteroidetes and Firmicutes were the two dominant phyla among all age groups. Phylum Actinobacteria was significantly higher in RM05 group, phyla Spirochaetes, and Tenericutes were highly abundant in RM18 group, and phyla Proteobacteria and Epsilonbacteraeota were enriched in RM36 group. Genera Prevotella_1, Bacteroides, and Bifidobacterium were abundant in RM05 group. The short chain fatty acid (SCFA) producing bacteria Rikenellaceae_RC9_gut_group showed high abundance in RM18 group and fiber degrading genus Alloprevotella was highly abundant in RM36 group. Random forest analysis identified Alloprevotella, Ileibacterium, and Helicobacter as important age discriminatory genera. In particular, the genera Ruminococcaceae_UCG-005, Bacteroides, Saccharofermentans, and Fibrobacter in RM05, genera [Eubacterium] coprostanoligenes_group, Erysipelotrichaceae_UCG-004, Helicobacter, Saccharofermentans, Papillibacter, and Turicibacter in RM18, and genera Rikenellaceae_RC9_gut_group, Lachnospiraceae_AC2044_group, and Papillibacter in RM36 showed the top interactions values in the intra-group interaction network. CONCLUSIONS: The results showed that rumen microbiota of Mongolian cattle reached to stability and maturity with age after weaning. This study provides some theoretical evidence about the importance of functional specific rumen bacteria in different age groups. Further studies are needed to determine their actual roles and interactions with the host.

Recombineering using RecET-like recombinases from Xenorhabdus and its application in mining of natural products.

Xenorhabdus can produce a large number of secondary metabolites with insecticidal, bacteriostatic, and antitumor activities. Efficient gene editing tools will undoubtedly facilitate the functional genomics research and bioprospecting in Xenorhabdus. In this study, BlastP analysis using the amino acid sequences of Redαß or RecET recombinases as queries resulted in the identification of an operon (XBJ1_operon 0213) containing RecET-like recombinases encoding genes from the genome of Xenorhabdus bovienii strain SS-2004. Three proteins encoded by this operon was indispensable for full activity of recombineering, namely XBJ1-1173 (RecE-like protein), XBJ1-1172 (RecT-like protein), and XBJ1-1171 (single-strand annealing protein). Using this newly developed recombineering system, a gene cluster responsible for biosynthesis of a novel secondary metabolite (Min16) was identified from X. stockiae HN_xs01 strain. Min16 which exhibited antibacterial and cytotoxic activities was determined to be a cyclopeptide composed of Acyl-Phe-Thr-Phe-Pro-Pro-Leu-Val by using high-resolution mass spectrometry and nuclear magnetic resonance analysis, and was designated as changshamycin. This host-specific recombineering system was proven to be effective for gene editing in Xenorhabdus, allowing for efficient discovery of novel natural products with attractive bioactivities. KEY POINTS: • Screening and identification of efficient gene editing tools from Xenorhabdus • Optimization of the Xenorhabdus electroporation parameters • Discovery of a novel cyclopeptide compound with multiple biological activities.

ARTP and NTG compound mutations improved Cry protein production and virulence of Bacillus thuringiensis X023.

A high production mutated strain Bacillus thuringiensis X023PN (BtX023PN) was screened from the wild strain Bacillus thuringiensis X023 (BtX023) after atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG) mutation. BtX023PN grows faster than the wild strain, and its lysis of mother cell was 6 h ahead BtX023, but the ability of sporulation was significantly reduced. Bioassay indicated that compared with the wild type strain, the virulence of BtX023PN against Plutella xylostella (P. xylostella) and Mythimna seperata (M. seperata) increased to 2.33-fold and 2.13-fold respectively. qRT-PCR and SDS-PAGE demonstrated that the production of Cry1Ac increased by 61%. Resequence indicated that the mutated sites enriched on the key carbohydrate metabolism and amino acid metabolism. This study provides a new strain resource for the development of Bt insecticides and a feasible technical strategy for the breeding of Bt. KEY POINTS: • Atmospheric and room temperature plasma used in breeding of Bacillus thuringiensis. • Less stationary phase time with more ICP production. • Semi-lethal concentration against Plutella xylostella reduced by about 57.

Detection of InDel and CNV of SPAG17 gene and their associations with bovine growth traits.

Sperm-associated antigen 17 (SPAG17) gene encodes a central pair protein, which is involved in flagellar motility, male fertility and skeletal growth in ruminants. The insertions/deletions (indels) and copy number variations (CNVs) influence phenotypic traits by altering the sequences and copy numbers of functional genes, respectively. This study identified a novel 8-bp indel of SPAG17 gene in 1520 individuals from eight different cattle breeds, as well as a novel CNV region in 355 animals. The correlation analysis of indel showed that the individuals of ID genotype had superior performance traits such as body height (p = 0.038) and body slanting length (p = 0.041) as compared to other genotypes in Xianan cattle. For the CNV, different copy numbers were closely related to the body height in Qinchuan (p = 0.045) and body weight in Xianan (p = 0.036) breeds. Importantly, significant difference was observed between the 8-bp indel and the copy number loss in Xianan breed (p < 0.01). These findings indicated that the variations within the bovine SPAG17 gene can be considered as an effective DNA molecular marker for beef cattle breeding.

Neutrophil Extracellular Traps Mediate Bovine Endometrial Epithelial Cell Pyroptosis in Dairy Cows with Endometritis.

Neutrophils are involved in the development of endometritis, but it remains unknown how neutrophils induce inflammation and tissue damage. Neutrophil extracellular traps (NETs) clear invading pathogens during infection but induce pyroptosis, leading to inflammation and tissue damage. Thus, our objective was to investigate whether NETs participate in bovine endometrial epithelial cell (BEEC) pyroptosis during endometritis. To confirm this, NETs and caspase-1/4; apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC); nod-like receptor protein-3 (NLRP3); and gasdermin D N-terminal (GSDMD-N), TNF-a, IL-1ß, IL-6, and IL-18 in endometrial tissue were detected. Pathological section and vaginal discharge smears were performed to visually determine endometritis in the uterus. BEECs were stimulated with NETs to induce pyroptosis, which was treated with DNase I against pyroptosis. Caspase-1/4, ASC, NLRP3, GSDMD-N, TNF-a, IL-1ß, IL-6, and IL-18 in BEECs were analyzed in endometrial tissue. The results showed that NET formation, as well as pyroptosis-related proteins and proinflammatory, cytokines were elevated in the endometrial tissue of cows with endometritis. Pathological sections and vaginal discharge smears showed increased neutrophils and plasma cells in the uterus, as well as tissue congestion. In BEECs, NETs increased the level of pyroptosis-related proteins and proinflammatory cytokines and were diminished by DNase I. In summary NETs participate BEEC pyroptosis during endometritis in dairy cows.

Lactobacillus rhamnosus CY12 Enhances Intestinal Barrier Function by Regulating Tight Junction Protein Expression, Oxidative Stress, and Inflammation Response in Lipopolysaccharide-Induced Caco-2 Cells.

The intestinal barrier is vital for preventing inflammatory bowel disease (IBD). The objectives of this study were to assess whether the Lactobacillus rhamnosus CY12 could alleviate oxidative stress, inflammation, and the disruption of tight junction (TJ) barrier functions induced by lipopolysaccharide (LPS), and therefore to explore the potential underlying molecular mechanisms. Our results showed that LPS-induced Cancer coli-2 (Caco-2) cells significantly increased the levels of reactive oxygen species (ROS), lactate dehydrogenase, inflammatory cytokines interleukin-1ß, interleukin-6, interleukin-8, and tumor necrosis factor-α (IL-1ß, IL-6, IL-8, and TNF-α), and the cell apoptosis rate while decreasing the levels of TJ proteins occludin, zonula occludens-1 (ZO-1), and claudin and antioxidant enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase(CAT, SOD, and GSH-Px) (p < 0.05). However, Lactobacillus rhamnosus CY12 could relieve cytotoxicity, apoptosis, oxidative stress, and pro-inflammatory cytokine expressions, and also inhibit the Toll-like receptor 4/nuclear factor kappa-B(TLR4/NF-κB) signaling pathway. Furthermore, the gene expression of antioxidant enzymes, as well as the mRNA and protein expressions of TJ proteins, was improved. Particularly, the concentration of 108 cfu/mL significantly prevented the inflammatory injury induced by LPS in Caco-2 cells (p < 0.05). These findings support a potential application of Lactobacillus rhamnosus CY12 as a probiotic to prevent LPS-induced intestinal injury and treat intestinal barrier dysfunction.

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis.

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.

Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa.

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.

Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

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.

Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona.

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.

The seasonal development dynamics of the yak hair cycle transcriptome.

BACKGROUND: Mammalian hair play an important role in mammals' ability to adapt to changing climatic environments. The seasonal circulation of yak hair helps them adapt to high altitude but the regulation mechanisms of the proliferation and differentiation of hair follicles (HFs) cells during development are still unknown. Here, using time series data for transcriptome and hormone contents, we systematically analyzed the mechanism regulating the periodic expression of hair development in the yak and reviewed how different combinations of genetic pathways regulate HFs development and cycling. RESULTS: This study used high-throughput RNA sequencing to provide a detailed description of global gene expression in 15 samples from five developmental time points during the yak hair cycle. According to clustering analysis, we found that these 15 samples could be significantly grouped into three phases, which represent different developmental periods in the hair cycle. A total of 2316 genes were identified in these three consecutive developmental periods and their expression patterns could be divided into 9 clusters. In the anagen, genes involved in activating hair follicle growth are highly expressed, such as the WNT pathway, FGF pathway, and some genes related to hair follicle differentiation. In the catagen, genes that inhibit differentiation and promote hair follicle cell apoptosis are highly expressed, such as BMP4, and Wise. In the telogen, genes that inhibit hair follicle activity are highly expressed, such as DKK1 and BMP1. Through co-expression analysis, we revealed a number of modular hub genes highly associated with hormones, such as SLF2, BOP1 and DPP8. They may play unique roles in hormonal regulation of events associated with the hair cycle. CONCLUSIONS: Our results revealed the expression pattern and molecular mechanisms of the seasonal hair cycle in the yak. The findings will be valuable in further understanding the alpine adaptation mechanism in the yak, which is important in order to make full use of yak hair resources and promote the economic development of pastoral plateau areas.

Alteration of the gut microbiome and immune factors of grass carp infected with Aeromonas veronii and screening of an antagonistic bacterial strain (Streptomyces flavotricini).

Aeromonas veronii is a widely distributed novel pathogen that can affect humans and animals, it can cause sepsis in fish with high mortality and serious economic losses to aquaculture. In the study, the gut microbiome of the infected and uninfected grass carp with Aeromonas veronii were analyzed probiotics and pathogenic bacteria by the Miseq high-throughput sequencing, the results showed that the infected fish were significantly higher in Proteobacteria, Firmicutes, Fusobacteria, and the immune factors in liver and kidney were up-regulated by qRT-PCR. In order to effectively inhibit the pathogen, we screened an actinomycete strain and had good antibacterial effect on Aeromonas veronii. The new antagonistic bacteria was named as Streptomyces flavotricini X101, the whole genome sequencing revealed that the metabolic process was most active. After grass carp was inoculated with the minimum inhibitory concentration of 900 µg/mL of the strain's fermentation supernatant, then Aeromonas veronii was injected, we found that the pathological symptoms such as body surface, anus and abdominal congestion were alleviated by H&E staining. Cellular experiments showed that it wasn't toxic to liver cells of grass carp. Overall, this is the first study of changes in intestinal flora, phenotype, and immune factors in grass crap infected with Aeromonas veronii, it had important theoretical significance and application value for immunization and prevention.

Enhancing the insecticidal activity of new Bacillus thuringiensis X023 by copper ions.

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-ß-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-ß-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 cry1Ac and vip3Aa 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-ß-hydroxy butyrate synthesis.

Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora pogona.

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.

Comparison between Tibetan and Small-tailed Han sheep in adipocyte phenotype, lipid metabolism and energy homoeostasis regulation of adipose tissues when consuming diets of different energy levels.

This study aimed to gain insight into how adipose tissue of Tibetan sheep regulates energy homoeostasis to cope with low energy intake under the harsh environment of the Qinghai-Tibetan Plateau (QTP). We compared Tibetan and Small-tailed Han sheep (n 24 of each breed), all wethers and 1·5 years of age, which were each divided randomly into four groups and offered diets of different digestible energy (DE) densities: 8·21, 9·33, 10·45 and 11·57 MJ DE/kg DM. When the sheep lost body mass and were assumed to be in negative energy balance: (1) adipocyte diameter in subcutaneous adipose tissue was smaller and decreased to a greater extent in Tibetan than in Small-tailed Han sheep, but the opposite occurred in the visceral adipose tissue; (2) Tibetan sheep showed higher insulin receptor mRNA expression and lower concentrations of catabolic hormones than Small-tailed Han sheep and (3) Tibetan sheep had lower capacity for glucose and fatty acid uptake than Small-tailed Han sheep. Moreover, Tibetan sheep had lower AMPKα mRNA expression but higher mammalian target of rapamycin mRNA expression in the adipocytes than Small-tailed Han sheep. We concluded that Tibetan sheep had lower catabolism but higher anabolism in adipose tissue and reduced the capacity for glucose and fatty acid uptake to a greater extent than Small-tailed Han sheep to maintain energy homoeostasis when in negative energy balance. These responses provide Tibetan sheep with a high ability to cope with low energy intake and with the harsh environment of the QTP.

Isolating a new Streptomyces amritsarensis N1-32 against fish pathogens and determining its effects on disease resistance of grass carp.

In this study, a Streptomyces strain was isolated from the soil samples of Yanghu Wetland Park in Changsha, Hunan Province. This strain showed excellent antimicrobial activity against 10 fish pathogens, as indicated by the results of the agar-diffusion and oxford cup assays. After 16s rDNA sequencing and physiological & biochemical analyses, it was identified as Streptomyces amritsarensis, namely for S. amritsarensis N1-32. Cytotoxicity test was performed, and the results exhibited that this strain had no toxicity to hepatic L8824 cell line from grass carp liver. The diets supplemented strain N1-32 at concentrations of 1 × 107 cfu/g and 1 × 109 cfu/g was used to feed fish. After 28 days, the expression levels of antioxidant-related genes Nrf2 and Keap1 in the liver and spleen were significantly up-regulated, and the expression of immune-related gene IgM was notably increased in the liver, kidney, head-kidney, and spleen. Toll-like receptor 4 (TLR4) gene expression was up-regulated in the spleen, and TLR4, myeloid differentiation factor 88 (MyD88) gene were up-regulated in the kidney. The survival rate of grass carp was significantly improved after pathogen infection. Whole-genome analysis of N1-32 showed that the strain harbored related genes, capability for producing substances that enhance the immunity of grass carp and inhibit pathogens. A total of 22 gene clusters were identified in the genome, including 5 terpene gene clusters, 4 nonribosomal peptide-synthetase (NRPS) gene clusters and 2 lantipeptide gene clusters. In summary, these results showed that strain N1-32 as a feed additive could regulate grass carp immunity and enhance the resistance of grass carp against fish pathogens.