Abstract:L-ornithine is a non-protein amino acid involved in urea metabolism and polyamines biosynthesis. Due to the positive functions in treating liver diseases and enhancing immunity, L-ornithine is widely used in medicine and food industry. Currently, L-ornithine production mainly depends on chemosynthesis, enzymatic catalysis and microbial fermentation. Among them, microbial fermentation has gradually become the focus for L-ornithine production owing to its superiority in cost and environmental protection. In this article, recent progress in the development of high L-ornithine producing strains through genetic engineering is summarized, the metabolic engineering strategies for improving L-ornithine production in Corynebacterium glutamicum are discussed, and the future direction is predicted.

Abstract:Porcine deltacoronavirus (PDCoV) is the only member of the Deltacoronavirus until now, which was found to infect mammals. PDCoV mainly infects porcine small intestine to induce severe atrophic enteritis by causing atrophy of villous epithelial cells in the small intestine, especially the jejunum and ileum. The severe clinical symptoms caused by PDCoV are mainly watery diarrhea, vomiting and death of newborn piglets, which brought out large economic losses in the pig industry. Since 2014, the detection rate of PDCoV single infection has a certain proportion in the global outbreak of porcine diarrhea disease. In addition, a high proportion was determined with other porcine coronaviruses co-infection. With the completion of full genome sequencing, successful isolation of PDCoV strains, as well as further research on the interaction with host have been gained more understanding about PDCoV. In this article, the prevalence of PDCoV, genetic diversity of genome structure, receptor of virus infection and the regulation mechanism of host innate immune response are reviewed, which based on the existing research literature and combined with our group progress. We hope that it will help the researchers to have a comprehensive and in-depth understanding of PDCoV.

Abstract:The prevention and treatment for gastrointestinal tumors have become an important public health issue due to the high morbidity and mortality. Microflora in the gastrointestinal tract usually participate in the metabolism and immune response to maintain body homeostasis. Recent studies have found that a variety of bacteria in the gastrointestinal tract play an important role in the occurrence and development of gastrointestinal tumors. Bacteria could induce tumors by virulence factors, biofilm, metabolites and other factors. The efficacy of chemotherapeutic agents could be affected as well. However, the involvement of bacteria in tumorigenesis remains unclear. Therefore, this paper reviews the mechanisms of gastrointestinal tumors induced by bacteria, thereby providing the theoretical basis for early prevention and treatment.

Abstract:[Objective] The purpose of this research is to test the effects of myxobacteria predation on the microbial community structure in microcosms and determine the role of myxobacteria predation in the regulation of artificial microecosystem.[Methods] Predation ability was determined by the predation diameters of myxobacteria EGB against nine species of prey bacteria using Lawn predation method. The microbial community structure changes caused by the predation of myxobacteria were analyzed through high-throughput sequencing technology.[Results] The predation abilities of myxobacteria EGB to 9 different species of prey bacteria were significantly different. The predation ability of myxobacteria to Bacillus tropicus was significantly higher than that of other bacteria (P<0.05). Adding different amounts of myxobacteria in an artificial microcosm system containing nine prey bacteria, the bacterial community diversity index (Shannon) was significantly reduced. The results of PCoA showed that the myxobacteria predation could affect the microcosm microbial community structure. The relative abundance of seven species of prey bacteria was reduced significantly (LefSe, P<0.05), while the relative abundance of Burkholderia cenocepacia was increased significantly (P<0.05) after 24-hour of artificial microcosm cultivation. The results of artificial microcosm experiments showed that the addition of myxobacteria EGB was the main factor influencing the microbial community structure, and the addition of the minimum dose of myxobacteria (1 mL) also had a significant effect. With the increase of tested time, Burkholderia was the only prey bacteria that could resist the predation of myxobacteria and kept its relative high abundance.[Conclusion] Myxobacteria predation could regulate the microbial community structure in the microcosm, which lays a theoretical foundation for applying in soil-ecosystem regulation in the future.

Abstract:[Objective] Mutations or abnormal expressions of START family proteins in mammals are the basis for diseases such as adrenal hyperplasia, breast cancer and colon cancer. START family proteins are key regulators of plant development. The mechanism of the essential START proteins in prokaryote is unclear. Rv0164 belongs to the START family and is an essential gene with unknown function in Mycobacterium tuberculosis. Therefore, exploring the function of Rv0164 will add a new theory to the molecular mechanisms of START family.[Methods] Bioinformatics method was used to analyze the sequence of Rv0164. Rv0164 was expressed in the model strain Mycobacterium smegmatis and analyzed the protein localization. The interaction proteins of Rv0164 were identified by Co-IP and mass spectrometry, then verified by yeast two hybrid and pull down.[Results] The N-terminal 17 residues of Rv0164 were not conserved in Mycobacterium. Rv0164 had no signal peptide. Rv0164 was localized in cytoplasm and regulated by some proteostasis mechanisms which were weaker in stationary phase than in logarithmic phase. Deletion of the N-terminus of Rv0164 produced protein instability in both stationary phase and logarithmic phase. Rv0164 interacted with several cytosolic proteins.[Conclusion] The N-terminus of Rv0164 enhances protein stability. Rv0164 localizes in cytoplasm and can bind essential proteins.

Abstract:[Objective] To reveal the functions of type III secretion system (T3SS) of peanut Bradyrhizobium sp. MM6 interacting with different host plants.[Methods] We analyzed its structural characteristics of T3SS gene clusters by BLASTn in NCBI related database, and constructed the T3SS regulatory gene ttsI mutant by homologous single exchange. We also compared the differences of symbiotic phenotype between the MM6 and its ttsI mutant by nodulating experiment.[Results] The T3SS gene clusters of MM6 included 3 regions containing 10 conserved structural genes and 8 effectors. The similarity was 83% to 93% with B. diazoefficiens USDA110 corresponding genes sequence. The total nodule number and fresh root nodule weight significantly increased in the ttsI mutant nodulating peanut and wild soybean. In soybean Zhonghuang 57, the MM6 formed red effective root nodules, whereas the ttsI mutant only formed 1 empty nodule, and the plant leaves turned yellow, and the dry weight of aboveground part was significantly lower than that of the wild type strain.[Conclusion] The T3SS of MM6 played a negative role in the symbiosis system of MM6 with peanut and wild soybean, whereas it played a positive role when nodulated with the soybean Zhonghuang 57.

Abstract:[Objective] To analyze the dominant serotype, class I integrons gene cassette, drug resistance characteristics and phylogenetic of Escherichia coli isolated from four large-scale healthy dairy farms in Shandong Province.[Methods] We collected 194 samples of fresh feces from dairy cows in 4 large-scale dairy farms. The isolation and identification of E. coli was tested by using laboratory method, and the serotype was identified by the E. coli diagnostic serums commonly used. The hemolysis of E. coli was detected by 10% sheep blood plate while the sensitivity of 14 conventional antibiotics was detected by K-B method and the ST type of E. coli was analyzed by MLST technique and the cloning relationship among the strains was analyzed by eBURST v3 software.[Results] The results showed that 171 strains of E. coli were isolated from 194 fresh feces samples, mainly pathogenic E. coli (19.9%) and invasive E. coli (17.0%), and the dominant serotypes were O128:K67 (12/171) and O143:K7 (12/171). In addition, the positive rate of hemolytic E. coli was 9.4% (16/171) and the drug susceptibility test showed that the ratio of multi-drug resistant strains was 22.2%, among which the highest resistance rate to ampicillin was 33.9%, followed by tetracycline, which was 24.0%. The results of PCR detection of drug resistance genes and integrons showed that 59.1% of the strains carried the β-lactam resistance gene bla_TEM, 59.1% of the strains carried the aminoglycoside resistance gene ant(2'), and no tetracycline resistance gene (tetA and tetB) was detected. The positive rate of integron I was 4.1% (7/171), dfrA12-aadA2-sul1 was the dominant gene cassette structure (4/171); MLST divided E. coli into 8 ST types, of which ST155 (10/171) and ST58 (45/171) formed a clonal complex and no new ST type was found.[Conclusion] This study confirmed that the dominant serotypes of E. coli isolated from fresh feces in large-scale healthy dairy farms in the region were O128:K67 and O143:K7. Some E. coli were hemolytic and the resistance rate was only high in ampicillin and tetracycline. Moreover, the dominant gene cassette structure is dfrA12-aadA2-sul1. The MLST classification showed that the isolates from different dairy farms were closely related, indicating that the distribution of E. coli in cattle origin was polymorphic, and there was no correlation between serotype and ST type. There is a multi-drug resistance phenomenon in E. coli from dairy cows which is potential dangers of food public safety. This research has certain theoretical guiding significance for improving the safe production and quality evaluation of dairy products in large-scale dairy farms.

Abstract:[Objective] To study the lipid metabolism mechanism regulated by Ubiquitin-conjugating Enzyme (E2) CrUBC23 in Chlamydomonas reinhardtii.[Methods] The mRNA levels of ubiquitin-conjugating enzyme CrUBC23 were analyzed by qRT-PCR under low nitrogen and low phosphorus conditions in C. reinhardtii. Fragments for RNAi interference and over-expression of CrUBC23 gene were cloned, and then the RNA interference vector and over-expression vector were constructed and transformed into C. reinhardtii. The biomass and lipid content of the transgenic strain was detected. The fusion expression vector CrUBC23-GFP was constructed also, and the subcellular localization of onion epidermal cells was carried out by using Agrobacterium tumefaciens.[Results] The level of RNA in C. reinhardtii were increased significantly under low nitrogen and low phosphorus conditions, with an increase of 4.98-5.80 times and 1.85-5.20 times as much as that in normal culture, respectively. RNA interference results showed that the neutral lipid content and total lipid content of transgenic algae decreased by 5.5% and 3.16%-17.6%. While, over-expression showed that the neutral lipid content increased by 8.8% and the total lipid content increased by 4.51%-14.03%.[Conclusion] CrUBC23 is a positive regulator in lipid metabolism, and may locate in the nucleus.

Abstract:[Objective] We studied the immunoreactivity of Mycoplasma synoviae lipoprotein P80 (MS P80) and applied it to detect ELISA antibody.[Methods] Firstly, we performed bioinformatics analysis, prokaryotic expression and purification on MS P80, and analyzed its immunoreactivity with 6 positive sera against different MS isolates and other avian pathogens by Western blotting. Then we used the purified rMS P80 as coating antigen to establish an indirect ELISA assay for detection of MS antibodies, and tested its specificity, sensitivity, reproducibility and coincidence rates compared with US IDEXX ELISA kit.[Results] The MS P80 protein was predicted as a lipoprotein that shared 98%-100% homology between different MS strains and 25% to 34% homology with other species. Western blotting analysis proved that rMS P80 protein had good immunoreactivity and specificity. An indirect ELISA assay based on rMS P80 was established and showed no cross-reaction with positive sera against other pathogens. The intra- and inter-assay variation coefficients were less than 5% and 10%, respectively. When compared with the commercial kit, the established ELISA assay was more sensitive, and the total coincidence rate was 86%.[Conclusion] MS P80 has good immunoreactivity, intraspecies conservation and interspecies specificity. Therefore, it can be used as a target antigen for MS antibody detection.

Abstract:[Objective] To understand the intraspecific genetic differentiation of mitochondrial genomes (mitogenomes) in Beauveria bassiana.[Methods] Mitogenomes of six isolates of B. bassiana were downloaded from GenBank. Composition and structure of these mitogenomes, nucleotide variations at exonic, intronic and intergenic regions were compared. Phylogenetic relationship of these isolates was analyzed based on common core protein-coding genes.[Results] The mitogenome size of different isolates of B. bassiana ranged from 28.8 to 32.3 kb. There were 14 common core protein-coding genes, 2 rRNA genes and 25 tRNA genes in all of these mitogenomes, showing a strong collinear relationship. However, the number of introns in different isolates varied (2 to 5 introns per isolate). Intron insertion/deletion polymorphism was found in cox1, cox2 and nad1, as the main factor contributing to variations of mitogenome size. Based on nucleotide variations at exonic, intronic and intergenic regions, the relative variability at intronic and intergenic regions was larger than that at exonic regions. Phylogenetic analysis revealed that these isolates of B. bassiana grouped together with a high support value, and isolates with identical intron distribution tended to cluster closely.[Conclusion] B. bassiana shows genetic differentiation in its mitogenome due to intron number variations, indel and single nucleotide polymorphism. Our data provide new evidence for understanding the differentiation of mitogenomes in fungal species.

Abstract:[Objective] This study aims to explore the effect of environmental factors on nitrogen-related microbial functional community structure of bioretention systems under the condition of drying-rewetting alternation.[Methods] Soil samples were collected from spatial distribution (planting layer, submerged layer) in vegetated and un-vegetated bioretention systems after drying and rewetting. The amoA and nirS functional genes were used as molecular marker. The diversity and community structure of nitrifying and denitrifying bacteria in different soil samples were studied by high-throughput sequencing technology named Illumina MiSeq PE300. Canonical correlation analysis (CCA) and redundancy analysis (RDA) were performed to assess the relationship between nitrogen-related microbial functional community structure and environmental factors was analyzed.[Results] The functional genes of microbial populations showed significant spatial differences. Nitrogen-related functional bacteria in the planting layer were more abundant and diverse than which in the submerged layer. However, the differences in proportion of operational taxonomic units (OTUs) between the two functional genes in planting layer and submerged layer were increased by rewetting. The results from the community composition analysis of nitrogen-related functional microbes showed that the dominant phylum of amoA-nitrifying and nirS-denitrifying bacteria were the Proteobacteria in all the soil samples. The root system of plants had no significant effect on the diversity index of nitrogen-related functional microbes, but in the plant system layer, the denitrifying bacteria species (at the genus level) were more than that in the submerged layer, and the opposite trend were showed in the plant-free system. The analysis of CCA and RDA showed that soil spatial distribution was the most important environmental factor on the distribution of nitrifying and denitrifying microbial communities.[Conclusion] The nitrogen-related functional microbial community in bioretention system under drying and rewetting alternation was controlled by the soil spatial distribution, water content and plant roots. However, the underlying reasons still await further investigation.

Abstract:[Objective] The aim of this study was to design a pair of absolute quantitative specific primers for detecting the number of the low content Bifidobacterium in different samples.[Methods] The complete genome sequence of 57 strains of Bifidobacterium was downloaded from NCBI, and the specific primers of Bifidobacterium were designed based on their common single-copy core genes. Primers were screened by PCR and rescreened by specificity. Then ddPCR (Droplet Digital PCR) was used to verify the specificity, sensitivity and practicability of the selected primers.[Results] The specificity of Bif-D-9 primer was best, and 4 strains Bifidobacterium were amplified but 20 strains of non-Bifidobacterium could not be amplified. The diluted DNA was quantitatively by ddPCR, and the amplification results show a linear decreasing trend, which proves that the sensitivity is better. At the same time, Bif-D-9 combined with ddPCR can quantitatively determine the copy number of Bifidobacterium in infant feces is 71 copies/μL, and the maternal feces contained 2.7 copies/μL, which proves the practicality is good.[Conclusion] The primer Bif-D-9 was high specificity, high sensitivity and accuracy of Bifidobacterium, and is suitable for the quantitative study of Bifidobacterium in complex environmental samples.

Abstract:[Objective] In this study, the difference of female and male Populus cathayana phyllosphere microbial community structure, and their key environmetal factors in driving the differences were investigated.[Methods] The natural Populus cathayana forest of Xiaowutai Mountain in Hebei province was chosen to explore the phyllosphere bacterial and fungal community composition of female and male Populus cathayana using 16S rRNA/ITS1 gene-based MiSeq sequencing, and their relationships with leaves physicochemical properties.[Results] The microbial diversity indexes showed no significant difference between female and male phyllosphere microbial communities (P>0.05). Significant differences in the relative abundance of bacterial genera Amnibacterium and Spingomonas (P<0.05), and fungal genera Aureobasidium, Elmerina, Exobasidium, Endoconidioma, Monilinia and Rhodotorula were detected between sexes by Metastats analysis. The microbiota analysis based on OTUs showed that both male and female of Populus cathayana had their unique phyllosphere bacteria and fungi microbiota, such as fungi Pringsheimia (0.15%) and bacteria Chitinophaga (0.04%) in the female phyllosphere. Redundancy analysis indicated the leaves moisture content significantly influenced phyllosphere fungal community structure of Populus cathayana (P<0.05), while no significant correlation was found between the phyllosphere bacterial community structure and the leaves physicochemical properties.[Conclusion] There were significant differences in relative abundance of phyllosphere bacterial and fungal at genus level between female and male Populus cathayana, and the structure of phyllosphere microbial community of different plants might be affected by the leaves physicochemical properties, which provided insight for exploring the differences of phyllosphere microorganism between dioecious plants.

Abstract:[Objective] This study aims to investigate the roles of ferredoxin and cystathionine γ-lyase genes located in cysteine metabolic pathway of Clostridium acetobutylicum.[Methods] ClosTron technology is used to inactivate target genes to obtain mutants. The mutants, cultured in the phosphate-limited medium, are maintained at acidogenesis and solventogenesis phase at controlled pH under continuous fermentation condition. The OD and glucose uptake of wild type and mutants are determined.[Results] The Δfer and ΔmccB mutants are obtained by using ClosTron system. The Δfer mutant cannot use sulfate as the sole sulfur source, but it will recover growth by adding sulfite or cysteine in batch fermentation. Moreover, cysteine is harmful to the growth of C. acetobutylicum and the Δfer mutant as the sole sulfur source, and it mainly influences the growth in solventogenic phase. The ΔmccB mutant also grows poorly during acidogenic and solventogenic phases with methionine or cysteine as the sole sulfur source.[Conclusion] Cysteine is a key sulfur-containing compound in the metabolic pathway of C. acetobutylicum and its metabolism is tightly controlled. Ferredoxin participates in its biosynthesis through reduction reactions of sulfate to sulfite; while the cystathionine γ-lyase participates in another cysteine biosynthesis pathway from methionine to cysteine, which is not essential.

Abstract:[Objective] The aim of this study was to isolate and identify plant pathogenic fungal strains with good herbicidal activity and to isolate active metabolites, for the development of new biological herbicides.[Methods] Phytopathogenic fungi were isolated and purified by solid medium inoculation. The targeted strain was identified by morphological observation and 5.8S rDNA sequencing analysis. The active components were separated and purified after screening and tracing, and the structure of the active monomer compounds was determined by spectral analysis. The herbicidal activity of active metabolite and its safety to common crops were determined by Petri dish biological analysis.[Results] Tea pathogen CY-H had good herbicidal activity. The inhibition rates of fermentation broth on radical growth of Echinochloa crusgalli and Amaranthus retroflexus were 94.6% and 77.3% respectively. CY-H was identified as Diaporthe sp.. The single compound CY1 was isolated from CY-H and identified as cytosporaphenones C. At the concentration of 100 μg/mL, CY1 had a good herbicidal activity against the A. retroflexus, with the inhibition rates of 57.1%, and it had a good safety against Triticum aestivum and Brassica napus with the inhibition rate of about 20%.[Conclusion] The tea pathogen CY-H could be potentially developed as a microbial herbicide.

Abstract:[Objective] Root hemiparasites should be taken into account as another biotic stress of grasses as they take directly water and nutrients from their hosts through haustoria. Cool-seasonal grasses have established mutualistic symbioses with Epichloë endophytes:the grass-Epichloë symbionts perform better than endophyte-free plants in biotic or abiotic stresses. However, few studies have considered the benefits of Epichloë endophyte on the physiological characteristics of host grasses under parasitic stress.[Methods] We performed a manipulated pot experiment with endophyte-infected (E+) and endophyte-free (E-) Stipa purpurea. Host grasses were planted with different densities of the hemiparasitic plant Pedicularis kansuensis. The antioxidant enzymes, osmotic materials and root vitality of S. purpurea were determined during different parasitising periods.[Results] The presence of P. kansuensis increased the malondialdehyde (MDA) and proline content and activities of antioxidant enzymes but decreased the root vitality of S. purpurea, and the physiological parameters of S. purpurea parasitised by high hemiparasite density were higher than low hemiparasite density. Meanwhile, activities of antioxidant enzymes, proline content and root vitality of E+ S. purpurea were significantly higher than in E-counterparts; in contrast, the MDA content of E+ plants was lower than E-counterparts.[Conclusion] Our results suggest that Epichloë endophyte can improve S. purpurea tolerance to parasitic stress by increasing activities of antioxidant enzymes, adjusting cell membrane permeability and strengthening the root vitality of host plants. Consequently, endophyte-infected S. purpurea may be a potential candidate as a biological method for effective and sustainable management of the hemiparasite weed-P. kansuensis.

Abstract:[Objective] In order to study the endophytic bacterial community structure and diversity of tobacco seeds.[Methods] The V3-V4 region of endophytic bacteria 16S rRNA gene of three varieties tobacco seeds was amplified, and the amplified fragments were sequenced using Illumina MiSeq high-throughput sequencing technology. The endophytic bacterial community structure and diversity of three varieties tobacco seeds were analyzed.[Results] We obtained a total of 128558 high-quality sequences in V3-V4 region from three varieties seeds, and the Shannon indexes varied in the range from 2.03 to 3.73. The endophytic bacterial community diversity indexes of K326 and Yunyan 85 were both higher than that of Yunyan 87. Proteobacteria, Actinobacteria, Firmicute and Bacteroidete were the dominant bacterial phylum of endophytic bacteria of three varieties tobacco seeds. The total number of endophytic bacteria genus in three varieties tobacco seeds was 27. Pseudomonas was the most dominant genera of endophytic bacteria of K326 and Yunyan 85, Escherichia-shigella was the most dominant genera of endophytic bacteria of Yunyan 87. 16S function prediction revealed that a large amount of beneficial function information about synthesis of proteins, nucleotides, sugars, coenzymes and metabolites showed higher abundance in tobacco seeds.[Conclusion] The diversity of endophytic bacteria of tobacco seeds was rich, the composition was basically similar, but their abundances showed some differences. Potential beneficial bacteria presented in seeds included Pseudomonas, Paenibacillus, Rhizobium, Massilia, Luteimonas, Salana and Lelliottia, which have a large number of beneficial metabolism-related functions. These research results could provide some reference information for the functional research and utilization of tobacco seed endophyte and biological control of seed diseases.