Abstract:[Background] Bottleneck problems such as poor yield and low productivity had been hindering the progress in commercial production of polymalic acid from glucose by Aureobasidium pullulans. [Objective] To enhance polymalic acid yield and productivity from Aureobasidium pullulans BK-10 by optimizing the fermentation medium and conditions. [Methods] Single-factor experiment, Plackett-Burman design and orthogonal experiment were adopted to evaluate the importance of various fermentation medium ingredients and optimize kinds of fermentation conditions. A 5 L fermentor experiment was conducted to verify the above optimized conditions. [Results] The optimum fermentation medium consisted of 100 g/L glucose, 1.5 g/L urea, 0.20 g/L KH2PO4, 0.20 g/L ZnSO4, 0.05 g/L MgSO4, 0.75 g/L KCl, 30 g/L CaCO3, 0.01% Tween-80, the optimal fermentation temperature was found 26 °C and the optimal working volume was 50 mL of fermentation medium in a 250 mL flask. [Conclusion] Yield of polymalic acid from glucose reached 0.71 g/g with an increase of 18.33%, and the productivity was 0.89 g/(L·h) which was increased by 71.15% as compared with the unoptimized conditions. These results show great economical potential for commercial production of polymalic acid even or L-malic acid by fermentation from glucose.

Abstract:[Background] In addition to the general function of lactic acid bacteria, Bacillus coagulans have strong biological properties of resistance to acid, bile salts, heat and easy storage. [Objective] Screen a strain of Bacillus coagulans from pickle to apply for the preparation of probiotics and optimize its sporulation rate as a guide for further industrial production. [Methods] Bacillus with good antibacterial effect screened by specific culture conditions, are identified by specific primers, 16S rRNA gene sequence analysis and physiological & biochemical experiments. The single-factor and orthogonal experiments are used to optimize the spore-producing conditions. [Results] Bacillus coagulans BC01 with a strong inhibitory effect to Escherichia coli CVCC 1527, Salmonella typhimurium CVCC 2228, Clostridium perfringens CVCC 46 and Salmonella choleraesuis CVCC 503 was screened. Its survival rate under 120 min of gastric acid treatment is up to 94%. 84.3% after 6 h in the 0.3% bile salt solution. The best medium is obtained by single factor and orthogonal experiment optimization. The optimized formula includes, molasses 10.0 g/L, yeast leaching powder 20.0 g/L, NaCl 5.0 g/L, K2HPO4 5.0 g/L and MnSO4 10.0 mg/L. The optimum culture conditions are as follows, inoculation quantity 4%, temperature 45 °C, initial pH 7.0, rotational speed 200 r/min, culture time 36 h. Under the conditions, the number of viable cells up to 6.7×109 CFU/mL and the rate of sporulation 89.2%. [Conclusion] Bacillus coagulans BC01 for microecological preparation was screened. The sporulation rate of Bacillus coagulans BC01 was optimized. It will lay down a foundation for industrial scale production of Bacillus coagulans.

Abstract:[Background] Nujiang Prefecture (Nujiang Grand Canyon), which possess unique geographic conditions and abundant flora and fauna diversity, making great value to study its biodiversity. However, little research have been done to investigate the actinomycetal diversity in this area. [Objective] Research the diversity of soil actinomycetes in Nujiang Grand Canyon. [Methods] High throughput sequencing (Illumina HiSeq platform) was employed to investigate the actinomycetal culture-independent diversity of soil samples. We used several bioinformational figures to making comparison of the differences in actinomycetes composition structure among the seven samples. Meanwhile, the suited culture-dependent isolation methods were determined by tested different pre-treatment methods, undesired bacteria inhibitors and multiple isolation media. [Results] A total of 474 203 bacteria genes and 15 671 OTUs were obtained, the results distributed in 47 phyla, 90 classes, 170 orders, 320 families, 561 genera. Specific to the actinomycetal phylum, high throughput sequencing detected 9 classes, 53 families, 80 genera. The analysis of the diversity between different samples showed that the samples, which called BS, F-G, F-L, L, exhibited better condition than BB，BT，BH. The results showed that 351 actinomycetal strains were obtained from seven soil samples. It belonged to 8 orders, 14 families, 26 genera. YIM171 medium showed the greatest isolation results. The sample of BH exhibited the most rich actinomycetal diversity obviously. [Conclusion] Abundant actinomycetal diversity in this area was mined out by this research. Further bioactivity potential should be tested to offer much more actinomycetal strains for the exploitation of secondary metabolite.

Abstract:[Background] Myxobacteria are a group of higher prokaryotes with social behavior. They have great research value in screening natural drugs because their metabolites have rich, diverse and novel biological activities. [Objective] We studied the diversity of myxobacteria in Daxing’an Mountains of Inner Mongolia, and isolated and purified the culturable myxobacteria, and studied their antimicrobial activities and screened strains against Phytophthora infestans. [Methods] The diversity of myxobacteria was studied by PCR-denaturing gradient gel electrophoresis (DGGE). Culturable myxobacteria were isolated from 15 soil samples collected from Daxing’an Mountains by rabbit dung pellets inducing method, Escherichia coli inducing method, and filter paper inducing method. The purified strains were identified by morphological observation, physiological and biochemical characteristics, and the 16S rRNA gene sequence analysis. Then, the antimicrobial activities of the strains were determined by plate confrontation method. [Results] The results of DGGE showed that 13 genera of myxobacteria were identified from 15 soil samples, covering most of the known genera of myxobacteria and some of the unclassified myxobacteria. 88 strains were isolated in this study and 22 of them were purified. The purified strains belonged to two genera, Myxococcus and Corallococcus, and 8 species, Myxococcus xanthus, Myxococcus flavescens, Myxococcus virescens, Myxococcus coralloides, Myxococcus stipitatus, Corallococcus macrosporus, Corallococcus exiguous, Corallococcus coralloides. All of the purified myxobacterial strains exhibited one or more kinds of antimicrobial activities. 19 strains had the activity against E. coli. 14 strains had the activity against Phytophthora infestans. Eight strains showed the activity against Staphylococcus aureus. 13 strains had the activity against Saccharomyces cerevisiae. Seven strains showed the activity against Bacillus subtilis. [Conclusion] Myxobacterial resource in Daxing’an Mountains of Inner Mongolia is rich. Myxococcus and Corallococcus could be the dominant populations of myxobacterial community in this region. The purified myxobacterial strains exhibited one or more kinds of antimicrobial activities and 64% of them have the activity against Phytophthora infestans, which has the potential value for further research in the future.

Abstract:[Background] The culturable rate of environmental bacteria is only about 1%, and the exchange of information among bacteria is an important reason for limiting the rate of culture. Cyclic adenosine monophosphate (cAMP), the second messenger known as intercellular information transmission, is a widely-used substance for bacteria to perceive and respond to the environmental changes. [Objective] The study is aimed to research the changes of bacterial population composition induced by cAMP and investigate the effect on microbial culturability, further to provide reference for improving the culturability of bacteria and the exploration of strain resources. [Methods] Using the high-throughput sequencing and isolation methods to analyze the bacterial diversity in sediment of Taihu Lake. [Results] Results of high-throughput sequencing showed that 60 OTUs (Operational taxonomic units) which were different from the control group appeared. Among them, Proteobacteria and Bacteroides were the significant differences, the former increased by 4.08%, but the latter decreased by 3.36%, and Verrucomicrobia was high significant at p<0.01. At the genus level, there were 8 significant different genera, and 2 (Aeromonas and Trichococcus) very significant statistic differences. Results of isolation and culture showed that, under cAMP’s effect, the population diversity of the bacteria could be increased, with CFU (Colony-forming units) up to 1.6 times as that of the control group, and the bacteria of Exiquobacterium, Microbacterium, Thermomonas, Lacibacter, Pedobacter, Massilia, Kocuria and Arthrobacter were induced. [Conclusion] From high throughput results, cAMP can significantly promote the growth of strains of Proteobacteria, but the results from isolation and culture show that cAMP can improve the culturability of Exiquobacterium, Microbacterium, Thermomonas, Lacibacter, Pedobacter, Massilia, Kocuria and Arthrobacter.

Abstract:[Background] Desertification is a major environmental problem, biological soil crusts (BSCs) can inhibit desertification, and the diazotrophs play an important role in the formation and development of BSCs. However, the community structure and diversity of diazotrophs in BSCs are not clear enough. [Objective] To clarify the community structure and diversity of diazotrophs in different types of biological soil crusts (BSCs) and soils under them and the effectors of soil environmental factors. [Methods] Using dilution heat method and alkali solution diffusion to measure the content of organic matter (OM) and the available nitrogen (AN) of soil, respectively; nifH gene was sequenced via the high-throughput sequencing platform, and the community structure, diversity and the variance analysis of diazotrophs were analyzed by bioinformatics analysis; Canonical Correlation Analysis (CCA) were used to analyze the correlation among the community structure, samples and the physico-chemical parameters of soil. [Results] Dominant phylum were Proteobacteria and Cyanobacteria in moss crusts (HSM), Cyanobacteria in the other types of BSCs; the class of Alphaproteobacteria and Betaproteobacteria accounted for the most fraction in the soil underneath moss crusts (nifH was only detected in soil underneath moss crusts, HSMs); significant differences existed in community structure at genus level, algae crusts (HSA) was predominated by unclassified_f_Nostocaceae (90.99%), Lichen crusts (HSL) by Scytonema (45.85%) and unclassified_f_Nostocaceae (44.14%), HSM by unclassified_f_Nostocaceae (29.21%), Scytonema (22.57%), Nostoc (15.34%), Skermanella (14.74%), unclassified_o_Nostocales (10.60%), and HSMs by Skermanella (33.80%), Azohydromonas (25.66%), unclassified_p_Proteobacteria (18.20%) and unclassified_c_Alphaproteobacteria (10.62%). [Conclusion] The community structure and diversity of diazotrophs in Algae, Lichen and Moss crusts and soil underneath crusts were markedly different, and the community species and the diversity increased with the development of the BSCs. This study provides a basis for appreciation and utilization of the diazotrophs in the BSCs.

Abstract:[Background] Plateau lakes are a type of special environments due to the conditions of high altitude, low air pressure, high ultraviolet radiation and lower oxygen content; while microorganisms in the plateau lakes are important participants in substance circulation and energy flow of lake ecosystems, and the situations/performances of their extracellular enzymatic activities formulate their adaptation ways and abilities to the special environment. [Objective] To screen yeasts with extracellular enzymatic activities, which were isolated from Fuxian Lake and Xingyun Lake in Yunnan Plateau, and to obtain active yeasts with potential application values. [Methods] Yeasts exhibiting activities of extracellular proteinase, cellulase, amylase, lipase, chitinase, xylanase, phytase, inulase, laccase, manganese-dependent peroxidase and lignin peroxidase were detected by using screening plate method at 5 °C and 25 °C. [Results] All yeast strains exhibited at least one extracellular enzymatic activity, mainly exhibited activities of phytase, inulase and amylase, secondly exhibited activities of lipase, cellulase, xylanas and manganese-dependent peroxidase and lignin peroxidase. The number of strains exhibited activities of chitinase, proteinase and laccase activities were lower, and the yeast strains isolated from Xingyun Lake did not exhibited laccase activity. The number of yeast strains exhibited at least five extracellular enzymatic activities was higher at 5 °C than that at 25 °C. [Conclusion] The rich diversity of yeasts with extracellular enzymatic activities exists in the two lakes, and hence these yeasts might participate in the circulation of substances in the plateau lake ecosystems. For further exploitation and utilization of extracellular enzymes, these active yeasts can be used as the resources of extracellular enzymes, and it is worth to further explore their application potential.

Abstract:[Background] Cardamine hupingshanensis is a novel selenium (Se)-hyperaccumulating plant in China with the predominate Se species as selenocystine (SeCys2), which is different with other Se-hyperaccumulating plants in USA, eg. Astragalus bisulcatus, Stanleya pinnata. [Objective] To explore the role of microorganism during Se-hyperaccumulation in Cardamine hupingshanensis, isolation, identification and Se metabolism in vitro of a Se-tolerant endophyte from C. hupingshanensis were conducted. [Methods] A Se-tolerant endophyte was isolated and purified from fresh leaves of C. hupingshanensis as CSN-1. The physiological and biochemical analysis and 16S rRNA gene sequence analysis were carried out. Moreover, the endophyte CSN-1 was cultured in selenite medium to study its Se metabolism. [Results] The Se-tolerant endophyte CSN-1 was identified as Bacillus methylotrophicus. The selenite culture revealed that the medium absorbance was higher than that in controls under 1.5 mg Se/L medium, but lower under 10 mg Se/L medium. The metabolized Se speciation was Se4+ in supernants, but SeCys2 in bacteria deposits. [Conclusion] The Se-tolerant endophyte Bacillus methylotrophicus CSN-1 presented in Se-hyperaccumulating plant, Cardamine hupingshanensis, and could transform selenite into SeCys2. The low concentration of Se could promote the growth of Bacillus methylotrophicu CSN-1, but negative effects could be observed at high Se levels.

Abstract:[Background] This project is based on the methods of detection and identification of Alicyclobacillus in our laboratory. We hope to establish a good economic value and practical value, more convenient, fast, accurate, specific and sensitive detection methods. [Objective] In order to detect and identify Alicyclibacillus rapidly in raw materials or finished products in fruit juice production enterprises. [Methods] We immunized BALB/c mice with A. acidoterrestris (ATCC49025) and established indirect ELISA to screen hybridoma cells. using hybridoma technology, we obtained 3 hybridoma cell lines that could stably secrete A. acidoterrestris antibody. Two of the three were IgG1 and their biological characteristics were identified. [Results] Two monoclonal antibodies 3F7 and 9C4 had different antigenic sites and the stability remained unchanged after multiple passages. Specificity experiments showed that the two monoclonal antibodies did not cross react with A. acidocaldarius (NCIMB11725), Bacillus cereus (ATCC11778), Bacillus subtilis (ATCC11774) and A. cycloheptanicus (ATCC49029). [Conclusion] These two monoclonal antibodies can be further used in the development of colloidal gold test strips.

Abstract:[Background] Pamamycins are macrolid compounds with anti-infectin bioactivities. Their unique structural features and promising biological activity stimulated extensive efforts towards their various investigations. Furtehermore, we discovered that the biosynthetic gene cluster of pamamycins was located in the chromosome of Streptomyces lincolnensis NRRL 2936. [Objective] The biosynthetic pathway had been investigated, and the function of two regulatory genes in the gene cluster was elusive. In this research, the plasmid pJQK450 containing a full length of pamamycin biosynthetic gene cluster was cloned from the genomic library of S. lincolnensis NRRL 2936 and introduced into heterologous hosts by intergeneric conjugation. Subsequently, the function of two regulatory genes was elucidated by targeted gene disruption and complementaion. [Methods] The Fosmid, named pJQK450, containing of the completed pamamycin gene cluster was captured by narrow-down polymerase chain reaction strategy from Streptomyces lincolnensis NRRL 2936 Fosmid genome library. Plasmid pJQK450 was transferred into the E. coli ET12567/pUZ8002 and then introduced into the heterologous hosts by intergeneric conjugation. Then, the validated-exconjugants were fermented, and the biosynthetic capacity of pamamycin of the recombinant strains was evaluated by bioactivity analysis against Mycobacteriun smegmatis mc2155 and LC-MS detection. Furthermore, the regulation roles of PamR1 and PamR2 were determined by targeted gene inactivation and complementation experiments. [Results] The entire gene cluster of pamamycin was successful expressed in S. coelicolor M1154. Through genetic validation, both PamR1 and PamR2 were found to be negative regulators in pamamycin biosynthesis. [Conclusion] Heterologous expression of pamamycin facilitates the biosynthesis engineering and targeted accumulating pamamycin component. Additionally, the determination of the two negative regulatory genes paved way for the yield improvement of pamamycin.

Abstract:[Background] Escherichia coli topoisomerase I (E. coli TopA) plays essential role in DNA replication, transcription, recombination and regulation of gene expression. It is active only if it is bound with zinc. However, it remains unknown if it could bind other metals, especially heavy metals. [Objective] This study is aimed to characterize the binding activity of E. coli TopA to some toxic heavy metals in the environment, and to verify the potential effect of heavy metals on the topoisomerase activity of E. coli TopA. [Methods] We expressed and purified E. coli TopA from E. coli cells grown in M9 minimal media supplemented with exogenous zinc, cobalt, nickel, cadmium, iron, mercury, arsenic, chromium, lead or copper separately. The metal contents of purified TopA proteins were determined by ICP-MS (Inductively coupled plasma mass spectrometry). By using E. coli TopA mutants in which the zinc-finger motifs were disrupted, the binding sites of these metals in TopA were identified. The in vitro DNA relaxation assay was conducted to study the topoisomerase activity of E. coli TopA bound with different metals. Finally, intrinsic fluorescences of E. coli TopA bound with different metals were also measured to discover potential conformation diversity among them. [Results] We found that E. coli TopA could maximally bind three atoms of cobalt, nickel or cadmium per protein monomer respectively, while could not bind mercury, arsenic, chromium, lead or copper. The binding sites of these three metals in TopA were just located in the zinc-finger motifs, in which one atom of metal iron was coordinated with each zinc-finger motif respectively. It also showed that the DNA topoisomerase activity of TopA remained active when bound with cobalt, nickel or cadmium. Moreover, intrinsic fluorescence measurement showed that the protein conformation of cobalt, nickel or cadmium bound TopA would be similar as that of zinc bound. [Conclusion] In light of the crucial role of DNA topoisomerase in maintenance of cell viability, these results indicate that the topoisomerase activity of TopA would not be disrupted by common heavy metals (no binding or binding without inhibition of enzyme activity), which could be considered as an evolutionary mechanism for E. coli in resistance to toxicity of heavy metals in environment.

Abstract:[Background] Bacterial wilt was a destructive soil borne disease without effective chemical pesticides to prevent. While antagonistic actinomycetes had many successful application on a variety of plant disease with environmental protection and no residual, which provided a new way for the biological control of bacterial wilt in eggplant. [Objective] We isolate and screen antagonistic actinomycetes strains with inhibitory activity on Ralstonia solanacearum from the rhizosphere soil of healthy eggplants. [Methods] Actinomycete was isolated by Pour Plate method. Antagonistic actinomycete was screened by double agar layer plaque technique, agar diffusion assay and confrontation culture in vitro. Strain XL-6 was identified by morphological, culture, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The fermentation condition was optimized by single factor and orthogonal experiment. [Results] Strain XL-6 showed antagonistic to Ralstonia solanacearum and three other pathogens. Strain XL-6 belonged to Streptomyces rochei. The optimum culture conditions of strain XL-6 was with a medium of 30.0 g cornmeal, 5.0 g yeast powder, 2.0 g K2HPO4, 2.0 g MgCl2, 1.0 g NaCl in 1 000 mL filtrate water at initial pH of 7.0, liquid volume 70 ml in 250 ml flask, 180 r/min at 28 °C, inoculation size of 6% for 6 d. [Conclusion] Strain XL-6 was identified as Streptomyces rochei and showed stronger antagonistic action to Ralstonia solanacearum under optimized fermentation conditions.

Abstract:[Background] Ascosphaera apis is a special fungal pathogen that specially infect honeybee larvae. Currently, information related to gene expression of A. apis during the infection process is limited. [Objective] This study was designed to analyze highly-expressed genes (HEGs) differences between A. apis stressing the 6-day-old larval gut of Apis mellifera ligustica and the pure culture of A. apis, and to explore the gene expression of A. apis during the late stage of stress and the pure culture of A. apis. [Methods] In our study, the 6-day-old larval gut of A. m. ligustica under the stress of A. apis and the pure culture of A. apis were sequenced using RNA-Seq, and the HEGs were obtained based on FPKM value, followed by function prediction and exploration of biological significance via GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment as well as Venn analyses. [Results] A total of 105 447 578 raw reads were produced from RNA-Seq, and 88 466 344 clean reads with a mean Q20 of 97.50% and a mean Q30 of 93.81% were obtained after filtration. GO enrichment analysis showed that the HEGs of AaCK were enriched in 26 GO terms, among them the cell (22 unigenes), cell part (22 unigenes) and metabolism (21 unigenes) were mostly enriched; the HEGs of AamT were enriched in 22 GO terms, and the mostly enriched ones were the catalytic activity (23 unigenes), cell processes (18 unigenes) and metabolic processes (18 unigenes). KEGG pathway enrichment analysis showed that the HEGs of AaCK were involved in 109 pathways, among them the largest group was ribosome (179 unigenes) followed by biosynthesis of amino acids (70 unigenes) and carbon metabolism (62 unigenes); the HEGs of AaCK were involved in 114 pathways, and the mostly enriched one was ribosome (178 unigenes) followed by carbon metabolism (116 unigenes) and oxidative phosphorylation (112 unigenes). Furthermore, Venn analysis suggested that there are 260 shared HEGs, while 2 161 and 4 445 HEGs were specially expressed in AaCK and AamT, respectively. [Conclusion] Findings in the present study can offer the expression profiles of HEGs of A. apis stressing the 6-day-old larval gut of A. m. ligustica and the pure culture of A. apis, reveal the gene expression rules of A. apis before stress and A. apis during the late stage of stress, and provide helpful information for uncovering molecular mechanisms regulating the pathogenesis of A. apis.

Abstract:[Background] Verticillium wilt has seriously restricted the sustainable yield of cotton in Xinjiang, China. Endophyte in cotton (Gossypium hirsutum L.) have great potential for biological control of cotton verticillium Wilt. There are the close relation to cotton verticillium wilt and endophytic microorganisms, but no study has investigated the content of fungal endophyte in different branches and leaves as well as roots of Verticillium Wilt affected cotton. [Objective] To study the spatiotemporal dynamic of endophytic fungi number and the relationship between the number of endophytic fungi and the pathogen of Verticillium Wilt in cotton infected by Verticillium Wilt. [Methods] Primers and probes specific to Internal Transcribed Spacer (ITS) of fungi were designed. A TaqMan probe-based, Real-time fluorescence quantitative PCR was established. Taqman probe real-time fluorescence quantitative PCR method was used to determine the total amount of endophytic fungi in cotton Verticillium Wilt, and to analyze the relationship between the number of endophytic fungi and the pathogen of Verticillium dahlia. [Results] The number of total endophytic fungi in cotton roots infected by Verticillium Wilt showed different trends in different growth period of cotton in Korla, Xinjiang. The maximum number of total endophytic fungi in cotton roots at boll opening stage infected by Verticillium Wilt was 1.46×109 copies/g in Korla cotton root fresh fungi (FRW). The number of total endophytic fungi showed increased slowly in bud stage, and reached the maximum value, 8.30×107 copies/g FRW of cotton roots in boll opening stage. The number of endophytic fungi was the highest in South Xinjiang Korla, and was average up to 1.46×109 copies/g FRW in boll opening period; followed by an average of 8.30×107 copies/g FRW in flower stage; at least by an average of 1.85×104 copies/g FRW in seedling stage, Jinghe. The spatial variation trend of number of endophytic fungi is decreasing in the Southern, Eastern, Northern Xinjiang; the number of endophytic fungi was higher in Korla and Alar, the number of endophytic fungi was the highest in South Xinjiang Korla, and was average up to 1.46×109 copies/g FRW in boll opening period; followed by an average of 8.30×107 copies/g FRW in flower stage; at least by an average of 1.85×104 copies/g FRW in seedling stage, Jinghe. The correlation between number of endophytic fungi and of Verticillium dahliae was significantly positive in cotton infected by Verticillium Wilt in Jinhe, Xinjiang. The correlation index PCC value is as high as 0.639. The correlation between number of endophytic fungi and of V. dahliae was negative correlation in cotton infected by Verticillium Wilt in Shihezi and Hami, Xinjiang. The correlation index PCC value was ?0.180 and ?0.275, respectively. The correlation between number of the other endophytic fungi and of V. dahliae was positive, but the correlation is not significant. [Conclusion] There were numerous endophytic fungi in cotton infected by Verticillium Wilt in Xinjiang. The higher concentration of endophytic fungi was in roots. The number of total endophytic fungi in cotton roots infected by Verticillium Wilt showed different trends in different growth period and sampling sites of cotton in Xinjiang. The maximum value number of total endophytic fungi in cotton roots infected by Verticillium Wilt appeared during the blooming period in Korla, Xinjiang.

Abstract:[Background] The suppurative disease, a bacterial infection caused by Pseudomonas aeruginosa and relatively difficult to prevent, has been a difficult problem in the domestication and breeding of forest musk deer. Currently, no vaccine is available. [Objective] To study the status of infection and molecular epidemiology of P. aeruginosa. [Methods] P. aeruginosa strains were isolated from two forest musk breeding centers in Zhenping County, Shaanxi Province and Baoxing County, Sichuan Province from October 2014 to October 2015, as well as their drug resistance was tested by traditional bacteriological methods. The fingerprint data bank of all strains was established by pulsed field gel electrophoresis (PFGE) typing, PFGE data were analyzed by BioNumerice software to reveal the prevalence pattern, and the pathogenicity of some isolates was analyzed. [Results] In total 60 P. aeruginosa strains were isolated, of which 34 were isolated from Zhenping, and 26 were isolated from Baoxing. The isolated P. aeruginosa strains were resistant to 17 antimicrobial agents at different levels, and the isolates from different area or different samples exhibited similar resistance phenotype. Multi-resistance was observed among the P. aeruginosa isolates, most of them exhibited multidrug-resistance to 5 and 6 antimicrobial agents. PFGE spectrum similarity of the 60 strains was between 49.1% and 100%, and A to O 15 PFGE types were obtained through cluster analysis, C, E, G, J were the dominant types. The virulence experiment on mice (LD50) suggested that the strains from animals were far more virulent than those isolated from the environmental, and the dominant bacterial type (E, F, J gene type) were more virulent than other strains. [Conclusion] There was horizontal transmission path with P. aeruginosa in different areas. The data provide a basis for prevention and treatment of suppurative inflammation in the captive musk deer.

Abstract:[Background] Dietary fiber has been regarded as the seventh nutrients and can be utilized by the microbes in the hindgut of monogastric animals. [Objective] The present study was conducted to investigate the influence of oat β-glucan (a typical soluble dietary fiber) and microcrystalline cellulose (MCC, an insoluble dietary fiber) on the structure and composition of colonic bacteria in BALB/c mice. Results of current study can provide a reference for the formulating of dietary fiber containing feed in animal production and the rational utilization of different types of dietary fibers in human food. [Methods] A total of 27 healthy male BALB/c mice (18.13±0.95 g) at the age of six weeks were selected and randomly allocated to three groups. Mice in the three groups were fed diet containing 20% MCC (the purity≥99%, M), 28% oat β-glucan (the purity is 70%, G), and control diet without fibrous supplement, respectively. The experiment lasted for 21 days. At the end of the experiment, three mice from each group were sacrificed and the colonic digesta of each mouse was collected. The bacterial community of the digesta samples from the three groups were compared using PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) and high-throughput sequencing methods. [Results] PCR-DGGE analysis showed significant differences on the richness and Shannon index of the three groups, of which group G presented lower than groups M and control (P=0.027, 0.035). The cluster analysis showed that there were two samples of each group clustered into separate clade and the similarities of bands for group G, M and control were 71%, 55% and 67%, respectively. The results of high-throughput sequencing showed a significant difference on the bacterial Shannon index and β-diversity among the three groups (P=0.047, 0.035). In all samples, Bacteroidetes, Firmicutes and Proteobacteria were identified as the most three predominant phyla, comprising 95.9% to 99.4% of the total reads. Compared to control group, the relative abundance of phylum Bacteroidetes in group G showed a 26.78% increase, while it showed a 15.62% decrease in group M of those genera in Bacteroidetes, an unclassified genus belonging to family S27_4 and Bacteroides made a maximum contribution to the change of this phylum (P=0.099, 0.051). On the other hand, the relative abundance of Firmicutes in group G showed a 28.99% decrease than control group, while it showed a 15.82% increase in group M, and this change was found mainly due to the change of relative abundance of order Clostridiales, Ruminococcaceae and Lactobacillus (P=0.027, 0.061 and 0.079, respectively). [Conclusion] Therefore, both of the two dietary fibers influenced the bacterial community in the colon of BALB/c mice. The supplement of high-level oat β-glucan in the diet decreased the bacterial diversity in the colon of the mice. Core bacteria groups specifically utilizing the two types of dietary fibers were found in the colon of BALB/c mice. Bacteria belonging to family S27_4 may prefer to utilize plant polysaccharide, such as oat β-glucan, while some bacteria belonging to order Clostridiales may specifically use MCC.

Abstract:[Background] Lactobacillus paraplantarum L-ZS9, which could produce bacteriocin, would be used in food preservative and fermentation. [Objective] To understand expression characters of regulated genes of bacteriocin synthesis under environment stress conditions for Lactobacillus paraplantarum L-ZS9. [Methods] We used high-throughput sequencing technology to sequence its transcriptome. Based on the Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we carried out functional annotation and classification, pathway annotation. [Results] The results suggested that distribution of genes’ coverage is beyond 90% of two samples, the total genes with expression difference were 927, of which 744 were up regulation and 183 down regulation. KEGG pathway analysis showed that 68 out of the 649 genes with different expression levels were classed into ABC transport pathway, accounting for 10.48% and three genes were up regulated beyond 16 times and one was down regulated beyond 1/16. In addition, expression of some orphan genes changed with 16 times up or down-regulation, some even could increase more than 10 000 times. [Conclusion] This study further improved the gene information and laid the foundation of bacteriocin metabolic pathway and stress respond research for Lactobacillus paraplantarum.

Abstract:[Background] Tripterygium is a traditional Chinese medicine with great value of clinical application for its diversity pharmacological activity. However, its lack of output and complicated extraction process limit the clinical application. Secondary metabolites of plant endophytic fungi can produce similar pharmacological activity, which is expected to solve the problem of lack of plant and become a potential resource for screening new drugs. [Objective] To isolate endophytic fungi from tripterygium wilfordii Hook.f., and screen their anti-microbial and anti-tumor activities in vitro. To transform the crude extracts of tripterygium wilfordii Hook.f. with active strains to achive increasing efficiency and reducing toxicity. [Methods] Endophytic fungi were isolated from tripterygium wilfordii Hook.f. with tissue block method; antibacterial activity against Staphylococcus aureus and Escherichia coli was tested with disk diffusion method; anti-tumor activity to two cell lines (MCF-7/SKOV3) was tested with MTT (Thiazolyl blue) assay. The ITS (Internal transcribed spacer) sequence sequencing analysis was used to identify the active strains species. [Results] 43 strains of endophytic fungi were isolated from Tripterygium wilfordii Hook.f. Five of them showed bacteriostasis to Staphylococcus aureus and the activity of LGT7 was similar to that of 1 μg/mL of penicillin sodium; 6 of them showed cytotoxicity to MCF-7 as well as SKOV3 and the growth inhibition ratio of LGT7 and LGT41 was above 90%; 4 of them showed transforming activity to crude extracts from Tripterygium wilfordii. The active strains were identified as Phomopsis sp. and Diaporthe sp.. [Conclusion] Endophytic fungi isolated from Tripterygium wilfordii Hook.f. have anti-microbial and anti-tumor activity in vitro. They can enhance the activity and reduce the toxicity by transformation.

Abstract:[Background] Phellinus baumii is a fungus with a high medicinal value and wide application prospect. The main active ingredient of Phellinus baumii is polysaccharide. Besides, lectin is another physiologically active substance which has the functions of immunomodulation, anti-tumor and antibacterial. [Objective] A novel lectin, SHL24, is isolated and purigied from the liquid fermentation of medicinal fungus Phellinus baumii and its bioactivity is studied. [Methods] Sephadex G-50, DEAE Sephadex A-25, MPLC-MonoQ and LPLC-Sephadex G-75 were used to purify this lectin. The molecular mass was determined by SDS-PAGE and LPLC-Sephadex G-75. The effects of different pH, metal ions, sugars, fermentation times, red blood cells, and temperatures on coagulation activity were studied. [Results] SHL24 is a single-subunit protein with the relative molecular weight of 24 kD. SHL24 can agglutinate red blood cells of mouse and human, while it has a variety of activities for different resources of red blood cells. SHL24 cannot be inhibited by D-mannose, D-maltose, dextrose, cane sugars, D-lactose, arabinose and L-rhamnose. SHL24 has good thermal stability and its coagulation activity isn’t inhibited by cations, such as Ca2+, Mg2+ and Zn2+. [Conclusion] The stable physical-chemical properties and biological activity of SHL24 make it worth to further study on its pharmacological action.

Abstract:[Background] The secreted proteins of Chlamydia trachomatis play important roles in chlamydia interaction with host cell, development cycle and pathogenesis. Our previously study have found that C. trachomatis glycogen synthase (GlgA) to be a newly chlamydia secreted protein, however, the secretion mechanism and the roles of GlgA in chlamydial pathogenesis is still unknown. [Objective] We studied regulatory mechanism of C. trachomatis GlgA protein expression and secretion, to provide experimental basis for studying chlamydia pathogenic mechanism. [Methods] SignalP 4.1 software was amplified to predict GlgA protein N terminal signal peptide. C. trachomatis infected HeLa cells were treated individually with C16 compounds, C1 compounds, or their combination to observe the effect of blocking type II or type III secretion system on GlgA secretion. Novobiocin treatment, plaque-forming assay, as well as shuttle plasmid transformation technique were used to construct either plasmid-free or plasmid-compensate C. trachomatis strains, then the strains were further identified by detecting both the plasmid encoding gene and protein. Indirect immunofluorescence assay was done to evaluate the effects of the plasmid deletion on GlgA protein expression and secretion. [Results] The full length GlgA is predicted to contain no putative signal peptide at its N terminus. Neither C16 nor C1 compounds inhibited GlgA secretion into the cytosol of chlamydia-infected cells. The plasmid-encoded gene pgp7, the plasmid-encoded protein Pgp3, the genome-encoded protein GlgA were all detected in the cultures infected with either wild type serovar D or CTD1-pGFP::SW2 transformant but not the plasmid-free CTD1 organisms. [Conclusion] The expression and secretion of GlgA protein is not dependent on bacteria type II or III secretion system, but closely related to the plasmid of C. trachomatis.

Abstract:[Background] Staphylococcus aureus is an important pathogen, and procoagulation is one of the most important pathogenic mechanism, possibly novel genes in this approach. [Objective] To screen and identify procoagulant genes of Staphylococcus aureus by observing the decreased agglutination of colony phenotype. [Methods] Random mutagenesis library was constructed by using Tn917 transposon system, and mutants would be screened by combining dynamic turbidimetry and tube agglutination. The related genes were identified, and putative function of these genes was predicted through bioinformatics analysis. [Results] A total of 82 mutants were selected in a way of decreased agglutination phenotypes, and insertion sites of 76 mutants were successfully identified with 13 mutated genes, including 4 were reported in previous studies (which is 30.8% of these genes identified). [Conclusion] We acquired related genes based on decreased agglutination of mutants, and this was a new method of identifying potential genes involved in procoagulant ability, at the same time, new agglutination candidates obtained were reserved for further study.

Abstract:Baculoviruses are enveloped viruses with double stranded DNA, and specifically infect invertebrates. It produces two types of virions during the life cycle: budded viruses (BVs) and occlusion-derived viruses (ODVs). Baculouvirus has been used as an efficient eukaryotic expression vector to produce recombinant viruses for the expression of heterologous proteins in insect cells. Furthermore, heterologous proteins displayed on the surface of BVs have been widely used to screen specific targets in the fields of medicine, clinical treatment, biology and so on. Moreover, Baculovirus is unable to proliferate in mammalian cells, so it won’t trigger strong immune response in mammals and won?t cause function and tissue damage. Due to these unique advantages, Baculovirus has been developed as a gene therapy vector with a bright prospect in the concerned fields of oncotherapy, tissue regeneration and targeting drugs delivery. This review summarizes the developments of genetically modified baculoviruses in the fields of protein expression, surface display and gene therapy, which will provide theoretical basis for the improvement and wide application of genetically modified baculoviruses.

Abstract:CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is an acquired immune system found in bacteria and archaea that degrade target sequences of DNA from invading viruses or plasmids in a RNA-guided manner. CRISPR/Cas9 technology modified from type II CRISPR/Cas system has been developed as a strong genome editing and expression regulation tool, and widely used for studying gene function, metabolic engineering, synthetic biology, and other related fields. In this review, we summarize recent progress in development, classification, functional principle, applications and prospects of CRISPR/Cas9 technology in microbes.