Abstract:Abstract:Genetic instability, such as morphological variation, is a common occurrence in Streptomyces. It causes lots of trouble in fermentation industry and research, such as strain degeneration and reduction of antibiotic productivity. The relationship between genetic instability and chromosome instability in Streptomyces has been well studied. It is very important to reveal the molecular mechanism of genetic instability for strain improvement and for construction of stable overproducing strains. In this review, the present status and prospect on the study of the genetic instability in Streptomyces are presented.

Abstract:Abstract: Phage endolysin targets the integrate cell wall and attack bonds in the peptidoglycan, resulting in degradation of bacteria. It features two or three domain structures, involving one or two catalytic domains and one binding domain. Endolysin is a promising antibiotic agent against gram-positive bacteria pathogen, such as Streptococcus pneumonia, Streptococcus pyogenes and Staphylococcus aureus. Compared to antibiotics, it is more specific and tested bacteria show no resistance to lysin. Therefore, it's a feasible measure for solving drug resistant problem. Beyond this, it is highly active and rapid lysis efficiency and has synergy effect when used together or with other antibiotics. Antibody against endolysin will not neutralize its activity. So endolysin treatment may be a new approach for preventing and controlling of bacteria pathogen.

Abstract:Abstract: The alternative sigma factor Sigma B plays important roles in both virulence and stress tolerance in Listeria monocytogenes. It is now clear that there is a strong link between the virulence potential of Listeria monocytogenes and its ability to tolerate stress. Several studies have identified genes that play important roles in stress tolerance and virulence. For example, genes involved in osmotic stress tolerance, acid and alkaline tolerance, oxidative stress tolerance, extreme temperature tolerance, and bile salt tolerance have all been implicated in the virulence of L. monocytogenes. We reviewed the role of Sigma B in several environmental stress conditions to understand the physical character of this microorganism, to discuss the best preservation conditions of food, and to prevent bacterial infection.

Abstract:Abstract: [Objective] Our study aimed at searching for the pathogenic factor causing Siberian sturgeon (Acipenser baerii) disease. [Methods] A pathogenetic bacterial strain X-1-06909 was isolated from naturally infected Siberian sturgeon in Beijing. The strain was identified according to its physiological and biochemical properties, and the sequence analysis of 16S rRNA gene. The drugs sensitivity was detected with Kirby-Bauer’s agar diffusion method. [Results] Based on the result of 16S rRNA sequence analysis，the strain X-1-06909 shares 99.6% sequence identity with the type strain ATTCC35624T of Aeromonas veronii.The morphological characteristics of the strain were gram-negative, polar single-flagella.The results of physiological and biochemical tests were that the strain could ferment glucose and produce gas , methyl red (M-R) and Voges-Proskauer (V-P) tests were positive, arginine dihydrolase test was negative, it grew on 3% sodium chloride culture medium, the tests for hydrolysis of gelatin and hydrolysis of esculin were negative. The result for drug sensitive tests showed that among 21 antibiotics, cefadroxil，neomycin etc. had better inhibitive effect on the strain. [Conclusion] The isolated strain X-1-06909 was identified as A. veronii. The results will provide evidences for further caring the diseases of sturgeons.

Abstract:Abstracts: Biocontrol Bacillus subtilis strain Bs-916 has obvious effects against Rhizoctonia solani. We demonstrated that the mutant strain, M49 obtained by means of low energy ion implantation in strain Bs-916, which produces significantly lower levels of surfactin, had no obvious effects against R. solani. [Objective] In order to identify the influencing factors of surfactin-decrease mutant strain M49, its phenotype and related gene expression levels were studied. [Methods] Strains to be tested for sporulation were grown for 24 h in sporulation medium. Plasmid pDG1728(1 μg/mL) was used for DNA transformation to test competence development of M49 and Bs-916 strains. RT-PCR (Semi-quantitative reverse transcriptase-polymerase chain-reaction) was used to determine the expression levels of selected genes involved in competence and sporulation in both the wildtype Bs-916 and mutant M49 strains, such as comS, rapA, rapC gene. [Results] Our data showed that mutant strain M49 confers a leaky competence phenotype, typified by a ten-fold reduction. This indicated a fact that DNA fragments are more easily transformed to wildtype strain than M49 mutant. The M49 strain also appeared to exhibit a sporulation-deficient phenotype, compared with the wild-type Bs-916, its spore’s number declined by about 75%. RT-PCR results showed that both the comS and rapC genes were expressed in the Bs-916 strain but not in the M49 strain. [Conclusions] These results indicate that surfactin production, spore formation and competence development of Bacillus subtilis mutant strain M49 are severely decreased. And then, our data implicated that the cause of spore formation and antifungal activity may be the mutation of three key amino acids in ComA protein.

Abstract:Abstract: [Objective] This study was to identify genes involved in thermotolerance and osmotolerance of Beauveria bassiana. [Methods] A collection of T-DNA random insertion mutagenesis was constructed and thermosensitive and osmosensitive mutants were screened. The DNA fragments flanking to inserted T-DNA sequence in the mutants were isolated by using Y-shaped adaptor dependent extension (YADE) method. [Results] Five mutants impaired in thermotolerance or osmotolerance were identified and characterized. Mutant 212 and 2550 could not grow at 32oC, while mutants 139, 2737 and 2812 showed depressed growth under osmotic stress. Conidial viabilities of 212 was decreased by 23%, 52% and 21% after stressed by ultraviolet, heat shock, and H2O2 oxidation, respectively, compared to the wild-type strain. Virulence of mutant 212 and 2737 against aphids Myzus persicae were reduced by 12% and 40%, respectively. Sequencing analysis indicated that two function-unknown genes were interrupted by insertion of T-DNA in mutant 212 and 2737. In other three mutants, the insertions are located in the non-encoding regions. [Conclusion] The results demonstrate the successful use of T-DNA insertion mutagenesis in identification of genes involved in thermotolerance and osmotolerance of B. bassiana.

Abstract:Abstract: [Objective] The regulatory function investigation of two component system gacS/gacA on two phz gene clusters expression and quorum sensing system (QS) in Pseu-domonas. sp. M18. [Methods] The two phz gene clusters were sequenced and the expres-sional features by GacA were analyzed using RT-PCR and phzA-lacZ transcriptional fu-sions, the regulation of GacA over QS system was studied also in P. sp. M18. [Results] Two phenazine sturctural clusters, namely, phzA1-G1 and phzA2-G2 in P. sp. M18 shared 99% identities with those in P. aeruginosa PAO1. However, in the non-coding region downstream the phzA2-G2, P. sp. M18 has a three-144 bp-repeat sequence which does not exist in P. aeruginosa PAO1. PhzA1-G1 expressed at a higher level than phzA2-G2 in the wide type M18 strain. GacA functioned differently over these two phz gene clusters but negatively regulating the two clusters expression at the whole level, which was opposite to that in P. aeruginosa PAO1. The inactivation of gacA gene can reduce rhlI expression while has no effect on lasI expression, indicating that the phz gene expression regulated by GacA via QS was a minor part and the major phz expression was regulated by GacA through an unknown pathway instead of QS in P. sp. M18. [Conclusion] The different regulation of GacA activity on secondary metabolites and QS in P. sp. M18 and P. aeruginosa PAO1 may be the results imposed by the environmental selective pressure during evolution pathway.

Abstract:Abstract: [Objective] The storage disease was the important problem in jujube storage. The main problem was that Microorganism cause jujube disease. [Methods] In order to find out the characteristic of microorganism, I used the method of Biolog in the experiment. The experimental material was Lingwu long jujube. The test treatment were CKⅠ, CKⅡ, TREATⅠ and TREATⅡ. I used two kinds of micro-plate which were FF and ECO. [Results] Depending on the test results, I found that there was great difference on functional diversity of jujube fruit surface microorganism in storage. The storage time was longer the microorganism on the jujube fruit surface were richer and utilized different C source higher. The TREATⅠ and TREATⅡ used by which diversity, Homogeneity and AWCD were lower than CKⅠand CKⅡ. There were six kinds of the main C source. They were Carbohydrate-based, Carboxylic Acids, the Polymer, Phenolic Compounds, Amino Acids and Amine. [Conclusion] All of the test results were showed that the method of Biolog could be applied for the research on functional diversity of jujube fruit surface microorganism in storage.

Abstract:Abstract: [Objective] In order to identify the function of acpD in azoreduction, the gene was obtained from Shewanella decolorationis S12 and expressed in Escherichia coli TOP10 which showed little azoreduction activity. [Methods] Gene cloning and expression of acpD were performed by pGM-T vector. Sequences were analyzed by DNAMAN software and azoreduction activity was assayed spectrophotometrically. [Results] The deduced amino acid sequences from acpD of S. decolorationis S12 showed 61％ identity to FMN-dependent NADH-azoreductase from E. coli JM109 with extremely conservative amino acid sequences at FMN binding sites. Moreover, the resulting recombinant E. coli TOP10 (pGMT-N) exhibited high azoreduction activity for azo dyes of different polarity by intact cells and their extracts. On the other hand, NADH was used as electron donor to drive azoreduction and FMN could enhance the reduction activity. [Conclusion] The product of acpD from S. decolorationis S12 is a nonspecific FMN-dependent NADH-azoreductase, which transfers electrons from NADH via FMN to azo bond, and exhibits high azoreduction activity under in vivo or in vitro conditions.

Abstract:Abstract: [Objective] The aim was to identify the anti-tumor compounds of the submerger fermentation broth of Paecilomyces militaris (strain RCEF0927). [Methods] Anti-tumor activity was tested with a resazurin cytotoxicity assay model with Chinese hamster ovary (CHO) cells. Bioactive compounds were analysed with combined method of High Performance Liquid Chromatography (HPLC), High Resolution Mass Spectrometer (HRMS) and anti-tumor assay. [Results] Activity test showed that the submerger fermentation broth had strong cytotoxicity against CHO cells. Extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD- HRMS-Cytotoxicity assay revealed that the molecular formulars of the active compounds in the extract were possibly C10H13N5O3, C22H22O9, C23H24O10, C22H22O10, C15H10O4, C15H10O, C36H70O11, C36H68O12, C38H74O11, C40H76O13 and C44H82O14. [Conclusion]The possible structures of the bioactibe compounds were deduced with bioassay, HRMS, Uv and database inquiry as cordycepin, daizein, genistein 7,4′-dihydroxyisoflavone-7-O-(4″-O-methyl)-?-D-glucopyranoside, 7,4′-dihydroxy-6- methoxyisoflavone-7-O-(4″-O-methyl)-?-D-glucopyranoside, 5,7,4′-trihydroxy- isoflavone-7-O-(4″- O-methyl)- ?-D- glucopyranoside, polyoxyethylene ether oleate, dehydro products of polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxypropylene or polyoxybutene contained derivants of polyoxyethylene sorbitan fatty acid ester. Cytotoxicity of the compounds was revealed for the first time.

Abstract:Abstract: [Objective] Streptococcus oligofermentans was a newly characterized streptococcal species, which we isolated from dental plaques of carious-free humans. Our previous work indicated that in aerobic environments, it produced, as well as survived high concentration of hydrogen peroxide (4.4 mmol/L). This study analyzed the function of dpr gene in hydrogen peroxide tolerance of Streptococcus oligofermentans. [Methods] Cloned and expressed the dpr gene, and characterized Dpr protein. We constructed the dpr mutant strain of S. oligfermentans and detected its susceptivity to hydrogen peroxide. Furthermore, S. oligofermentans dpr gene was cloned into S. mutans, and its susceptivity to hydrogen peroxide was tested. [Results] S. oligofermentans dpr mutant strain was much more susceptive to hydrogen peroxide than the wild type, and introduction of its dpr gene into S. mutans could enhance the later’s hydrogen peroxide tolerant ability. Dpr proteins could bind irons, but not to DNA. [Conclusion] dpr gene of S. oligofermentans has significant role in hydrogen peroxide tolerance of the host strain, while Dpr protein may have other oxidant-damage protective mechanisms except for iron-binding.

Abstract:Abstract: [Objective] We studied the effects of arbuscular mycorrhizal (AM) fungus (Glomus versiforme) on iron species at different trifoliate orange [Poncirus trifoliata (L.) Raf.] rhizospheric soil with pot culture. [Methods] The soil range in horizon was separated into 0-2 cm, 2-4 cm and 4-8 cm away from the citrus taproot by nylon bags with 32 μm sieve, which citrus roots could not across but AM hyphae could. Mycorrhizal colonization was testified by trypan blue staining method. Iron species contents were quantified by atomic spectrometry, and phosphorus contents was through phospho-vanado- molybdate colorimetry. [Results] Available iron contents in AM treatment were followed the order by 0-2 cm > 2-4 cm > 4-8 cm. AM fungus decreased the contents of Exch-Fe, OM-Fe, RES-Fe. The concentration of Exch-Fe, Carb-Fe and MnOX-Fe became zero one year later. AM colonization was significantly positive relation with RES-Fe (P<0.01) [Conclusion] Arbuscular mycorrhizal fungi could affect the activation on mineral elements, and improve the available iron contents through the changes on iron species in soil.

Abstract:Abstract: [Objective] We cloned xylanase-encoding gene xynA and its promoter from Bacillus pumilus, expressed in Bacillus megaterium and characterized the recombinant xylanase. [Methods] We inserted the xylanase-encoding gene xynA and its promoter in Bacillus expression vector pWH1520 and pWG03 which was modified from pWH1520. We transformed the recombinant plasmid pWTEJX and pWGXYN into Bacillus megaterium, and obtained the recombinant stains BMJXH9 and BMGpp12. Enzymes produced by recombinant strains expressing xynA were produced in the medium. The xylanase activity produced by recombinant BMGpp12 was three times higher than that of BMJXH9. The recombinant xylanase had the original enzyme alkali-tolerant properties. [Conclusion] Alkali-tolerant xylanase gene was successfully expressed and this provided a basis for further study of xylanase applied.

Abstract:Abstract: [Objective] Isolation and characterization of rhizosphere copper-resistant bacteria from a copper accumulator plant Elsholtzia splendens were investigated. [Methods] Cultivable Cu-resistant bacteria were isolated by plating and screening from rhizosphere soils of Elsholtzia splendens growing on a copper mine tailing. Bacteria were characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction—Cu tolerance, phosphate-solubilizing, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophore and indoleacetic acid production, and further classified by restriction analysis of 16S rDNA (ARDRA). Strains that produced ACC deaminase were identified by 16S rDNA sequence analysis. [Results] Twenty-seven Cu-resistant strains were isolated from rhizosphere soil of Elsholtzia splendens and classified by ARDRA in 7 different taxonomic groups at the similarity level of 60%. All strains produced IAA or their derivatives, 44.4% of the strains produced a very high level of siderophores, and five strains were able to grow on ACC as the sole nitrogen source. Strains 2EBS12, 2EBS13, 2EBS15 and 3EBS11 were identified as Acinetobacter, strain 2EBS14 was essentially consistent Alcaligenes. [Conclusion] Cu-resistant rhizobacteria isolated from Elsholtzia splendens have abundant characteristics relative to promoting plant growth and genetic diversity, rhizobacteria Acinetobacter sp. and Alcaligenes sp. contained ACC deaminase activity.

Abstract:Abstract: [Objective] To compare the halometabolite producing capability between actinomycetes of earth origin and marine origin, based on genetic screening of the 1,5-dihydroflavin adenine dinucleotide (FADH2-dependent) halogenase gene. [Methods] We used 141 actinomycete isolates that were dereplicated by phenotype, 70 of earth origin and 71 of marine origin, and obtained halogenase gene fragments from them by PCR screening. We then sequenced the PCR products and analyzed corresponding amino acid sequences phylogenetically. We made further comparison of the halogenase sequences between actinomycetes of different origins, and between marine-origin streptomycetes and marine-origin Micromonospora isolates. In addition, we detected polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes by PCR in the halogenase gene-positive isolates. [Results] We observed higher occurrence of the halogenase gene in marine-origin actinomycetes (36.6%) than in earth-origin actinomycetes (14.3%), and in marine-origin streptomycetes (69.0%) than in marine-origin Micromonospora isolates (14.3%). Most (86.1%) of the halogenase gene-positive isolates contained PKS and/or NRPS genes. Moreover, the halogenase sequences of marine-origin isolates differed largely from the known ones, and clustered into a couple of distinct clades in the phylogenetic tree. In addition, we found greater diversity of the halogenase genes in marine-origin Micromonospora isolates than in marine-origin streptomycetes. [Conclusion] Based on the results of this study, we propose that actinomycetes, especially streptomycetes, from marine habitat could serve as a good source for new bioactive halometabolite discovery in the future.

Abstract:Abstract: [Objective] We studied the effects of arbuscular mycorrhizal fungus (Glomus versiforme) on growth and iron uptake of trifoliate orange [Poncirus trifoliata (L.) Raf.] at different pH levels of nutrient solution. [Methods] P. trifoliata seedlings were grown in substrates watered by nutrient solution with 50 μM Fe- ethylenediaminetetraacetic acid (EDTA) at pH values of 5.0, 6.0 (as control), 7.0 and 8.0 with a sand culture. Mycorrhizal colonization was tested by trypan blue staining method. Chlorophyll concentratration and root Fe (III) chelate reductase activity were determined by spectrophotometer. Potassium and active iron contents were quantified by atomic spectrometry, and phosphorus contents was through phospho-vanado- molybdate colorimetry. [Results] The colonization of Glomus versiforme significantly increased the plant height, stem diameter, leaf numbers, and dry mass. Arbuscular mycorrhizal fungus significantly enhanced the accumulation of chlorophyll, active iron, total iron and root Fe (III) chelate reductase activity, and decreased P/Fe and 50(10P+K)/Fe ratios. All biomass, iron contents and root Fe (III) chelate reductase activities of P. trifoliata seedlings at pH 6.0 level were the maximum both in inoculated and non-inoculated treatments. [Conclusion] Arbuscular mycorrhizal fungi could remedy chlorosis caused by iron-deficiency in citrus, and 6.0 was the optimal pH value for the growth of P. trifoliata seedlings.

Abstract:Abstract: [Objective] To obtain the recombinant gp90 protein of Reticuloendotheliosis virus (REV) and the anti-gp90 serum with high titer. [Methods] Using the plasmid pMD18T-env as template, we amplified the gp90 gene and then cloned it into pET-28a (+). The recombinant plasmid pET28a-gp90 was transformed into Escherichia coli BL21 (DE3) ,which was induced with isopropylthio-β-D-galactoside(IPTG). After identification by SDS-PAGE and Western blotting, the purified gp90 protein was injected into Balb/c mice to prepare anti-gp90 serum. The specificity and titer of the antiserum were evaluated by IFA and the enzyme-linked immunosorbant assay (ELISA). [Results] SDS-PAGE and Western blotting showed that the gp90 protein was expressed successfully in the form of inclusion body in the recombinant E coli. ELISA showed the mouse anti-gp90 serum had a titer of 1:12800. Successful expression of recombinant gp90 protein and preparation of its antiserum laid the foundation for the development of diagnostic reagent of REV.

Abstract:Abstract: [Objective] To isolate large genomic DNA fragments from Monascus ruber for the construction of cosmid libraries. [Methods] Modified phenol-chloroform method was used to isolate genomic DNA. The isolated genomic DNA was digested by Sau3AI to 40kb fragments on average. Then, the fragments were packaged by Stratagene’s Gigapack III XL packaging extract. A pair of degenerate primers were used to amplify a fragment of PKS (polyketide synthase) gene from this cosmid library. [Results] The average size of genomic DNA isolated by this method was larger than 48kb, with a concentration of 5μg/μl. The constructed cosmid libraries had 10 folders coverage of the Monascus spp. genome. A cosmid containing the homologue of PKS gene was obtained by PCR screening. [Conclusion] This modified method of isolating large fragments genomic DNA of Monascus ruber was efficient and feasible.

Abstract:Abstract: [Objective] A selective enrichment broth (SSL) was formulated to allow simultaneous growth of Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogens. [Methods] Suitable additive agents were selected by single factor experiment, the enrichment effect of the broth for the three pathogens were evaluated by conventional detection method and real-time PCR. [Results] A selective enrichment broth, SSL, was obtained by adding the selective agents, including nalidixic acid, lithium chloride, and potassium tellurite, in the basic broth, and sodium pyruvate and mannitol as the supplemented elements. Recovery of three target pathogens in SSL was obtained within 24 h of incubation at 37 ℃, yielding cell dnesities of 107-108 CFU/mL. Meanwhile, SSL broth effectively inhibited the growth of non-target organisms. 710 samples were detected by SSL with real-time PCR, and there is no error report. [Conclusion] SSL is demonstrated to be a promising new multiplex selective enrichment broth for simultaneous detection of the three most prominent foodborn pathogens by multipathogen detection on a single assay platform.

Abstract:Abstract: [Objective] Staphylococcus aureus small colony variants (S. aureus SCVs) could lead to persistent, recurrent infection with the characteristics of aminoglycosides antibiotics resistance, making them a big challenge for clinical diagnosis and therapy. We aimed at isolating and identifying isolates of S. aureus SCVs and providing the biological material of SCVs study in China. [Methods] The combination assays of observing colony phenotype, identification of the species-specific gene nuc of S. aureus by PCR amplification and a series of biochemical tests were conducted on 104 clinical isolates originally isolated from human, cow and environment. The suspected isolates were confirmed as S. aureus SCVs by complementation assay with supplementation of menadione, thiamine, thymine and haemin. [Results] One of the isolates from environment was identified as SCVs, named CDC54 with the species-specific gene nuc of Staphylococcus aureus (S. aureus) confirmed by PCR amplification, whose major phenotypes included smaller colony, decreased pigmentation, decreased coagulase, reduced fermentation of lactose, decreased haemolytic activity, increased resistance to aminoglycosides.[Conclusion] The CDC54 will play an important role in studying prevention, control and pathogenesis for S. aureus SCVs infection.

Abstract:Abstract: [Methods, objective] We amplified the 1026 bp hrp(hypersensitive response and pathogenicity ) gene from Pseudomonas syringae pv. glycinea isolate Psg12 genomic DNA by PCR technique, and then constructed expression vector pGEX-hrpZPsg12 with regular molecular cloning operation. The recombinant plasmid was transformed into BL21(DE3). Recombinant protein was induced by Isopropylthio-β-D-Galacgoside (IPTG).[Results] The molecular mass of the fusion protein is 61kDa analyzed by SDS-PAGE. The protein, similar to the other known harpins, was heat-stable, which contained abundant glycine(G), but had no cysteine. Furthermore, this protein was sensitive to protease K and able to trigger hypersensitive response (HR) in common tobacco. The HR elicitation by the protein in tobacco was inhibited by eukayotic metabolic inhibitors, NH4VO3 and LaCl3. The hrpZ gene showed 79% identity to hrpZPsg which cloned from P. syringae pv. glycinea(Psg r0) in Japan and 79-99% identity to other hrpZ in GenBank. However, it did not show any sequence identity with those of other genus of gram-negative plant pathogenic bacteria. [Conclusion] In summary, hrpZPsg12 was a novel gene that was cloned by us from P. syringae pv. glycinea, and this is the first report to express hrpZPsg12 gene in BL21.