Abstract:Abstract: Type Ⅵ secretion system (T6SS) is a newly found secretion system, which distributes widely in Gram-negative bacteria. It consists of structural proteins (constitute secretion system), translocated proteins (form transmembrane pipe structure), secretory proteins and a few auxiliary function proteins for secretion systems. T6SS can enhance the adaptability of bacteria on the environment, mediate pathogenicity to host cells and has some other functions.

Abstract:Abstract: Biosynthesis of nanomaterials, as a novel “green synthesis technology”, has attracted increasing interests. Many microorganisms such as bacteria, actinomycetes (eukaryotes), as well as yeasts and fungi (prokaryotes) have been found to have ability to biosynthesize nanomaterials intracellularly or extracellularly. A series of metal and semiconductor nanomaterials with the excellent features of high monodispersity, stabilization, adjustable size, and biological activity, such as Au, Ag, Au-Ag, Fe3O4/ Fe3S, Titanium and CdS, CdSe, and Silica, have been biosynthesized. Here, we provided a brief overview of the current research on the use of microorganisms in the biosynthesis of nanomaterials. The biological procedures, biosynthesis mechanism, controlled biosynthesis on the shape and size, and their applications were summarized. The prospects of biosynthesis using microorganism have also been addressed.

Abstract:Abstract: Nicotinamide - adenine dinucleotide (phosphate) (NAD(P)) metabolism involves many fundamental cellular events, such as energy metabolism, maintenance of redox homeostasis and regulation of cell longevity. Inhibitors of essential enzymes of NAD(P) biosynthetic pathways might be promising leads for novel antibiotics, such as the NAD synthase inhibitors. This review described the crystal structural, functional properties, regulator and structure-based inhibitors design for NAD synthase. This might provide the basis for developing NAD-based therapeutics.

Abstract:Abstract:[Objective] The aim of this study was to isolate efficient high-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs) degrading bacterial strains, and to study their degradation potential.[Methods] We used sublimation method to enrich and isolate the degrading bacteria from coking plant samples. Morphological properties, the sequence homology of 16S rRNA and gyrb genes were used to identify the isolated strains. GC-MS was used to analyze the degradation potential against some HMW-PAHs. [Results] A HMW-PAHs degrading bacterium, HBS1, was obtained. Strain HBS1 could use several HMW-PAHs such as pyrene,benzoanthracene,benzopyrene,chyrsene,indeno[1,2,3-cd]pyrene,benzo[g,h,i]perylene and fluoranthene as sole carbon source for growth. Strain HBS1 was identified as Gordonia sp.. Based on the high sequence similarities (more than 99%) of both 16S rRNA gene and gyrb gene to those of Gordonia amicalis. When the initial concentration of pyrene was 50 mg/L, strain HBS1 could consume 97% of the pyrene in 17 days. One fragment of the dioxygenase gene was obtained by PCR with size about 300 bp, which was closest to the counterpart from Mycobacterium sp. with 93.8% similarity. [Conclusion] We isolated a strain HBS1 from seriously PAHs-polluted soils and identified it belong to Gordonia sp.. The isolate got a great degradation potential of high-molecular weight polycyclic aromatic hydrocarbons.

Abstract:Abstract: [Objective] To characterize the genes of Legionella pneumophila isolated from different water source in Guangzhou from 2006 to 2009. To genotype the strains by using sequence-based typing (SBT) scheme. [Methods] In total 44 L. pneumophila strains were identified by SBT with 7 diversifying genes of flaA、asd、mip、pilE、mompS、proA and neuA. Analysis of the amplicons sequence was taken in the European Working Group for Legionella Infections (EWGLI) international SBT database to obtain the allelic profiles and sequence types (STs). Serogroups were typed by latex agglutination test. [Results] Data from SBT revealed a high diversity among the strains and ST01 accounts for 30% (13/44). Fifteen new STs were discovered from 20 STs and 2 of them were newly assigned (ST887 and ST888) by EWGLI. SBT Phylogenetic tree was generated by SplitsTree and BURST programs. [Conclusion] High diversity and specificity were observed of the L. pneumophila strains in Guangzhou. SBT is useful for L. pneumophila genomic study and epidemiological surveillance.

Abstract:Abstract: [Objective] To determine the chromosome DNA ploidy of Candida glycerinogenes. [Methods] We screened the haploid cell of Saccharomyces cerevisiae and the diploid of Candida tropicalis as the reference cell to identify the ploidy of Candida glycerinogenes. The concentrations of chromosome DNA extracted from both haploid and diploid cells were determined by the diphenylamine reaction method. Meanwhile, the cell number of Candida tropicalis, Candida glycerinogenes, Saccharomyces cerevisiae and its haploid were accounted by hemocytometer measurement method, respectively. Because in the same UV irradiation conditions, the haploid cells was more easy died than diploid cells, so we used the UV irradiation test to further verification. [Results] The results of the content of chromosome DNA in a single cell showed that Candida glycerinogenes was a diploid cell. Moreover, it was also further confirmed because the haploid cell of Saccharomyces cerevisiae was more sensitive than Candida glycerinogenes under UV treatment. [Conclusion] By this study, we confirmed that Candida glycerinogenes was a diploid cell.

Abstract:Abstract: [Objective] To isolate a new osmophilic yeast for producing D-arabitol and research its optimal fermentation conditions for highest yield of D-arabitol from glucose. [Methods] The isolated strain was characterized by electron microscopy, Biolog (GN) test, G+C content measurement and 26S rDNA D1/D2 domain sequences analysis. The purified fermentation product was identified by IR, 1H-NMR, 13C-NMR, MS and optical rotation analysis. Then the fermentation conditions for D-arabitol production were optimized. [Results] A new osmophilic yeast was isolated and identified as Candida sp. H2. Through the single factor experiment, the optimum conditions of 250 g/L glucose, 10 g/L yeast extract, initial pH 6.0, 35 °C of culture temperature, 200 rpm of agitation, 200 mL medium in a 1000-mL flask of broth content, 1% (v/v) of inoculum size, 96 h of fermentation time were achieved. Based on the conditions above, weight yield of 35% (86.55 g D-arabitol from 250 g glucose) was obtained and 10% higher than the conditions not optimized. [Conclusions] Candida sp. H2 was a novel strain for producing D-arabitol and valuable for further study.

Abstract:Abstract: [Objective] With the aim of elucidating the physiological characteristics of a low-pH tolerant strain Torulopsis glabrata RT-6. [Methods] The intracellular pH, ATP level, the membrane bound H+-ATPase activity, the membrane fatty acid composition and the intracellular polyphosphate content of the parent strain CCTCC M202019 and the mutant strain RT-6 were determined and compared under different pH conditions. [Results] Compared to that of the parent strain, the cell growth and pyruvate concentration of the mutant strain RT-6 were increased by 60.6 % and 85.4 % (56 h), respectively. Similarly, the strain RT-6 had higher intracellular pH compared to the control strain at external pH5.0, 4.5, and 4.0. The ATP content, the membrane bound H+-ATPase activity and the intracellular polyphosphate content of the mutant strain RT-6 were increased by 11.7 %, 13.6 %, and 3.5 % at external pH 5.5, while at external pH 4.0, increased by 61 %, 38.6 %, and 30.8 % , respectively. Furthermore, the mutant strain RT-6 exhibited higher content of the unsaturated fatty acids and higher membrane fluidity. [Conclusion] Discharging more intracellular H+ and inhibiting the intracellular H+ production contributed to the strain RT-6’s higher intracellular pH, and therefore the acid tolerance.

Abstract:Abstract: [Objective] The amphipathic α-helical peptide is an important class of antimicrobial peptides. In this study, 16-residue-long peptide (VGR16) composed of 8 Val residues in the nonpolar face and 5 Arg residues in the polar face were designed based on the helical wheel projection to produce antimicrobial peptide with improved antibacterial activity accompanied by decreased toxicity. [Methods] Antimicrobial activity and toxicity against red blood cells and mammalian cells were investigated to evaluate the biological function of the peptide. In addition, bactericidal kinetics was tested. [Results] Antimicrobial assays revealed that the peptide VGR16 showed antimicrobial activity and their MICs against gram-negative and gram-positive bacteria ranged from 16μg/ml to 64μg/ml, respectively. VGR16 also exhibited rapid bactericidal action. It was surprisingly found that the peptide displayed no hemolytic activity even at a concentration of 256 μg/ml. Cell culture assays indicated that the peptide VGR16 had no cytotoxicity against mammalian cells for its MICs. [Conclusion] The results showed that the peptide could be a likely candidate for future antimicrobial applications.

Abstract:Abstract: ［Objective］To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. [Methods] We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions；We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. ［Results］Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase；Under the N-S conditions，pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2、MnP 252.2 U/L and LiP 1169.5、MnP 172.4 U/L respectively). [Conclusion] The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results posses high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.

Abstract:Abstract: [Objective] To clone Ser/Thr protein phosphatase type 5 gene (PP5) from Metarhizium anisopliae, analyze the structure features of PP5 gene with its encoding protein and expression profile during two conidiation program (microcycle conidiation and normal conidiation). [Methods] The DNA sequence of PP5 was isolated by Blasting the EST sequence of PP5 in subtracted library with genomic data of M. anisopliae. Primers were designed based on the DNA sequence to clone the full length cDNA of PP5 by PCR, and the characteristics of the encoded protein was analyzed by online tools and biological softwares. The PP5 expression profile was quantified by real time PCR at different stages of microcycle conidiation and hyphal stage of normal conidiation in M. anisopliae. [Results] The genomic DNA, which was interrupted by six introns, was 2100 bp long. The cDNA, encoding 325 amino acid residues, is 1428 bp. Analysis to Ser/Thr protein phosphatase type 5 in M. anisopliae show a conserved structure features. Quantitative real time PCR analysis showed that PP5 expression varied obviously in different stages of microcycle conidiation. Expression was sharply up-regulated after 16 h, with the highest transcript levels at 24 h in microcycle conidiation, but lowly expressed in normal conidiation. [Conclusion] This work presents the first report about the detailed sequence and structure of PP5 from entomopathogenic fungi. Comparison of expression profile of microcycle conidiation and normal conidiation reveals that PP5 is principally involved in microcycle conidiation in M. anisopliae, and it provides ideal candidate for further studies to PP5 and its molecular regulation.

Abstract:Abstract: [Objective] Plant-parasite nematode is one of the most important pathogens in plant. Our objective is to screen endophytic bacteria against plant-parasitic nematodes from plant. [Methods] Endophytic bacteria were isolated and screened by testing their metabolite against Bursaphelenchus xylophilus in vitro. Those strains inhibiting B. xylophilus were selected to culture in liquid medium and fermentation conditions were optimized by orthogonal test. The stability of the antinematode substances was evaluated by various. In addition, four strains were identified by 16SrDNA sequence analysis. [Results] In total 13 strains of endophytic bacteria secreting antinematode metabolite were isolated from 6 species of plant. The supernatant of the fermentation broth of these endophytic bacteria gave 100% mortality of nematodes after treated as the follows:1ml each was mixed with 0.2ml of the suspension of nematodes (2000 nematodes/ml) then incubated at 25℃ for 24 h, some of which could led to leakage or dissolution of nematodes. Among them, four strains, BCM2、SZ5、CCM7 and DP1, showed stronger activity than others. The supernatants diluted three times also gave not less than 95% mortality after 24 h treatment, and those from DP1 and SZ5 even gave 100% mortality. The fermentation conditions of the four strains were optimized and the antinematode activity grew up four times after optimization. The antinematode substances of these strains were found stable when treated with protease or heating or stored at 4℃ after 100 days, while instable when treated with acid or alkali. DP1 and CCM7 were identified to be Bacillus subtilis, while SZ5 and BCM2 to be Bacillus cereus. [Conclusion] Endophytic bacteria secreting antinematode metabolite were found in economic crops. The metabolite of some strains showed strong and stable antinematode activity. Our results indicate the real potential of biocontrol by endophytic bacteria.

Abstract:Abatract [Objective] By using brominated flame retardant we compared the bacterial diversity of highly polluted river sediment with that of nearby unpolluted lake. [Method] Total DNA was extracted from unpolluted and highly polluted sediment sample by brominated flame retardant in Guiyu of China. The 16S rRNA gene was amplified by PCR using bacterial primer 27F and 1500R. The plasmid libraries of the amplicons were constructed. The positive clones with insert were screened on plates with IPTG/X-gal/Ap. Amplified ribosmal DNA restriction analysis (ARDRA) was carried out with restriction enzymes HhaⅠand HinfⅠ. Representative clones of each operational taxonomic unit based on the ARDRA patterns were selected to be sequenced. After proof reading and careful comparison to remove the chimeric sequences, the partial sequence of 16S rRNA gene were used for construction of the phylogenetic tree. [Result] The result of blast searching showed that clones from unpolluted sediment sample belonged to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Verrucomicrobia, Firmicutes, the predominant bacteria (30.2% of total clones) is Acidobacteria; most clones from polluted sediment belonged to α-Proteobacteria, β-Proteobacteria, ε-Proteobacteria, δ-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, candidate division OP01, candidate division OP08, the predominant bacteria (44.9% of total clones) are ε-Proteobacteria and Chloroflexi. [Conclusion] Bacterial community structure of polluted sediment has distinguished feature and obviously different from the unpolluted sediment sample, which is mainly reflected in the dominant position of ε-Proteobacteria and Chloroflexi in the bacterial flora.

Abstract:Abstract: [Objective] Streptococcus suis 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. In this study, we investigated the functionality of one of Streptococcus suis 2 sortases, known as the srtBCD. [Methods] To obtain the isogenic mutant srtBCD, the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination. The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR. To better assess the role of srtBCD gene in the virulence of 05ZYH33, cell adherence assays and experimental infection of mice was adopted. [Results] A SrtBCD defective mutant of 05ZYH33 was found to be associated with growth curve upon cultivation in standard laboratory used in our in vitro assays. Furthermore, abolishment of the expression of srtBCD result in impaired interactions of S.suis with human laryngeal epithelial cell line. However, there is no differences when infection mice by the WT and mutant strain. [Conclusion] These results suggest that srtBCD are critical for the pathogen-host interaction of S. suis 2,but abolishment of srtBCD does not impair the full virulence of 05ZYH33. It is to expect that future study carried out with S.suis 2 to verification the conclusions.

Abstract:Abstract: [Objective] To study the bactericidal effect and the possible mechanisms of the three components system [soybean peroxidases (SBP)-hydrogen peroxide (H2O2)-potassium iodide (KI), SBP-H2O2-KI]. [Methods] The inhibition and bactericidal effect of SBP-H2O2-KI system to bacteria were detected by OD600 and the number of live bacteria (CFU). The sensitivity was tested by comparing the minimum inhibitory concentration (MIC) of bacterial cultures before and after cultured under sub-lethal dose of SBP-H2O2-KI system. Oxidizing activity groups were detected with physical and chemical methods in order to explain the bactericidal mechanisms of SBP-H2O2-KI system. [Results] SBP-H2O2-KI ternary system had rapid and high efficient bactericidal effect to a variety of bacterial strains in just several minus. The MICs had no significant changes when bacterial cultures continuously cultured in sub-lethal dose of SBP-H2O2-KI system, and no resistance / tolerance mutant strains could be isolated from them. Both physical and chemical test results showed that no hydroxyl radical produced in SBP- H2O2-KI reaction system, chemical test results showed that no superoxide anion but a singlet oxygen and iodine produced in SBP-H2O2-KI reaction system. [Conclusion] These results suggested that singlet oxygen and iodine or the iodine intermediate state may possible be the main sterilization factors for SBP-H2O2-KI system, and hydroxyl radical and superoxide anion not. In addition, the both characteristics of SBP-H2O2-KI system: rapid and high efficient bactericidal effect, and bacteria difficultly resisting to it, indicated it would have a good potential application in medical and plant protection area.

Abstract:Abstract: [Objective] The transcription factor SoxR, which serves as a cellular redox sensor，is a global activiator of anti-oxidative stress in Escherichia coli. The granaticin biosynthetic gene orf20 from Streptomyces vietnamensis was found to be a soxR-like gene. This study was carried out towards understanding the physiological function of this gene. [Methods] The orf20 gene was cloned and expressed in E. coli. An orf20 deletion mutant of S. vietnamensis was constructed using a modified PCR-targeting disruption method. The growth curves of the recombinant E. coli strains and the mutant S. vietnamensis at various paraquat concentraions were assayed to resolve any changes of resistance levels. [Results] Soluble expression of orf20 gene in E. coli was achieved, and the recombinant ORF20 protein was tagged by seven histidines at the carboxylic end. An orf20 deletion mutant of S. vietnamensis DMR20 was successfully constructed. The DMR20 mutant showed no growth changes to the wild, and the ability of sporulation was retained, too. But, unexpectedly, the production of granaticin was improved for more than three folds. Compared to the wild type, the DMR20 mutant had no visible changes of resistance to paraquat, however, E. coli carrying the recombinant plasmid pET28b-orf20 received an elevated resistance to paraquat. [Conclusion] The orf20 gene can complement the soxR gene in E. coli, but is not involved in the regulation of anti-oxidative stress response in S. vietnamensis. Instead, it imposes a negative effect on granaticin production.

Abstract:Abstract: [Objective] We cloned and expressed a cytochrome P450 gene cyp107z from Streptomyces ahygroscopicus ZB01, and determined the kinetic parameters of the recombinant enzyme in vitro. [Method] Degenerate primers were designed utilizing the conserved sequence of cyp genes and were used to amplify partial sequence of cyp107z gene from Streptomyces ahygroscopicus ZB01 genome. The full-length cyp107z gene sequence was obtained by genome walking, and linked with pET28a to construct pET-cyp107z13 expressing vector which was then transferred into Escherichia coli, and the expressed recombinant protein was purified by Ni-NTA affinity chromatography. The catalysis system of the recombinant protein was constructed with avermectin as substrate, and the kinetic parameters of the recombinant protein were determined by monitoring the consumption of NADPH in the system in vitro. [Results] A cyp107z homologous gene named cyp107z13 was cloned from Streptomyces ahygroscopicus ZB01 genome, which was 1290 bp in length encoding 429 amino acid residues. The Km of purified recombinant protein of CYP107Z13 expressed in E.coli was 1.4 μmol/L, the Vmax was 0.041 μmol/min?mg and the kcat was 0.033 s-1 in a reaction system with avermectin as substrate. [Conclusion] A cyp10z13 gene from Streptomyces ahygroscopicus ZB01 was cloned, the heterologous expressed recombinant protein can catalyze the oxidizing reaction with avermectin as substrate.

Abstract:Abstact:[Objective] The research object is to induce hydroxyapatite (HAP) synthesis using fungus. [Methods] Using the PDA (Potato Dextrose Agar Medium) liquid medium containing different concentrations of Na2HPO4 and CaCO3 to study the way Aspergillus niger synthesize HAP, and observe the induced mineral crystal structure and analysis the induced mineral type with transmission electron microscopy (TEM) and X-ray diffraction (XRD). [Resluts] The main results are as follows: (1) A. niger can induce HAP synthesis in PDA liquid medium with the proper concentration of Na2HPO4 and CaCO3. (2) The reaction of A. niger induced HAP synthesis depends on the time of the response system. Longer time is more advantageous in producing HAP. [Conclusion]The main reason for A. niger inducing HAP crystals formation are as follows: fungal metabolism produces the acidic substances to dissolve CaCO3 and the growth mycelia absorbing Ca2＋ lead to Ca2＋enriched on the surface, to promote the production of secondary mineral apatite and further transform into HAP in the mycelia spheres. Because the response condition for bio-induction HAP is temperate, the preparation craft is simple, the cost is low, the method has the potential application prospect.

Abstract:Abstract：[objective] Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via a highly conserved Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. However, FMDV is an RNA virus, which harbors the evolutionary potential to render the RGD motif dispensable upon changes in constant environment. We studied mutation of RGD motif upon short-time passages FMDV Asia1/JS/China/2005 field strain in different host. [Methods] VP1 gene was amplified from Asia1/JS/China/05 field strain, the fourth passage virus in sulking mice (MF4) of the above strain and the virus isolated from a pig housing with cattle inoculated with the above strain followed eight passages in BHK-21 cell (PBF8) by RT-PCR, and the VP1 genes were sequenced and their deduced amino acid sequences were compared with each other. [Result] Dominant population with RGD and Arg-Ser-Asp (RSD) receptor recognition site motif was generated after four passages of Asia1/JS/China/2005 field virus in sulking mice and another dominant population with Arg-Asp-Asp (RDD) motif was produced after eight passages the virus isolated from housing pig with cattle inoculated with the above field strain. [Conclusion] This study indicated that the dominant FMDV virus populations with RSD or RDD receptor binding site instead of original RGD motif were produced upon short-time evolution of FMDV field isolates with RGD motif in different environment. These studies not only increase number of viable mutants with substitutions in the RGD region, but also these profoundly altered, but viable, mutants with different receptor recognition site will provide useful tools for studies of cell recognition by FMDV and host tropism modifications.