Abstract:Abstract: Quorum sensing as an extracellular signal transduction system is distributed widely among many bacteria to coordinate their behaviors or actions by mediating gene expression, and plays key roles in many physiological processes and pathogenicity. Quorum sensing was also observed among many streptomycetes, as an important regulatory mechanism of secondary metabolite biosynthesis and/or cell differentiation, and displayed certain diversity of the autoinducer structures and action mechanisms. The participation of A-factor-driven quorum sensing systems in the secondary metabolism has been extensively studied, and triggered the identification of a major signal class featured with γ-butyrolactone core. Additionally, PI-factor, M-factor and certain small antibiotic molecules recently found in streptomycetes clearly could play important roles in the biosynthetic pathways of some antibiotics, and might represent extracellular autoinducer classes with novel structures. Meanwhile, some specific products of streptomycetes including cholesterol oxidase and glycerol have been identified to function as cell-signaling molecules which modulate the secondary metabolic activities in streptomycetes, probably by the mode of quorum sensing. Here, we reviewed research advances on quorum sensing systems involved in the accumulation of secondary metabolites in streptomycetes, mainly focusing on the clarification of their action modes and structural diversity of autoinducers. We also prospected the research trends in this field and application of autoinducers through quorum-sensing in metabolic engineering of natural products.

Abstract:Abstract Environmental pollutants are a major concern worldwide. Bioremediation mediated by microorganisms is a highly promising technology that is environmentally friendly, safe and effective. However, successful execution of these bioremediation strategies requires a complete understanding of factors governing the growth, metabolism, dynamics and functions of indigenous microbial communities at contaminated sites. The combination of genomics, transcriptomics, proteomics and metabolomics has provided a crucial insight into microbial communities and their mechanisms in bioremediation of polluted environment. This current review is focused on application of these technologies in bioremediation at contaminated sites. Limitations of each “-omics” analysis are briefly discussed and integration of these “-omics” technologies in study the processes of harmful algal bloom and the microbial degradation metabolism of organic pollutants are presented.

Abstract:Abstract: Ibuprofen has been recognized as an environmental endocrine disruptor due to its ability to interfere with prostaglandin synthesis. In order to address the myriad challenges faced by the issue of ibuprofen in the environment, the recent research progress was summarized in this paper to characterize the ibuprofen consumption, its potential hazard, and biodegradation and degradation mechanisms. The importance and urgency to carry out the ibuprofen degradable gene cloning, its function analysis and its molecular degradation mechanisms were emphasized.

Abstract:Abstract: [Objective] In order to investigated composition and diversity of bacterial in a cold sulfur spring in Xinjiang faulting zone. [Methods] Environmental total DNA was directly extracted from the water of the No.10 cold sulfur spring. The 16S rRNA genes were amplified from the total DNA by PCR with bacteria-speci?c primers and construction a clone library. Positive clones were randomly selected from the library and identified by restriction fragment length polymorphism (RFLP). The unique RFLP pattern corresponded sequences were sequenced, BLAST and then constructed phylogenetic tree. [Result] In total, 228 positive clones were screened and grouped into 33 Operational Taxonomic Units (OTUs). The clone coverage C value was 92%.33 Operational Taxonomic Units were divided into 3 phyla with Blast analysis and RDP classifer: Proteobacteria, Bacteroidetes, and Firmicutes. Proteobacteria (98%) was the absolutely dominant group, of which 20% of the clones were highly related to the known photoautotrophic and chemoautotrophic bacteria (> 97 % sequence similarity). Besides, 64% of the clones showed less than 96% of sequence similarity with sequence deposited in GenBank database, of them 54% sequences were affiliated to genus Legionella spp. [Conclusion] Bacterial diversity in No.10 cold sulfur spring was low, but maybe have a diversity of novel species and lineages. In addition, large number of novel species of Legionella were detected in the spring water may suggest the water potentially a source of Legionnaires disease and may constitute a menace to the health of human and livestock that lived down the spring.

Abstract:Abstract: [Objective] To study adaptive mechanism in hypersaline environments of extreme halotolerant filamentous actinomycetes. [Methods] Using HPLC we analyzed compatible solutes from extreme halotolerant filamentous actinomycete strain Prauserella alba YIM 90005T that was cultivated at different NaCl concentrations. [Results] Ectoine and 5-hydroxyectoine were two major compatible solutes for strain Prauserella alba YIM 90005T. Ectoine accumulated to the maximum content of 18.77 μg/mg dry cell weight after being inoculated in 10% NaCl (w/v). And 5-hydroxyectoine reached 22.98 μg/mg dry cell weight after being inoculated in 24% NaCl (w/v). The ectA (acyltransferase), ectB (aminotransferase), ectC (ectoine synthase) and ectD (ectoine hydroxylase) genes cluster encoding genes on ectoine and hydroxyectoine synthesis were further cloned by designing the degenerate primer and genome walking methods. The sequence analysis indicated that ectABCD was an operon. Furthermore, the expression of ectB and ectD inoculated at different salt concentrations was quantified by real-time PCR, and the results indicated that the expression of the gene cluster would be increasing as the salt concentration increased. [Conclusion] 5-hydroxyectoine was the major compatible solute for osmotic regulation of strain Prauserella alba YIM 90005T to adapt high salt concentration.

Abstract:Abstract: [Objective] Adhering to the intestinal epithelial cells is one of the beneficial functions exerting by probiotics. We tested and verified the expression of adhesion-related genes in vivo and in vitro to understand the influence from Lactobacillus acidophilus NCFM on host cells. [Method] We selected the adhesion-related genes through GO (gene ontology) category from the Human Genome U133 Plus 2.0 Array analysis. These genes were verified by in vitro Caco-2 cells culture model and in vivo mouse model using Real-time PCR method. [Results] After L. acidophilus NCFM adhering to Caco-2, we found that 12 adhesion-related genes were up-regulated. The up-regulation was confirmed in vivo and in vitro by Real-time PCR assay. Among them, the up-regulated expression of chemokine (C-C motif) ligand 2 (CCL2) gene was the most distinguished one. [Conclusion] Our research showed that L. acidophilus NCFM, adhering to Caco-2 cells, could cause the differential expression of the host adhesion-related genes. Results of this study seemed to provide some useful data for further revealing its effect on intestinal epithelial cells.

Abstract:Abstract: [Objective] The regulator protein H-NS of Yersinia pestis was expressed using the Escherichia coli BL21λDE3 protein expression system, and its DNA-binding activity was characterized. [Methods] The entire coding region of the hns gene was amplified by PCR from Y. pestis strain 201, and then cloned into the BamHI and SalI sites of the vector pET28a. The recombinant plasmid pET28a-hns was transformed into BL21λDE3. Over-expression of His-H-NS in the LB medium was induced by addition of 1 mM IPTG (isopropyl-b-D-thiogalactoside). The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). The electrophoretic mobility shift assay and DNase I footprinting experiments were carried out to analyze the DNA-binding activity of His-H-NS in vitro. [Results] The purified His-H-NS protein could bind to the upstream DNA regions of psaA, psaE and rovA of Y. pestis, and the H-NS-binding sites were determined at the single nucleotide resolution. [Conclusion] The purified His-H-NS protein could bind to target DNA fragments, suggesting that H-NS would regulate the transcription of relevant genes in Y. pests.

Abstract:Abstract: [Objective] Glycoproteins derived from yeast expression systems are usually hyperglycosylated and contain non human N-glycans of the high mannose type. α-1,6-mannosyltransfe rase(och1p) plays a key role in modifying glycoproteins with high mannose-type N-glycans. Therefore, we engineered a P. pastoris X-33 strain with OCH1 gene deletion. This strain was further used as a host for production of glycoproteins with smaller N-glycans. [Methods] First, we knockout the URA3 gene of P.pastoris X-33 which encoding orotidine-5’-phosphate decarboxylase by double homologous recombination. Then, we knockout OCH1 gene using URA3 gene as a selecting marker, and obtained X-33( och1-) strain. After that, this mutant strain was used to expression the glycoprotein granulocyte-macrophage colony-stimulating factor (GM-CSF). [Results] Different from hyperglycosylated GM-CSF expressed in wild type P.pastori X-33, the glycoprotein expressed in X-33(och1-), containing smaller N-glycan. [Conclusion] The results suggested that X-33(och1-) strain can be used as an expression host for production of glycoproteins lacking the outer-chain hypermannoses, and a host could be used for further N-glycosylation engineering.

Abstract:Abstract:［Objective］To construct prokaryotic fusion gene expression vector pET-28a- cag pathogenicity island protein 4(cag4)，express the recombinant fusion protein cag4 and analyze the enzyme activity of recombinant protein.［Methods］We cloned cag4 gene, this encoded lytic transglycosylase in CagPAI of Helicobacter pylori NCTC11637 via PCR method. After TA cloning and restriction enzyme digested confirmed, we constructed prokaryotic expression plasmid pET-28a- cag4 and transformed the plasmid into E.coli BL21 (DE3) for heterogenesis expression after it is sequenced. We performed SDS-PAGE and Western blot. We purified and collected the recombinant protein by using Ni2＋-NTA columns under denaturation condition.We renatured the recombinant protein via dialysis. Besides, we isolated peptidoglycan from Micrococcus lysodeikticus by SDS boiling method.We detected the enzyme activity of recombinant protein by zymogram analysis and the effect of pH values by turbidimetric analysis.［Results］The recombinant protein has the enzyme activity of lytic transglycosylase.The enzyme activity, which defined as the rate of degraded (DA?min-1mg-1), was higher at pH 6.0 group than other groups (pH 5.0 and 7.0).［Conclusion］The cag4 of Helicobacter pylori NCTC11637 has the enzyme activity of lytic transglycosylase.

Abstract:Abstract: [Objective] To study dehydrogenation by short ethoxy chain nonylphenols dehydrogenase (sNPEO-DH) from Ensifer sp. AS08. [Methods] We screened four amino acid residues of sNPEO-DH that are adjacent to the isoalloxazine ring of the coenzyme flavin adenine dinucleotide (FAD) by using multiple sequence alignment and homology modeling. Mutations were introduced by site-directed mutagenesis. The recombinant proteins were expressed, purified and assayed. [Results] The relative activities of mutants N90A and N509A against the hydrophilic substrate PEG1000 decreased to 51% and 89%, respectively, and against the hydrophobic substrate NPEOav10 decreased to 26% and 40%, respectively; indicating that N90 and N509 might be related to substrate binding. The relative activity of mutant H465A and N507A lost 90% and 100%, respectively; “stop-flow” experiments revealed that the processes of proton transfer from substrate to FAD and from FAD to enzyme were blocked in mutant N507A and H465A, respectively. [Conclusion] Amino acid residues N507 and H465 located at the activity center of sNPEO-DH and play roles as catalytic sites for the oxidative dehydrogenation of the substrates and FADH2, respectively.

Abstract:Abstract：[Objective] To elucidate the co-localization characteristic between porcine epidemic diarrhea virus (PEDV) N protein and B23.1 phosphoprotein. [Methods] Two pairs of primers used to amplify N gene and B23.1 gene were designed and synthesized according to CV777 N gene sequence (AF353511) and human nucleolar phosphoprotein B23.1 gene sequence (BC050628.1), respectively. The PEDV N gene and B23.1 gene were amplified by RT-PCR from PEDV strain CV777 and Vero E6 cells, respectively; then cloned into eukaryotic expression vector pAcGFP1-C1 and pDsRed2-N1, to generate the recombinant plasmids pAcGFP1-C1 / N and pDsRed2-N1/B23.1, respectively. Vero E6 cells were transfected with plasmids pAcGFP1-C1 / N and pDsRed2-N1/B23.1. [Results] The fusion proteins successfully expressed in transfected Vero E6 cells by western blot analysis, and the PEDV N protein and the B23.1 phosphoprotein showed co-localization features in co-transfected cells through confocal microscopy analysis. [Conclusion] The results will help to identify the nucleolar localization signals in PEDV N protein and to elucidate the mechanism of N protein located in nucleus.

Abstract:Abstracts: [Objective] To identify and characterize a hydrocarbon-degrading bacterium isolated from the sediment of the Yellow Sea. [Methods] We used 16S rRNA gene sequences based phylogenetic analysis, physiological and biochemical characterization, DNA G+C content assaying, determination of cellular fatty acids, testing of carbon sources and respiratory lipoquinone and experiment of DNA–DNA relatedness. Its capability of degrading aliphatic hydrocarbons in ONR7a media supplemented with nine n-alkanes, separately, as sole source of carbon and energy was further determined. [Results] The Gram-negative isolate PY97S was a member of the genus Marinobacter, catalase- and oxidase-positive, and with Q-9 as its predominant respiratory lipoquinone. The similarity between its 16S rRNA gene and that of its most closely related type strain in GenBank Marinobacter koreensis DD-M3T was 96.93%, and their level of DNA relatedness was 46.7%. The appropriate temperature for its growth ranged from 15℃ to 35℃ with the optimum of 30℃, the appropriate initial acidity from pH 6.0 to 9.5 with the optimum of pH 7.0, and the appropriate salinity (NaCl) from 0% to 10% with the optimum of 0%. It metabolized many carbohydrates and organic acids and was sensitive to diverse antibiotics including ampicillin and piperacillin. The G+C content of its genomic DNA was 48.2 mol%. The major fatty acids were 2-methyl C15:0 (29.97%), C16:1ω7c (27.22%), C12:0 (22.22%) and C16:1ω9c (5.73%). [Conclusion] The isolate PY97S was identified as a petroleum hydrocarbon-degrading novel species of genus Marinobacter, holding the potential of being applied in the bioremediation of oil spill.

Abstract:Abstract：[Objective] To understand the impacts of anthropogenic activities on structure and composition of bacterial communities and to evaluate how bacterial communities respond to environmental gradients at coastal sediments. [Methods] The diversity of bacterial communities in sediments form tourist and mariculture zones at coastal area of Dalian Changshan Islands was assessed using terminal restriction fragment length polymorphism (t-RFLP) and denaturing gradient gel electrophoresis(DGGE) approaches. Meanwhile, 16S rRNA clone library was constructed to reveal the composition and structure of bacterial communities in the most seriously polluted site (D4). [Results] There were much higher values of richness, Shannon-wiener and evenness index at D4 site by the analysis of terminal restriction fragments (t-RFs). The clustering result on the t-RFs areas and DGGE patterns showed that the bacterial diversity of tourist zone were more similar, while the distinction was increased with pollution levels among the tourist and mariculture zones. The 16S rRNA clone of D4 revealed that the Proteobacteria were the dominant phylum, and γ-proteobacteria was the main class within Proteobacteria. [Conclusion] The study documented changes in bacterial community structure by human impacts of mariculture than geographical location.

Abstract:Abstract: [Objective] The study aims to investigate the phylogeny diversity of the denitrification bacteria communities in the sediments of the eutrophic East Lake, Wuhan based on nitrite reductase gene (nirS) restriction fragment length polymorphism (RFLP) method and sequencing analysis, and to analyse community variation according to the environment parameters. [Methods] We collected the sediment samples from the four typical sub-lake of East Lake in Wuhan, Guozheng Lake, Tangling Lake, Tuan Lake and Miao Lake, and measured the environmental parameters appropriately. After extracted the genomic DNA from the sediment, four nirS gene clone libraries were successfully constructed. The operation taxonomy units (OTUs) were determined by RFLP method and the representative fragment of every OTU was sequenced. The diversity, richness and evenness statistics of the NirS-like communities were calculated by using DOTUR software. Neighbour-joining phylogenetic tree was constructed basing on the amino acid sequences of NirS from the East Lake sediments and reference sequences retrieved from the GenBank database. The relationship between tested NirS communities and the references from different environments was also discussed. [Results] Environmental parameters showed that Miao Lake sediment contains the highest amount of total nitrogen (TN) and NH4+-N, while the Tuan Lake sediments contains the lowest amount. Among the four sub-lakes, Tuan Lake harbours the highest diversity and richness of NirS-like denitrifiers, while the denitrifiers in Miao Lake was the lowest. Phylogenic analyses suggested that sedimentary NirS-like denitrifiers in the East Lake could be distributed into three groups, Group I to III. Group I accounts for 67.7% of all tested communities. Eighty-one percent of sequences from Guozheng Lake were clustered into Group I, while 67.7% of sequences from Miao Lake were clustered into Group II. Comparative analysis of communities from East Lake and artificial wetland found there are phylogenetically related. [Conclusion] There are diverse and abundant NirS-like denitrifiers inhabited in the sediments of East Lake, Wuhan. The diversity indices and spatial distribution of these communities are affected by the content of TN, NH4+ and NO3- nutrients in the sediments.

Abstract:Abstract: [Objective] In order to investigate the distribution of shiga toxin-producing Escherichia coli (STEC) among healthy sheep in a farm and the pathogenicity to mice and Vero cells of these STEC isolates. [Methods] We used polymerase chain reaction (PCR) to detect genes of eaeA, stx1, stx2, hlyA, which had been developed in this laboratory previously, combing the selective cultivation and Chrom-Agar (CA) O157 plates to isolate STEC strains. [Results] A total of 107 STEC strains were isolated in a sheep farm during six visits from August, 2008 to January, 2009. The isolation rate was 19.8% (107/550). These isolates belonged to 41 O serotypes and 60 O: H serotypes, except that 21 were O non- typable and 1 was rough. O93 was the common serotype. Some isolates such as O5, O91, O103, which are reported in other countries originated from healthy sheep were also isolated in this study. Stx2 positive rate was higher than that of stx1. 50% lethal dose assay in mice indicated the pathogenicity of isolates was low and none of the 3 tested isolates caused mice death. We selected 107 stx gene positive STEC strains to induce the lambdoid bacteriophages. The results showed that 71 out of 107 isolates formed plagues, while 28 did not after induction. Detection of Shiga toxins for three tested isolates in Vero cell assay indicated one stx gene positive strain lacked the toxigenicity to Vero cells. [Conclusions] Sheep are the natural reservoirs of STEC and they are healthy to carry STEC. Although these STEC isolates experience low pathogenicity to mice, they are potential threat to human health. Shiga toxin gene positive were not equal to production of Shiga toxins, so we need to further study the Shiga toxin expression and regulation mechanism.

Abstract:Abstract: [Objective] To set up suitable kinetic models for the evaluation of heat inactivation rate of Listeria monocytogenes in ground beef. [Methods] Ground beef, inoculated with 3 strains of L. monocytogenes, was subjected to heating at 55℃, 57.5℃, 60℃, 63℃, 66℃ or 70℃ to develop the isothermal kinetic models. The survival curves were fitted with modified Gompertz model after the bacterial counts were decreased from 109CFU×g-1 to 103CFU×g-1. Natural logarithm of μ (the relative inactivation rate) and M (time constant) were fitted with linear regression model. The kinetic models were validated at 59℃ and 64℃. [Results] We established one first-level model and one secondary-level model to predict the inactivation rate. In addition, we validated the accuracy and deviation of the models to guarantee their feasibility. [Conclusion] The models should be suitable for describing the survival of L. monocytogenes in ground beef at different temperatures, which are helpful for the control of the pathogens.

Abstract:Abstract [Objective] To reduce immunogenicity of recombined staphylokinase (r-Sak), site-directed mutagenesis of Arg77 and Glu80 residue was performed to simultaneously remove T and B cell epitope in r-Sak molecule. [Methods] The solvent accessible surface areas of residues 77 and 80 in r-Sak were used to analyze rational design of Sak mutation. The Sak mutants were expressed in E coli DH5α. After purified by a 3-step chromatography, their fibrinolytic activities and immunological properties were analyzed. [Results] Immunogenicity tests suggested that Sak induced a Th2-type immune response. Substitution of Glu80 with alanine or serine successfully reduced its solvent accessible surface area while simultaneously removing part of the T and B cell epitope. Changing Arg77 to glutamine, asparagine, or lysine removed only part of the T cell epitope. Of six dually substituted variants, Sak(R77Q/E80A) and Sak(R77Q/E80S) variants effectively eliminated part of the B and T cell epitopes, which markedly reduced their immunogenicity.

Abstract:Abstract: [Objective] Porcine β-defensin-2 mature peptide gene fragments were correctly integrated into genome chromosome of the yeast KM71, which was stable Pichia pastoris strains expressing porcine β-defensin-2 mature peptide. Porcine β-defensin-2 mature peptide was expressed successfully. [Method] Considering the biased codon usage of Pichia pastoris, we designed three primers and used PCR technology to get the gene fragment of pBD-2 mature peptide. We obtained the recombinant vectors of pPIC9K-pBD-2 and pPIC9k-GST-pBD-2. The recombinant plasmids linearized were transformed into yeast KM71 by electroporation. By changing the parameters of expression environment, screening yeast clones induced by methanol expressed the Porcine β-defensin-2 mature peptide finally. [Result] GST-Porcine β-defensin-2 and Porcine β-defensin-2 gene fragments were successfully integrated into genome chromosome of the yeast KM71, We finally constructed the recombinant yeast; Protein GST-Porcine β-defensin-2 and Porcine β-defensin-2 were expressed successfully. The peptide of Porcine β-defensin-2 can restrain the Salmonella choleraesuis (C500-). [Conclusion] We got the recombinant yeast which can express porcine β-defensin-2 mature peptide. This study is an exploration to express Porcine β-defensin-2 in eukaryotic cells, It established foundation for the subsequent study of large-scale expression of Porcine β-defensin-2 mature peptide.