Abstract:Abstract: Bioluminescence imaging is one of the bio-optical imaging techniques which report definite biological event in living animals with genetic modification. With high sensitivity, simple operation and high precision, it is particularly applied in observing vital processes like viral infection and tumor growth in vivo. We summarize the principle of bioluminescence imaging, introduce its application in finding virus replication site, study of interferon（IFN） inhibiting-virus effect and real-time visualization of viral latent infection and reactivation, and preview the trend of bioluminescence imaging technological development.

Abstract:Astract: Peptaibols are a family of antimicrobial peptides, which are synthesized by non-ribosomal peptide synthetases (NRPS) and contain high proportions of α-aminoisobytyric acid (Aib). Up to now, 317 peptaibols have been identified, and the majority of them are produced by Trichoderma strains. In this review, we described the diversity, fermation, purification, identification and biosynthesis of peptaibols from Trichoderma.

Abstract:Abstract: Since its invention in 1970s, nucleic acid sequencing technology has contributed tremendously to the genomics advances. The next-generation sequencing technologies, represented by Solexa from Illumina, SOLiD from Applied Biosystems and 454 from Roche, re-energized the application of genomics. In this review, we first introduced the next-generation sequencing technologies, then, described their potential applications in the field of microbiology.

Abstract:Abstract: [Objective] To investigate co-existence of resistance genes (β-lactamases, BLs, and aminoglycoside-modifying enzymes, AMEs) and their association with the genetic marker genes of ClassⅠ, Ⅱ, Ⅲ integrons carried by multiresistant Escherichia coli isolates. [Methods] We used VITEK-GNS to determine the susceptibility of 136 isolates to 14 antibiotics, disc agar diffusion test to confirm ESBL-producing isolates, PCR to analyze BLs, AMEs and integrons genes, conjugation and plasmids extraction to locate the methylase genes. [Results] We found that 70.59% of the isolates produced ESBLs. They showed stronger resistance against 9 antibiotics than isolates without ESBLs in 14 antibiotics. PCR amplification showed that the positive rate of BLs, AMEs and qacE△1-sul1 was 96.32%, 100% and 94.12%, respectively, but ClassⅡ, Ⅲ integrons genes were negative. Only one strain was oprD2 gene negative. 90.44% of the isolates were both positive for BLs and qacE△1-sul1 genes, and 94.12% for AMEs and qacE△1-sul1 genes, but there was no statistical significance. 90.44% of the isolates were all positive for the 3 genes. 12 strains carried 16S rRNA methylase genes including armA (2.21%), rmtB (7.35%) while rmtA, rmtC, rmtD were negative. The conjugation assay and plasmids mapping results showed that the methylase genes were located on the 23 kb plasmid, and the efficiency of transformation was 83.3%. [Conclusions] The results suggested that there was a tight correlation between the 3 genes (BLs, AMEs and qacE△1-sul1)and the incidences of multi-resistance of Escherichia coli, but there was no correlation of the incidence of multi-resistance with ClassⅡ, Ⅲ integrons. 16S rRNA methylase genes harboured plasmids of ~23 kb which transformed other isolates within the same strains efficiently.

Abstract:Abstract: [Objective] As one of the most abundant and cheapest recyclable bio-resources, cellulosic biomass appear to be the promising new energy if the two major components, glucose and xylose, are efficiently used. [Methods] We compared the metabolism of Rhizopus oryzae growing on glucose or xylose. We measured biomass accumulation, intercellular metabolite contents, and organic acids production. [Results] When cultured on xylose, the rate of bio-transformation reached 35.2% compared to 24.3% in the glucose. Intercellular contents accumulated much more by using xylose as sole carbon than using glucose. However, the quantity of organic acid was less with xylose than with glucose. In addition, the NADH/NAD+ and ATP quantity also differed. [Conclusion] With our data analysis, solution is suggested to the all-around utilization of the cellulous in the fermentation industry, which means increasing biomass by xylose at first stage, and producing organic acids or other ferment products at the second stage. Such suggestion may be a reasonable guideline to the economic application of the cellulosic material in the near future.

Abstract:Abstract: [Objective] To improve the tolerance of main metabolites, we used genome shuffling to achieve high 1,3-propanediol producing mutants. [Methods] Based on 96 deep-well palates containing prepared ended fed-batch broth as an efficient selection method, genome shuffling has been applied in strain improvement. [Results] Five high producers were obtained after genome shuffling (LSG1, LSG2, LSG4, LSG5 and LSG6). During batch fermentation (3 L), the 1,3-propanediol production of the five mutants were improved 17.0%，19.0%，12.9%，23.9% and18.0%, compared with the parent strain; the conservations from glycerol were improved 17.7%，20.0%，13.3%，24.4% and 17.7%. [Conclusion] Genome shuffling was an efficient approach for strain improvement, and 96 deep-well palates containing fed-batch broth has been demonstrated as an efficient selection approach.

Abstract:Abstract: [Objective] To study the effects of gene pta disruption on biosynthesis of L-tryptophan. [Methods] The pta gene of the L-tryptophan producing strain E. coli TRTH was disrupted by Red recombination technology and a pta mutant E. coli TRTHΔpta was constructed. Fed-batch fermentation of E. coli TRTHΔpta was carried out in 30-Liter fermentor to investigate the biomass, L-tryptophan production, organic acid content and the concentration of NH4+, lactate, pyruvate and succinate. The metabolic flux balance model of L-tryptophan synthesis by E. coli was established. Based on this model, the practical metabolic flux distribution of E. coli and its pta mutant were determined with the linear program planted in MATLAB software. [Results] Compared with E. coli TRTH, the pta mutant was able to maintain higher growth rate at exponential phase, the final biomass and the L-tryptophan production were increased by 52.7% and 46.8% respectively. Meanwhile, the data analysis of organic acids accumulated during fed-batch culture showed that the concentration of acetate was decreased to 2.5 g/L, which was only 19.5% of that of the parental strain; as the decreased concentration of succinate, the accumulation of pyruvate and lactate was increased. The concentration of Na+, K+, PO43- were consistent with E. coli TRTH during the fed-batch culture, the concentration of NH4+ was decreased by 33.2%. The metabolic flux analysis indicated that EMP pathway and TCA cycle were reduced by 7.4% and 32.2% respectively, but PP pathway was increased by 8.4% compared with E. coli TRTH during the middle and late period of the fed-batch culture. [Conclusion] In the process of L-tryptophan fermentation, pta gene deletion in E. coli TRTH led to change in metabolic flux and acetate content, which derepressed its inhibition on cell growth and production of L-tryptophan and finally made a substantial increase of bacterial biomass and L-tryptophan production.

Abstract:Abstract: [Objective] To screen lipases applied to biodiesel production, a lipase-producing microorganism was isolated and the enzyme was characterized. [Methods] A strain with lipolytic activity against Jatropha oil was isolated from the soil pretreated by Jatropha curcas L. seed and cultivated on Jatropha oil as sole carbon source. The organic solvent tolerance of the isolated strain and its lipase were measured. The esterification and transesterification catalyzed by the isolated lipase were surveyed. The isolated strain was identified according to the physiological and biochemical characteristics as well as 16S rDNA sequences analysis. [Results] The lipolytic activity of the strain LP-2 was 3.03 U/mL. The relative biomass of strain LP-2 in the media containing 5%(v/v) methanol was 87.3%. The residual activity of LP-2 lipase in 10%(v/v) hexane was 80.9%. LP-2 lipase could catalyze esterification between lauric acid or palmitic acid and n-butanol, n-octanol, dodecanol or glycerol; stearic acid and n-octanol, dodecanol or glycerol; oleic acid and methanol, n-butanol, n-octanol or dodecanol. The transesterification of Jatropha oil with methanol could be catalyzed by LP-2 lipase. Strain LP-2 was identified as Staphylococcus epidermidis and named Staphylococcus epidermidis LP-2. [Conclusion] S. epidermidis LP-2 lipase had the ability to catalyze esterification and transesterification reactions, which suggested that it had potential of producing biodiesel.

Abstract:Abstract: [Objective] To obtain new fungi producing dextranase, we identified a strain F1001 showing high dextranase activities. [Methods] Morphological and ITS rDNA sequences homology analysis were performed to identify the strain F1001. The enzyme was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation and Sepharose 6B column chromatography. We studied the catalytic properties and the mechanism of the dextranase. [Results]The isolated strain F1001 was identi?ed as Penicillium aculeatum precisely by ITS rDNA sequences homology analysis. Its molecular mass was estimated to be about 66 kDa by SDS-PAGE. Activities of dextranase were measured with dextran 70 kDa as substrate, the optimal reaction temperature was 35℃, and the optimum pH was 5.0, it was relatively stable in the condition of pH 4.0 ? 7.0 and under 50℃. The optimum substrate concentration was 3% (w/v). The final dextranase hydrolysis product was isomaltose, it was proved that the enzyme was endodextranase and only had activity with dextran joined mainly by continual α,1-6 glucosidic linkages. The Km for dextranase was calculated to be 3.55×10-5 mol/L, and the Vmax was 4.29×10-2 mol（Glu）/min?L. The enzyme activity was enhanced by Zn2+and Cu2+, and the low concentration of Cu2+ could improve the dextranase activity to 134.7%. However, the enzyme was strongly inhibited by Mn2+. [Conclusion]We isolated a new strain F1001 producing high dextranase activity and the enzyme was relatively stable. These results may provide an important basis for industrial applications.

Abstract:Abstract: [Objective] To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization. [Methods] For immediately downstream region of initiation codon in gldA, we designed optimized sequence by choosing higher AT-content synonymous, in order that this region’s AT-content was increased without changing the corresponding amino acids. Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR, so that physically optimized gene gldA-4 was acquired. We cloned gldA-4 into pET-32a(+) to construct expression plasmid pET-gldA-4, which was transformed into Escherichia coli BL21(DE3) for gaining engineering bacteria E. coli-4, by contrast engineering bacteria involved gldA-WT named E. coli-WT. After E. coli-4 and E. coli-WT were fermented in shake flasks, we measured enzyme activities of expression products with glycerol as substrate. [Results] Four gldA-4’s bases in the second, fifth and sixth codon were different with gldA-WT, so AT-content of the optimized gene was up to 80.0% higher than the wild gene’s 53.3%. Furthermore, enzyme activity of E. coli-4’s crude extract was 191.3 U/mL more three times than E. coli-WT’s 48.3 U/mL. [Conclusion] This optimization scheme was quick and easy, but indeed increased dehydrogenase’s activity. It possible becomes a universal method to improve heterogenous expression level of target genes.

Abstract:Abstract: [Objective] To isolate and characterize bacteria to degrade cypermethrin (CP). [Methods] We used enrichment culture to isolate and characterize bacterial strains based on the observation of morphological and biochemical characters and analysis of 16S rRNA gene sequences. We determined the concentration of CP, metabolite, densities of strain cells and cell surface hydrophobicity (CSH) in pure culture liquid. [Results] We isolated a bacterium able to effectively degrade CP from polluted wastes of pesticide plant and assigned it as strain L12. Phylogenetic analysis based on 16S rRNA gene showed the similarity of 99% between strain L12 and Bacillus cereus. In pure culture with CP as the sole carbon and energy source, 85.6% of CP at initial concentration of 50 mg /L was degraded in 5 days. Thin layer chromatography (TLC) analysis of the culture extracts revealed presence of a metabolite (Rf 0.13), and HPLC analysis confirmed the retention time of the metabolite (2.26min) corresponded with 3-phenoxybenzoic acid (3-PBA) standard. The method of microorganism adhering to hydrocarbon (MATH) was used to analyze strain L12 of the highest CSH of 68.91%. [Conclusion] The results showed that strain L12 belonged to Bacillus cereus, which showed CSH and capacity to degrade CP to 3-PBA.

Abstract:Abstract: [Objective] To analyze the serological and genetic divergence in the Vibrio parahaemolyticus from environment and cases of foodborne disease, and to compare these two groups in terms of virulence and other biological traits. [Methods] Serotyping, multi-PCR, and pulse-field gel electrophoresis (PFGE) were carried out. [Results] The main serotypes of cases isolates were O3:K6 (40.8%), O1:KUT (7.1%), O4:K8 (7.1%), and the main serotypes of environmental isolates were O3:KUT (14.2%),O1:KUT (11.4%) and O2:K3 (11.4%）. No O3:K6 strain was isolated from environment. Most cases isolates were tdh positive and trh negative, which account for 83.6%, while most environmental isolates were tdh negative and trh negative, which account for 88.5%. PFGE indicated that Clone P1 was the dominant clone cluster, including serotype O3:K6 (29 isolates), O4:K68 (4 isolates）, O11:K36 (3 isolates), O1:K25 (2 isolates), and these strains were all obtained from cases of foodborne disease. No dominant clone cluster was found in environmental isolates. [Conclusions] V. parahaemolyticus from cases of foodborne disease in Shenzhen mostly were hemolytic, tdh positive and trh negative，there were dominant PFGE type in cases isolates, the main serotype was O3:K6, while environmental isolates mostly were non-hemolytic, tdh negative and trh negative, no dominant PFGE type was found.

Abstract:Abstract:[Obiective] Finding a series of archaeal 16S rRNA gene universal primer applied strategies to detect complex microbial diversity in environmental samples, especially with rapidly development of next generation sequencing technology challenge. [Methods] We used Oligocheck soft to simulate two pairs of archaeal 16S rRNA gene universal primers with RDP (Ribosomal database project) database 16S rRNA gene sequences matching percentage. In succession, the sediment sample was constructed for two clone libraries by using two pairs of archaeal 16S rRNA gene universal primers. [Results] The soft simulation matched percentage result suggests that primer f109/r958 is better than primer f21/r958. This result is consistent with RFLP and diversity index analyses by two clone libraries. [Conclusion] Multi-primers and properly primer are used to recovery environmental microbiology diversity, which will be advanced in environmental microbial resolution.

Abstract:Abstract: As a transhydrogen in the mitochondrial respiratory chain, Coenzyme Q (CoQ) has a critical role in the metabolism. 4-hydroxybenzoate polyprenyltransferase (UbiA), which coded by ubiA gene, is the rate-limiting enzyme in the CoQ biosynthesis progress. However, the relationship between structure and function remains unclear. [Objective] To set up a synthetic oligonucleotide-based shuffling model and apply in the random mutation of DNA sequence of ubiA gene which codes putative active site. [Methods] By using ubiA knockout mutant of E. coli. MC4100 as receptor, we set up a local Shuffling model by replacing target sequence with a random sequence fragment. Sequences of mutants and their function were analyzed. [Results] After local Shuffling and two cycle of screening, we gained seven mutants. Compared to the wild type, most of the mutant amino sequences showed obvious change, and had different change trend of CoQ production. Sequencing result showed that three aspartic acid sites may be highly related to UbiA catalyze activity. [Conclusion] The local Shuffling model we set up is feasible. By applying this model, we preliminary verified several location of key amino acid sites of UbiA.

Abstract:Abstract: [Objective] To explore a new rapid detection method for detecting of Food pathogens. [Method] We used the Smartongue, to determine the composition informations of the liquid culture samples and combined with soft independent modelling of class analogies (SIMCA) to analyze their respective species, then set up a Smartongue -SIMCA model to discriminate the V. parahaemolyticus.[Results]The Smartongue has 6 working electrodes and three frequency segments, we can built 18 discrimination models in one detection. After comparing all the 18 discrimination models，the optimal working electrodes and frequency segments were selected out, they were：palladium electrode in 1 Hz frequency segment, tungsten electrode in 100 Hz and silver electrode in 100 Hz. Then 10 species of pathogenic Vibrio were discriminated by the 3 models. The V.damsela, V. metschnikovii, V. alginalyticus, V. cincinnatiensis, V. metschnikovii and V. cholerae O serogroup samples could be discriminated by the SIMCA model of V. parahaemolyticus with palladium electrode 1 Hz frequency segment; V. mimicus and V. vulnincus samples could be discriminated by the SIMCA model of V. parahaemolyticus with tungsten electrode 100 Hz frequency segment; V. carcariae and V. cholerae non-O serogroup samples could be discriminated with the SIMCA model of V. parahaemolyticus in silver electrode 100 Hz frequency segment. The accurate discrimination of ten species of Vibrio samples is 100%. [Conclusion]The Smartongue combined with SIMCA can discriminate V. parahaemolyticus with other pathogenic Vibrio effectively. It has a promising future as a new rapid detection method for V. parahaemolyticus.

Abstract:Abstract: [Objective] The aims of this study were to investigate yeast diversity in the plateau lake and to explore the value of these yeasts in such ecological environment. [Methods]Yeasts were isolated from 9 water and 4 soil samples collected in Chenghai lake in Yunnan province. The isolates were identified by using large-subunit (26S) rDNA gene D1/D2 domain sequence analysis and traditional methods. The ability of the yeast strains to produced various enzymes was tested. [Results] In total 64 yeast strains were isolated, these strains were identified as 22 species in 8 genera, including 4 suspected new species or variety. Genera Cryptococcus and Geotrichum were shared in the water and soil samples. Nine strains with secreted enzyme activities were selected. One of them could produce both protease and amylase .[Conclusion] The preliminary result showed the biodiversity of yeasts in Chenghai lake and the application potential of the yeasts isolated.

Abstract:Abstract：[Objective] To study the expression of immunity and inflammatory mediator factor PTX3 in intestinal epithelial cells treated with Lactobacillus acidophilus NCFM and further to reveal the regulatory mechanism. [Methods] Caco-2 cells were cocultured with Lactobacillus acidophilus NCFM for 0, 2, 4, 8, 12 h and 0, 0.5, 1, 2, 4 h respectively, then the total RNA and protein were extracted. The expression of PTX3 gene was analyzed by Real Time RT-PCR. The phosphorylation levels of NF-κB was analyzed by Western Blot. Caco-2 cells were pretreated with PDTC for 30 min before cocultured with Lactobacillus acidophilus NCFM for 2 h, then the total RNA was extracted and the expression of PTX3 gene was analyzed by Real Time RT-PCR. [Results] Lactobacillus acidophilus NCFM could induce the expression of PTX3 in Caco-2 cells. The PTX3 expression peaked at 4 h after coculture. Then its expression gradually waned out. Lactobacillus acidophilus NCFM could rapidly activate the phosphorylation of NF-κB, and the expression of PTX3 was decreased notably after pretreated with PDTC for 30 min. [Conclusion] Lactobacillus acidophilus NCFM could transiently regulate the immunity and inflammatory mediator factor PTX3 expression through rapidly activating NF-κB signaling pathway in Caco-2 cells.

Abstract:Abstract: [Objective] To explore the mechanism of cholesterol-degrading by Lactobacillus plantarum LpT1 and LpT2 in vitro. [Methods] After Lactobacillus plantarum LpT1 and LpT2 being inoculated and cultured in the medium MRS, MRS+CH, MRS+CH+S and MRS+CH+N, the cholesterol content in supernatant, deposit and cells, and the total cholesterol content before and after inoculation were determined and compared to primarily predict the mechanism of cholesterol-degrading by L. plantarum. [Results] The mechanism of cholesterol- degrading by L. plantarum LpT1 and LpT2 in vitro involved in metabolic and non-metabolic pathway. Non-metabolic pathway was related to co-precipitation and cell absorption. Metabolic pathway was due to the production of the special enzymes during the growth of L. plantarum which made the cholesterol degrade to be other materials and caused its content reduction. [Conclusion] L. plantarum has shown its ability to degrade cholesterol in vitro.