Abstract:As a ubiquitous metalloid in the environment, arsenic (As) has attracted concerns by humans due to its strong carcinogenic property. Environmental As fate is often affected by physico-chemical and biological factors, of which microbe-mediated arsenic transformation predominates. Arsenic uptake, redox, methylation, sequestration and efflux have been verified to be involved in As resistance in microbes. In some microbes, As transformation contributes to detoxification, while some can obtain the energy for their growth and reproduction from this process. Here we reviewed the mechanisms of microbe-mediated As uptake, transformation, sequestration and efflux, which may help elucidate As geochemical cycling and provide the methods to remediate As-contaminated soils and waters, and to reduce As uptake by crops.

Abstract:Outer membrane vesicles (OMVs) are vesicle-like structures, widely present in gram-negative bacteria and even in some gram-positive bacteria. OMVs contain biological active substances and their sizes are normally between 20 to 250 nm. Components of OMVs include lipopolysaccharide, outer membrane protein, phospholipids, DNA, as well as the periplasmic components produced during their formation. OMVs are non-viable vesicles that contain multiple antigenic proteins from the bacterial outer membrane, and are capable of activating the immune system, therefore they are considered to be potential vaccine candidates. Although outer membrane vesicles were discoverd more than 50 years ago, hardly any reports were published in China. In this review, we summarized the progress of outer membrane vesicles as a novel strategy for disease prevention and control in two aspects: the mechanism of the outer membrane vesicle-induced immune response and the advances in OMVs vaccine. This review provides some information on outer membrane vesicles as vaccine development.

Abstract:[Objective] Bioethanol is a new type of green energy, and predominantly produced currently from starch such as corn and sweet potato. However, pectin contained in the substrates will increase the viscosity of ethanol fermentation broth, which will affect the mass transfer and increase the burden of equipment. In this study, we expressed pectinesterase on the Saccharomyces cerevisiae cell surface and evaluated its effect on fermentation viscosity decrease. [Methods] Pectinesterase gene was fused with the C-terminal-half region of α-agglutinin and then inserted into the downstream of the secretion signal gene, to generate a yeast surface-display expression vector pMGK-AG-PE, which was then transformed into the industrial S. cerevisiae. [Results] Recombinant yeast strain PE successfully displayed pectinesterase on the surface of cells with 2.6 U/g wet cells. The recombinant enzyme performed the maximal activity at 60 ℃, pH 5.0, and this enzyme had high activity and stability from pH 4.0 to 5.5. In the simultaneous saccharification and fermentation of sweet potato, the ethanol production of the PE strain was 95 g/L. The viscosity of fermentation broth using the PE strain was lower than that of the parent strain, 120 mPa.s compared to 145 mPa.s after 12 h of fermentation. [Conclusion] Expressing pectinesterase on yeast cells surface decreased viscosity of fermentation broth, which is beneficial to starch degradation and ethanol production.

Abstract:[Objective] A new lactic acid bacterium was isolated from Chinese traditional pickles and its potential probiotic properties were analyzed. [Methods] The strain was identified by morphological observation, physiological and biochemical tests and 16S rRNA gene comparison. Its probiotic properties were evaluated, including resistance to NaCl, the minimum inhibitory concentration of antibiotics, nitrite degradation ability and cholesterol removal ability. [Results] This strain was identified as Lactobacillus alimentarius W369 (No. CGMCC 7.180). L. alimentarius W369 could survive under the concentration of 10% NaCl and it was sensitive to some common antibiotics. By studying its probiotic properties, we found that its nitrite degradation rate reached 92.92% after 72 h and the cholesterol removal rate reached 31.80%. The fermented supernatant showed strong abilities of DPPH· and the superoxide anion radical scavenging capacity, as well as Fe2+ chelating ability. The clearance rate were (88.02±1.48)%, (43.75±3.10)% and (29.99±2.34)% respectively. Strain W369 also exhibited strong reducing capacity, hydroxyl radical scavenging action and lipid peroxidation inhibition capacity. Both the fermented supernatant and cell-free extracts exhibited glutathione peroxidase (GSH-Px) and superoxide dismutase (Mn-SOD) activities. [Conclusion] L. alimentariusW369 possessed many probiotic properties including nitrite degradation ability, cholesterol removal ability and antioxidant activity. It has many potential application values.

Abstract:[Objective] Sediment bacteria are the important biological factors for remediating of eutrophic environments. To enrich our understanding of the bacteria communities in eutrophic urban lake sediments for better environment protection and pollution control in urban lake eco-systems, we resolved the composition of bacteria communities and their spatial variation in the sediments of a middle-size eutrophic urban lake, East Lake. [Methods] We used 16S rRNA gene RFLP and sequencing methods to generate the phylogeny information of the bacteria community, used principal coordinates analysis (PCoA) and canonical correspondence analysis (CCA) methods to resolve the relationship between East Lake and other lakes, and the relationship between environmental factors and the bacteria communities. [Results] Sediments inhabited 13 phyla and 2 unclassified clusters. PCoA further revealed that the bacteria communities in three sub-lakes of East Lake sediments were closely related to the communities in similar eutropic lake environments, and divergent from the hypereutrophic sub-lake Miao Lake, which was also found to inhabit a relative abundant amount of Thermogymnomonas-type archaea. CCA further revealed that the distribution of bacteria was closely correlated with the carbon, nitrogen and phosphate contents in the sediments. [Conclusion] The environment factors regulated the bacteria community composition and distribution. The results of this study providereference to the research, protection and pollution control on urban lake eco-systems.

Abstract:[Objective] To study nitrogen metabolism of Zygosaccharomyces rouxii and its relationship with the formation of soy sauce ethyl carbamate precursors. [Methods] Z. rouxii ZQ02 was cultivated with single source of nitrogen, preferred nitrogen sources or under salt stress, to investigate its ability of using arginine, citrulline and urea. [Results] Alanine, glycine and asparaginate were confirmed to be the preferred nitrogen sources of Z. rouxii ZQ02. Addition of preferred nitrogen sources did not inhibit the use of urea and citrulline, on the contrary, the consumption of urea and citrulline by Z. rouxii ZQ02 was stimulated with the addition of alanine and glycine. Z. rouxii ZQ02 did not accumulate any citrulline and urea from degradation of arginine. Urea and citrulline were used by Z. rouxii ZQ02 in the medium with single source of nitrogen. However, use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under saline stress, resulting in the incomplete use of ethyl carbamate precursors. [Conclusion] Use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under high salt stress, resulting in the accumulation of ethyl carbamate precursors produced by other microorganisms during soy sauce fermentation.

Abstract:[Objective] To study the inhibitory effect of 3' untranslated region (UTR)-targeted artificial microRNA (amiRNA) against porcine reproductive and respiratory syndrome virus (PRRSV) replication in porcine alveolar macrophages (PAM). [Methods] Recombinant adenovirus (rAd) expressing the 3' UTR-targeted or control amiRNA and green fluorescent protein (GFP) reporter gene were generated by transfecting AAV-293 cells with the transfer vector. The expression of sequence-specific amiRNA was detected by quantitative RT-PCR. The anti-PRRSV effect of amiRNA was detected by quantitative RT-PCR, Western blotting and viral titration assay. [Results] Two rAds, namely rAd-amiR3UTR-GFP and rAd-amiRcon-GFP, were generated. Both primary PAM and 3D4/163 cells could be transduced by rAd with different transduction efficiencies. The amiR3UTR was expressed in dose-and timedependent manners in rAd-transduced PAM cells. The amiR3UTR, but not amiRcon, had significant and stable inhibitory effects against replication of three different PRRSV strains in a dose-dependent manner. [Conclusion] The rAd-delivered amiR3UTR had strong anti-PRRSV effect against different PRRSV strains and rAd-amiR3UTR-GFP could be explored further as the alternative strategy against PRRS.

Abstract:[Objective] To understand the molecular biology information of ibeB gene of meningitic Escherichia coli isolates in calves. [Methods] The strain used was isolated from the brain and liver tissue of calves died from Meningitis. It was identified to be an O161-K99-STa pathogenic Escherichia coli strain and named as bovine-EN and bovine-EG. Based on the sequence of ibeB gene of meningitic Escherichia coli K1 RS218 strain in GenBank, a pair of primers was designed and the ibeB gene was cloned from isolates by PCR. Part molecular biology information of ibeB among different strains was compared. [Results] The sequence length of isolates ibeB gene was 1500 bp, containing a 1371 bp open reading frame (ORF) encoding 457 amino acids. Bioinformatics analysis showed that the nucleotide and amino acid homology of ibeB gene of bovine-EN strain shared 90.5% and 96.9% identity with Escherichia coli K1 RS218 ibeB gene, respectively, while bovine-EG strain shared 99.4% and 100.0% identity with Escherichia coli K12 respectively. The ibeB gene of bovine-E strains encoded water-soluble protein whose molecular weight was 50.26 kDa and isoelectric point was 6.05. This protein contained a signal peptide A but no transmembrane domain. Subcellular localization of ibeB belonged to the secreted protein, which secretory signal path site (SP) proportion was 0.939. [Conclusion] The ibeB gene was cloned from meningitic E. coli isolates and had higher homology and similar biological characteristics with meningitis E. coli K1 RS218ibeB, which belongs to extraintestinal pathogenic Escherichia coli.

Abstract:[Objective] This study was aimed to elucidate the effect of periodic flooding-drying to ecological processes of ammonia oxidizers in the hydro-fluctuation belt of the Three Gorges Reservoir. [Methods] Soil samples were collected at thee altitudes in regions of Wanzhou, Fengdu and Changshou, representing 8, 5 and 0 times floodingdrying management, respectively. Soil physiochemical properties were analyzed and microcosms were constructed to monitor nitrification activity by fertilizing soils with ammonium substrate. Real-time PCR was used to quantify the population size of ammonia-oxidizing archaea (AOA) and bacteria (AOB). DGGE fingerprints and clone libraries were conducted to study the shift of AOA and AOB compositions in nitrifying soils. [Results] Among the physiochemical characteristics of the soils, soil organic matter and total phosphates increased along with cycle increasing. After incubation for 13 days, the net nitrification rates of the samples with 8 cycles exceeded those with 5 cycles. The quantities of both AOA and AOB have increased during the incubation. Phylogenetic analysis showed that AOA were placed within the soil group 1.1b and soil group 1.1a, while bacterial ammonia oxidizers were closely related to Nitrosospira and Cluster 0. [Conclusion] Periodical flooding-drying increased soil organic matter, enhanced soil nitrification activity and likely played important roles in shaping community structures of soil ammonia oxidizers.

Abstract:[Objective] This study aimed to describe the composition of the endophytic bacterial communities in Salicornia europaea root, and to examine how endophytic bacteria vary across host growth periods. [Methods] PCRbased Roche FLX 454 pyrosequencing was applied to reveal the diversity and succession of endophytic bacteria. [Results] A total of 20363 partial 16S rRNA gene sequences were obtained. These sequences revealed huge amount of operational taxonomic units (OTUs), that is, 552-941 OTUs in a root sample. Endophytes in roots mainly comprised four phyla, among which Proteobacteria was the most represented, followed by Firmicutes, Actinobacteria, and Bacteroidetes. Gammaproteobacteria was the most abundant class of Proteobacteria, followed by Betaproteobacteria of this phylum. Genus Azomonas, Serratia, Pantoea, Serpens, Pseudomonas, Halomonas, and Kushneria were shared by all growth stages. Gammaproteobacteria increased during the five stages. The dominant bacterial genera during five periods were related to Delftia, Kushneria, Serratia, Pantoea, Erwinia, respectively. Five libraries contained 2108 unique OTUs with 5 OTUs in common. The greatest number of OTUs was detected during flowering stage. Endophytic bacteria diversity was reduced during fruiting stage. A combination of soil pH, average monthly temperature and soil salt concentration has significant effects on the endophytic bacterial community structure during the five stages. [Conclusion] As a whole, the diversity of endophytic bacteria was high inroot of Salicornia europaea. The distribution of endophytic bacteria showed obvious dynamic changes, and the host growth stages determined the endophytic bacterial community.

Abstract:[Objective] We screened endophytic bacteria containing ACC (1-aminocyclopropane-1-carboxylate) deaminase from soybean nodules, and evaluated salt-alkaline resistance, phylogenetic status and the growthpromoting of representative strain. [Methods] The features of strains producing ACC deaminase were determined by using the ACC as a sole nitrogen source, adopting standard curve method, colorimetric method, solid medium screening method, bacterial morphology, physio-biochemical characteristics, similarity analysis of 16S rRNA gene, inoculation tests. [Results] Eight endophytic bacteria containing ACC deaminase were screened from soybean nodules collected from 36 points of 13 cities (regions)in Henan province. Enzymaticactivity of DD132 was the highest (15.712 U/mg). Screened strain tolerated to medium of 4%-6% NaCl concentration. Among of them, DD165 and DD132 could tolerate 9% NaCl concentration. Five bacteria growing well under pH 11, showing that these strains had stronger alkali resistance. Eight strains containing ACC deaminase activity were affiliated to four genera: Bacillus, Enterobacter, Stenotrophomonas and Pantoea. Inoculation test showed that DD132 had a significant growthpromoting effect on wheat seedlings. [Conclusions] Endophytic bacteria containing high ACC deaminase activity from soybean nodules have stronger salt-alkaline resistance. DD132 has obvious growth-promoting effect on wheat seedlings.

Abstract:[Objectives] Siderophore is a low molecular iron chelate produced by microorganisms. It has broad application prospects in medicine, environmental restoration, health food and other fields. According to the literature survey so far, no siderophore was found from alkaline environment eukaryotes. Therefore, screening of fungi with high siderophore activity is of great significance. [Methods] By chromium azure S coloration, we screened 99 fungi isolated from Cheng Hai (an alkaline lakes in Yunnan province) and Datun alkaline tailings (Gejiu, Yunnan province). By spectrophotometric detection, we investigated the strain capacity of siderophore and type of siderophore. By solid phase extraction, we investigated the siderophore enrichment effect. Based on electron microscopy morphologic observation and ITS gene phylogenetic tree construction, we identified the strain. [Results] Strains FEDT-866, FEDT-145, FECH-998 and FECH-595 were siderophore high-yield ones. Except for strain FEDT-866, the siderophore active substances were suitable for solid phase extraction (SPE). Strains FEDT-866 and FECH-998 belong to Aspergillus and have higher similarity with Aspergillus tubigensis and A. nomius, respectively. Strains FECH-595, FEDT-145 belong to Penicillium and have higher similarity with P. svalbardense and P. chrysogenum. [Conclusion] We isolated and identified four fungi for possible siderophore production.

Abstract:[Objective] The purpose of this study was to isolate novel strains from the soil nearby meat processing factories to produce collagenase. After the yield of collagenase from the strain improved, the collagenase was purified and used for hydrolyzing collagen. [Methods] The strain was identified based on morphological features, physiological and biochemical characteristics and 16S rRNA gene phylogenetic tree analysis. The yield of collagenase was increased by optimizing the fermentation condition, and the collagenase isolated from the fermentation supernatant of the strain was finally purified with strong anion exchange resins. [Results] The collagenase-producing strain was identified as Bacillus cereus. The optimized fermentation conditions of the strain were: 2.0% glucose as optimum carbon source, 1.5% tryptone as optimum nitrogen source, 0.005% of Ca²⁺ as optimum metal ion. The optimum temperature and pH were 37 ℃ and 7.5, respectively. Under the optimum conditions, the enzyme activity of collagenase was (65.81±2.06) U/mL, 1.5-fold increased than that before the optimization. After purified with strong anion exchange resins, a collagenase with the purity higher than 90%, the molecular weight about 100 kDa, and the specific activity of 7615.0±78.7 U/mg was obtained. [Conclusion] The activity of Bacillus cereus collagenase was higher than the reported collagenases. Using this novel collagenase, collagen could be degraded into short biological peptides in a short time. Hence, this collagenase has application prospects in many fields, such as food, medical, health care products and cosmetics.

Abstract:[Objective] To understand the biochemical role of white rot fungus Trametes sp. SQ01 manganese peroxidase (MnP) towards 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoates (HOPDA)/HOPDA derivatives and to reveal the new catalytic features of MnP, white rot fungus Trametes sp. SQ01 MnP was extracted, and the purified enzymes were used in the oxidation of HOPDAs. [Methods] UV-vis spectrophotometry was used to study the transformation of 10 substituted HOPDAs by manganese peroxidase and measure the steady-state kinetics parameters of manganese peroxidase against parts of HOPDAs. The molecular structures of HOPDA and HOPDA oxidation product were analyzed by infrared spectroscopy. [Results] Manganese peroxidase exhibited catalytic activity towards both HOPDA and halogenated HOPDA. Especially, our manganese peroxidase used 3,8,11-3Cl HOPDA as substrate, while biphenyl hydrolase (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) and Rhodococcus sp. R04 showed negligible activity towards this substrate. The steady-state kinetic analysis indicated that HOPDA displayed the lowest K_m among 5 HOPDAs, the catalytic efficiency (K_cat/K_m) of 3, 10-2F HOPDA was the highest. UV-visible spectroscopy analysis indicated that the maximum absorption of products of HOPDA showed blue-shift with increasing the reaction time in the visible region. Infrared analysis showed that MnP converted conjugated diene of HOPDA to monoethylenically, and cause hydroxyl on C_β to disappear. [Conclusion] Manganese peroxidase can effectively degrade HOPDA and its derivatives. Such catalytic properties of manganese peroxidase provide a new strategy for successfully degrading biphenyl and its intermediate metabolites.