Abstract:Abstract:In recent years，with the development of biotechnology，the molecular mechanism of the secretion pathways in Escherichia coli is clearer and clearer，and it becomes one of the most effective methods to use E.coli expression system for extracellular production of recombinant proteins． This review discusses recent advances in the secretion mechanism and application of E.coli α-hemolysin secretion pathway．

Abstract:Abstract:The intestinal environment is dependent on the host-microbiota interaction．Antigens of gut microbiota could affect the intestinal immunological homeostasis．Gut microbiota also participate in the metabolism of carbohydrate，lipid and protein． In return，the metabolites from these metabolic processes could regulate the bacterial metabolism，community composition and intestinal nutrient assimilation． MicroRNA serves as an important intracellular factor that regulates host gene expression．Gut microbiota could affect the host mRNA transcription and the post-transcription modification of certain genes by microRNA．Recent researches have revealed that gut microbiota could change the expression of microRNAs involved in gut inflammation and mucosal barrier via host-microbiota interaction．In this paper，we summarized recent advances in host-microbiota interaction and highlighted the research about microRNA regulation on gut homeostasis and host-microbiota interaction．

Abstract:Abstract:In survival competition with phage，bacteria and archaea gradually evolved the acquired immune system-Clustered regularly interspaced short palindromic repeats (CRISPR)，presenting the trait of transcribing the crRNA and the CRISPR-associated protein (Cas) to silence or cleaving the foreign double-stranded DNA specifically．In recent years，strong interest arises in prokaryotes primitive immune system and many in-depth researches are going on．Recently，researchers successfully repurposed CRISPR as an RNA-guided platform for sequence-specific gene expression，which provides a simple approach for selectively perturbing gene expression on a genome-wide scale． It will undoubtedly bring genome engineering into a more convenient and accurate new era．

Abstract:Abstract:［Objective］ We aimed to create a novel report system for screening the mutation of the negative regulatory genes，especially for those repressing the expression of cryptic antibiotics clusters.［Methods］We used marker-free gene disruption strategy，which combines with the “REDIRECT ( Rapid Efficient Directed Recombination Time Saving)” technology and in vivo site-specific recombination by Streptomyces phage BT1 integrase，to construct a scbR2/inoA double mutant strain of S．coelicolor M145． This strain was used as the host of the report system．For the construction of the reporter plasmid，the ScbR2 repressed promoter of cpkO from CPK (cryptic polyketide) cluster was used to drive the expression of a promoterless conserved gene inoA of S．coelicolor．Then the reporter plasmid was introduced into the host strain described above to test the availability of inoA as a reporter gene in this system.［Results］The scbR2/inoA double mutant strain gave rise to a bald pheno type on MM medium in the absence of inositol，and produced yellow pigmented secondary metabolite by the disruption of scbR2 to release the repression of cpkO，a pathway specific activator gene situated in CPK cluster． After introducing the reporter plasmid into this test stain，the resulting strain recovered the phenotype as wild-type strain，indicating that the promoter of cpkO can drive the expression of inoA in scbR2 mutant and consequently restore the biosynthesis of inositol． ［Conclusion］Our results indicated that inoA can be used as a novel reporter gene for Streptomyces，especially for detecting the activation of the“silent”promoter．This report system might be available for screening the mutation of the negative regulatory genes for the cryptic secondary metabolic gene clusters．

Abstract:Abstract:［Objective］We characterized the hypothetical gene pair ssl2138 and sll1092 that constitute a vapBC-family toxin-antitoxin (TA) system on the chromosome of Synechocystis PCC 6803.［Methods］An RT-PCR analysis was conducted to confirm the co-transcription of this gene pair，a selection-expression system was constructed to demonstrate the effect of their encoded proteins on E．coli growth． To validate interaction between these two proteins，an affinity capture analysis was performed．［Results］ ssl2138 and sll1092 were co-transcribed under normal growth conditions．Ectopic expression of sll1092 inhibited the growth of E．coli，which could be overcome by simultaneous or subsequent expression of ssl2138． Both proteins Ssl2138 and Sll1092 in vivo formed a protein complex due to their specific interaction．［Conclusion］ ssl2138 and sll1092 constitute a functional vapBC-family TA system of Synechocystis PCC 6803．

Abstract:Abstract: Methanolobus psychrophilus R15，isolated from the Zogei wetland at Tibetan plateau，is a cold-active methanogenic archaeon growing from 0 to 30℃ and optimally at 18℃．R15 grew in the NaCl concentrations ranging from 5 to 800 mmol/L.［Objective］This study aimed to find compatible solutes that can improve the growth of R15 at cold，and the possible function as cryoprotectant.［Methods］Using LC-MC we determined the accumulated substances in the R15 cells growing at lower temperatures，as well as in the cold-shocked cells; by supplementing the accumulated substances and the chemicals known as the bacterial compatible solutes in the R15 culture，we detected their functions of assisting the cold-growth of R15; by adding the detected compatible solutes into the glutamate dehydrogenase (GDH)，we determined the enzymatic stabilities at lower temperatures.［Results］Choline and betaine were accumulated both in the 4℃ -cultured and 4℃ -shocked 30℃ culture of R15． It was determined that choline，betaine，glycine，carnitine，acetoin and ectoine all improved the growth of R15 at cold． Choline，betaine and glycine could enhance the stability of GDH at low temperature.［Conclusion］Some compatible solutes can act as the cryoprotectant for methanogenic archaea，which expands our knowledge of the physiological functions of the compatible solutes．

Abstract:Abstract:［Objective］To characterize rmlA gene inavian pathogenic Escherichia coli (APEC).［Methods］We constructed the rmlA mutant of APEC by the Red recombination system．Then we analyzed the differences of growth，movement and biofilm formation between mutant strain and wild strain．We compared the differences of virulence gene transcription between mutant strain and wild strain by real-time PCR.［Results］The rmlA mutant did not affect the growth and motility of APEC，but enhanced the biofilm formation significantly．In addition，the transcription level of some virulence genes in rmlA mutant showed that the luxS ，irp2 were raised 2 and 1.6 times respectively，but decreased the iucD and fyuA by 25 times compared to the wild strain． ［Conclusion］These data indicate that the rmlA gene could strengthen the ability of APEC biofilm formation，and affect the transcription level of some virulence genes，but not the growth and motility．

Abstract:Abstract:［Objective］The present study aims to isolate and identify actinobacteria associated with the soft coral Nephthea sp.,and to isolate natural products from these actinobacteria under the guidance of PCR screening for polyketides synthase (PKS) genes.［Methods］Eleven selective media were used to isolate actinobacteria associated with the soft coral Nephthea sp. collected from Yongxin Island． The isolated actinobacteria were classified on the basis of phylogenetic tree analysis of their 16S rRNA genes. Degenerated primers targeted on conserved KS (ketoacyl-synthase) domain of type I PKS genes were used to screen for potential isolates. The positive isolates were cultured in three different media to check their producing profiles. One bioactive strain that is rich in metabolites was subjected to larger scale fermentation for isolating bioactive natural products.［Results］A total of 20 strains were isolated from Nephthea sp., and were categorized into 3 genera including Streptomyces，Dietzia and Salinospora，among which18 strains were positive in screening with type I PKS genes. Two bioactive compounds rifamycin S and rifamycin W were isolated and identified from Salinospora arenicola SH04． ［Conclusion］This is the first report of isolating indigenous marine actinobacteria Salinospora from the soft coral Nephthea sp. It provides an example of isolating bioactive secondary metabolites from cultivable actinobacteria associated with Nephthea sp. by PCR screening.

Abstract:Abstract:［Objective］ We investigated the mechanism of Src homology and collagen homology (Shc) in autophagy caused by troglitazone (TZ).［Methods］To reveal the regulatory role of p52Shc in autophagy，we used confocal microscopy and immunoblotting to examine autophagy induced by TZ. Then we used small RNA interference (siRNA) to deplete Shc and plasmids transfection to overexpress wtShc as well as 3mShc in PAE cells. Finally，we reached conclusion by detecting autophagic status following the deprivation of UNC-51-like kinase -1(Ulk1) by siRNA.［Results］We found that the deprivation of Shc showed to enhance autophagy，whereas p52Shc over expression suppressed TZ-depended autophagy concurring with an attenuated AMP-activated protein kinase (AMPK) and Ulk1 signaling. Besides，it demonstrated that p52Shc tyrosine sites of 239，240 and 317 implemented a critical role in the process.［Conclusion］ Collectively，Shc adaptor protein was involved in TZ-inducing autophagy likely via affecting AMPK and Ulk1 signaling．

Abstract:Abstract:［Objective］To investigate the prevalence and transmission of plasmid-mediated quinolone resistance qnrS gene among Escherichia coli isolates in a poultry farm and its environment.［Methods］E.coli isolates from fecal samples in a poultry farm and its environment from February to June 2010 were screened for the prevalence and dynamic changes of qnrS gene． Susceptibility testing，conjugant experiments，and pulsed field gel electrophoresis of qnrS-positive isolates were also performed.［Results］A total of 379 isolates were randomly obtained from feces samples of chickens，ducks and geese in a poultry farm and its environment. The qnrS positive strains were detected in all sources of isolates and two alleles of qnrS were prevalent on this farm．The positive rate of qnrS gene in environmental strains was 29.2%，which was significantly higher than that in the avian strains (13.4%). Chicken can quickly acquire qnrS gene after they live on this farm．Transconjugants of the qnrS gene can elevate ciprofloxacin Minimun inhibitory concentrations (MICs) by 16－32 fold compared with the recipient. Various determinants for resistance to other antimicrobial agents were also transferred with the qnrS plasmid. The XbaⅠ PFGE analysis of the qnrS positive strains showed that the dissemination of qnrS was not mainly due to the clonal dissemination of positive strains. However，qnrS-positive strains with indistinguishable PFGE patterns were found in ducks and environment.［Conclusion］Both horizontal gene transfer and clonal spread could be responsible for the dissemination of the qnrS gene in the poultry farm and its environment，but it is mainly disseminated by horizontal transmission.

Abstract:Abstract:［Objective］Pyropia haitanensis is of great commercial importance and wildly cultivated in Zhejiang and Fujian provinces． To observe the characteristics and changes of phycosphere microbial communities during cultivation can help us monitor the potential pathogens and microbial factors affecting the health of cultivated seaweeds.［Methods］The morphological characteristics and the diversity of phycosphere and surrounding seawater microbes were studied by pure culture method and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Similarity analysis was carried out online with the 16S rDNA (bacteria) and 18S rDNA (fungi) sequences in GenBank. The phycosphere microbial diversity during different growth stages，cultivated areas and periods was studied.［Results］Totally 467 bacteria and 55 fungi were isolated during P. haitanensis cultivation． The diversity of fungi was smaller than that of bacteria. The bacteria were classified into 41 genera，belonging to Alphaproteobacteria, Gammaproteobacteria，Actinobacteria，Firmicutes and Bacteroidetes. The dominant bacterial communities were Alphaproteobacteria and Gammaproteobacteria． Most of the fungi were classified into Ascomycota，only one strain belonging to the Basidiomycota，Agaricomycetes. Bacteria of 19 specific genera were isolated from P. haitanensis while 13 specific genera were isolated from the surrounding seawater. Most actinomycetes and fungi were isolated from the conchocelis cultured indoors，which was different from the microbial communities of the thalli in intertidal zone. Within the isolated microbes，we found that some strains had very high similarity with those pathogens such as Cobetia marina (C. marina，P. haitanensis red-rotting disease)，Phoma porphyrae (P. yezoensis disease) and saprotrophic fungi Fusarium sp. and Aspergillus sp.［Conclusion］The diversity of Pyropia phycoshpere microbes during cultivation was affected by the seaweed morphology，culture time and environmental factors. The strains that shared high similarity with Pyropia pathogens were found in this study，which would cause our great attention to these potential pathogens for Pyropia diseases.

Abstract:Abstract:［Objective］This study was aimed to obtain a key gene of osmo-adaptation and glycerol synthesis regulation in Candida glycerinogenes，namely mitogen-activated protein kinase HOG1 gene (CgHOG1)，and to verify its function of osmo-regulation.［Methods］The gene CgHOG1 was amplified by Degenerate PCR and Self-Formed Adaptor PCR from the C. glycerinogenes genome and the bioinformatic analysis of CgHOG1 gene was conducted. The CgHOG1 gene was transformed in Saccharomyces cerevisiae hog1Δ null mutant and its salt tolerance characteristics was investigated.［Results］The gene CgHOG1 encoded a protein of 387 amino acids with an open reading frame of 1164 bp and the amino acid sequence showed 86% identity to Hog1p of Ogataea parapolymorpha． Heterologous expression of CgHOG1 gene in S.cerevisiae W303 hog1Δ null mutant showed an increase in salt tolerance and glycerol production compared to S.cerevisiae W303 hog1Δ null mutant.［Conclusion］The CgHOG1 obtained in this study is a novel HOG1 gene from C. glycerinogenes，which plays an essential role in the yeast hyperosmotic response and glycerol synthesis. We supplied a new gene for the osmo-adaptation mechanism in C. glycerinogenes and molecular modification of the salt-tolerant and droughtresistant crops.

Abstract:Abstract:［Objective］DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB，C，DandE． Ourstudy is designed to clone and express dptC，to optimize the expressing condition，and then to purify the DptC.［Methods］dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites. The positive clone was verified by antibiotics resistance gene screening and sequenced，and then transferred into host strain E.coli BL21 (DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC，we purified DptC from Anxh103 by kta FPLC with a GST-Trap column.［Results］A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18℃ for 8－18 h; 0.6 mmol/L IPTG，LB with 50 μmol/L Fe2+.［Conclusion］The anchor redeemed for high throughput expression of dptC．The TEV site in pJTU3622 made the process of purifying DptC easier and simpler．This helps lay the ground work for future study on the function of DptC. Also，the light brown color of DptC and the medium with 50 μmol/L Fe2 + showed us DptChas the same character with DndC which belongs to an iron-sufur protein with 4Fe－4S.

Abstract:Abstract:［Objective］The removal of phenolic compounds was a key problem for coking wastewater treatment. This study focused on the isolation and characterization of anefficient phenol-degrading bacterial strain from coking wastewater.［Methods］Phenol-degrading bacterium was screened by using phenol as the sole carbon source，strain was identified according to the morphological properties and 16S rRNA sequence analysis，the phenol-degrading characteristics and the potential for removal of phenol in the coking wastewater were evaluated.［Results］Strain P1 was identified as the genus Rhodococcus sp. with morphological properties and 16S rRNA gene sequence. This strain could survive at the presence of phenol with concentration up to 1400 mg/L. The optimal conditions for biodegradation of 600 mg/L phenol in mineral medium were at 32－42℃，pH 7.0 and 0－4% NaCl. The phenol-degrading efficiency by P1 strain was different in response to various heavy metal ions，Ni2+，Mn2+ and low concentration of Pb2 + had no effect on the phenol degradation．However，Cu2+ ，Ni2+ and Cd2+ seriously inhibited phenol degradation by strain P1. The 279.9 mg/L phenols from 1/3 coking wastewater were completely degraded after incubation for 2days.［Conclusion］Strain P1 is an efficient phenoldegrading bacterium and has of the potential for removing phenolic compounds in coking wastewater.