Abstract:Abstract:We provide here an overview of proposals applied and projects funded by the division of microbiology，department of life sciences，National Natural Science Foundation of China in 2012． We analyzed the traits and problems in different sub-disciplines，and illustrated the stimulating policy for future funding． This overview provides reference for Chinese researchers to apply relevant funding for projects in microbiology.

Abstract:Abstract:Nitric oxide is a highly reactive molecule with dichotomous regulatory roles in numerous physiological and pathological events． It has been recognized as an intra-and inter-cellular signaling molecule in animals，plants and microorganisms． Recent research data indicate that fungi are capable of synthesizing nitric oxide． Appropriate amounts of nitric oxide play important biological roles in fungal cells． However，excessive amounts of nitric oxide will damage cells and evoke apoptosis． Nitric oxide regulates the synthesis of cGMP，an important intracellular secondary messenger molecule，involved in the control of a variety of signal transduction pathways in fungal cells． Nitric oxide regulates the cellular development，morphogenesis，sporulation，spore germination，reproduction and apoptosis in fungi． Nitric oxide affects the physiological function of fungi throughout the life cycle． Although the mechanism of nitric oxide in plants and animals has been widely studied，there are limited reports about nitric oxide in fungi; and further investigation is needed to illustrate the nitric oxide synthesis，degradation pathways and the mechanism of signal transduction in the fungal system.

Abstract:Abstract:［Objective］To isolate，incubate and characterize cultivable endophytic antinobacteria from medicinal plants，and analyze the diversity of the endophytic antinobacteria，then explore the novel microbial resources．［Methods］Ten media were used to isolate endophytic antinobacteria from 37 fresh medicinal plant tissue samples． The optimal cultivation conditions for endophytic antinobacteria were determined by comparison． Based on the morphology of the colonies and cells of the new isolates，we chose174 isolates to analyze their 16S rRNA gene sequences and the diversity of the medicinal plant endophytic antinobacteria． The physiological characteristics of 27 representative strains were studied using Biolog GEN III MicroPlates，API 50CH and API ZYM kits．［Results］In total 940 endophytics affiliated to 47 genera of 30 families were isolated，among which more than 600 actinobacteria belonged to 34 genera and 7 unknown taxa． Good growth of the endophytic antinobacteria on PYG ( peptone-yeast-glycerol) medium with pH 7.2 at 28－32℃ was observed．Physiological characteristics differences of these isolates related to their phylogenetic relationships． Greater differences were shown among the strains from the same host plants than those from different plants grown in the same area．［Conclusion］There are great diverse endophytic actinobacteria inside the medicinal plants． No direct relationship of the endophytic actinobacteria from medicinal plants with the host plants in the sole carbon source utilization，fermentation of carbon sources to produce acid and the enzyme activities was found，while it seemed that the physiological characteristics of the isolates related to the geographical distribution of their host．

Abstract:Abstract:［Objective］Structure-similar ATP-binding cassette (ABC) transporter proteins，BbT1 in Beauveria bassiana (Bb) and IfT1 in Isaria fumosorosea (If)，were characterized．［Methods］ Various phenotypes including cellular antioxidant response，multidrug resistance and virulence were compared between wild-type strain and the constructed mutants ΔBbT1，ΔBbT1/BbT1 and ΔBbT1/IfT1．［Results］Compared with the wild-type，ΔBbT1/BbT1 and ΔBbT1/IfT1 strains showing no significant changes in examined phenotypes，ΔBbT1 became 27% to 2.1-fold less tolerant to the oxidative stress of 20－40 mmol/L H2 O2 and 2－8 mmol/L menadione，28% to 4.7-fold less resistant to carbendazim，itraconazole，dimetachlone，cycloheximide，ethirimol and 4-nitroquinoline N-oxide，and about 20% less virulent to the second-instar larvae of Spodoptera litura． ［Conclusion］The ABC transporter proteins BbT1 and IfT1 are similar not only in sequence structure but also in biological function，act as one of the determinants for the multidrug resistance of Bb and If，and also contribute to the fungal virulence due to involvement in cellular antioxidant response．

Abstract:Abstract:［Objective］To establish the kinetic inactivation models of Vibrio parahaemolytius in saline and peeled shrimp treated with acetic acid，lactic acid and citric acid for guidance of their potential application in shrimp decontamination．［Methods］To determine the survival rate (P) of V． parahaemolyticus in saline and peeled shrimp treated with organic acids，dose-response in peeled shrimp between P and concentrations of organic acids was modeled directly． Logit (P) was transformed from survival P with the formula ln ［P/(1－P)］for linear modeling． Both linear models were used to interpolate 50% and 90% effective inhibitory concentrations ( EC50 and EC90 )，which were then used to compare the difference of inhibitory potency between saline and peeled shrimp．［Results］Organic acids in saline were more inhibitory to V． parahaemolyticus in saline than in peeled shrimp，seen as 160 to 200-fold increase of EC50 and EC90 for lactic acid and citric acid，and more than 70-fold increase for acetic acid． These results indicate that food matrix had significant impact on the antimicrobial activity of organic acids． We also found that EC90 of the tested organic acids in peeled shrimp was far below the 2. 5% limit for use as food ingredients regulated by USDA． With equimolar concentration in the test solutions，the order of inhibitory potency is citric acid ＞ lactic acid ＞ acetic acid． ［Conclusion］Food matrix could have negative impact on antimicrobial activity of organic acids． Concentrations of organic acids around 2% could lead to significant reduction of V． parahaemolyticus contamination of peeled shrimp for improved food safety．

Abstract:Abstract:［Objective］AfMp1p is a glycosylphosphatidylinositol (GPI) anchored cell wall protein (GPI-CWP) identified in filamentous fungus Aspergillus fumigatus，which contains a specific C terminal signal for cell wall localization．Trichoderma reesei is known as a safe fungal species (GRAS) and widely used in the industry．Thus，developing of the cell-surface expression systems in T．reesei is of industrial interest．［Methods］The GPI signal from the A．fumigatus AfMp1p was fused to the C-terminal of green fluorescent protein (GFP) and transformed into T．reesei． The optimization of T．reesei transformation and the expression profiles of the GFP were investigated in detail．The cellular location of the GFP fusion protein was detected．［Results］Fluorescent image analysis and Western blot analysis indicate that the GFP fusion protein locates on the cell wall of T．reesei．［Conclusion］According to these results，the GPI signal from A．fumigatus can be recognized in T．reesei and the expression system constructed in this study can be used to express heterogeneous protein in the cell wall of T．reesei.

Abstract:Abstract:［Objective］This study is aimed to isolate and identify an aerobic denitrifying bacterium with high ability for nitrogen removing，and optimize its growing and denitrifying conditions to obtain the theory basis for controlling the eutrophic artificial lake． ［Methods］Aerobic denitrifying bacterium strain A-13 was screened by denitrifying medium．Strain identification was carried out through morphological，biochemical and physiological characteristics，16S rRNA gene and periplasm nitrate reductase gene analysis．The optimal pH，temperature，carbon source，dissolved oxygen (DO)，inoculum ratio were tested as well． ［Results］Strain A-13 isolated from a drain outlet located in Gaoqi village，Shangjie town，Minhou county，Fuzhou，China，was a member of Pseudomonas stutzeri and its DNA sequence was most closely related to Pseudomonas stutzeri DSM 50283．The optimal conditions for its growth and denitrification were followed as: pH 6.5，33℃，150 r/min，5% inoculum rate and the best carbon source was sodium succinate． Under these conditions，the maximum removal capacity for NO－3 was approximately 1900 mg/L．The strain could grow well in the medium with high salinity(10%) and could also use NO－2 and NH+4-H as the sole nitrogen source．［Conclusion］The isolated P．stutzeri，A-13 is a potential strain to treat wastewater with high salinity and/or eutrophication．

Abstract:Abstract:［Objective］To study the effect of RsbV gene deletion on the virulence of Listeria monocytogenes.［Methods］The fragment with RsbV deletion was generated by gene overlap extension PCR (SOE-PCR) and the mutant with RsbV deletion was obtained by homologous recombination based on the wild strain LM-XS5．The differences in virulence between the two strains were determined by LD50，bacterial counts in liver and spleen，and qRT-PCR experiments．［Results］LD50 of the RsbV gene-deleted strain was 104higher than that of the wild strain．The numbers of gene-deleted strain in the mouse's liver and spleen were significantly fewer than that of the wild strain (P＜0.05)．Results of qRT-PCR show that four virulence factors' expression levels of the RsbV gene-deleted strain were significantly lower than that of the wild strain (P＜0.05). The RsbV gene-deleted strain induced a strong immune response in mice against the wild strain．［Conclusion］RsbV regulates the expression of four virulence gene (inlA，LLO，PlcA and PrfA) of Listeria monocytogenes; The virulence of the RsbV genedeleted strain is significantly reduced，but it still has good immunogenicity．

Abstract:Abstract:［Objective］To study the role of the outer membrane protein H (OmpH) in pathogenicity of avian Pasteurella multocida.［Methods］ The ompH knock-out mutant of avian P. multocida C48-3 was constructed by homologous recombination. The DNA replacement was confirmed by PCR，RT-PCR and Western blot. We compared the differences of biological characteristics such as growth rate，capsular structure，adhesion ability and virulence between the ompH knockout mutant of C48-3ΔompH and parent strain C48-3，as well as the complemented strain C48-3C.［Results］ C48- 3ΔompH was successfully constructed． Electron microscopy examination of C48-3ΔompH shows the absence of capsular material compared to the parent strain C48-3 and complemented strain C48-3C．The adhesion assay shows that the number of C48-3ΔompH adhered to CEF cells was significantly lower than that of C48-3 and C48-3C. C48-3ΔompH was relatively attenuated in mice by intraperitoneal injection.［Conclusion］The construction of C48-3ΔompH would facilitate further study on pathogenesis of avian Pasteurella multocida.

Abstract:Abstract:［Objective］ To study the relationship between CD4+CD25+Foxp3+regulatory T cells and Th17 responses during pulmonary infection of Chlamydia muridarum (Cm) in BALB/c mice.［Methods］ BALB/c mice aged 6－8weeks were inoculated intranasally with 5×103 IFU of Cm to set up the murine model of Chlamydial pneumonia． The body weight changes，the growth of Cm and the pathology in the lung were monitored at different time post-infection． To determine the CD4+CD25+Foxp3+regulatory T cells responses in BALB/c mice，intracellular cytokine staining was used to assay the percentage of CD4+CD25+Foxp3+T cells in the spleen and mediastinum lymph node (MLN)．The production of cytokines/chemokines in the lung were monitored，including IL-6、TGF-beta、IL-17、IL-2 (by ELISA)，KC and MIP-2 (by RT-PCR).［Results］ Intranasally infected with 5×103 IFU of Cm in mice resulted in chlamydial pneumonitis featured by body weight lost，chlamydia growth and pathological damage in the lung compared with their uninfected counterparts．On day 3 post-infection，the percentage of CD4+CD25+Foxp3+T cells in the spleen and MLN were significantly decreased than the control mice; then began to increase and recover to the original level on day 7 postinfection．The production of Thl7 associated cytokines/chemokines such as IL-6，IL-17，KCand MIP-2 increased，which peaked on day 7 post-infection，then gradually reduced． The production of TGF-beta and IL-2 was consistent with the trend of CD4+CD25+Foxp3+T cells．［Conclusion］ During pulmonary infection of Cm in BALB/c mice，CD4+CD25+Foxp3+regulatory T cells may promote type 17 T cell immunity through providing TGF-beta in the presence of IL-6.

Abstract:Abstract:［Objective］The purpose of this study was to select cold-adapted lactic acid bacteria (LAB) from the intestinal tract of cold-water fishes from the Eerqisi river Alatai． Xinjiang.［Methods］By using culture medium MRS and Elliker，isolation of lactic acid bacteria (LAB) from 9 intestinal canal of cold-water fishes was carried out. Taxonomic identity and genetic diversity of strains isolated were determined by partial 16S rRNA gene sequences and rep-PCR with three different kinds of primers named BOX，(GTG) 5，ERIC.［Results］A total of 78 ychrotrophic isolates were obtained．Among them，24 isolates had characteristics of LAB and showed the optimal growth temperature ranging from 15 to 24℃ ． The phylogenesis result showed that 24 strains belonged to 6 Genuses，Carnobacterium (3 strains)，Lactococcus (9 strains)，Enterococcus (7 strains)，Brochothrix (1 strain)，Weissella (2 strains)，Streptococcus (2 strains). rep-PCR clustering analysis showed that Lactococcus and Enterococcus strains were from different Species． Lactococcus were belonged to 4 species while Enterococcus assigned to 2 species． ［Conclusion］The phylogenetic diversity of cold-adapted LAB from the intestinal tract of cold-water fishes from the Eerqisi river in the Alatai Xinjiang was relatively abundant.

Abstract:Abstract:［Objective］ ArgR，coded by the argR gene from Corynebacterium crenatum AS 1.542，acts as a negative regulator in arginine biosynthetic pathway. However，the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported． Here，we constructed a deletion mutant of argR gene: C. crenatum AS 1.542ΔargR using marker-less knockout technology，and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain.［Methods］We used marker-less knockout technology to construct C. crenatum AS 1. 542ΔargR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR.［Results］C. crenatum AS 1. 542ΔargR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds.［Conclusion］ The arginine biosynthetic genes in C crenatum are clearly controlled by the negative regulator ArgR. However，the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Abstract:Abstract:［Objective］To investigate the fitness costs of the New Delhi Metallo-β-lactamase-1 (blaNDM-1) bearing plasmid pNDM-BJ01 in Acinetobacter lwoffii strain 10621，and to evaluate the potentials of the sustainability and expansions among the Acinetobacter lwoffii population.［Methods］We obtained the spontaneous mutant of A. lwoffii missing pNDM-BJ01，10621NDM-1(－) via serial passages of the wild strain 10621NDM-1(+) on the antibiotics-free media. We then compared the in vitro fitness of these two strains by measuring the growth curves，the ability of biofilm formation and the survival rates in nutrient-free PBS buffer．［Results］Plasmid pNDM-BJ01 was unstable in A． lwoffii strain 10621，and 11 passages will be enough to get it deleted from the ancestral strain．We found no significant difference in the growth curves either at 26 or 37°C for 10621NDM-1(+) and 10621NDM-1(－)．The biofilm formation ability of the plasmid free derivate 10621NDM-1(－) was significant higher than its resistant ancestor 10621NDM-1(+) at 26°C，whereas the latter showed higher ability of biofilm formation at 37°C． Strain 10621NDM-1(+) was vulnerable in the nutrient-free PBS buffer，with less than 5% survival rate on the first day and dying out on the sixth day，whereas 10621NDM-1(－) survived till the seventh day.［Conclusion］blaNDM-1 bearing plasmid pNDM-BJ01 possesses significant fitness costs in A. lwoffii strain 10621，and it will get depleted easily if the antibiotic press released. pNDM-BJ01-free 10621NDM-1(－) strain has higher fitness than its ancestor，10621NDM-1(+)，which implies the limited expansion potential of plasmid pNDM-BJ01 in A. lwoffii.