Abstract:摘要：本文详细介绍了2010年度国家自然科学基金委员会生命科学部微生物学科的项目申请、受理和资助的情况，并对项目申请中的新情况、新特征进行了初步的分析，希望这些分析能够为今后科研人员的项目申请提供有用的参考。

Abstract:Abstract: The transcription factor SoxR is a typical member of the family of mercury resistance operon regulator (MerR), extensively distributed across several bacterial phyla, such as Proteobacteria, Actinobacteria and Acidobacteria. SoxR, which is initially regarded as a cellular redox sensor, is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and then enhances the expression of its target genes. SoxR responds to the oxidative stress mediated by superoxides in Escherichia coli in a global manner, whereas it coordinates the bacterial community growth adapted to the environment in Pseudomonas aeruginosa by sensing pyocyanin, which is a terminal signaling factor in the quorum sensing network. In this review, we focused on the structure, mechanism and roles of the transcription factor SoxR. Although compelling achievements have been made in recent years, some aspects still remain to be unraveled. A widely accepted mechanism of activation of SoxR has not yet been established or validated. And there is much and urgent call for intensive studies from more taxonomic groups rather than E. coli and P. aeruginosa. With more and more work done on these aspects, one can not only understand SoxR more comprehensively, but also gain deeper insights into the bacterial complex regulation networks, and even achieve milestones in this field.

Abstract:Abstract: Bacterial polysaccharides play important roles both in medicine and industry. Due to the development of bacterial genome sequencing, many gene clusters related to the biosynthesis of bacterial polysaccharide have been found, aligned and analyzed. Despite of their complex composition and structures, different bacterial polysaccharides are biosynthesized via similar pathways. This review discussed the research development of the biosynthetic mechanism of different bacterial polysaccharides, with emphasis on the glycosyltransferases and polymerases involved in the biosynthetic pathway.

Abstract:Abstract: We reviewed existing literature concerning the effects of probiotics on serum cholesterol levels in animals and humans, with particular attention to the possible mechanisms of their action. Probiotics are live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host. One specific benefit that has been reported is that certain probiotic strains (e.g. lactobacilli, bifidobacteria and enterococci) can lower serum cholesterol levels. However, conclusions regarding such hypocholesterollemic effects can vary from studies on animals and humans due to differences in their physiology. As for the cholesterol-lowering mechanisms, different hypotheses have been proposed, including: (I) cholesterol is absorbed into the cellular membrane or cytoplasm; (II) cholesterol is bound to the cellular surface; (III) cholesterol is co-precipitated with free bile acids; (IV) conjugated bile acids were hydrolyzed by probiotics and the resulting free bile acids are more likely than are conjugated ones to be excreted from the body; (V) free bile acids were bound to the cellular surface by capsule exocellular polysaccharides produced by probiotics; (VI) food-derived indigestible carbohydrates were fermented by probiotics to produce propanoic acid in the gut, which can then decrease systemic levels of serum cholesterol by inhibiting hepatic cholesterol synthesis; (VII) a reduction in cholesterol absorption by probiotics through the down-regulation of NPC1L1 gene expression of cells; and (VIII) cholesterol micelle is disrupted by probiotics. The future research is needed to further confirm these hypotheses.

Abstract:Abstract: 【Objective】To study the structure and function of a newly found virus strain Spodoptera litura multicapsid nucleopolyhedrovirus II (SpltMNPVII) ORF146 gene. 【Methods】The primers were designed according to the sequence of SpltMNPVII genome. The promoter of ORF146 was amplified by PCR. The promoter activities and the time course of mRNA transcription were analyzed. The fragment of the ORF146 gene was then cloned into the vector of pET28a(+) and expressed. The polyclonal antibody was prepared by using the purified fusion protein. Titration determination of anti-ORF146 antibody was evaluated by ELISA.【Result】Nucleotide sequence analysis demonstrated that this gene has a 1383 bp ORF, encoding 460 amino acids with a predicted molecular weight of 50.4 kDa. Analysis of both promoter activities and the time course of mRNA transcription of the ORF146 gene showed that ORF146 was transcripted in early stage as well as in late stage. The transcription began at 2 h post infection (hpi) and reached two peaks at 8 and 18 hpi and then the transcription level was slightly decreased from 24 hpi. pET-28a-ORF146 fusion protein expressed in prokaryotic and purified polyclonal antibody was with good specificity, with the titer above 1:3200. 【Conclusion】SpltMNPV II ORF146 gene is a composition structure protein, which was expressed at both early and late stage. ORF146 might be involved in viral DNA replication. The polyclonal antibody can be used to further study the biological characteristics and functions of proteins.

Abstract:Abstract: [Objective] The objectives of this work were (i) to construct a yeast two-hybrid AD-cDNA library of Astragalus sinicus and provide a fundamental system to screen target proteins involved symbiotic nitrogen fixation, and (ii) to isolate the target proteins interacting with the leghemoglobin. [Methods] By using the MatchmakerTM Library Construction & Screening Kit (Clontech), we constructed a yeast AD-cDNA library basing on the total RNA, which was isolated from the root and nodule tissues of A. sinicus at different developmental stages infected by Mesorhizobium huakuii 7653R. [Results] The quality examination of the AD-cDNA library showed that the transformation efficiency was 1.0×106 transformants/3μg pGADT7-Rec DNA, and the average length of cDNA inserts was around 1.0 kb. The library was then screened with the leghemoglobin AsB2510 as bait by yeast two-hybrid system, and 26 positive clones was obtained on SD/-Leu/-Trp/-His/-Ade containing X-gal. 10 of them were individually further confirmed by resuing the plasmid, amplifying the cDNA insert and retesting the protein-interacting phenotype. [Conclusions] The cDNA inserts of positive clones were sequenced and undertaken a blast analysis in NCBI database, it was found that clone LY-53 contained a tify domain and divergent CCT motif, which was an important transcription factor needs in-depth investigation.

Abstract:Abstract:[Objective] In order to study the diversity and distribution of Benzoyl coenzyme A reductase (bcrA) and oxygenase components of 1H-2-oxoquinoline 8-monooxygenase(oxoO) gene in a lab scale denitrifying bioreactors for treating quinoline-containing wastewater. [Methods] Genomic microbial DNA was extracted from the biofilm samples of bioreactor. Based on the known oxoO genes sequences in GenBank, we designed primers for oxoO gene amplification. Using our designed oxoO gene primers and a pair of degenerate primers of bcrA gene obtained from literature, we amplified the DNA samples. And the amplicons were used for constructing clone libraries of oxoO and bcrA gene. We also performed quantification analysis of these two genes in bioreactor by using Real-time qPCR method. [Results] The quantification analysis showed gradual increase of the bcrA gene abundance and reduction of the oxoO gene abundance along with time. The clone library analysis of these two genes indicated that most clones in bcrA gene clone library having more than 97% sequence similarities to the known bcrA gene of Thauera bacteria and others only having 74%~86% sequence similarities to bcrA gene sequences. Whereas, only a few clones in the oxoO gene clone library have 99% sequence similarities to that gene of Pseudomonas putida. But the sequences of most clones only distantly related with known oxoO genes. [Conclusions] This study showed a high bcrA and oxoO gene diversity, with some new gene sequences, in the lab scale denitrifying bioreactors. The abundance of bcrA and oxoO gene changed remarkably during the running of bioreactor, and closely related with the performance of bioreactor. Therefore, bcrA and oxoO genes have potential to be used as molecule markers to monitor the process of treating quinoline containing wastewater.

Abstract:Abstract: [Objective] To obtain phosphate-dissolving strains which can be suitable for corn production. [Methods] We screened phosphate-dissolving strains from soil samples. We conducted the plate experiment and soil experiment in laboratory, pot experiment in greenhouse and field experiment to test the insoluble phosphate dissolving ability of the strains. [Results] Fifteen strains of phosphate-dissolving fungi were isolated from soil samples, including 9 Penicillium oxalicum, 2 Penicillium variabile, 1 Penicillium aculeatum, 1 Trichoderma viride, 1 Aspergillus niger, and 1 Aspergillus nidulans. The 15 strains could obviously drop the pH in culture media. Five strains (Z15+、ZQ3、ZI1、Zh and Z30) reduced the pH value from 7.0 to below 2.0 in 18 h. The plate experiment and soil experiment in laboratory indicated that these 5 strains could propagate in plates and soil by using the root secretion of corn as sole carbon source. The pot experiment inoculating with the 5 strains was conducted in greenhouse by planting corn. The result demonstrated that the content of available phosphorous was increased remarkably with strains ZI1 and Zh. The content of available phosphorous at 49th day increased 28.05% and 37.04%, respectively, than initial content in strains ZI1 and Zh treatment. The dry matter yield of corn significantly increased 26.04% and 20.21% than the control, respectively. Then strains ZI1 and Zh were used in field experiment as microbial inoculums. The yield of corn treated with strain Zh increased 13.22% than that of the control, reaching 10873.05 kg/ha. However, the yield of corn treated with strain ZI1 presented no significant difference with the control. [Conclusion] The phosphate－dissolving strain Zh obtained in this experiment was identified as Aspergillus nidulans ,which is suitable for corn production.

Abstract:Abstract: [Objective]To provide theoretical basis for understanding the physiological function of transglutaminase in Streptomyces, which would benefit for improving the production of this enzyme, transglutaminase gene was disrupted in Streptomyces hygroscopicus.【Methods】First, the interruption plasmid pKC1139-TG1 was constructed by inserting a segment of transglutaminase gene in the temperature-sensitive vector pKC1139. Second, pKC1139-TG1 was transformed into S. hygroscopicus protoplast and the interruption plasmid was inserted into the chromosome by homogenous recombination .Third, the strain with disrupted transglutaminase gene was screened by apramycin resistance and named as S. h-△TG. 【Results】Compared with the parent strain, S. h-△TG was able to grow as substrate mycelium, but not able to form aerial hyphae.【Conclusion】Transglutaminase may be involved in the aerial hyphae formation of S. hygroscopicus.

Abstract:Abstract: [Objective] Biphenyl hydrolase (BphD) is an enzyme of the biphenyl biodegradation pathway in Rhodococcus sp. R04. Expression, purification and properties of BphD, together with chemical modification were investigated. [Method] Using heat-induced expression vector pBV220, we expressed bphD heterologously in E. coli BL21 (DE3). The products were purified by chromatography, and the purified enzyme was used to study its functions and properties by circular dichroism. [Results] BphD has been purified to homogeneity with a final purification fold of 5.02. With 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA ) as a substrate, the optimum pH and temperature of BphD are 9 and 80 ℃, respectively and the Km is 0.44 μmol/L. The enzyme had remaining 90% of activity incubating for 1 hour at 60 ℃, and had a half-life of 1 hour at 70 ℃, making it the most thermostable biphenyl hydrolase reported in Rhodococcus. Secondary structure of BphD has undergone great changes with the temperature up to 70 ℃, and has been severely damaged at 80 ℃. The sequence alignment between BphD and other homologous hydrolases as well as chemical modification of BphD indicated that Ser, His and Asp play important roles in the hydrolysis of HOPDA. In addition, Trp residue may be near the active center of enzyme as the key amino acid involved in substrate hydrolysis, which is not previously reported in the literature. [Conclusion] BphD with pH stability and thermostability is quite rare in the homologous hydrolases in Rhodococcus. The key status of Ser, His, Asp and Trp will build a basis for more learnings of the transformation mechanism of BphD.

Abstract:Abstract：[Objective] To provide new atrazine-degrading strains for atrazine-polluted soil, we isolated the high-efficiency degradation bacterium from contaminated soil, identified with taxonomy, and studied the degrading characteristics and remediation capability of the strain in black soil. [Methods] We used morphological, physiological and biochemical characteristics and 16S rDNA sequences analysis to identify the strain. We studied the effect of environmental factors such as incubation time, temperature and pH on the strain growth and atrazine degradation to confirm the optimum degradation conditions. The strain’s degradation range was studied by using different herbicides as sole carbon and nitrogen source. The strain’s remediation capability to atrazine-polluted soil was tested by sensitive crop bioassay after inoculating in pot soil. [Results] We isolated a high-efficiency degradation strain T3AB1 that used atrazine as sole carbon and nitrogen source from maize field suffering atrazine in Nehe, Heilongjiang province. It was initially identified as a member of the genus Arthrobacter sp.. The strain could degrade more than 99% of 500mg/L atrazine (pH8.0) within 72 h. We determined the best degradation conditions as follows: pH7.0－8.0, 25－30℃, 72－108h. In addition, the strain used other herbicides as sole carbon and nitrogen source such as imazamox, imazethapyr, trifluralinm, clomazone and fomesafen, and the degradation rate reached 12.66%－40.54% after 168h. The strain could significantly remedy the sensitive rice crop biomass indexes after treating 21 days, and as time extending, its role in the remediation gradually increased. [Conclusion] The screened atrazine-degrading strain T3AB1 could be a suitable candidate for bioremediation of atrazine-polluted soil.

Abstract:Abstract: [Objective] Hepatitis B virus (HBV) is a major human pathogen that chronically infects 400 million people worldwide. Chronic infection of HBV plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). To clarify the mechanism of HBV-related HCC and immune escape of HBV, we investigated the relationship between infection of HBV and basal autophagy. [Methods] To examine the number of autophagosomes upon transfection with HBV, HepG2.2.15 cells were transfected with green fluorescent protein-microtubule-associated protein 3(GFP-LC3) and observed by fluorescence microscopy. Huh7 or HepG2 cells was transiently transfected with HBV expression vector pHBV1.3, then the phosphatidylethanol -amine conjugation of microtubule-associated protein 3 (LC3) and the degradation of p62, both of which are specific indictors of autophagy, were evaluated by western blot. Moreover, HepG2 or Chang liver cells were transiently transfected with the constructed HBV X protein(HBx) expression vector in order to evaluate autophagic status of the cells. [Results] HBV and HBx were both able to increase the autophagosomes formation as well as the enhancement of autophagic flux. Notably, C type HBx had a more increment of autophagy than B type does. [Conclusion] HBV can enhance basal autophagy and the increment is dependent on HBx. Different genotypes of HBx had different effects on basal autophagy. Take together, these findings will help us to clarify the mechanism of HBV infection and the development of Hepatitis B to HCC in the future study.

Abstract:Abstract:[Objective] To optimize the isolation and culture technique of Chlamydophila psittaci avian strains and to establish an animal model infected with C. psittaci. [Methods] C. psittaci ompA gene was amplified from DNA extracted from bird livers by polymerase chain reactions (PCR). For the PCR positive avian samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 72 h in different dilutions，and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the avian strains(2×104, 2×105, 2×106 IFUs) were used to attack C57BL/6 mice by intranasal injection，mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. [Results] Six of one hundred avian samples were positive by C.psittaci ompA gene amplification, and three were positive by cell culture. The C.psittaci avian strains were cultured in Vero or HeLa cells. Vero cells showed stronger tolerance of cytolysis after chlamydia infection and chlamydia inclusion bodies were larger and more dense. Successfully establish a murine model of intranasal infection with C.psittaci, and 2×105 IFU is the suitable amount of organisms to induce respiratory chlamydia infection．[Conclusion] The isolation and culture condition was optimized for C.psittaci avian strains, and a murine model of respiratory tract infection by C.psittaci was successfully established, which can be applied to the clincal diagnosis of C.psittaci and epidemiological or pathogenetic study.

Abstract:Abstract: [Objective] To study the antiviral activity of probiotics and analyze its possible mechanism. [Methods] We used the methods of NDV-HA and MTT to evaluate the NDV hemagglutination titer and inhibitory rate treated by probiotics in vitro and on CEF respectively. [Results] Five probiotics and its metabolites could reduce the titer of NDV significantly, but the two pathogenic bacterium and their metabolites could not, these results indicated that probiotics may destruct the structure of NDV directly and this effect was strain-specific. Probiotics could increase the inhibitory rate of NDV significantly, and depended on concentration (P<0.01). The result of NDV inhibitory rate increased when the cells pre-treated with probiotics reflected that the probiotics may block NDV adsorbing to cells; from the phenomenon that probiotics and NDV added to cells at the same time reduced the CPE of cells, we could see that probiotics can destruct the structure of virus directly; the lowest inhibitory rate of NDV when cells infected with NDV before probiotics added showed that the probiotics will work hardly after the cells infected. [Conclusion] Probiotics not only can destruct the virus, but also can block the virus infecting the cells and inhibit the proliferation of virus in cells.

Abstract:Abstract: [Objective] To compare six molecular methods for differentiation of Lactococcus lactis subsp. lactis and cremoris. [Methods] Six molecular methods, such as 16S rRNA gene analysis, 16S-23S rRNA Intergenic spacer region polymorphism, Denaturing Gradient Gel Electrophoresis (DGGE), Random Amplified Polymorphic DNA (RAPD), Repetitive Extragenic Palindromic-PCR and Restricted Fragment Length Polymorphisms were used to differentiate the reference strains. [Results] Except for 16S rRNA gene analysis and 16S-23S rRNA Intergenic spacer region polymorphism, the other protocols were competent. [Conclusion] The methods of DGGE and RAPD were more appropriate for differentiation of Lactococcus lactis subsp. lactis and cremoris.

Abstract:Abstract: [Objective] To construct Brucella vaccine strain M5-90ΔvirB2 deletion mutants with attenuated virulence. [Methods] We constructed the homologous recombination plasmid pGEM-7zf-ΔvirB2-sacB by using traditional molecular biology technology and then obtained M5-90ΔvirB2 strains by ampicillin and sucrose screens after electroporation. The deletion mutants were identified by PCR. Its stability were detected by continuous bacteria culture. [Results] The virB2 deletion mutant strains were successfully constructed. The reverse mutation did not occur within 10 passages. [Conclusion] The results of this study will based on the development of gene deletion attenuated vaccine of Brucella.

Abstract:Abstract: [Objective] To find the primary function of aes-31 fragment through construction of defined mutation of Avian Pathogenic Escherichia coli strain E058 and animal experiments. [Methods] The fragment of aes-31 was generated by PCR and cloned into pGEM-T-easy vector. A resultant suicide vector containing the aes-31 fragment named pMEG375-aes-31 was constructed and transformed to a receptor strain SM10. Then recombinant strain SM10 was hybridized with E058 strain in solid state. Mutant derivatives of strain E058 were generated by homologous recombination and were named E058(?aes-31). The 50% lethal dose (LD50) of E058 and E058(?aes-31) in commercial day-old chickens experimentally inoculated via intratrachea were 104.3CFU and 103.5CFU, respectively. The same way was used to inoculate with 108 CFU to obtain the pathogenic ability of E058 and E058(?aes-31) in 35-days-old SPF chickens. In the chicken challenge model, the mutant was tested to determine the individual function for virulence and persistence in 2-week-old SPF chicks. [Results] The pathogenicity test for E058 strain and E058(?aes-31) strain showed that the mutant had a higher mortality (75%) to 35-day-old specific pathogen-free (SPF) chicks than that of E058 (62.5%). In the chicken challenge model, there was no obviously CFUs difference in blood and lung in chicks of E058 group and E058(?aes-31) group 6 hours after inoculation. After 24 hours there was obvious CFUs difference in heart, liver, spleen, lung and blood in chicks of E058 group and E058(?aes-31) group. After 48 hours, there was also obvious CFUs difference in heart, liver and spleen in chicks of E058 group and E058(?aes-31) group E058(?aes-31) had a trend of increasing virulence in chicks. [Conclusion] Aes-31 might be associated with negative regulatory gene for E058 virulence and its actual function needed further study.