Abstract:Abstract:Lignin is complex heteropolymer produced from hydroxycinnamyl alcohols through radical coupling．In nature，white-rot fungi are assumed initially to attack native lignin and release lignin-derived-low-molecular-weight compounds，and soil bacteria play an importent role for completely degradation of these compounds．Study on the soil bacteria degrading lignin-derived-low-molecular-weight compounds will give way to understand how aromatic compounds recycle in nature，and to utilize lignin compounds as the renewable materials for valuable materials production． Sphingobium sp．SYK-6 that grows on lignin biphenyl (5，5’-dehydrodivanillate) had been isolated from pulp effluent in 1987．We have researched this bacterium more than 25 years，a serious aromatic metabolic pathway has been determined，and related genes have been isolated．As the complete genome sequence of SYK-6 has been opened to the public in 2012，the entire aromatic compounds degradation mechanisms become more clear．Main contents in our review cover: (1) genome information; (2) aryl metabolism; (3) biphenyl metabolism; (4) ferulate metabolism; (5) tetrahydrofolate-dependent O-demethylation system for lignin compound degrdation; (6) protocatechuate 4，5-cleavage pathway; (7) multiple pathways for 3-O-methylgallate metabolism．

Abstract:Abstract:［Objective］ To analyze the endophytic bacteria in symptoms and symptomless tissues，reveal dominant bacterial population which may interact with Ca. Las. ［Method］ The population structure and diversity of endophytic bacteria in symptoms and symptomless tissues of single HLB-affectedcitrus plant were studied based on 16S rDNA sequences analyzed by PCＲ-DGGE (PCR-denaturing gradient gel electrophoresis). Quantitative analysis of Ca．Las，dominant bacteria and total amount of bacterial population in symptoms and symptomless tissues of branches，leaves and fruits was done by quantitative PCR．［Result］ The amount of Ca．Las is significantly different in symptoms and symptomless tissues; bacterial population in symptoms citrus tissues was significantly higher than in symptomless tissues．PCR-DGGE analysis result shows that endophytic bacterial structures are basically the same in symptoms and symptomless tissues．Extraction，cloning and sequencing of 17 obvious bands in DGGE electrophoretogram reveal that 8 of 17 bands belonged to the genus Serratia，which accounted for 47.06%．Sequence alignment shows that they belonged to different strains of Serratia marcescens with the 99. 63% similarity．Quantitative analysis of the total bacteria and Serratia marcescens shows that there is no significant difference in total bacterial count between symptoms and symptomless tissues whereas the amount of Serratia marcescens was negatively correlated with the amount of Las.［Conclusion］The Ca. Las is heterogeneously distributed among various tissues of HLB-affected citrus plant．The amount of Ca．Las was negatively correlated with Serratia marcescens and total amount of endophytic bacteria have no relevance with symptom of HLB disease．How the interaction of Serratia marcescens and Ca．Las in tissue of citrus needs further study.

Abstract:Abstract:［Objective］To achieve the high-efficiency expression of feruloyl estrase gene (AnfaeA) from Aspergillus niger h408 in Pichia pastoris and characterize the recombinant feruloyl esterase (FAE).［Methods］ Using gene splicing by overlap extension (SOE)，we cloned AnfaeA gene from A．niger h408 and subcloned into T vector for sequencing analysis．The expression vector pPIC9K-Anfae was constructed by the ligation of the Anfae A gene into the shuttle vector pPIC9K．The plasmid pPIC9K-Anfae was linearized and then electrotransformed into P．pastoris GS115． The recombinant strain with high level of FAE activity was obtained through plate screening． Effects of pH and temperature on recombinant FAE were determined by ultraviolet (UV) methods．［Results］We have successfully cloned and high-efficiently expressed the AnfaeA gene (GenBank: KF911349) from A．niger h408 in P．pastoris GS115．The sequencing result showed that the length of Anfae A was 783bp．The gene contained an Open Ｒeading Frame encoding 260 amino acids and was similar to feruloyl esterase A from A．niger by homology analysis． The deduced amino acids contained a typical active lid and catalytic triad of lipase．The SDS-PAGE result indicated that molecular weight of the recombinant FAE was about 30 kDa and the activity of the recombinant enzyme was 24. 72 U/mL．The specific activity of the recombinant FAE was 40.84 U/mg．Compared with A．niger h408，the recombinant enzyme activity increased about to 1100 times．The optimal temperature and pH for recombinant FAE was 50℃ and 5.0，respectively．Recombinant FAE showed nearly 80% of its maximal activity at 60℃ and was active in the pH range 4.0-9.0.［Conclusions］The high-efficient expression of AnfaeA gene in P．pastoris provided a prerequisite for achieving industrial application in feed and paper-making industry．In addition，the results established the experimental basis for further improvement of recombinant feruloyl esterase by directed evolution．

Abstract:Abstract:［Objective］We studied the metabolome responses to different relative virulence gene expression of pathogenic Vibrio parahaemolyticus based on reverse transcription real-time PCR (RT-PCR) and metabolomics.［Methods］ We extracted total RNA and metabolites of V．parahaemolyticus ATCC33846 cultured at different temperatures (4，25 and 37℃)．We applied relative quantification to analyze the tdh expression and Ultra Performance Liquid Chromatography ＆ Quadrupole-Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS) platform to determine the low-molecular-weight metabolites．Then，we compared the metabolic profiling of V．parahaemolyticus by principal components analysis．The correlation between relative gene expression and metabolome was obtained by Pearson and Spearman correlation coefficient (r) analysis.［Results］The expression level of tdh at different temperatures ranked 25℃＞4℃＞37℃．The main metabolites that changed significantly (P＜0.05) were organic acids，amino acids，alcohols，ketones，and esters．The results from correlation coefficient suggested that 11 metabolites were highly correlated with tdh expression，including 3 negative correlations and 8 positive correlations，at the threshold of |r|=1，P＜0.05．Further analysis revealed that alcohols were highly significant correlated with tdh expression．［Conclusion］ The results suggested that there were significant correlation between metabolome and virulence genes expression，which might contribute to understanding the pathogenic mechanism of V．parahaemolyticus.

Abstract:Abstract:［Objective］L-isoleucine-4-hydroxylase (IDO) encoding gene ido from Bacillus thuringiensis TCCC 11826 was cloned and expressed，followed by enzyme characterization．In addition，recombinant strain was tested for its 4-Hydroxyisoleucine (4-HIL) biotransformation． ［Methods］ Ido gene was amplified from B．thuringiensis TCCC 11826 genomic DNA and expressed in BL-IDO．Recombinant IDO was extracted，purified and characterized．Recombinant strain used for biotransformation of 4-HIL was constructed．［Results］Composed of 723 nucleotides encoding 240 amino acids (sharing 97.47% and 97.91% identities with that of B.thuringiensis 2-e-2)，ido gene was cloned from B. thuringiensis TCCC 11826. The recombinant IDO contained a His1-X-Asp/Glu-Xn-His2 motif that is specific for Fe(II)/α-ketoglutaratedependent hydroxylases and catalyzed L-isoleucine to 4-HIL．Normal hyperbolic kinetics was observed with L-Ile in the reaction by recombinant IDO．Lineweaver-Burk treatment of the data yielded apparent Km and the Vmax was 0.18 mmol/L and 2.10 μmol/min/mg，respectively．The optimum temperature and pH for the recombinant IDO was 35℃ and 7.0 respectively; moreover，the relative activity of the enzyme remain 85.10% after 5 h incubation at 35℃．In all，ecombinant strain harboring ido transformed 89.28% of L-isoleucine to 4-HIL．［Conclusion］ In this study，an ido (Accession No．KC884243) with novel sequence was isolated and enzymatic characteristics of recombinant IDO was systematically analyzed．In addition，we successfully achieved the biotransformation of 4-HIL from L-isoleucine． This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives．This work will lay theoretical foundation and practical basis on the microbial manufacture technology of 4-HIL and other amino acid derivatives．

Abstract:Abstract:［Objective］ To characterize D-amino acid oxidase from Arthrobacter protophormiae (DSM 20168)．［Methods］ Genes apdaao-1 and apdaao-2 from A．protophormiae (DSM 15035 ＆ 20168) were cloned by PCR;expression vectors were constructed and expressed in E.coli BL21 (DE3)．The mutant was constructed by site-directed mutagenesis using plasmid pET-ApDAAO-2 as the template．After Ni-NTA column chromatography purification，the protein was characterized.［Results］ Protein ApDAAO-1，ApDAAO-2 and 4 mutants were expressed and purified successfully．The apparent molecular masses of all purified proteins were about 36 kDa by SDS-PAGE．The optimum temperature of ApDAAO-2 and 4 mutants was 30℃ similar to ApDAAO-1． ApDAAO-2 and its mutants exhibited much broader optimal pH than ApDAAO-1，and they revealed broad substrate specificity and high specificity to D-Met (100%) except T256K，which showed the substrate preference for D-Phe (108%)．For substrates D-Met and D-Phe，the secondorder rate constants kcat/Km of ApDAAO-2 and 4 mutants were several-fold higher than ApDAAO-1 and pKDAAO，respectively.［Conclusion］Comparing with ApDAAO-1 and pKDAAO，ApDAAO-2 and its mutants had much broader substrate specificity and higher catalytic efficiency，which suggested that they might have much higher commercial value.

Abstract:Abstract:［Objective］ To enhance enzymatic activity and thermostability of N-acylhomoserine lactonase (AiiA)．［Methods］ We performed site-directed mutagenesis based on AiiA homologous 3-D protein structure，and analyzed enzymatic activity and thermostability of both wild type and mutated AiiA．［Results］ The wild type AiiA lost its Nacylhomoserine lactone (AHL) degrading activity after being incubated at 45℃ for 30 min or after being stored at 4℃ for 5 days．By comparison，the AHL-degrading activities of three types of mutated AiiA (N65K，T195Ｒ，and A206E) were enhanced，and their storage periods at 4℃ were extended to 7 days．In addition，the N65K mutant acquired higher temperature tolerance with remain of more than 45% of its enzymatic activity after being incubated at 45℃ and 5.0% enzymatic activity after being incubated at 55℃ as compared to the wild type．［Conclusion］Molecular modulation by site-directed mutagenesis could significantly improve enzymatic activity and thermostability of AiiA．

Abstract:Abstract:［Objective］ To study the transformation of 2-hydroxy-6-oxo-6-phenylhexa-2，4-dienoates (HOPDAs) by laccase from Trametes sp．SQ01 so to further understand the new catalytic properties of laccase and solve the problem of accumulations of HOPDAs in polychlorinated biphenyls (PCBs) degradation. ［Methods］ With UV-vis spectrophotometer，we studied the transformations of 8 substituted HOPDAs by laccase，and measured the steady-state kinetics parameters of laccase against parts of HOPDAs.［Results］ Laccase catalyzed HOPDAs to colorless substances without any mediators; among them，especially 3，8，11-3Cl HOPDA that was barely transformed by 2-hydroxy-6-oxo-6-phenylhexa-2，4-dienoate hydrolase (BphD) and Rhodococcus sp．R04，also could be transformed by this laccase．The analysis of the steady-state kinetics indicated that 10-Cl HOPDA was the optimal substrate of laccase among 5 HOPDAs，and the Km was lower than that of HOPDA and 8-Cl HOPDA．Although 3，10-2F HOPDA was not the optimal substrate (Km=17.02 μmol/L)，its transformation efficiency (kcat/Km) was the highest.［Conclusion］Laccase from Trametes sp. SQ01could transform various HOPDAs effectively，and has its potential in eliminating PCB pollution.

Abstract:Abstract:［Objective］To clone and characterize the malate-CoA ligase in the polymalic acid biosynthetic pathway from Aureobasidium pullulans CCTCC M2012223.［Methods］ The malate-CoA ligase gene was cloned into the expression vector pET-Mcl by IPCR technique，and expressed in Escherichia coli BL21 (DE3)．After purified with Ni-NTA column chromatography，the protein was characterized.［Result］The full-length of malate-CoA ligase gene was 1498 bp，and composed with 440 amino acids containing 4 exons and 3 introns. The optimal temperature and pH was 25℃ and 8.0，respectively，but the high substrate concentration of ATP could obviously inhibited the enzyme activity．The monomer selectivity showed that the enzyme catalyzed the substrates of oxalic acid，oxaloacetic acid，butyric acid，and malonic acid.［Conclusion］ The malate-CoA ligase gene in the polymerization pathway of polymalic acid from Aureobasidium pullulans CCTCC M2012223 was successfully cloned，which will be helpful in deeply understanding the polymerization pathway and producing new polymers．

Abstract:Abstract: ［Objective］The aim of this study was to investigate endophytic bacterial diversity of wild soybean varieties with different resistance to soybean cyst nematode(Heterodera glycines)，for deciphering the interactions of soybean cyst nematode with endophytic bacteria． ［Methods］After screening wild soybean varieties against race 3 of H．glycines，we investigated endophytic bacterial diversity in root tissues of wild soybean varieties with different resistance to H．glycines using 16S rDNA cloning library and amplified ribosomal DNA restriction analysis.［Results］Endophytic bacteria of wild soybean root belonged to 6 bacterial groups，the clones belonging to group Proteobacteria and Firmicutes were the endophyte dominants in wild soybean with 46.8% and 13.6% of total clones， respectively．Actinobacteria，Bacteroidetes，Acidobacteria，Deincoccus-Thermus and Archaea were less represented． 18.8% of clone sequences were similar to those of uncultured bacteria in the environment．The bacterial diversity was higher in H． glycines-Ｒesistant than-Susceptible wild soybean varieties，and the dominant group was different between H．glycines-Ｒesistant and -Susceptible wild soybean varieties．Mesorhizobium tamadayense，Enterobacter ludwigii and Bacillus megaterium were the main bacterial groups in special operational taxonomic units (OTUs) of H．glycines-Ｒesistant wild soybean variety．［Conclusions］By 16S rDNA cloning library and amplified ribosomal DNA restriction analysis，the diversity of dominant group of endophytic bacteria in root tissues has difference among H．glycines-Ｒesistant and -Susceptible wild soybean varieties.

Abstract:Abstract:［Objective］We studied the diversity of endophytic fungi associated with Ferula sinkiangensis K．M．Shen．［Method］Endophytic fungi from different years (1-2 years，3-4 years and＞5 years) and different parts (root，stemand leaf) of Ferula sinkiangensis K．M．Shen were isolated by tissue expand method．Strains were classified by morphology and similarity of internal transcribed spacer (ITS) sequence by Clustal X method． Composition，diversity and preference of endophytic fungal community were analyzed by the isolation rate (IR)，isolation frequency ( IF)，Shannon-Wiener biodiversity index (H’)，Margalef Ｒichness index (R)．［Results］In total 140 endophytic fungi were isolated from F．sinkiangensis K．M．Shen and classified into 18 genera．Among the 140 isolates，Aureobasidium( 25.7%)，Alternaria (16.4%) and Phyllosticta (15. 7%) were the dominant genera．The isolation results show that there were some notable differences between distribution and composition of the endophytic fungi isolated from different years and different parts of Ferula sinkiangensis K．M．Shen．Meanwhile，a certain degree of years and tissue preference were also obvious.［Conclusion］ The results obtained in this study will be helpful to exploit the endophytic fungal resources of Ferula sinkiangensis K．M．Shen，which can also provide a new way for the realization of the artificial breeding of Ferula sinkiangensis K．M．Shen．

Abstract:Abstract:［Objective］ To optimize the method of isolating a small amount of metagenomic DNA efficiently from bronchoalveolar lavage fluids (BALF) of patients with stable chronic obstructive pulmonary diseases (COPD)，which will facilitate subsequent PCR and DNA sequencing． ［Methods］BALF (5mL) of stable COPD patients was spun down to collect the cells． To extract genomic DNA from Gram-positive bacteria more efficiently，QIAGEN' s DNA extraction protocol was optimized as follows: Added Buffer ATL to the pellets and used bead tubes and tissue homogenizers to break cell walls; then added proteinase K and incubated; after adding Buffer AL and ethanol，pipetted the mixture into a DNeasy spin column then centrifuged; washed the column with Buffer AW1 and Buffer AW2，finally added 50μL Buffer AE to elute DNA．After measuring the total DNA concentration，the bacterial 16S rDNA was amplified by PCR and amplicon libraries were created for further etermination. ［Results］The DNA content of BALF with optimized protocols was 467.5 (135.0-1697.5) ng，which was significantly higher than those extracted with phenol-chloroform 95.0 (0－612.5)ng．After optimizing，more 16S rDNA PCＲ production can be obtained for future analysis (P=0.002)．［Conclusion］The optimized DNA extraction methods combining DNA isolation kits with bead-beating were more efficient in isolating tiny metagenomic DNA from BALF.

Abstract:Abstract:［Objective］To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein，and to study their antitoxic effects.［Methods］The C．jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a(+) and pGEX-6p-1．The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein．The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA)，and their specificity with a Dot-ELISA and western blotting analysis．We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells．［Results］Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-1-cdtB were successfully constructed，and fusion proteins rHis-CdtB and rGST-CdtB expressed，respectively．Five hybridoma cell lines，designated 1F3，1F5，2E4，2E11，and 2F2，were screened for the stable secretion of mAbs against CdtB． The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1．The mAb titers in the ascites fluids were 1:1 108 on indirect ELISA．Dot-ELISA demonstrated that the five mAbs reacted specifically with C．jejuni．Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein．The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P＜0.01)．［Conclusion］The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C．jejuni．

Abstract:Abstract:［Objective］ The DNA-binding domain of ToxR protein of Vibrio parahaemolyticus was expressed using the Escherichia coli BL21λDE3 protein expression system，and its DNA-binding activity was characterized．［Methods］ The fragment of DNA-binding domain at N-terminal of ToxR (ToxR-N) was amplified by PCR from V．parahaemolyticus strain RIMD2210633，and then cloned into the BamHI and Hind III sites of the vector pET28a．The recombinant plasmid pET28a was transformed into BL21λDE3．Over-expression of His-ToxR-N in the LB medium was induced by adding 1 mmol/L IPTG (isopropyl-b-D-thiogalactoside)．The over-expressed protein was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham)，and then the His-tag was removed by using restricted thrombin．The electrophoretic mobility shift assay was used to analyze the DNA-binding activity of ToxR-N to the promoter-proximal DNA regions of calR and VP1687，respectively．The promoter-proximal regions of calR and VP1687 were separately cloned into the pHRP309 vector containing a promoterless lacZ gene．Then，each of the two recombinant LacZ reporter plasmids was transformed into the wide-type strain (WT) and the toxＲ null mutant strain (ΔtoxR)，respectively，to measure the promoter activity (the β-Galactosidase activity) of the target genes in WT and ΔtoxR by using the β-Galactosidase Enzyme Assay System．［Results］The purified ToxR-N protein had the ability to bind to the upstream DNA regions of calR but not VP1687．The LacZ fusion results showed that the transcription of calR and VP1687 was positively and negatively regulated by ToxR in V．parahaemolyticus，respectively． ［Conclusion］The recombinant ToxR-N protein could be used for studying the transcriptional regulation mechanism in V．parahaemolyticus．ToxR fulfills a mechanism of negative regulation of T3SS1 genes by activating the expression of calR through protein- proximal promoter DNA association.

Abstract:Abstract:Microbial oxidation in soil is the only biological sink for atmospheric methane (about 1.8ppmv)．Two groups of atmospheric methane oxidizing bacteria are existed in aerobic soils: obligate and alternative atmospheric methane oxidizing bacteria．The former，such as upland soil cluster α (USCα) and upland soil cluster γ (USCγ)，are widely distributed in a variety of aerobic upland soils，and their particulate methane monooxygenase (pMMO) have very high affinity for methane in low concentration． Bacteria in this group are probably genuine oligotr6ophs．However，so far，there is still no cultivated strain of this group．The latter (Methylocystis/Methylosinus) belongs to traditional methane-oxidizing bacteria，and are widely distributed in soil environments with periodic high methane emission． Most strains of these two genera are known to possess two pMMO isozymes with low and high affinity to methane respectively，and these strains can keep atmospheric methane oxidizing activity for relative long periods (＞3 months) relying on the high affinity pMMO (pMMO2)．However，the growth and reproduction of bacteria in this group are still dependent on endogenous highconcentration methane which is periodically produced within the soils． We reviewed the research progress of these two groups of atmospheric methane-oxidizing bacteria，their possible living strategies，and the effects of several key environmental factors (e.g.soil temperature and moisture，soil pH，vegetation，land use，nitrogen input) on their community composition and methane oxidizing activity．Several important research directions of atmospheric methane oxidizing bacteria have also been proposed.