Abstract:Abstract：ClpX is a member of Hsp100（heat-shock protein） family which is conserved among organisms. Hsp100/ Clp implicates in stress resistance, intracellular protein turn-over, DNA replication and regulation of gene expression. Tuberculosis remains one of the major threats to human health. In pathogens, ClpX protease plays an important role in the gene expression regulation, pathogenesis, and resistance of immune stress. The structure, substrates and target genes of ClpX are summarized in this study. The biological function of M. tuberculosis ClpX, such as gene expression regulation, pathogenesis, intracellular survival and persistence, evolution and structural feature, substrates is the focus of this summary.

Abstract:Abstract: Two models of domestication and cultivation of termite-mushroom were discussed: the cultivation of termitomyces model, which method of woodrotting fungi cultivation was emphasized and the original ecological model, which multiplication of symbiotic termites was focused. The problems and possible solutions during termite-mushroom cultivation were also discussed.

Abstract:Abstract: [Objective] To elucidate phylogenetic relationships of modular polyketide synthases (PKS) in actinomycetes, and to provide a guide for screening and discovery of polyketides. [Methods] We retrieved amino acid sequences of 190 ketosynthases (KS) and 195 acyltransferase (AT) of 20 modular polyketide synthases from database PKSDB, and constructed Neighbor-Joining trees based on amino acid sequences of KS, AT and KS+AT respectively using MEGA 4.0. We computed the mean distances within and between groups of KSs. We designed a pair of degenerate primers based on two conserved regions of KS, and screened 20 bioactive isolates by PCR. After sequencing the KS genes of positive strains, we constructed a Neighbor-joining tree of the 89 KSs identified in this study with the 160 known ones. We also fermented 13 KS-positive isolates and carried out chemical analysis of the fermentation extracts. [Results] KSs from the same PKSs of actinomycetes tended to group respectively into clades, and KSs responsible for synthesis of products with similar structures tended to cluster together. The mean distances within groups of KSs from each PKS pathway were less than 0.300, and the mean distances between groups of KSs from different PKS pathways were generally more than 0.300. All the ATs grouped into two big clades according to the substrate specificity. Some ATs from the same pathway were grouped within the two big clades respectively, and the rest were scattered. The tree of KS+AT integrated the topological structures of both KS tree and AT tree. The majority KSs from individual isolates tended to group into clades. Most KSs from four of the isolates grouped into four distinct clades, and most KSs from another five isolates respectively fell into four clades of known pathways. We isolated and identified three predicted compounds as expected. [Conclusion] Vertical evolution is likely to be the dominant evolutionary mode of KS genes in actinomycetes, while AT genes may have evolved mainly through horizontal gene transfer. Considering that KSs cluster according to the corresponding product structures, and that the mean distances 0.300 between groups of KSs could sever as an evaluation criteria for different PKS pathways, the phylogenomic analysis of KS is more suitable than that of AT or KS+AT for guiding the screening of polyketide compouds from actinomycetes.

Abstract:Abstract: [Objective] Identification and characterization of the genes involved in precursor supplying for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in the haloarchaeon Haloferax mediterranei. [Methods] By using BLAST (Basic Local Alignment Search Tool) search methodology, we obtained five genes (phaB1, phaB2, phaJ1, phaJ2 and phaJ3) that were possibly involved in the 3-hydroxyacyl-CoA precursor supplying for PHBV biosynthesis in H. mediterranei. Firstly, we proved that these five genes were all transcribed under the PHBV-accumulating condition in H. mediterranei. Then, we knocked out these genes individually or in combination, by double-crossover homologous recombination, resulting in the following mutants: ΔphaB1, ΔphaB2, ΔphaJ1, ΔphaJ2, ΔphaJ3, ΔphaB1phaB2, ΔphaJ1phaJ2 and ΔphaJ1phaJ2phaJ3. Finally, we performed the complementation analysis of the ΔphaB1phaB2 strain, with the phaB1 and phaB2 genes, respectively. [Results] Whenever the three phaJ genes were knocked out individually or in combination, there was no obvious influence on PHBV accumulation in H. mediterranei. Knockout of phaB1 also did not affect the PHBV accumulation obviously. However, when phaB2 was knocked out, the yield of PHBV and the fraction of the 3-HV monomer decreased significantly. Notably, when the phaB1 and phaB2 were knocked out in combination, the mutant ΔphaB1phaB2 no longer produced PHBV. [Conclusions] The PHBV-specific acetoacetyl-CoA reductases (PhaB) involved in the precursor supplying for PHBV biosynthesis are encoded by phaB1 and phaB2 in H. mediterranei.

Abstract:Abstract: [Objective] Jujube witches’-broom (JWB) is an important plant disease caused by phytoplasma. The major objective of our research was to classify JWB in Beijing and Hebei districts and to provide reference for classification in subgroup level. [Methods] By use of PCR, the elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs fTufu/rTufu and rp(v)F1A/rp(v)R1A. Partial tuf gene and rp gene were sequenced and similarity analysed with other phytoplasmas. [Results] We obtained partial tuf gene sequence (824bp) and complete rp gene (1196bp) from the diseased sample. In tuf gene, JWB in Beijing shared most similarity (92.84%) with Flavescence dorée (FD) phytoplasma (Candidatus Phytoplasma vitis) , however, shared a low similarity (57.29%) with JWB in Shaanxi district which had been already reported .The similarity analysis for sequences of rp gene showed a high identity(﹥96 %) with members of the 16SrV group phytoplasmas. It shared most identity (99.83%) with JWB strain Taishan and Hemp fiber witches’-broom phytoplasma (HFWB) of the 16SrV group. [Conclusion] The JWB strains in Beijing and Hebei are members of 16Sr V; JWB in Beijing and Hebei share high similarity, and show a diversity with JWB in Shaanxi.

Abstract:Abstract: [Objective] AsE246 was a novel nodule-specific expressed nodulin gene encoding non-specific lipid transfer protein 1 (nsLTP1) of A. sinicus. We screened and identified host plant target protein interacting with AsE246, and characterized the expression patterns of target gene under symbiotic and stress conditions. [Methods] Yeast two-hybrid system, small-scale yeast hybridization test and real-time PCR technique were used to capture the host target protein that interacts with the bait protein AsE246, and to quantitatively analyze the temporal and spatial expression characteristics of target gene during root nodule development and nitrogen fixation process. [Results] One positive clone was obtained, its cDNA insert sequence and Blast analysis showed that: the target protein was a DnaJ-like protein, thus the corresponding encoding gene was named as AsDJL1. AsDJL1 was specifically-enhancing expressed in nitrogen-fixing root nodules, and significantly increased under NaCl stress while significantly decreased under (NH4)2SO4 stress. [Conclusion] This work was the first report on the isolation of proteins interacting with LTP. The work obtained some direct and convincing evidences to show the interacting gene demonstrated high similarity as AsE246 in expression patterns and functions involved. The present progress provided a basic foundation and theoretical basis to undertake any further investigation into their interaction, and regulation mechanism associated with symbiotic nitrogen fixation or response to environmental stress.

Abstract:Abstract: [Objective] To investigate the organic carbon metabolism during sludge anaerobic digestion and to clarify the mechanism of acetate accumulation. [Methods] We Used inhibitors 2-bromoethanesulfonate (BES) and chloroform (CHCl3) to block the methanogesis to investigate the accumulation of various fermentation intermediates. We determined the bacterial number of homoacetogen and syntrophic acetogen and calculated the thermodynamics of the acetogenesis reaction to identify the directions of organic carbon metabolism and acetate accumulation during sludge anaerobic digestion. [Results] With the addition of BES, the acetate concentration was 27 mmol/L, the gene copy number of fhs was 2-3 folds of the control group and the number of sytroph acetogen was slightly decreased. In the group of chloroform addition, the acetate was 22 mmol/L, while the copy number of fhs gene was one order of magnitude lower than that in the BES model. [Conclusion] The two inhibitors lead to acetate accumulation from the anaerobic digestion of sludge. BES inhibited the methanogen and had no effect on other anaerobic acetogens. The accumulation of the acetate came from the hydrolysis acidogenesis, homoacetogenesis and syntrophic acetogenesis. Chloroform inhibited not only methanogen but also the homoacetogen and syntrophic acetogen severely. The acetate accumulation mainly came from the hydrolysis acidogenesis fermentation.

Abstract:Abstract:[Objective] In order to optimize precursor supply for L-tryptophan biosynthesis, a Corynebacterium pekinense PD-67 mutant with phosphoenolpyruvate carboxylase gene(ppc) in-frame deletion was constructed. The effect of ppc knock-out on physiological characteristics of the mutant was investigated. [Methods] The upstream and downstream fragments of ppc were cloned from C. pekinense PD-67 chromosome and ligated to integration vector. The mutant C. pekinense PD-67-Δppc was screened by homologous recombination. The physiological characteristics of the mutant were investigated by fermentation experiments and measurement of pyruvate carboxylase (PCx) and pyruvate kinase (PK). [Results] The mutant with ppc gene in-frame deletion was screened and confirmed by PCR check and phosphoenolpyruvate carboxylase determination. The mutant exhibited slow growth and less cell mass, 80% as much as the parent strain. The ppc knock-out resulted in decrease of L-tryptophan accumulation and overproduction of pyruvate-related amino acids, which accompanied by increase of PK activity and the decrease of PCx activity, in C. pekinense PD-67. [Conclusion] The knock-out of ppc gene affected the metabolism of the strain to some extent. Only by blocking the anaplerotic pathway PEPCx participated was insufficient to increase the accumulation of L-tryptophan in C. pekinense PD-67.

Abstract:Abstract:【Objective】Our aim was to enhance nisin production by overexpression of nisin immunity gene nisI in nisin-producing strains.【Methods】Nisin immunity gene nisI with a strong promoter P59 was cloned into vector pHJ201 and introduced into Lacotococcus lactis NZ9800, resulting in a recombinant strain L. lactis NZ9800/pHMI. Then the differences between the recombinant strain and the control strain L. lactis NZ9800/pHJ201 were analyzed in several aspects, including their growth curves, nisin resistance level and antibacterial activity against indicator strain Microccus flavus NCIB 8166.【Results】The overexpression of nisI had no significant difference in growth rate between recombinant strain and contrast strain. However, it promoted recombinant strain tolerance 25% higer nisin resistance level and stronger antibacterial activity against M. flavus NCIB 8166, which was increased by 32% and 25% when fermented for 6 and 8 hours, respectively. 【Conclusion】These results indicated that overexpression of nisI gene in the nisin producing strain can effectively enhance nisin resistence level and thus improve nisin production.

Abstract:Abstract：[Objective] We studied the reduction of soluble and highly toxic selenite to elemental red selenium, under aerobic condition by Pseudomonas alcaliphila MBR, with organic carbon as electron donor. [Results] The strain could grow under pH 6-11 and resist to high concentration of selenite with the minimal inhibitory concentration of 50 mmol/L. After 5 days, the strain used sodium citrate as electron donor, and reduced 2.0 mmol/L selenite to elemental red selenium from the culture fluid, the elemental red selenium was stored outside the cells. The glutathione and nitrate could increase the number of reduction rate. [Conclusion] This study implies the application of Pseudomonas alcaliphila MBR to convert selenite to elemental red selenium.

Abstract:Abstract: [Objective] In order to obtain the antagonistic protein of Bacillus subtilis G87 and definitude its characterization. [Methods] Methods of ammonium sulfate precipitating and column chromatography analyzing were used to isolate and purify the protein. [Results] A purified protein (peak 6-2-1) was obtained which molecular weight was 50.8kD by SDS-PAGE and isoelectric point was 5.90 by IEF-PAGE. The antifungal protein contained 0.62% saccharide and some proline or hydroxyproline, but no lipid and aromatic amino acid. The inhibitory activity of the antifungal protein would decreased distinctly at the higher temperature (≥60℃) and in the condition of alkalinity (pH>8), but tolerant to ultraviolet radiation, chloroform, trypsin, proteinase K and pepsin. [Conclusion] Antifungal protein of Bacillus subtilis G87 was a kind of glycoprotein without aromatic hydrocarbon. It was sensitive to higher temperature and tight alkalinity but not to proteinase analog and ultraviolet radiation et al.

Abstract:Abstract：[Objective] The present study was to identify four strains of plant growth-promoting bacteria (PGPB) and their plant growth-promoting ability through inoculation. [Methods] Four strains of PGPB were genetically analyzed by PCR detection of nifH and 16S rRNA gene. Both phosphate-solubilizing and nitrogen-fixation capacity were examined by spectrophotometric quantification and acetylene reduction assay, respectively. Effect of strain inoculation on plant growth were also evaluated. [Results] Phylogenetic analysis based on nifH and 16S rRNA gene sequences indicated that HN011 was mostly related to Vibrio natriegens, and SZ7-1 and SZ7-2 resembled Klebsiella oxytoca. Although similarity of 16S rRNA sequence showed that SZ002 belongs to Paenibacillus sp., nifH gene of SZ002 had high sequence similarity with Klebsiella genus. Phosphate solubilization showed that insoluble phosphate was well solubilized in the liquid medium by all four strains of PGPB, which also had high nitrogen-fixation capacity. Plant dry weight, total N and total P were generally significantly higher in some inoculated than in the non-inoculated plants (P<0.05). [Conclusion] Our results showed that all four strains of PGPB isolated from mangrove had dual phosphate solubilization and nitrogen fixation ability, resulting in beneficial effects to mangrove growth. This work suggest that these PGPB strains could be used as inoculants for mangrove reforestation.

Abstract:Abstract: [Objective ] To investigate the kinds and characteristics of spiroplasma in honeybees, as well as to study the taxonomy and transmission of honeybee spiroplasma under natural conditions. [Methods] We examined the morphology of spiroplasma isolates by dark field and transmission electron microscopy and studied the biological characteristics by using conventional culture-dependent methods and molecular biology and serological methods. [Results] Three spiroplasma isolates were obtained from healthy Apis mellifera. All isolates exhibited helicity during their growth phase, with one isolate (MF0905) being shorter and having less helicity. This isolate also differed from the other two (MF0903 and MF0904) in having larger colonies with an irregular margin instead of being round. In addition, isolate MF0905 could not hydrolyze arginine whereas MF0903 and MF0904 could. All three isolates could use glucose and D-fructose as a carbon source but did not hydrolyse urea. Phylogenetic analysis based on the 16S rDNA, ITS and the rpoB gene showed that MF0903 and MF0904 had a close relationship with Spiroplasma melliferum, and MF0905 was close to S. clarkii. Serological studies including the growth inhibition test, metabolic inhibition test and deformation test gave the same result as the phylogenetic analysis. [Conclusion] The spiroplasma isolate MF0905 might be S. clarkii and other two isolates were S. melliferum. This result indicated that Spiroplasma melliferum is not the only spiroplasma species in honeybees in China.

Abstract:Abstract: [Objective] To investigate the change of Clostridium cluster Ⅳ community in the colon of piglets from 7 to 35 days of age, and its correlation with butyrate concentration. [Methods] Three litters of neonatal piglets were used in this study. One piglet from each litter was sacrificed randomly at the age of 7, 14, 21（weaning day）, 24 and 35 days, digestive samples in the colon were collected. The concentration of volatile fatty acid(VFA) were determined by gas chromatography. 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE)and real-time PCR were used for qualitative and quantitative analysis of Clostridium cluster Ⅳ community. [Results] Similarity analysis of DGGE profile revealed that samples from piglets at the age of 7 days formed a coherent cluster with indices above 90%, no significant changes in Clostridium cluster Ⅳ community were found around weaning period. Real-time PCR analysis showed that 16S rRNA gene copies of total bacteria in the colon of piglets decreased significantly 3 days after weaning, this tendency was in accordance with the changes in concentration of total VFA and butyrate in colon, while there was no significant difference in copies of Clostridium cluster Ⅳ group. Sequencing analysis indicated that Clostridium cluster Ⅳ group in the colon of piglets were dominated by Faecalibacterium prausnitzii, Subdoligranulum variabile and other uncultured bacteria. [Conclusion] Changes in Clostridium cluster Ⅳ community in the colon of piglets were found from the age of 7 days to 14 days, while there was no significant difference during the weaning transition.

Abstract:Abstract: [Objective] To confirm the involvement of pqqL gene of Escherichia coli in PQQ biosynthesis, a pqqL deletion mutant of E. coli DH5α was constructed and investigated. [Methods] pqqL and kan gene were cloned and a linear targeting fragment pqqL-kan-pqqL was constructed in vitro. Then pqqL gene was knocked out and DH5α?pqqL mutant was constructed by Red homologous recombination. The PQQ biosynthesis was compared between the mutant and its parential strain by detection of bio-activity of sorbose dehydrogenase with DCIP method. [Results] The DH5α?pqqL deletion mutant was successfully constructed, and the result indicated that PQQ would be synthesized in pBCP162/DH5α?pqqL and pMD19T Simple-pqqABCDE/DH5α, but not in pMD19T Simple-pqqABCDE/DH5α?pqqL. [Conclusion] The function of pqqL gene in Escherichia coli is the same to that of pqqF gene.

Abstract:Abstract: [Objective] To identify Aspergillus awamori strain F12 isolated from rhizospheric soil of Rhizophora stylosa Griff and to characterize antibacterial compounds from the ethyl acetate extracts of its fermentation broth. [Methods] Strain F12 was identified based on its morphological characters and internal transcribed spacer (ITS) sequence. Its secondary metabolites were purified by chromatography, and elucidated by mass spectroscopy, 1H-NMR, 13C-NMR and physicochemical characters. The antibacterial activities of these compounds were tested against Staphyloccocus aureus and Bacillus subtilis. [Results] Strain F12 was identified as Aspergillus awamori. Three compounds, including 1,4-dimethoxybenzene (1), emodin (2) and 3, 6-dibenzylpiperazine-2, 5-dione (3), were purified and elucidated from the ethyl acetate extracts of fermentation broth, among which compound 1 was first reported for the genus of Aspergillus. Compound 2 suppressed the growth of Staphyloccocus aureus and Bacillus subtilis with MIC values of 16 μg/mL and 32 μg/mL respectively, while the other two compounds have no effects on these bacteria. [Conclusion] Aspergillus awamori strain F12 isolated from rhizospheric soil of Rhizophora stylosa Griff can produce 1, 4-dimethoxybenzene and emodin, among which the latter can suppress the growth of bacteria apparently.

Abstract:Abstract: [Objective] The aim of this study is to assess the diversity of polycyclic aromatic carbon (PAH)-degrading bacteria in the coastal seawater of Xiamen Island.. [Methods] The phenanthrene-degrading bacteria were enriched by suspending phenanthrene-coated Polyvinylchloride (PVC) plates in the seawater close to Xiamen International Cruise Dock. PCR-DGGE and 16S rRNA gene clone library were used to analyze the bacteria colonizing on the PVC plates. Further, PAH-degrading bacteria were re-enriched in lab after the in situ enrichment and subjected to diversity analysis and key member isolation. [Results] After 6 days incubation, the genus Cycloclasticus was shown to the dominant bacterium on the phenanthrene (Phe)-coated plates, which accounted for 50% of the total clones in 16S rRNA gene clone library. However, on the control plates without Phe-coating, bacteria of Rhodobacteraceae were the dominant member, which accounted for 47% of the total clones. PCR-DGGE results revealed that the genus Cycloclasticus was the dominant member in Phe-degrading consortium growing on the Phe-coated plates in situ. After re-enrichment with Phe in laboratory, 14 strains were isolated from the consortium, in which a potential novel species of belonging to Novosphingobium was identified as a Phe degrader, named strain B-14. However, the most predominant member-Cycloclasticus can not be cultivated into pure culture. [Conclusion] Genus Cycloclasticus is the most important PAH-degrading bacterium in the coast sea water of Xiamen Island. This is the first evidence to our knowledge about the in situ enrichment PAH-degrading bacterium in seawater.

Abstract:Abstract: [Objective] MicroRNAs (miRNAs) play an important role in the process of infection and replication of virus in host cells. In this study, we cloned two miRNAs expression vectors and examined their effects on the replication of H1N1 type influenza virus in MDCK (Madin dardy canine kidney) cells. [Methods]We constructed the plasmids expressing miR26a and miR939 and performed the transfection study in MDCK cells. Subsequently, the cells were infected with H1N1 type influenza virus 24 h after transfection. Then we tested the hemagglutination titer at 72 h time point to investigate the effect of miR26a and miR939 on the replication H1N1 type influenza virus in MDCK cells. [Results] The transfection study showed that miR26a and miR939 can achieve efficiently expression in MDCK cells. miR26a and miR939 can influence the replication of H1N1 influenza virus differently in two different ways. miR26a inhibits the replication, whereas miR939 stimulates the process. [Conclusion]Cellular miRNAs can regulate the replication of H1N1 influenza virus in host cells, and our paper should report the role of miR26a and miR939 in this regulation for the first time.

Abstract:Abstract: [objective] To prepare high-affinity anti-aflatoxin M1 monoclonal antibodies by High Throughput Screening ELISA (HTS-ELISA) [Methods] Balb/C mice were immunized by aflatoxin M1-bovine serum albumin conjugate, and screen secret anti-aflatoxin M1 monoclonal antibody hybridoma by HTS-ELISA. The antibody was characterized. [Results] Fourteen hybridoma cell lines which could secret high activity anti-aflatoxin M1 monoclonal antibodies were obtained. The affinity of the purified monoclonal antibody was 5.5x10-10 mol/L. The cross-reactivity of the monoclonal antibody clone against aflatoxin M1, aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol and BSA was 100%, 4.5%, 21.5%, 1.0%, 16.6%, 1.0%, 0%, 0%, respectively. The sensitivity of the anti-AFM1 monoclonal antibody binding to aflatoxin M1 was 0.01 μg/L and the linear range for developed indirect competitive ELISA was 0.1 - 10μg/L aflatoxin M1. The binding inhibition IC50 of the anti-aflatoxin M1 monoclonal antibody was 0.82μg/L. Assays of milk samples mixed with AFM1 ranging in concentration from 0.25 to 5.0 μg/L gave mean indirect competitive ELISA recovery of 60.3%-152.8%. [Conclusion] HTS-ELISA can be used for the preparation of the high-affinity anti-aflatoxin M1 monoclonal antibodies. The anti-aflatoxin M1 monoclonal antibody could be provided as the high quality material in the system of aflatoxin M1 immune detection.