Abstract:Abstract: Petroleum reservoir has a variety of microbes with diverse metabolic characteristics and great diversity. These microbes play an important role in geochemical cycle. Research on their metabolism and ecological relationships enables the understanding of Microbial Enhanced Oil Recovery (MEOR). Based on the present researches, we review the microbial metabolic characteristics and ecological relationships in oil reservoir.

Abstract:Abstract: ε-Poly-L-lysine (ε-PL) is a polymer with strong antimicrobial activity and consists of 25-35 L-lysine monomers linked via ε-amino-α-carboxyl peptide bond. This polymer is one of only two homo-poly(amino acid) polymers known in nature. To date, the biosynthesis mechanism of ε-PL was remained unclear. Recently, ε-PL synthetase was identified as a nonribosomal peptide synthetase and it utilized L-lysine to synthesize ε-PLs of various chain length by iteratively condensing reaction, which was similar to typeⅠpolyketide synthase and was not determined by ε-PL-degrading enzyme. Meanwhile, the shutter vectors pLAE001 and pLAE003, special for ε-PL producer have been constructed, which is significant for studying ε-PL biosynthesis. In this review, the biosynthesis of ε-PL and the genetic transformation were introduced. Also, the study in our group was presented and the views related to ε-PL study were raised. Finally, the prospect of application of combinational biosynthesis in ε-PL-producing strain improvement was also discussed considering ε-PL synthetase is a nonribosomal peptide synthetase containing several modules.

Abstract:Abstract: Due to its ability of hydrolyzing α-1,6-linkages, pullulanase plays a significant role in the industries where starch is used as raw material, including food processing and bioenergy. Exploitation of this enzyme focuses not only on screening novel strains, but also on its molecular structure as well as its secretion pathway and improvement of the properties. Therefore, researches on the gene regulation and the proteins involved in the secretory pathway seem especially important. This review introduced the advances in the study of these fields.

Abstract:Abstract: During the past two decades, the genetic diversities of bacterial and fungal communities and their relationship with the inhabited environments were intensively studied with the development of molecular biological techniques. However, although bacteriophages are ubiquitous and more abundant than their hosts in biosphere, their diversity is still little known. In this paper, we targeted the major capsid genes (g23) of T4-type bacteriophages and reviewed the recent progress on their genetic diversity. The distribution of g23 of T4-type bacteriophages was distinctly different among natural environments of marines, lakes and paddy fields, and the majority of g23 were grouped into several novel clusters according to their obtained environments. In addition, several research tips and future research tendencies for the study of environmental g23 were also addressed.

Abstract:Abstract: [Objective] The Snf1/AMPK family of protein kinases is highly conserved among eukaryotes. Our previous study showed that Cryptococcus neoformans SNF1 played critical roles in the production of virulence factors and virulence itself. In this paper, we report a novel function of SNF1 in cell wall integrity. [Methods] We used Calcofluor white staining epifluorescence microscopy to evaluate the cell wall integrity and cell segregation; tap water with constant flow rate and pressure to wash yeast colonies to evaluate cell-to-agar adhesion capability; growth on Sodium dodecyl sulfate (SDS), Congo red and Fluorescent Brightener 28-containing agar to examine cell wall integrity. [Results] The disruption mutant of SNF1 was sensitive to SDS and Congo red, suggesting impairments in the cell wall. The mutant cells showed abnormal separation, defects in adhesion to agar surface, and growth defects at high temperature which could be suppressed by osmotic stabilization. [Conclusion] C. neoformans SNF1 was essential for cell wall integrity that was likely responsible for normal adhesion of the cells to agar and resistance to heat.

Abstract:Abstract: [Objective] Prokaryotes synthesize phosphotidylcholine by using phospholipid N-methylation or phosphatidylcholine synthase pathway or both. To confirm which pathway the soil bacterium Pseudomonas sp. 593 utilizes, we tested its phosphotidylcholine synthesis, cloned the pcs gene encoding phosphatidylcholine synthase, examined Pcs activity, and constructed a pcs- mutant. [Methods] To clone the pcs gene from Pseudomonas sp. 593 genomic DNA, we firstly aligned amino acid sequences of phosphatidylcholine synthases in different pseudomonas strains reported in databases. Then we designed degenerate primers based on two amino acid segments conserved in sequences of phosphatidylcholine synthases. A partial fragment of the pcs gene was finally amplified from Pseudomonas sp. 593 genomic DNA. The amplified partial fragment was labeled with digoxigenin-dUTP (DIG) as a probe, sub-cloning library of Pseudomonas sp. 593 genomic DNA was prepared and then screened using DIG-labelled probe via in situ colony hybridization. DNA homologous recombination in vivo was preformed to delete pcs gene of Pseudomonas sp. 593. Thin-layer chromatography (TLC) assay was used to analyze total phospholipids, detect phosphotidylcholine content and determine pcs gene activity. [Results] TLC analysis revealed that Pseudomonas sp. 593 growing in the M9 or LB medium with choline was able to synthesize phosphotidylcholine, but wasn’t without addition of choline. A 894 bp DNA fragment coded a protein with phosphatidylcholine synthase activity was cloned from Pseudomonas sp. 593. The pcs- mutant obtained from in vivo mutagenesis was unable to form phosphotidylcholine, no matter choline was presented in the medium or not. [Conclusion] Phosphatidylcholine synthase pathway is a sole way for phosphotidylcholine synthesis in soil bacterium Pseudomonas sp. 593 or other Pseudomonas strains.

Abstract:Abstract：[Objective] In order to understand the weathering on potassium-bearing mineral by Aspergillus niger, we studied the formation of A. niger-mineral aggregation and polysaccharide in the revolving and fermenting mode and their role in the process of weathering on potassium-bearing mineral. [Methods] We used four different media to study the morphology of A. niger -mineral aggregation; ultraviolet-visible spectrum (UV-Vis), fourier transform infrared spectrum (IR), gas chromatography(GC), scanning electron microscopy(SEM) and energy dispersive spectrometer(EDS) were combined to research the changes of polysaccharide and their significances in the micro-environment forming fungal-mineral aggregation. [Results] A. niger myclia intertwined, adsorbed, chelated and bonded mineral powder to form aggregation by the assistance of polysaccharide and other metabolites. After formation of the aggregation, the concentration and structure of polysaccharide were changed significantly. [Conclusion] The changes of polysaccharide would enhance the adsorption on minerals, chelation on metal ions and adsorption on water molecules, which provided a favorable micro-environment for the fungal using mineral nutrients effectively.

Abstract:Abstract: [Objective] Ganoderma lucidum was cultivated on non-medicinal parts of Salvia miltiorrhiza, Chrysanthemum morifolium, Ptatycodgn grandlfiorum, as all are Chinese traditional herbal medicines. We studied the changes of active ingredients and efficacies of the Ganoderma lucidum fruit bodies. [Methods] The agronomic characters, polysaccharide and terpene contents, acute toxicity and efficacy of Ganoderma lucidum grown on the non-medicinal part of the three materials were compared with that grown on the ordinary formula group (OF.G) which was composed of corn cob, cotton seed shell.[Results] Biological conversion efficiencies of the Ganoderma lucidum fruit body using non-medicinal parts were higher than that of using the ordinary formula group (OF.G), though growth periods became longer; Contents of active ingredients were all improved except that the terpene content of the Salvia miltiorrhiza group was decreased. Both polysaccharide and terpene from the Chrysanthemum morifolium group were the highest, contents of which were respectively 2.47% and 0.79%; Acute toxicity test showed that Ganoderma lucidum fruit bodies were all with low toxicities. Mice maximum tolerance dose were 100 g/kg weight. In hemolysin test and sleeping promotion test, the Chrysanthemum morifolium group showed better effect than the ordinary formula group (OF.G). In anti-fatigue test, only the ordinary formula group (OF.G) proved to be more effective. [Conclusion] It’s feasible to cultivate Ganoderma lucidum and active ingredients and efficacies of Ganoderma lucidum have been changed using the non-medicinal parts of Chinese medicinal herbs.

Abstract:Abstract: Quorum-sensing (QS) is a regulatory mechanism with which bacteria regulate the gene expression according to their population density. Pseudomonas aeruginosa regulates the expression of multiple genes via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). [Objective] It aims to explore the regulation of QS on biosynthesis of polyhydroxyalkanoates (PHA) in P. aeruginosa. [Methods] Wild-type P. aeruginosa PAO1 and its QS mutants were used to investigate the effects of quorum-sensing on biosynthesis of PHA by GC and real-time PCR at physiological and molecular level. [Results] After treated with QS signal molecule synthesis inhibitor azithromycin, the accumulation of PHA significantly decreased in P. aeruginosa PAO1 and its QS mutant strains. The content of PHA in C4-HSL synthase gene rhlI mutant strain PAO210 had no significant difference compared with that of the wild type. However, the PHA contents were significantly affected in 3OC12-HSL synthase gene lasI mutant strain PAO55, 3OC12-HSL transcriptional regulator gene lasR mutant strain PAO56 and lasI / lasR double mutant strain PAO57. PHA synthase gene phaC1 expression exhibited a significant reduction in lasI mutant and lasR mutant strains. 3OC12-HSL signal molecules complementary experiment shows that the expression of phaC1 can be recovered to the level of the wild type, but the synthesis of PHA is only partially restored in lasI mutant strain. [Conclusion] The results implicates that lasI/lasR system might be involved in the regulation of intracellular PHA biosynthesis in P. aeruginosa PAO1.

Abstract:Abstract: [Objective] We investigated the reactivity and function of two cysteine residues in imidase (CIH) by site-directed mutagenesis. [Methods] Three variants of imidase (CIH) were constructed with Cys7 and Cys108 or only one of them substituted with Gly. The two thiol groups of Cys7 and Cys108 of imidase were specifically modified separately or collectively by dithio-bis-nitrobenzoic acid (DTNB) in the native state. It was also confirmed by SDS-PAGE analysis. To further verify the above results, the oligomeric structure and sulfhedryl groups of native and mutant CIH were also examined by measuring zinc binding ability and molecular size under different concentrations of H2O2. [Results] Compared with CIH, CIH108 retained 72% activity, while CIH7,108 and CIH7 had no activity using DL-hydantoin as substrate. The spectral detection result shown that the two thiol groups were both in a free state. It is indicated that CIH and CIH108 are tetramer, CIH7,108 is multimer, and CIH7 is a mixture of monomer and multimer. The zinc binding ability of CIH108 was still relative high, while CIH7 and CIH7,108 decreased obviously. The increased concentration of H2O2 could increase the intrachain disulfide bond of CIH and the interchain disulfide bond of CIH108. [Conclusion] All data imply that the Cys7 is required for binding zinc ion and maintaining the stable structure of enzymatic molecule.

Abstract:Abstract:［Objective］ The function for catalyzing acetophenone derivatives of short-chain carbonyl reductase Ⅱ ( SCR Ⅱ) from Candida parapsilosis was modified by site-directed mutagenesis． ［Methods］ An important site ( E228 ) was selected for mutagenesis through amino acid sequence and protein structure alignment，and the corresponding variant E228S was constructed in E． coli． Using the acetophenone derivatives as substrates，we determined specific activities and biotransformation function of the variant． ［Results］The specific activity of the variant E228S was reduced to 25% of the wild type for 2-hydroxyacetophenone reduction． However，it increased approximately 7-20 times for acetophenone，4'- methylacetophenone and 4'-chloroacetophenone． The biotransformation results showed that the variant catalyzed the transformation of ( S) - 1-phenyl-1，2-ethanediol in a yield of less than 10% ，while exhibited excellent performance to afford ( R ) - 1-phenethylethanol， ( R ) - 1-( 4-methylpheyl ) ethanol and ( R ) - 1-( 4-chlorophenyl ) ethanol from acetophenone，4'-methylacetophenone and 4'-chloroacetophenone with high optical purity of 99% in a yield of above 80% ． ［Conclusion］We broadened the substrate specificity and catalytic function of SCRⅡ by replacement of the critical amino acid ( E228 ) inside the substrate binding pocket，which provided a novel approach for rational modification of short-chain carbonyl reductases，making it as a potential tool for asymmetric reduction and chiral alcohol preparation．

Abstract:Abstract：[Objective? The (R)- and (S)- specific carbonyl reductases (RCR and SCR) with enhanced green fluorescence protein (EGFP) were expressed in Saccharomyces cerevisiae W303-1A. By analysis of EGFP expression spectrum, the protein distribution and subcellular localization of two enzymes were determined. [Methods? By SOE-PCR method, the fused genes of RCR and SCR with EGFP were cloned and constructed on an eukaryotic expression vector pYX212, and transformed into S. cerevisiae by electroporation. With fluorescent protein as selection marker, the expression and distribution of RCR and SCR were observed. [Results? The EGFP expression in S. cerevisiae cells was observed under the laser scanning confocal microscopy．It was showed that the two enzymes expressed stably in S. cerevisiae and mainly distributed in the intracellular membrane and cytoplasm, while minority were dotted in the center of cells. According to the fluorescent intensity, the expression level of SCR was much higher than that of RCR in S. cerevisiae. The biotransformation results showed that the fused proteins EGFP-RCR and EGFP-SCR reduced 2-hydroxyacetophenone to give (R)- and (S)-1-phenyl-1,2-ethanediol (PED) respectively, with the optical purity of 86.6% in a yield of 70.4% for the former enzyme and with the optical purity of 92.3% in a yield of 81.8% for the latter one. [Discussion? The fusion of RCR or SCR with EGFP showed no effect in protein conformation and biological activity. When compared with the recombinant Escherichia coli harboring RCR or SCR, the genetically engineered S. cerevisiae had obvious advantages in the biological function. The study provided the solid foundations for visible research of functional expression controlling and subcellular localization of carbonyl reductases.

Abstract:Abstract: [Objective] The dynamic characteristics of community structure and relative abundance of Geobacteraceae were investigated to understand their response to microbial iron(III) reducing in flooded paddy soil. [Methods] The paddy soil was incubated anaerobically and the amount of Fe(II) was determined during the flooding incubation. We retrieved Geobacteraceae sequences from clone libraries constructed for different time points (1 h and day 1, 5, 10, 20 and 30) after flooding of the paddy soil. The diversity and community structure were analyzed by using RFLP method, and the relative abundance of Geobacteraceae was detected by real-time PCR. [Results] Microbial reduction of iron(III) changed greatly in early time and was stable after incubated for 20 d in paddy soil. The largest iron reduction potential was 10.16 mg/g with a Vmax of 1.064 mg/(g.d) at the time of 4.84 d whereas this process achieved plateau after 20 days flooding. Diversity of Geobacteraceae, given by alpha indices, fluctuated during the flooding incubation. Two peaks of diversity were observed in treatments of 5 d and 20 d respectively, while significant low diversity appeared in samples of 10 d and 30 d. Beta indices described the differences between community structures of Geobacteraceae and hence reflected the variation of the flooding situation over time. In all samples, 10 RFLP-based preponderant types were found, which fell into clade 1 and clade 2 of Geobacteraceae. The relative abundance of Geobacteraceae was the lowest in 1 d (1.20%) and the highest in 20 d (4.54%). [Conclusion] The dynamic variation of Geobacteraceae diversity, community structure and abundance are closely related to microbial iron(III) reducing in flooding paddy soil.

Abstract:Abstract: [Objective] We used tri5-PCR technique to identify the toxin-producing Fusarium strains, the toxogenic characteristics and toxicogenic condition of tri5 positive strains. We also evaluated the potential trichothecene-producing level of the Fusarium strains from the air and solid materials in poultry houses. [Methods] Tri5 gene, the start gene encoding trichothecene synthase, was taken to detect 139 Fusarium isolates by PCR. Toxogenic culture was carried out for tri5 positive Fusarium strains, and the quantities of T-2 and HT-2 toxins in toxogenic cultures were measured by high performance liquid chromatography (HPLC) after immune-affinity column clear-up. [Results] Among the 42 tri5 positive strains determined by tri5-PCR, all 10 tri5 positive strains from the air in poultry houses were found to produce T-2 toxin (1.36ng/mL~5ng/mL) or HT-2 toxin (6.1ng/mL~17.1ng/mL) after culture. The optimal conditions for toxogenic culture were 9 days of culture with 5℃-20℃ temperature fluctuation and light-dark alternating at 24h intervals, shaking at early stage and non-shaking at later stage. The toxin production of tri5 positive strains was significantly affected by temperature and time, but it has no correlation with the dry mass of mycelium. [Conclusion] In comparison with the traditional method, tri5-PCR is a rapid method for accurate detection of toxogenic Fusarium isolates in large amounts of environmental samples from poultry houses. The results provide a technical support and theoretical basis for early hazard warning and control of toxogenic Fusarium strains in animal raising environments.

Abstract:Abstract：[Objective] To investigate the relationship between microbial community and the blight diseases in rhizosphere of carnation for biological control. [Methods] Bacterial strains isolated from the rhizosphere of healthy and blight carnation plants in greenhouse were replicated by morphology and 16S rRNA gene similarity, and investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. [Results] Isolates belonged to 4 phyla of bacteria, 65 isolates from the samples of healthy carnation plants belonged to 9 genera and Bacillus, Streptomyces, Mortierella as the dominant bacteria. However, 33 isolates from the samples of blight carnation plants belonged to 12 genera and Stenotrophomonas, Sphingobacterium, Pseudomonas, Chryseobacterium, Amycolatopsis and Fusarium only from the sample of blight carnation plants. At least 13 isolates should represent potential novel species based on lower similarites of 16 S rRNA gene (90-98%). [Conclusion] The result showed that either the percentage of the fungus in the total strains or the abundance of Bacillus groups in the total strains can be the referential targets to evaluate whether the carnation soil would be healthy for the carnational growth or lead to fusarium wilt diseases, accurately forecasting potential risks of the disease.

Abstract:Abstract: [Objective] To compare the specificity of three 16S rRNA gene-based PCR primer pairs (FPR-1/FPR-2, FPR-2F/Fprau645R and Fprau223F/Fprau420R), which are used to specifically detect and quantify the important human gut bacterium Feacalibacterium prausnitzii. [Methods] Clustal X was used to align the sequences of individual primer and the 16S rRNA gene of F. prausnitzii and other bacteria. The number of Faecalibacterium spp. sequences in Ribosomal Database Project (RDP) matched by each primer was obtained by the Probe Match tool. With the full-length 16S rRNA gene clone library constructed by our laboratory which contained 7255 clones from the gut microbiota of 7 Chinese people, the Simulated PCR (SPCR) program was applied to predict the clone number of F. prausnitzii and other gut bacteria matched by every primer pair; PCR amplification was performed with three primer pairs and representative clones to verify the SPCR prediction. Real-time quantitative PCR was performed with three primer pairs, respectively, for fecal samples from 14 healthy individuals. [Results] The first base at the 3’ end of Fprau645R showed the highest mismatch level for non-F. prausnitzii bacteria. The percentage of the number of Faecalibacterium spp. sequences matched by Fprau645R accounting for that of matched bacteria sequences in the RDP database was 97.6%, which was significant higher than that of other primers. As SPCR predicted, all three primer pairs can detect about 1171 clones from F. prausnitzii; the clones of non-Faecalibacterium spp. detected by FPR-2F/Fprau645R mainly were Subdoligranulum spp., but the non-Faecalibacterium spp. clones detected by FPR-1/FPR-2 and Fprau223F/Fprau420R were mainly Subdoligranulum spp., Oscillibacter spp., Ruminococcus spp. and unclassified Ruminococcaceae etc. The real PCR showed the same results with SPCR. The real-time quantitative PCR showed FPR-1/FPR-2 and Fprau223F/Fprau420R detected more bacteria than FPR-2F/Fprau645R. [Conclusion] The three primer pairs can detect F. prausnitzii and Subdoligranulum spp., however, the specificity of FPR-2F/Fprau645R is better than FPR-1/FPR-2 and Fprau223F/Fprau420R.

Abstract:Abstract: [Objective] To determine the species and genotypes of Balantidium isolated from pigs in Henan province, China. [Methods] Scatoscopy and the modified DMEM media were used to isolate trophozoites of Balantidium from pig feces. The ITS1-5.8S rRNA-ITS2-based molecular marker method, Acridine orange staining (AO) and microscopic observation were used to determine the population characteristics among different isolates of B. coli from various pigs farms. [Results] We isolated 15 isolates from the pigs at diagnosis in the Animal Hospital of Henan University of Science and Technology from the pig farms of the 8 counties or cities of the west of Henan province in total, and all of them belong to the same species B. coli. MJ-2 and SX-1 isolates were genotype A of B. coli, and the remaining 13 isolates were genotype B. Trophozoites of MJ-2 and SX-1 were bigger, moved more slowly and lower density in feces in vitro than other 13 isolates, while structures of their nuclei were not different. [Conclusion] Both genotype A and B of B. coli are present in the pig farms of the west of Henan province, China, and genotype B is the determinant population in pigs farms. These findings could provide an important implication for the effective control of balantidiosis of human and other hosts.

Abstract:Abstract:[Objective] We isolated and identified dominant microorganisms from the rhizosphere of continuous cropping with peanut, to study the relationship between dominant microorganisms and peanut continuous cropping. [Methods] By using dilution-plate method we isolated dominant bacteria, fungi and actinomycetes from the rhizosphere of continuous cropping with peanut. Morphological specificity, culture shape, physiological-biochemical characteristic and partial 16S rDNA sequences were used to identify bacteria and actinomycetes. Morphology, growth on various media, and ITS rDNA sequences homology analysis were performed to identify dominant fungi. [Results] We isolated seven dominant bacteria strains, seven dominant fungi and seven dominant actinomycetes. Dominant bacteria were identified as Leifsonia xyli, Arthrobacter chlorophenolicus, Microbacterium flavescens, Sphingomonas sp., Pasteurella sp. , Bacillus simplex and Bacillus megaterium. Dominant fungi were identified as Cladosporium cladosporioides, Penicillium purpurogenum, Hypocrea lixii, Exophiala pisciphila, Penicillium janthinellum,Aspergillus sp. and Verticillium dahliae. Dominant actinomycetes were identified as Streptomyces violaceoruber, Streptomyces flaveus, Streptomyces panaciterrae, Streptomyces achromogenes, Streptomyces pseudogriseolus, Streptomyces cellulosae and Streptomyces aureus. [Conclusion] This study was the first time to isolate and identify dominant microorganisms from the rhizosphere of continuous cropping with peanut. The type of dominant microorganisms changed obviously after planting peanut, although the change was without regularity.