Abstract:Abstract: Heavy-metal pollution in soil is a major threat to human health and the entire ecosystem. Legumes and their associated rhizobial microorganisms are important for the biogeochemical cycles in agriculture and natural ecosystems. Legume-rhizobia symbiosis is very important in restoration of heavy-metal contaminated soils because the fixation of atmospheric nitrogen by legume-rhizobium symbiosis can increase the accumulation of nitrogen and organic matter. We reviewed the importance of remediation of heavy-metal contamination in soil and the current situation of remediation techniques, analysed the advantages and disadvantages of each remedial techniques. We address especially the superiority, recent advances and potential application of the legume-rhizobium symbiosis in remediation of heavy-metal contaminated soils.

Abstract:Abstract: Hypocrea jecorina（anamorph：Trichoderma reesei）is the main industrial fungi that can produce large amounts of extracellular cellulases and hemicellulases. It also represents a model system to study the mechanism of transcriptional regulation in eukaryotes. The expression of these hydrolases genes in Hypocrea jecorina can be triggered rapidly in the presence of inducers, but differences in the inducing mode of various soluble inducers have been reported. At present, three models have been offered to explain the question of “how an insoluble inducer such as cellulose would initiate the transcription of cellulases?” Moreover, interactions between the identified positive (Xyr1, Ace2, Hap2/3/5) and negative transcriptional regulators (Ace1, Cre1), as well as the interactions between these proteins and the promoters of cellelase and hemicellulase genes have also been primarily characterized. This review focuses on the key factors and the current understanding on the regulation of expression of cellulase and hemicellulase genes in Hypocrea jecorina.

Abstract:Abstract: Diterpenoid is a huge group of nature products isolated from plants and fungi. Diterpene cyclase, which is responsible for the diterpene carbon skeleton formation from geranylgeranyl diphosphate (GGPP), is a key enzyme in the biosynthetic pathway of diterpene. The specificity of diterpene cyclase in different species results in structural diversity and bioactivity variety of diterpenoid. Isolation and characterization of the diterpene cyclase in various species will facilitate studies on the biosynthesis and regulation of diterpenoid in future. Compared to plant diterpenoids, few fungal diterpenoid and diterpene cyclase were studied. This article reviews the research advancement of fungal diterpene cyclase in recent years, especially describes the biosynthesis pathway of diterpenoid, the characteristics and cloning strategies of fungal diterpene cyclase, and the metabolic engineering of diterpenoid.

Abstract:Abstract: The powerful weapon of Shigella spp. is Type Three Secretion System, which remains the focus of pathogenic research. The regulation and function of Type Three Secretion System of Shigella spp. are summarized in this review.

Abstract:Abstract: [Objective] To investigate the diversity of cultivable halophilic and halotolerant bacteria isolated from ordinary non-saline soil samples collected from Xiaoxi National Natural Reserve (28°42′15′′–28°53′15′′ N, 110°6′50′′–110°21′35′′ E), Hunan Province, China. [Methods] Bacterial strains were isolated from the samples by using the conventional culture-dependent method and investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. [Results] We isolated 114 bacterial strains (8 moderately halophilic, 19 slightly halophilic, 87 halotolerant) from the samples on media (marine agar 2216, International Streotomyces Project medium 2 and 5, nutrient and humic acid agars) supplemented with 5% to 20% (w/v) NaCl. On the basis of morphological, physiological and biochemical characteristics, we selected 61 strains to perform a phylogenetic analysis based on 16S rRNA gene sequences. Results showed that 61 isolates represented 41 species, belonging to 18 genera (Actinomadura, Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, Erwinia, Halobacillus, Jeotgalibacillus, Microbacterium, Microbulbifer, Nocardia, Pseudomonas, Rheinheimera, Rhodococcus, Sphingomonas, Staphylococcus, Streptomyces, Yaniella) of 16 families (Alteromonadaceae, Bacillaceae, Brevibacteriaceae, Chromatiaceae, Dermabacteraceae, Enterobacteriaceae, Microbacteriaceae, Micrococcaceae, Nocardiaceae, Planococcaceae, Pseudomonadaceae, Sphingomonadaceae, Staphylococcaceae, Streptomycetaceae, Thermomonosporaceae, Yaniellaceae) in three phyla (Actinobacteria, Firmicutes, Proteobacteria). The most abundant and diverse isolates were within the phylum Firmicutes (38 strains; 62.3%) and the phylum Actinobacteria (18 strains; 29.5%). The phylogenetic distance matrix results suggested that there were obvious genetic divergences between most isolates and their closestly related type strains (16S rRNA gene sequence similarities ranged from 96.9% to 99.8%), and that, out of 61 isolates, at least 7 strains (JSM 070026, JSM 081004, JSM 081006, JSM 081008, JSM 083058, JSM 083085, JSM 084035) should represent 7 potential novel species within 6 characterized genera (Yaniella, Bacillus, Jeotgalibacillus, Sphingomonas, Rheinheimera, Microbulbifer). [Conclusion] The results presented here showed that there is abundant diversity of halophilic and halotolerant bacteria, as well as a number of novel species in non-saline soil collected from Xiaoxi National Natural Reserve, Hunan Province, China.

Abstract:Abstract: [Objective] To characterize the molecular and phenotypic traits of the VGII genotype of Cryptococcus gattii isolate XH91 firstly isolated in China. [Methods] The serotype was identified by molecular method; multi-locus sequence typing (MLST) was performed based on 16 gene fragments both of nuclear and mitochondria genomes; the abilities of haploid fruiting, same-sex mating and opposite sex mating were all evaluated; the phenotypic traits including melanin production, capsule size, and growth at 37℃ were characterized. [Results] The isolate XH91 firstly isolated in China was serotype B. The isolate shared the same MLST genotype with the minor outbreak genotype VGIIb from Vancouver islands. Strain XH91 could mate with the reference strain of opposite mating type and produced basidiospores, but could not mate with the reference strain of same mating type and had no ability of haploid fruiting. We did not observe obvious difference between XH91 and reference strains for melanin production, capsule size, and growth at 37℃. [Conclusion] Based on the results from MLST and phenotypic analysis, the Cryptococcus gattii strain XH91 is identical with the minor outbreak genotype VGIIb from Vancouver islands. This study will be critical to gain further insight into the emergence and molecular epidemiology of the VGII genotype of Cryptococcus gattii from China.

Abstract:Abstract: [Objective] We investigated the genetic diversity and phylogeny of soybean rhizobia isolated from the regions of Loess Plateau in China. [Methods] We analyzed 130 soybean rhizobia isolated from 15 regions in 4 provinces of Loess Plateau through BOX-PCR, 16S rDNA PCR- RFLP, 16S-23S IGS PCR-RFLP and 16S rRNA gene sequencing. [Results] BOX-PCR, 16S rDNA PCR- RFLP and 16S-23S IGS PCR-RFLP were in good agreement with the results which showed that all strains tested ascribed to two groups: the genus of Sinorhizobium and Bradyrhizobium phylogenetically. The analysis of 16S rRNA gene of 5 representative strains indicated that they were related to type strains S. fredii, B. japonicum and B. liaoningense, homology coefficient with type strains was 100% respectively. [Conclusion] Soybean rhizobia isolated from the regions of Loess Plateau in China showed rich genetic diversity. S. fredii was the dominant species. Bradyrhizobium accounted for 10% of the strains tested only, of which, two strains were B. liaoningense.

Abstract:Abstract: [Objective] Transketolase (EC 2.2.1.1；TKT) is the key enzyme in non-oxidative phosphate pentose pathway. We cloned tkt gene from Corynebacterium pekinense AS 1.299 and its mutant PD-67 in order to investigate the effect of gene expression on physiological characteristics of C. pekinense. PD-67. [Methods] According to the homology between Corynebacterium glutamicum ATCC13032 and C. pekinense, we designed a pair of PCR primers to clone the tkt gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant tkt gene was expressed in C. pekinense PD-67 by subcloning the PCR fragment into plasmid pAK6. The physiological characteristics of the recombinant C. pekinense PD-67 was investigated by fermentation. [Results] Analysis of PCR fragments reveals that, besides the regulatory sequence, they contain the whole structure of tkt gene. There is no basic change all over the structure genes and regulatory sequences between C. pekinense AS1.299 and PD-67. Comparing with Corynebacterium glutamicum ATCC 13032, there exist 5 amino acids change in amino acid sequence. Four of them were located in the motifs involved in thiamine pyrophosphate binding sites. The tkt gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of TKT in C. pekinense PD-67(pTK3) is twice as much as that of the control strain C. pekinense PD-67(pAK6). The recombinant C. pekinense PD-67 exhibits higher cell mass and accumulation of more tryptophan. [Conclusion] The moderate amplification of TKT activity resulted in increase of L-tryptophan production without affecting the cell growth.

Abstract:Abstract: [Objective] The aim of this study was to optimize the property of nisin through altering its specific amino acid by site-directed mutagenesis method. [Methods] On the basis of M21K nisinZ, a former reported nisinZ mutant that exhibited antimicrobial activity against Gram-negative bacteria, the 29th amino acid of it was mutated from serine to lysine. The mutant M21K/S29K nisZ gene was cloned into vector pMG36e and the recombinant plasmid was introduced into Lacotococcus lactis NZ9800. The resulting M21K/S29K nisinZ was then isolated and purified, and its antibacterial activity, antibacterial spectrum and stability were analyzed and compared to those of M21K nisinZ and nisinZ. [Results] Compared with wild-type nisinZ and M21K nisinZ, the M21K/S29K nisinZ displayed reduced antimicrobial activity, but showed significantly increased stabilities to heat and pH stress. Moreover, M21K/S29K nisinZ also exhibited antimicrobial activity against Gram-negative bacteria as M21K nisinZ did. [Conclusion] By changing the 29th amino acid of nisin, we can optimize the property of nisin, especially its stability to heat and pH stress.

Abstract:Abstract: [Objective] We screened and isolated polyhydroxyalkanoate producing bacteria. [Methods] The strains were isolated from sludge from a beer brewery and screened by Sudan black B staining method. The isolated strains were identified according to their morphological features, physiological and biochemical analysis as well as 16S rRNA gene sequence analysis. The product extracted with hot chloroform from the isolated strain HG-B-1 was confirmed by Fourier transform infrared spectra. [Results] We isolated a bacterium, HG-B-1, from sludge collected from a beer brewery in Guangdong province, China. The yield of polyhydroxyalkanoates was 23.4％(w/w) based on dried weight of the bacterium cells when HG-B-1 grew in a medium containing saccharose. We analyzed 16S rRNA nucleotide sequence, and ascertained the phylogenetic position of the strain. [Conclusion] Strain HG-B-1 with PHAs biosynthesis ability was identified as Stenotrophomonas maltophlia.

Abstract:Abstract: [Objective] We investigated differences in morphology, physiology, periplasmic proteins and phagocytosis by macrophage among E. coli strains AD93(PE-PC-), AD93/ptac67(PE-PC+), Top10/ptac66(PE+PC+) and the wild types in order to understand if phosphatidylcholine (PC) can substitute phosphatidylethanolamine (PE) in vivo. [Methods] Bacterial cells were observed under microscope after staining with Gram-staining kit or by electron microscope. Bacterial growth under different conditions was monitored by measuring the absorbance at the wavelength of 600 nm. Periplasmic proteins were analyzed using SDS-PAGE and 2-D electrophoresis. Bacterial adherence and phagocytosis by macrophage were also examined by using murine RAW264.7 macrophage. [Results] 100% bacterial cells in AD93/pDD72 were bar-shaped but 25% AD93/ptac67 cells came out as long filaments. Different from AD93/pDD72, AD93/ptac67 and AD93 required Mg2+or Ca2+ for growth. Moreover, AD93/ptac67 displayed a different pattern of periplasmic proteins on a 2-D gel and a low relatively phagocytic efficiency in the phagocytosis test when compared to AD93/pDD72 and AD93. Both Top10/ptac66 and the wild-type Top10/ptac85 cells were bar-shaped under microscope, but the former showed noticeably difference in the outer-layer structure of cell wall, and its stress resistance and periplasmic protein composition were also different from those of the latter. [Conclusion] Substitution of phosphatidylethanolamine with phosphatidylcholine in E. coli cells is unable to restore the phonotype of PE- mutant to the wild type. Biological functions of PE and PC are different, and phosphatidylcholine cannot substitute phosphatidylethanolamine in vivo.

Abstract:Abstract: [Objective] We Isolated and characterized 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing endophytic bacteria from halophyte Suaeda salsa to understand the interactions between endophytes and halophyte. [Methods] ACC deaminase-containing endophytic bacteria were isolated from root, stalk and leaf of Suaeda salsa and were identified based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. Isolates were evaluated for their ACC deaminase, antifungal, protease activity, siderophores and phytohormones, such as indole-3-acetic acid, gibberellic acid and abscisic acid production, as well as atmospheric nitrogen fixation and phosphate solubilization. [Results] Four ACC deaminase-containing endophytic bacteria strains named as LP11, SS12, TW1 and TW2 were isolated and identified as Pseudomonas oryzihabitans, Pseudomonas sp., Pantoea agglomerans and Pseudomonas putida respectively. All the strains possessed the phosphate-solubilizing ability and could produce siderophores and phytohormones more or less. None of them could fix atmospheric nitrogen or produce protease. Only strain SS12 showed antagonism against two phytopathogenic fungi viz Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. cucumerinum. [Conclusion] ACC deaminase-containing endophytic bacteria of Pseudomonas sp. and Pantoea sp. isolated from halophyte Suaeda salsa have abundant biological characteristics related to plant growth promotion, stress homeostasis regulation and biocontrol activity.

Abstract:bstract: [Objective] We analyzed the prokaryotic microbial diversity of Eryuan Niujie hot spring, in Yunnan Province, to enrich our knowledge about thermo-stable microbes. [Method] We constructed bacterial and archaeal 16S rRNA gene libraries, analyzed the sequences and constructed phylogenetic trees to learn the prokaryotic microbial diversity. [Result] The majority of the prokaryotic microbes in this hot spring were bacteria, while β-Proteobacteria was the most abundant, next were Bacteroidetes and Chlorobi; the abundance and diversity of archaea were both less than that of bacteria, including Euryarchaeota and Crenarchaeota, while Euryarchaeota was the most abundant.

Abstract:Abstract: [Objective] In order to obtain the information about how irrigations with recycled water affecting the cultivable microbial population of the rhizosphere of turfgrasses, [Methods] We isolated strains from two irrigated areas with recycled water (RW) and drinking water (DW) in Taoranting Park in Beijing, China, by using diluting plate and counting method, and analyzing the 16S rDNA sequence of the isolates. [Results] We obtained 20 and 25 strains with different morphological character of colonies from the area irrigated with DW and area irrigated with RW, respectively. The sequence analysis of 16SrDNA showed that RW-irrigated system supported more complex communities of 18 genus and 24 species, whereas the DW-irrigated system only supported 15 genus and 20 species. Both samples had similar microbial population. There were 9.7% or 13.4% alphaproteobacteria, 8.1% or 12.3% betaproteobacteria, 17.9% or 42.0% gammaproteobacteria, 13.0% or 2.9% bacteroidetes, 23.6% or 10.1% firmicutes, and 27.6% or 19.6% actinobacteria at the rhizosphere of turfgrasses irrigated with DW or RW respectively. The dominant bacteria in DW area were the genera Bacillus whereas that of RW area was the genera Acinetobacter. Other than the gammaproteobacteria, the dominant genera in other groups of two areas were similar. [Conclusion] The result showed that the bacterial community composition of rhizosphere samples did not change, but the distribution of different types of bacteria does change after irrigation by reclaimed water, which were determined by the abundance increasing of the dominant species and the appearance or disappearance of non-dominant species. Finally, it is important that the control of the pathogen and metal should be enforced when using RW for irrigation.

Abstract:Abstract: [Objective] To find new microbial resources from a high-temperature oil reservoir.[Methods] Strain HL-3 was isolated by Hungate Anaerobic Technique from oil reservoir water sampled from Dagang oilfield, China. Through physiological, biochemical and phylogenetic analysis, the strain HL-3 was classified. [Results] Cells were Gram-positive. The temperature range for growth was 40℃－75℃ (optimum at 60℃) and the pH range was 5.0－8.0 (optimum at 6.5). The isolate could grow in the presence of 0%-3.2% NaCl (optimum at 0.25% ). Glucose, ribose, mannose, xylose and cellobiose could be metabolized. Metabolites of glucose were ethanol, acetate, CO2 and trace amount of propionate and butanol. The G+C content of DNA was 33.9mol%. Based on 16S rRNA studies，strain HL-3 was most close to T. uzonensis DSM 18761T(EF530067) with 98.8% similarity and to T. sulfurigignens DSM 17917T (AF234164) with the 98.1% similarity. Strain HL-3 tolerated to high sulfite (0.1mol/L) ions and extremely high concentration of thiosulfate (0.8 mol/L). When the concentration of thiosulfate was higher than 0.075 mol/L, the cell would generate S element granular. The presence of H2S gas was detected inside of space at the top of serum bottle. Strain HL-3 together with T. uzonensis DSM 18761T differed greatly in toleration of thiosulfate and sulfite. The toleration of strain HL-3 to thiosulfate and sulfite was most close to T. sulfurigignens DSM 17917T (AF234164). In addition, strain HL-3 to metabolite thiosulfate and sulfite was also similar with T. sulfurigignens DSM 17917T (AF234164). However, it differs largely from both of them to metabolize glucose.[Conclusion] Therefore, strain HL-3 may be a new spieces of the Thermoanaerobacter, and the definitive classification positioning is still awaiting for further verified with the method of determination of whole-genome DNA–DNA similarity[1].

Abstract:Abstract: [Objective] To evaluate polymerase chain reaction (PCR) combined with enzymatic digestion for identification of Legionella, and distribution of Legionella in environmental water systems in Guangzhou. [Methods] Forty-four water samples collected in Guangzhou were cultivated for Legionella were identified by PCR-enzymatic digestion, 16S rDNA and mip gene sequencing analysis. [Results] Sixty-six strains of Legionella pneumophila and 46 Non-L. pneumophila were identified by PCR-enzymatic digestion and sequencing analysis. Forty-six strains of Non-L. pneumophila included 20 strains of L. feeilei, 17 L. gormanii, 7 L. oakridgensis and 2 L. longbeachae. [Conclusion] PCR combined with enzymatic digestion is a simple, rapid, and specific method for the identification of Legionella. L. pneumophila was distributed widely, followed by L. feeilei, L. gormanii, L. oakridgensis and L. longbeachae, in environmental water in Guangzhou area.

Abstract:Abstract: [Objective] Salmonella species are important food-borne pathogens of human and animal. S. enterica serovar Enteritidis is the only serovar that routinely causes human infection through intact egg, the molecular basis of its ability to survive in egg is poorly understood. The importance of post-transcriptional regulation by small non-coding RNAs ( sRNA ) has recently been recognized. The sRNAs play diverse physiological roles in stress responses, regulation of metabolism, control of bacterial envelope composition and bacterial virulence. In this study, we studied regulatory function of salmonella sRNA sraB associated with survival ability of S. enterica serovar Enteritidis in egg albumen. [Methods] To study the contribution of sraB to the survival ability of S. Enteritidis in egg albumen, we constructed sraB deletion strain (SE2472△sraB) with wild type S. Enteritidis SE2472, using red recombination system. For complementation of sraB, complete fragment sraB was amplified from SE2472 and inserted into plasmid pHDB3 to overexpress sraB. We carried out the egg albumen bactericidal experiment with strains of SE2472, SE2472△sraB(sraB deletion), SE2472△sraB-comp(sraB complement) and control. To explore the regulatory role of sraB, we assayed the bactericidal activity of the two important antimicrobial components of egg albumen: lysozyme and transferrin. [Results] In the egg albumen bactericidal experiment, the survival rate of SE2472△sraB was only about 61%－70% of that of SE2472; while SE2472△sraB-comp improve the survival rate of SE2472△sraB by 10%－33% . In the tranferrin bactericidal experiment, the survival rate of SE2472△sraB was 38% of that of SE2472 at 8 h incubation, and 23% at 24 h incubation. SE2472△sraB-comp played an important role in improving the survival rate rescued the defect by 14% than SE2472△sraB at 8 h of incubation, but failed to rescue the defect at 24 h incubation. In the Lysozyme experiment, the survival rate of SE2472△sraB was 41% of that of SE2472 at 8 h incubation, and 27% at 24 h incubation, compared with SE2472△sraB, the expression of sraB of SE2472△sraB-comp has improved the survival rate by 35% after 8 h of incubation and 23% after 24 h of incubation. [Conclusion] Based on our results, we conclude that small RNA (sraB) plays important role during the survival of S. Enteritidis in egg albumen, and may contribute regulatory role in response to antimicrobial components of egg albumen such as lysozyme and transferrin.

Abstract:Abstract: [Objective] Cloning of ipaJ gene from Salmonella pullorum C79-13, and identification of expressed IpaJ protein as an immunogen of the pathogen. [Methods] With suppression subtractive hybridization (SSH) between Salmonella pullorum strain C79-13 (tester) and Salmonella enteritidis strain 50041 (driver), three subtracted fragments PEA3, PE31 and PE44 showed high homology with ipaJ in plasmid pSFD10 of Salmonella choleraesuis C500. The three subtracted sequences were spliced together into the whole sequence of ipaJ in Salmonella pullorum. Then the ipaJ gene was amplified from Salmonella pullorum by polymerase chain reaction (PCR) and cloned into prokaryotic expressive vector pET-30a(+). Western-blot was used to identify it as an immunogen. The distribution of the gene was also detected in Salmonella pullorum isolates. [Results] The ipaJ gene cloned from Salmonella pullorum was 840 bp, and the expressed fusion protein was 37 kDa. Specific reaction was found between Salmonella pullorum positive serum and expressed protein by Western-blot assay, confirming its identification as an immunogen of Salmonella pullorum. The PCR results showed that the gene exists in all Salmonella pullorum strains. [Conclusion] The ipaJ gene from Salmonella pullorum was first reported and cloned, and the expressed IpaJ protein was confirmed as an immunogen of Salmonella pullorum.

Abstract:Abstract: [Objective] The role of Quroum Sensing response regulator nprR on the expression of Cry protein in B. thuringiensis HD-73 was studied. [Methods] The nprR gene deletion mutant HD73 (ΔnprR) was constructed by using of homologous recombination. Beta-galactosidase assay of cry1Ac′-lacZ gene fusion and SDS-PAGE in both HD-73 and HD73 (ΔnprR) strains were performed to analyze the effect of nprR gene deletion on expression of cry1Ac gene. [Results] Beta-galactosidase assay of nprR′-lacZ in both LB and Schaeffer’s sporulation medium showed nprR gene in B. thuringiensis was initially transcripted at T0 (end of Logarithmic growth phase) and keeping expression in stationary phase. Beta-galactosidase assay of cry1Ac′-lacZ and SDS-PAGE indicated that expression of cry1Ac gene in HD73 (ΔnprR) was stronger than that in HD-73 during transition phase and early stationary phase. However, Cry expressed product between HD-73 and HD73 (ΔnprR） in LB medium has no significant difference when crystal and spore were released. [Conclusion] The deletion of nprR increased expression and transcription activity of cry1Ac during transition and early stationary phase in rich media.

Abstract:Abstract: [Objective] Streptomyces avermitilis NRRL8165 could serve as a good host for heterlogous expression of antibiotics biosynthetic gene clusters. However, it needs to improve its conjugation frequency to accept large plasmids. [Methods] We chose MgCl2 , NaCl, Ca(NO3)2 and CaCl2, to test whether they affected the conjugation frequency of large plasmids at salt concentration from 0-200 mmol/L. A complete random experiment was designed for optimization. [Results] We found that CaCl2 promoted conjugation dramatically, and MgCl2 did significantly too. The complete random experiment led to diclosure of an optimal combination of MgCl2 and CaCl2 by which the conjugation frequency was improved by 11 fold. In addition, a supplemented medium was found to lead to successful heterologous expression of actinorhodin in S. avermitilis. [Conclusion] Some inorganic salts can not only significantly improve the conjugation frequency of Streptomyces avermitilis NRRL8165 but also promote the expression of actinorhodin in Streptomyces avermitilis NRRL8165.