Abstract:Abstract: Acidithiobacillus caldus (A. caldus) is one of the predominant biomining bacteria, which shows application prospect in biological metallurgy. It can enhance the biomining efficiency together with iron oxidation bacteria in mixed biomining system. Based on the published papers and our study on this bacterium, we described the research progress on it from four aspects, including the biomining mechanism, arsenic-resistant mechanism, genome study and genetic reconstruction. Furthermore, we discussed the prospects of research on A. caldus.

Abstract:bstract: Genetic manipulation systems play an important role in the functional analysis of genes and gene products. The development of genetic system in hyperthermophilic archaea is lagging behind those in methanogenic and halophilic archaea. The main problem is the unavailability of efficient selective markers. However, in the past 10 years, researchers have made great progress in the development of genetic systems in the genus Sulfolobus and Thermococcus kodakaraensis, representatives of hyperthermophilic archaea. Here we summarize the latest progress in genetic systems and their application for hyperthermophilic archaea.

Abstract:Abstract: [Objective] To investigate the diversity of culturable bacteria isolated from the sea urchin Hemicentrotus pulcherrimus collected from a tidal flat of Naozhou Island (20°52′N–20°56′N 110°33′E–110°38′E), Leizhou Bay, South China Sea, China. [Methods] Bacterium strains were isolated from the sample by using conventional culture-dependent method and were then investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. [Results] We isolated 106 bacterium strains from the sample on media (Difco marine 2216, International Streotomyces Project medium 2, nutrient, sea water and humic acid agars) supplemented with 0~2 mol/L NaCl. On the basis of some morphological, physiological and biochemical characteristics, we selected 34 strains to perform a phylogenetic analysis based on 16S rRNA gene sequences. Our results showed that 34 isolates represented 21 species, belonging to 17 genera (Alteromonas, Bacillus, Brachybacterium, Brevibacterium, Halobacillus, Halomonas, Nocardiopsis, Oceanobacillus, Piscibacillus, Planococcus, Pontibacillus, Pseudoalteromonas, Pseudonocardia, Salinicoccus, Salinivibrio, Staphylococcus, Vibrio, Virgibacillus) of 10 families (Alteromonadaceae, Bacillaceae, Brevibacteriaceae, Dermabacteraceae, Halomonadaceae, Planococcaceae, Pseudoalteromonadaceae, Pseudonocardiaceae, Nocardiopsaceae, Staphylococcaceae, Vibrionaceae) in three phylogenetic groups (Actinobacteria, Firmicutes, Gamma-Proteobacteria). The most abundant and diverse isolates were within the phylum Firmicutes (58.8%) and the subphylum Gamma-Proteobacteria (26.5%). 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 99.6 to 99.9%), and that, out of 34 isolates, at least 5 strains (JSM076033, JSM076056, JSM076093, JSM078063, JSM078169) could represent 5 potential new species within 5 characterized genera (Jeotgalicoccus, Pontibacillu, Halomonas, Bacillus, Sporosarcina), in which the descriptions of 3 novel species [Jeotgalicoccus marinus (type strain, JSM 076033), Pontibacillus halophilus (JSM 076056), Halomonas zhanjiangensis (JSM 078169)] have been published in IJSEM (International Journal of Systematic and Evolutionary Microbiology). [Conclusion] The results presented above showed that there are abundant bacteria diversity in the sea urchin Hemicentrotus pulcherrimus collected from Naozhou Island.

Abstract:Abstract: [Objective] Vetiver zizanioides is a perennial grass of the Poaceae family, known as silage, soil and water conservation role. The aim is to collect and identify the resources of the nitrogen-fixing bacteria associated with Vetiver zizanioides. [Methods] Associated nitrogen-fixing bacteria isolated from Vetiver zizanioides, were studied by SDS-PAGE whole-cell protein patterns, insert sequence (IS)-PCR finger printing, utilization of sole carbon sources and 16S rRNA gene sequence analysis. [Results] Based on the results of finger printing analysis, protein patterns and biological test, isolates were grouped into 6 clusters, except 4 single strains. Phylogenetic analysis of 16S rDNA sequences indicated that isolates belonged to Herbaspirillum frisingense, Enterobacter ludwigii, Pseudacidovorax intermedius, Mitsuaria chitosanitabida, Pseudomonas putida, Pseudomonas fluorescens, Burkholderia vietnamiensis and Enterobacter cloacae. [Conclusion] The nitrogen fixers associated with Vetiver zizanioides showed great diversity and may have a potential application for the grass forage and agriculture.

Abstract:Abstract: [Objective] To obtain cholesterol-reducing lactic acid bacteria (LAB), we isolated and identified strains of LAB from indigenously fermented pickles and dried-sausage. [Methods] We screened original LAB strains based on the Calcium Carbonate-MRS medium (Calcium Carbonate-Man Rogosa and Sharp Medium) from pickles and sausage. The cholesterol-reducing strains were confirmed by screening with in vitro cholesterol levels. These strains were identified by methods of morphologic observation, catalase reaction, carbohydrate reaction and 16SrRNA sequencing. [Results] We obtained two strains of LAB (LpT1 and LpT2), which presented a comparatively high ability of cholesterol reducing. The two strains also showed high acid resistance and bile salt tolerance. The time of LpT1 entering the logarithm phase of growth and stationary phase of growth was 14 hours and 22 hours after cultivated, while that of LpT2 was 12 hours and 20 hours after cultivated. These two strains survived for at least 4 hours in the MRS broth with pH 2.0 and grew well in the MRS broth containing 0.2% bile salt, and we identified these two strains to be Lactobacillus plantarum by 16SrRNA sequencing. [Conclusion]The two LAB strains could reduce cholesterol in vitro and resist acid and bile salt.

Abstract:Abstract: [Objective] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widespread system that provides acquired resistance against phages in bacteria and archaea. Here we aim to genome-widely analyze the CRISPR in extreme halophilic archaea, of which the whole genome sequences are available at present time. [Methods] We used bioinformatics methods including alignment, conservation analysis, GC content and RNA structure prediction to analyze the CRISPR structures of 7 haloarchaeal genomes. [Results] We identified the CRISPR structures in 5 halophilic archaea and revealed a conserved palindromic motif in the flanking regions of these CRISPR structures. In addition, we found that the repeat sequences of large CRISPR structures in halophilic archaea were greatly conserved, and two types of predicted RNA secondary structures derived from the repeat sequences were likely determined by the fourth base of the repeat sequence. [Conclusion] Our results support the proposal that the leader sequence may function as recognition site by having palindromic structures in flanking regions, and the stem-loop secondary structure formed by repeat sequences may function in mediating the interaction between foreign genetic elements and CAS-encoded proteins.

Abstract:Abstract:[Objective] The mutated promoter of the erythromycin resistance gene(PermE*) is a strong promoter generally used in streptomycetes, and we evaluated the expression activities of a new promoter(Psf) and PermE* in Streptomycetes. [Methods] We used kanamycin resistance gene(neo) and catechol 2,3-dioxygenase gene(xylE) as reporters. [Results] Both promoters exhibited high level of promoter activities in Streptomyces clavuligerus NRRL3585, Streptomyces coelicolor M145, Streptomyces venezuelae ISP5230 and Streptomyces lividans TK54. The activities of Psf were higher than those of PermE* in S. clavuligerus and S. coelicolor. [Conclusion] Both Psf and PermE* are strong promoters suitable for gene over-expression in Streptomycetes and Psf will offer an alternative for high-level gene expression.

Abstract:Abstract: [Objective] Cryptococcus neoformans is an opportunistic human fungal pathogen that primarily infects immunocompromised patients. The most distinctive feature of C. neoformans is that it contains a highly regulated major virulence factor, the polysaccharide capsule. We demonstrate in this paper that a copper-dependent transcription factor gene CUF1 has a negative role in capsule biosynthesis. [Methods] Colony morphology, Indian inking staining microscopy, volume of cell suspension observation, weight of capsule produced by the disruption mutant of CUF1 and the wild type control. [Results] The mutant produced a mucoid large colonies compared to that of the wild type. Capsule size was larger as shown by microscopy. The mutant had a larger volume with the same number of cells. And the mutant produced more capsule as determined by weight of dried extracellular polysaccharides. Iron repletion could suppress the capsule overproduction phenotype of the Δcuf1 mutant. [Conclusion] The copper responsive transcription factor 1 (Cuf1) negatively regulates capsule biosynthesis in C. neoformans. Cuf1 may act via modulating iron acquisition through the high-affinity iron uptake pathway.

Abstract:Abstract: [Objective] The repC gene is the principal initiation protein gene for plasmid replication. We identified the repC-like sequences from Mesorhizobium huakuii strain HN3015 and its derivatives of plasmid curing. [Methods] Primers of RC1 and RC3 were used to amplify the repC-like sequences by a polymerase chain reaction, the PCR products were obtained and cloned into plasmid vector pMD-18T and then sequenced. Location of the repC sequences on different plasmids in the strains tested was carried out by Southern blotting. The nucleotides homology analysis of the repC gene was carried out using the BLAST. Amino acid sequences were deduced using the ExPASy. Multiple sequences alignments were performed using the ClustalW. Analysis of protein secondary structure was carried out using the PredictProtein. [Results] The repC-like sequences were obtained from the strains tested. The sizes of the PCR products were about 750 bp. The results of Southern blotting showed that the repC-like sequences were only associated with a plasmid in the stains tested. [Conclusions] The repC sequences of the strains tested showed 100% sequence similarity, but were obviously different from that of other rhizobia strains.

Abstract:Abstract: Recently a novel membrane-associated metalloprotease EGY1 was found to localize in the chloroplast of Arabidospis and is required for chloroplast development and fatty acid biosynthesis. The genes slr0643 and sll0862 from cyanobacteria Synechocystis sp. PCC6803 encode EGY1 homologs. [Objective] For functional characterization of these two genes, [Methods] disrupted mutants slr0643::km and sll0862::km were constructed by homologous recombination. [Results] Under 30 degree, normal light and autotrophic growth, compared with wild type, slr0643::km showed similar growth rate in the early stage, but emitted less photosystem I (PSI) fluorescence under 77K and the activity of the entire photosynthetic electron transfer chain was only 83% of the wild type, while cells contained normal thylakoid membranes. No significant difference was found between sll0862::km and wild type under this condition. [Conclusion] These results indicated that slr0643 was involved in PSI assembly and photosynthesis directly while sll0862 did not affect assembly of PSI and PSII directly at this stage, which lay a foundation for further exploring their function and mechnism in cyanobacteria.

Abstract:Abstract: [Objective] To study the effect of Pa-pex11 gene on penicillin production in Pencillium anrantiogriseum. [Method] Based on the conserved domain of pex11 from Penicillium chrysogenum, we designed primers pex01 and pex02 to amplify Pa-pex11 gene from P. anrantiogriseum. The complete Pa-pex11 gene was cloned via thermal asymmetric interlaced PCR (Tail-PCR). In order to obtain tranformants with additional Pa-pex11 gene copy, ATMT (Agrobacterium tumefaciens mediated transformation) was used to transform P. anrantiogriseum. The copy number of Pa-pex11 was measured by quantitative PCR. Penicillin production of the transformant was analyzed by bioassays and the microbodies were observed by transmission electron microscope (TEM). [Results] ATMT was successfully applied in P.anrantiogriseum. Increasing the copy number of Pa-pex11 gene resulted in a 1.7-fold increase of pencillin production, and the electron microscope graph showed that the number of microbodies increased obviously. [Conclusion] Our results suggest that increasing the copy number of Pa-pex11 can increase the number of microbodies, and stimulate the penicillin production.

Abstract:Abstract: [Objective] This study aimed at increasing the glycolytic flux and pyruvate productivity in Torulopsis glabrata with glucose as carbon source. [Methods] For this target, we introduced a mitochondrial located alternative oxidase encoded by Histoplasma capsulatum AOX1 gene into T. glabrata, and a mutant strain named as AOX with total NADH oxidase activity was 1.8-fold higher than that of the parent stain, was achieved. [Results] The heterologous expression of NADH alternative oxidase resulted in decrease of the dry cell weight and fermentation time by 20.3% and 10.7%, but the specific rate of glucose consumption and pyruvate production increased 34.7% and 54.1% higher, respectively. The reasons for high glycolytic flux were the intracellular NADH/NAD+ ratio and ATP concentration decreased 74.7% and 52.9% respectively, the specific activity of phosphofructokinase, pyruvate kinase increased 185.0% and 28.1%. [Conclusion] Introduction of a novel NADH oxidation pathway by alternative oxidase can efficiently increase the rate of glucose consumption and the target metabolite productivity.

Abstract:Abstract: [Objective] This study explored the mechanism of the microbial inactivation by high hydrostatic pressure . [Methods] We investigated the damages inflicted on the Vibrio Parahaemolyticus cells treated by high hydrostatic Pressure. The effect of ultra high pressure treatment on inactivation of Vibrio Parahaemolyticus cells was studied, the damages inflicted on the microstructure and the membrane proteins of the Vibrio Parahaemolyticus cells were also analyzed.[Results]The result indicated that after pressure treatment at 100 MPa,200 MPa and 20℃ for 10 min, the viable cell was reduced to 60 % and 15.3 % respectively, viable cell counts were not detected following pressure treatment at 300 MPa and 20℃ for 10 min.High pressure treatment at 300 MPa also caused changes in the microstructure of the cell, such as the cell wall of Vibrio Parahaemolyticus was shrinked, breached and leaked out cytoplasm ,the layer structure of cytoplasm disappeared ,and a large electron transmission area appeared in the cell after compression, the membrane disappeared ,the nucleoids condensed and the proteins aggregated after compression. At the same time, the membrane proteins of cells were damaged, and the cell membrane Permeability increased, resulting in leakage of the cell. [Conclusion]This study showed that the damaged cell membrane was the major reason of the mechanism of microbial inactivation by high hydrostatic pressure.

Abstract:Abstract: [Objective] We had a study on the antifungal effect of the metabolite BMME-1 from Bacillus marinus B-9987, in order to reveal the antifungal mechanism of it. [Methods] The permeability of the Alternaria solani was test by spectrophotometer after the treatment with crude extracts of B-9987. The composition of cell wall and the sterol components of the fungal plasmalemma of Alternaria solani was analyzed with Infrared Spectrum and GC-MS, respectively. [Results] We found that the metabolites of B-9987 had strong antifungal activity with MIC50 and MFC value being 6.2mg/L and 50mg/L. The absorbance in extracellular fluid detection showed that the tegument of the fungi was impaired. The detection of glucan and chitin indicated the change in the structure of the cell wall. The absorption peak of the carbon-hydrogen bond, β-glucosidic bond, carbon-oxygen bond was attenuated but the hydroxyl, carbonyl absorption was enhanced on the contrary. There were only one peak change in chitin chromatogram on the absorption of amide linkage comparing to the control. These changes on the structure may affect the stability of the fungal cell wall. Ergosterol was the predominant component of sterol with the proportion of 62.52±3.31% in control cells, but showed a decline during treatment with BMME-1 at a concentration of 56.36±2.52%. Accumulation of coprostanol, the precursor of ergosterol ,was found in the test. [Conclusion] From the result we can conclude that the antifungal mechanism of the crude extracts was interfering of ergosterol synthesis resulting in the change on permeability, and also mainly changed the structure of the cell wall, mainly acting on the glucan synthesis.

Abstract:Abstract: [Objective] To express and purify Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analyze the protease activity of Nsp2. [Methods] N-terminus and C-terminus of nsp2 gene were amplified by PCR and inserted into expression vector pET21a(+), respectively. The recombinant protein (Nsp2-N and Nsp2-C) were over expressed in E.coli BL21 and purified by Ni-NTA agarose affinity chromatogram and gel filtration. There is a cysteine protease domain (CP) in Nsp2-N through genetic alignment. In this study, the protease activity of Nsp2-N in cis was analyzed by western blot. Additionally, the predicted peptide substrate were synthesized, the protease activity in trans was analyzed by peptide cleavage assay in vitro. [Results] Two soluble recombinant protein were successfully expressed and purity reached up to 90% after purification. The putative protease domain of Nsp2-N couldn’t cleave the predicted substrate through cis cleavage and trans cleavage. [Conclusion] The putative protease domain in Nsp2-N may need other host factors to act as cofactor, which supply basis for further identification of biological activity and screening of anti-virus drug.

Abstract:Abstract：[Objective] Jujube witches’-broom is an important disease in jujube cultivation areas, which causes serious losses in jujube fruit production. To understand the genetic variability and diversity of jujube witches’-broom phytoplasma population from the different cultivars and various regions of China. [Method] We collected 32 samples from 14 cultivars or wild sour jujubes in 7 regions of China and detected them with PCR with the primers R16mF2／R16mR1 for phytoplasma 16S rDNA, SR1/SR for16S-23SrRNA space region (SR) and FD9f/r for secretion proteins (secY). The direct sequencing of PCR products and sequencing by cloned PCR products were used for sequence polymorphism and phylogenetic analyses by comparison to the databases of known conserved gene sequences. [Results] We detected phytoplasmas by PCR amplification of 16SrDNA from all the diseased jujube samples. All the phytoplasma isolates infected various jujube cultivars belonged to subgroup 16SrV-B of elm yellows group and had closer homology with Bischofia polycarpa witches’-broom and cherry lethal yellows phytoplasmas occurred in China than other 16SrV phytoplasmas in other countries. The sequence polymorphism at different extent in 16SrDNA, SR and secY gene and genetic diversity were revealed in phytoplasma strain population related to different habitats, among which the dominant strains were always detected by the direct sequencing of PCR products in all the diseased areas of China. The degree of variability on secY gene of collected phytoplasma strains was greater than that of 16SrDNA and SR sequences, and some base substitutions could not alter encoded amino acid, however certain single base deletions detected in a Shandong and a Beijing strains may have impact on the gene structure or function. [Conclusion] Phytoplasma strains from different cultivars and regions show dramatic genetic diversity. Compared with direct sequencing of PCR products, the sequencing by cloning PCR products was more useful for the displaying of variants and phylogeny in phytoplasma strain population.

Abstract:Abstract: [Objective] This study aimed to isolate, identify and clone degrading gene of a synthetic pyrethroids degrading bacterium. [Methods] A photosynthetic bacterial strain PSB07-21 capable of degrading several synthetic pyrethroids efficiently was isolated by an enrichment culture. PSB07-21 was identified based on its morphology, physiology and phylogenetic analysis of 16S rDNA sequence. The degradation ability of this strain was evaluated with gas chromatography.The degrading gene was cloned with PCR. [Results] PSB07-21 was closely related to Rhodopseudomonas sp. The optimum condition of degrading synthetic pyrethroidss was at 35℃, pH 7 and 3000lx. PSB07-21 could degrade fenpropathrin, cypermethrin and bipthenthrin by 66.63%, 43.25% and 50.18% in a concentration of 600 mg/L at day 15, respectively. We cloned a putative gene which was 326bp long with 37.0% identical to 2OG-Fe(II) oxygenase gene. When compensating low concentration Fe(II) in PSB medium with synthetic pyrethroids, the degradation efficiency of PSB07-21 was enhanced.[Conclusion] The strain has the potential application to synthetic pyrethroids bioremediation.

Abstract:Abstract: [Objective] To produce human papillomavirus type 11 virus-like particles (HPV11 VLPs) from Escherichia coli and to investigate its immunogenicity and type cross neutralization nature. [Methods] We expressed the major capsid protein of HPV11 (HPV11-L1) in Escherichia coli ER2566 in non fusion fashion and purified by amino sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography, sequentially. Then we removed the reductant DTT to have the purified HPV11-L1 self-assemble into VLPs in vitro. We investigated the morphology of these VLPs with dynamic light scattering and transmission electron microscopy. We assayed the immunogenicity of the resultant HPV11 VLPs by vaccinations on mice and evaluated by HPV6/11/16/18 pseudovirion neutralization cell models. [Results] We expressed HPV11 L1 in Escherichia coli with two forms, soluble and inclusion body. The soluble HPV11 L1 with over 95% purity can self assemble to VLPs in highly efficiency. Morphologically, these VLPs were globular, homogeneous and with a diameter of ~50 nm, which is quite similar with native HPV11 virions. The half effective dosage (ED50) of HPV11 VLPs is 0.031 μg, and the maximum titer of neutralizing antibody elicited is averaged to 106. The cross neutralization activity (against HPV6/16/18) of the anti-HPV11 serum was found to have exact correlation to the inter-type homology in amino acid alignment. [Conclusion] We can provide HPV11 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV11.

Abstract:Abstract：[Objective] QALPETGEE sortase’s substrate was anchored on the cell surface of yeast Pichia pastoris using EGFP for the detection of its expression. The sortase activity assay is based on interaction of sortase and substrate displaying on yeast. [Methods] The gene-encoding QALPETGEE- linker-EGFP was amplified by PCR using pcDNA/myc-his-EGFP as a template, and then inserted into shuttle vector pKFS. Next, the vectors were introduced into Pichia pastoris GS115. After cultivation, recombinant cells was verified with fluorescence microscopy and sortase activity was detected by fluorescence spectrophotometer from variety of free EGFP’s flurescence intensity. [Results] The green cells were observed by fluorescence microscopy, enhancing over time. Fluorescence spectrophotometer convinced that fluorescence intensity of free EGFP in the reaction supernatant has increased from 187.67±2.16 to 273.47±2.14 after interaction of sortase and its substrates. [Conclusion] The result suggests that sortase’s substrates have been display on yeast successfully, which could use for sortase activity assay high-effectively and economically.

Abstract:Abstract: [Objective] In order to study the Biodiversity of Lactobacilli which were from Northern Tibet in China, ERIC-PCR (enterobacterial repetitive intergenic consensus sequence polymerase chain reaction) technique was developed. [Methods] Seventy seven Lactobacillus strains were isolated from traditional fermented milk products which are made by herd man families in Northern Tibet of China. By ERIC-PCR technique method, using NTSYS-pc2.1 software, we investigated the polymorphism and types of the strains. [Results]The results suggested that the amplified bands of ERIC-PCR were clear and stable with high repetition and high polymorphisms. The cluster analysis showed that at level 0.73, 77 tested strains were clustered into 4 groups: A1: strains of Lactobacillus casei (35.06% of the strains), A2: strains of Lactobacillus fermentum (61.04% of the strains), A3: strains of Lactobacillus helveticus and A4: strains of Lactobacillus plantarum. The biodiversity of isolates from A1and A2 groups was further investigated. A1 could be divided into five genotypes at the similarity value of 80.0%. A2 could be divided into six genotypes at the similarity value of 84.0%. [Conclusion]Using ERIC-PCR technique, the Lactobacilli from Northen Tibet could be distinguished at specices level. Results from this study showed that there was no positive relationship between the ERIC-PCR gene types and the origin of the strains.