Abstract:[Objective] A lactate-utilizing, propionate-producing bacterium, strain L9, was isolated from rumen of goat fed with high concentrate by utilizing modified Hungate technique and anaerobic culture technique. The effect of the strain L9 culture on the rumen fermentation was further studied. [Methods] According to the characteristics of morphology, physiology, biochemistry tests and sequence comparison of 16S rRNA gene, strain L9 was identified as selenomonas ruminantium. The influence of strain L9 culture on in vitro rumen fermentation was studied using mixed rumen micro-organisms of goats as inoculums. [Results] The results of the metabolism experiment showed that it was capable of using lactate as the sole carbon source, and 90mmol/L lactate in LH medium could be completely utilized after 24h incubation. As compared with the control, strain L9 culture addition significantly increased the total volatile fatty acids ( TVFA) , the percentage of propionate and pH value, while reduced the ratio of acetate to propionate and lactate production (P<0.05). [Conclusion] The results suggested that strain L9 can reduce lactic acid production and enhance the TVFA and propionate production in in vitro fermentation, and thus could be beneficial for the fermentation of rumen microorganisms.

Abstract:[Objective] To isolate bacteria from Nansha area of South China sea, [Methods] Sediment samples of 22 sites were used. Bacterial isolation was conducted on plates of marine medium, followed by 16S rRNA identification and phylogenetic analysis. [Results] In total 349 bacteria were obtained, belonging to 87 species. Analyses of 16S rRNA sequence showed that Bacillus and other spore-forming bacteria occupied the majority of isolates in 10 sites. Bacillus was the most abundant bacterium and of high diversity; with 34 species and 8 possible novel species. Halobacillus also occurred frequently while other spore-forming bacteria including Brevibacillus, Paenibacillus, Pontibacillus and Thalassobacillus were also found, but less occurred in this area. In addition to these low-G+C-content bacteria, γ-Proteobacteria were the second subgroup of high occurrence, among which Pseudomonas, Marinobacter and Alcanivorax were relatively abundant. Generally, isolates of 750-2000m deep mainly consist of low-G+C-content bacteria, while mainly composed of gamma-Proteobacteria when the depth is over 2000m. [Conclusion] Marine sediments of South China Sea are rich in spore-forming bacteria, which deserve further study and exploitation.

Abstract:[Objective] Pseudomonas fluorescens 2P24 is a plant disease-suppressive bacterium isolated from wheat rhizosphere. Small RNA gene rsmZ in strain 2P24 plays an important role in the production of antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), a key factor in disease suppression. [Objective] The aim of this study was to determine the influence of upstream regulators on the transcription of rsmZ. [Methods] A reporting vector was constructed by fusing the promoter region of rsmZ gene with a promoterless lacZ gene on plasmid pRK970Km. The resulting plasmid was introduced into several regulatory gene mutants and the corresponding effects on RsmZ transcription were investigated. [Results] The results indicated that a response regulator gene gacA from GacS/GacA two-component system positively regulated the transcription of RsmZ, whereas the oxidoreductase gene dsbA negatively regulated RsmZ expression. Furthermore, mutation on PhoP/PhoQ two-component system resulted in the delay of RsmZ transcription. [Conclusion] Our study suggests that the rsmZ gene in strain 2P24 transcripts under the pleiotropic regulation of a number of upstream genes, and may be involved in a more complex signal transduction cascade than that we have known.

Abstract:Phenazine-1-carboxylic acid biosynthesized and secreted by Pseudomonas chlororaphis G-05 isolated from the rhizosphere of pepper in greenhouse (Huaian, China), contributes to its biological suppression of R. solani growth. [Objective] Our aim is to elucidate its biocontrol function and regulation mechanism. [Methods] We first identified the strain with biochemical method and homology comparison of 16S rDNA. A conservative DNA fragment of gacS gene was then obtained from the genomic DNA of the wild type strain G-05 by polymerase chain reaction (PCR). According to homologous recombination technology, a mutant G-05S was then created with insertional inactivation of gentamycin resistance cassette (aacC1). [Results] In comparison with the wild type strain G-05, the gacS-deficient mutant G-05S produced trace amount of phenazine-1-carboxylic acid in King’ s B（KMB）or Pigment Production Medium( PPM) medium, respectively. However, it produced indole-3-acetic acid (IAA) in the same way as the wild type strain. When the gacS gene was introduced with the shuttle vector pME6032, the mutant G-05S produced the same phenazine-1-carboxylic acid as the wild type strain. [Conclusion] The regulation mediated by gacS gene on secondary metabolites is specific and differential in some biocontrol agents.

Abstract:[Objective] Candida glycerinogenes, an excellent glycerol producer, has been used for commercial scale glycerol production. Recently, we cloned and sequenced the gene encoding NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) from C. glycerinogenes and this gene was named CgGPD, which plays an important role in glycerol production. However, compared with GPD1 and GPD2 from S. cerevisiae, the function of CgGPD was unclear to date. [Methods] In this study, a functional charaterization of CgGPD was undertaken, using S. cerevisiae and its isogenic gpd1/gpd2 mutant as expression host under high osmotic stress. [Results] Expression of CgGPD in wide type S. cerevisiae, using either TPI promoter from S. cerevisiae or upstream regulatory sequence of CgGPD accelerated glucose consumption rate and improved glycerol production signifcantly. In osmosensitive mutant, expresion of CgGPD including regulatory sequence increased cells osmotic tolernace and growth profile of transformants restored similar to wide type strain under the high osmotic stress condition. Furthermore, mutants harbouring CgGPD accumulated the intracellular glycerol content markedly and GPD specific enzyme activity increased abruptly when exposed to high osmolarity medium. [Conclusion] CgGPD from C. glycerinogenes compensate the GPD1 in S. cerevisiae functionally.

Abstract:[Objective] Sulfite, an intermediate metabolite in yeast sulfur-containing amino acid biosynthesis pathway, plays an important role in beer flavor stabilizing because of its antioxidant activity. In Saccharomyces cerevisiae, excretion of sulfite is regulated by sulfite transporter protein Ssu1p which is encoded by SSU1. We constructed a high sulfite excreting brewing yeast strain to solve the beer staling problem by SSU1 gene over-expressing. [Methods] Sulfite transporter gene SSU1-1 and SSU1-2 that contain different length of 5′ untranslated region were cloned by PCR, with the genomic DNA of an industrial mutant strain M8 as template. The amplified DNA was digested with BamHⅠand SalⅠ, and then inserted into plasmid YEp352 digested by BamHⅠ-SalⅠand constructed the expression vector pSU1 and pSU2. pSU1 and pSU2 were transformed into laboratory strain YS58 and tested the effect of SSU1 multi-copy expression on Saccharomyces cerevisiae sulfite production. Furthermore, pSU2 was transformed into industrial brewing yeast mutant strain M8, and SO2, H2S production and beer antioxidant abilities of transformant Y2, screened out by sulfite resistance plate, were tested. [Result] The SO2 production of laboratory yeast transformants pSU1-4 and pSU2-3 enhanced remarkably, but H2S production remained unchanged. Compared with the S. cerevisiae M8, SO2 production of transformant Y2 increased by 74.4%, TBA value decreased by 14.9%, DPPH radical scavenging ratio enhanced by 38.2%, but H2S production had little change. [Conclusion] Over-expression of SSU1 in both laboratory S. cerevisiae strain and industrial brewing yeast strain increased their sulphite excretion, enhanced beer antioxidant abilities and had no negative effect on sulfur-containing amino acids biosynthesis.

Abstract:[Objective] We explored whether a smart electronic tongue as a rapid technique, combining with specifically statistics analyze system, could monitor the growth of the three food-borne pathogens. [Methods] The smart electronic tongue, multi-frequency large amplitude pulse was based on voltammetry .We used it to detect the comprehensive information about a sample and the change process of the medium in 16 periods of time. We analyzed the complex data information in statistics with principal component analysis. According to principal component scores plot, we analyzed the sample. [Results] The growth of Staphylococcus aureus in liquid medium could be monitored by tungsten electrode in 100 Hz frequency segment of multi-frequency large amplitude pulse voltammetry, platinum electrode in 1 Hz, and both silver electrode and titanium electrode in 10 Hz. The growth of Escherichia coli O157:H7 could be measured by gold electrode in 100 Hz frequency segment, while platinum electrode in 1Hz, titanium electrode in 1Hz and tungsten electrode in 100 Hz. Then, palladium electrode was efficient to Bacillus subtilis in 1 Hz, 10 Hz, 100 Hz. [Conclusion] We used the multi-frequency large amplitude pulse sensor system combining with principal component analysis for the first time. It has a promising future as a modern rapid analytical technology for detecting the growth of microbe.

Abstract:[Objective] Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of acute and chronic infections. When growing in the host, it secrets a lot of virulence factors including elastase. This work aimed to explore the genes involved in hydrolyzing ability of Pseudomonas aeruginosa towards elastin. [Methods] We performed a transposon mutagenesis analysis of P. aeruginosa PA68 to identify candidate genes involved in elastin hydrolysis. We also monitored the promoter activity of the lasB, a gene encoding the elastase, in the mutants and the wild-type by introducing a PlasB-lacZ transcriptional fusion. [Results] Four mutants with altered levels of elastase production were isolated (elastase activity relative to wild-type was shown in parenthesis): 10 (51%), 17 (131%), 27 (8%) and 84 (13%). Locations of the transposon were mapped to the genome lasA, galU, xcpZ and ptsP, respectively. The results of the lasB promoter’s activity were consistent with the elastase activity data (b-galactosidase activity relative to wild-type was shown in parenthesis): 10(75%), 17(201%), 27(54%) and 84(7%). [Conclusion] Taken together, the data build up a connection of these four genes with elastase production. This is the first report that gene galU and ptsP may be employed in the regulation of the biosynthesis of elastase.

Abstract:[Objective] To overcome coenzyme restriction in the asymmetric reduction at high substrate concentration, we constructed a recombinant Escherichia coli with (R)-sepcificity carbonyl reductase coupled with formate dehydrogenase for cofactor regeneration. [Methods] The R-carbonyl reductase gene (rcr) and formate dehydrogenase gene (fdh) were amplified from Candida parapsilosis and Candida boidinii genomes by PCR technique respectively. Then the purified PCR products were inserted into a co-expression vector pETDuetTM-1 to construct plasmid pETDuet-rcr-fdh. The positive plasmid was transformed into codon optimized E. coli Rosetta, and a recombinant strain E. coli Rosetta/pETDuet-rcr-fdh was obtained. [Results] SDS-PAGE analysis showed that two enzymes were expressed simultaneously. Isopropyl-β-D-thiogalactopyranoside (1 mmol/L) induced at 30℃ the expression of both proteins encoded by rcr and fdh genes with the molecular weights of 37 kDa and 40 kDa. The biotransformation experiments were done using 2-hydroxyacetophenone and sodium formate as substrates. When the concentration of 2-hydroxyacetophenone was 6 g/L, the (R)-1-phenyl-1,2-ethanediol was produced with the high optical purity of 100% enantiomeric excess and a yield of 85.9%. Compared with the recombinant strain E. coli Rosetta/pETDuet-rcr without fdh gene for cofactor regeneration, the optical purity and yield of product from the asymmetric reduction of 2-hydroxyacetophenone by E. coli Rosetta/ pETDuet-rcr-fdh were increased by 1.3 and 2.7 times, respectively. [Conclusion] This method supplied a foundation for biosynthesis of (R)-1-phenyl-1,2-ethanediol for the cofactor regeneration by genetic engineering

Abstract:[Objective] We used specific-PCR and denaturing gradient gel electrophoresis (DGGE) to isolate Thauera spp. from a coking wastewater treatment plant. [Methods and results] To isolate Thauera from the denitrifying bioreactor of a coking wastewater treatment, biofilm was inoculated to six different media and cultured them under both aerobic and anaerobic conditions. We then compared the composition of Thauera spp. using Thauera-specific PCR-DGGE method. The media 1/10 NB and MMQ which grew higher diverse Thauera spp. and fewer colonies, were used to isolate Thauera sp. under aerobic condition. The colonies were then screened by Thauera-specific PCR. The purity of the colonies that shown Thauera-specific PCR positive signal was then checked by DGGE. The colonies with multiple species were further streaked on different media. DGGE analysis showed that Thauera in colony Q20 was enriched in medium MMP. The colony was finally purified by streaking on MMP medium for several rounds. The composition of the colonies were tracked by Thauera-specific PCR and DGGE at each step. Finally, three strains were purified, which were identified as Thauera sp. according to their 16S rRNA gene sequences. [Conclusion] Guiding with specific biomarker, the efficiency and sensitivity of bacteria isolation can be largely improved.

Abstract:[Objective] The trag (transfer gene G) was one of the novel infection-related factors identified by in vivo–induced antigen technology (IVIAT) from Streptococcus suis type 2 expression libraries with swine convalesecent

Abstract:[Objective] To study the influence of hemolytic and non-hemolytic enterococci on TNF-a expression in Murine macrophage cells. [Methods] The possibility of endotoxin contamination in the cell culture was excluded by polymyxin-B inhibition. RAW264.7 were infected with hemolyticor non-hemolytic enerococci at bacteria: cell ratio of 30:1 for 1h, followed by washing and re-incubation for 24h in complete medium containing 200 mg/mL Ampicillin. TNF-a concentrations in culture supernatants at different time intervals after infection for 3 h, 6 h, 9 h and 24 h were measured by ELISA. TNF-a mRNA expression by macrophages infected with hemolytic or non-hemolytic enerococci respectively for 6h was compared by RT-PCR. [Results] TNF-a was not detected in culture supernatants from uninfected RAW264.7 cells. The concentrations (pg/mL) of TNF-a present in culture supernatants from RAW264.7 cells stimulated by hemolytic enterococci were significantly higher than that stimulated by non-hemolytic enterococci at all time intervals tested, p<0.01, Student t-test. TNF-a mRNA expression measured by RT-PCR brought about similar results: more TNF-a mRNA was expressed in RAW 264.7 cells stimulated with hemolytic enterococci as compared with non-hemolytic enterococci stimulation (p<0.05, Student t-test). [Conclusion] hemolytic enterococci promoted the generation of inflammatory factor TNF-a.

Abstract:[Objective] Study the association of the length of poly(C) tract with virulence of foot-and-mouth disease virus. [Methods] The recombinant plasmids pGEM-XJ/AKT/69 containing the full-length cDNA of FMDV were linearized by NheⅠ/NotⅠand transcribed by T7 RNA polymerase. The in vitro transcripts were transfected into BHK-21 cells using Lipofectamine 2000 reagent. Then, the genetic engineering virus was rescued from BHK-21 cells. The poly(C) tracts were sequenced at different passages of rescued virus, and the pathogenicities were evaluated with 3-day-old mice and BHK-21 cells by detection of LD50 and TCID50. [Results] After six passages in BHK-21 cells, cytopathic effect was observed by microscopy. The rescued virus was rejuvenated once in unweaned mice, and then came back to cell passage. We found that the poly(C) tract of rescued virus was shortened when the virus was passaged twice in BHK-21 cells. Although the data of LD50 in mice and TCID50 in BHK-21 cells showed that the virulence and infectivity of genetic engineering viruses were lower than its parental virus, no significant difference was observed between the genetic engineering viruses with the different length poly(C) tract. [Conclusion] The length of poly(C) tract ranged from 12 to 17 nucleotides did not cause significant influence on the virulence and infectivity of genetic engineering virus.

Abstract:[Objective] To analyze the genetic relationship and predominate genotypes among Legionella pneumophila isolated from Guangdong province. [Methods] In total 43 Legionella pneumophila strains were genotyped according to the amplified fragment length polymorphism protocol, and the obtained electropherograms were contrasted to the published standard patterns of the European Working Group for Legionella Infections. [Results] In total 33 genotypes were identified from those 43 Legionella, the discriminative index was 99.79%, and the Kingmed AFLP type no. 011 was predominate. In total 18 clusters were discriminated by 0.8 dice coefficient, and the Kingmed AFLP cluster D was predominate. By comparison, 7 respective genotypes had high similarity to the EWGLI AFLP type no. 001 Lugano, 002 Lugano, 012 Rome, 013 London, 014 London, 015 Dresden and 021 Lyon, and it also existed some potential new genotypes. [Conclusion] The genetic polymorphism of Legionella pneumophila are diverse in the environmental water of Guangdong province, and amplified fragment length polymorphism is one of the effective means for the research of molecular epidemiology.

Abstract:Infectious bursal disease virus （IBDV) belongs to genus Avibirnavirus of the family Birnaviridae. The genome of IBDV consists of two segments of double-strand RNA, which encode four structural protein VP1-VP4 and one non-structural protein VP5. [Objective]To study the function of VP5 of IBDV, the recombinant virus, lack of VP5 gene, was constructed and rescused by reverse genetic technique. [Methods] We deleted the VP5 gene （on segment A) of IBDV Gt strain by silence the start codon （ATG-ATC) using site-directed mutagenesis. The full length cDNA of segment A was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme, which was introduced into an eukaryotic expression vector PCAGGS, under the strong chicken b actin promoter. The recombinant plasmid was named as pCAGGmGtA △VP5HRT. Co-transfection was carried with PCAGGmGtAdVP5HRT and PCAGGmGtBHRT in DF-I cells. [Results] Recombinant virus was successfully rescused, which was verified by RT-PCR and indirect immnuofluorescence assay. The rescusd virus could be a very helpful platform for further study of VP5 biological function.

Abstract:[Objective] To develop an efficient method to detect and analyze the endogenous plasmid in Stremptomyces Men-myco-93-63. [Methods] According to the property of ‘the linear chromosomes and plasmids of Stremptomyces spp.have proteins covalently bound to the 5′ends of the DNA′, a novel method was established to detect the type, configuration and size of the Streptomyces spp. plasmids by improving the preparation of agarose embedded block. The method can also show whether the linear plasmids have proteins covalently bound to the DNA from both the positive and negative aspects. [Results]The type and configuration of two plasmids were determined efficiently from Stremptomyces roseoflavus Men-myco-93-63 and verified by the two-dimensional electrophoresis. The speculated sizes of these plasmids were 47 kb and 53 kb, respectively. [Conclusion] Two linear plasmids were detected successfully from Stremptomyces roseoflavus Men-myco-93-63 by a new method.

Abstract:We summarized the key handicap and troubleshooting when proteomic techniques were used to investigate extremophilic microorganisms, and the actual state of their proteomes research in recent years. Up to now, proteomics techniques keep developing and improving rapidly, but they has not been widely used to explore proteome of extremophilic microorganisms including halophiles, thermophiles, psychophiles, acidophiles and alkaliphiles due to specific problems including incomplete dissociation of protein–protein complexes of extremophiles, and a lot of proteins synthesized by extremophiles are resistant to the conditions which dissociated and denatured proteins synthesized by mesophilic organisms. However, the foreground of potential application of the techniques draws people on attempting zealously multifarious methods. At the present time, several technical problems for separating halophilic proteins, integral membrane proteins and predicting the function of new proteins have been solved availably. Proteomics data have validated some conclusions of genome predictions, and revealed many novel proteins and a few properties of extremophiles can not be resolved fully by genome data. The investigation of extremophiles proteomes indicated that a comprehensive view of protein expression profiles should rely on more than one proteomic method. In addition, the mutual verification of conclusions on the basis of genome and proteome and combination of these two techniques must accelerate the study of extremophilic microorganisms, and redound to uncover deeply and wholly the unique mechanisms of microorganisms adaptation to extreme environments. Moreover, it would clarify the mechanisms of their survival, and point out new direction of survey for improving damage result from stresses, finally contribute to human survival and healthy.

Abstract:Peroxisome (P), a ubiquitous organelle in the eukaryotic cells, is involved in various important metabolic processes. Investigation of formation, proliferation and degradation of the peroxisome is an important part of study of organelles biogenesis. So far, mechanism of the peroxisome biogenesis is not completely clear, although over 30 related genes were identified and characterized. Filamentous fungus, a multi-cellular eukaryotic organism containing many plant pathogens and economic species, holds great value of studying function and mechanism in the peroxisome biogenesis. The peroxisome researches have been progressed in recent years quickly after availability of the genome data and development of the fungal bio-techniques. Meanwhile greater interests have been demonstrated in area of the peroxisome biogenesis and in its roles of pathogenicity of several phytopathogenic fungi. The summary of mechanism of the peroxisome biogenesis in filamentous fungi and relationship between peroxisome and pathogenicity were reviewed in this paper