Physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO2, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, beta-hydroxybutyrate was predominant (over 99 mol %); beta-hydroxyvalerate (0.25-0.72 mol %) and beta-hydroxyhexanoate (0.008-1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

The Autotrophic Synthesis of Polyhydroxyalkanoate by Alcaligenes eutrophusin the Presence of Carbon Monoxide

The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophuswas found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide. The gas mixtures that contained from 5 to 20 vol % CO did not inhibit the key enzymes of PHA synthesis–-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase. In the presence of CO, cells accumulated up to 70–75 wt % PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate. Chromatographic–mass spectrometric analysis showed that the PHA synthesized by A. eutrophusis a copolymer containing more than 99 mol % -hydroxybutyrate and trace amounts of -hydroxyvalerate. The PHA synthesized under the conditions described did not differ from that synthesized by A. eutrophuscells from electrolytic hydrogen.     

金枪鱼肉中组胺菌的分离及其理化性质分析

目的：对金枪鱼肉中的组胺生成菌进行生化实验和功能酶检测,进而对鱼肉中组胺菌的存在提供可参考数据。方法：利用组氨酸肉汤培养液,对金枪鱼肉中的组胺生成菌进行筛选,然后通过生物化学实验和形态学实验,进而对组胺生成菌进行分析。结果：分离得到2株组胺菌,菌种Ⅰ在5℃~30℃的培养温度范围内,随温度升高,组胺生成量逐渐增加,在30℃有最高组胺生成量,约2 700ppm。菌种Ⅱ在25℃有组胺最高生成量约为2 400ppm。结论：依据两种组胺菌的特征和生理生化特征,分离菌可能分别属于弧菌属、埃希氏菌属。     

Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha

A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP(+)-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of gamma-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD(+)-dependent succinate semialdehyde dehydrogenase in R. eutropha.     

Physiological,Biochemical, and Cytological Characteristics of a Haloalkalitolerant Methanotroph Grown on Methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h^–1) was twice as high as on methane (0.1 h^–1). The maximum growth rate increased with increasing medium salinity and pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。     

超大型烟草突变株的生理生化特征和分子生物学鉴定

超大型烟草突变株再生植株高度是野生型的2．2倍,叶片数是野生型的3．3倍,呈现晚花发育特征;叶片气孔保卫细胞中叶绿体数是野生型的1．3倍,叶绿素a、b和叶绿素总量均高于野生型,可溶性蛋白质含量是野生型的1．18倍,过氧化物酶、细胞色素氧化酶同工酶电泳图谱上有一定差异;可溶性蛋白SDS-PAGE电泳图谱上比野生型少4条谱带;RAPD结果表明突变体在DNA水平上确实发生了变化,DDRT-PCR结果显示出两者在基因表达上有差异。突变株再生植株可以开花结实,植株高大、叶数多,晚花的特征可以稳定遗传。     

Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha

A novel class of biopolymers referred to as polythioesters (PTE) was recently detected when the polyhydroxyalkanoate (PHA) accumulating bacterium Ralstonia eutropha was cultivated in the presence of 3-mercaptopropionic acid (3MP) or 3,3'-thiodipropionic acid (TDP). In this study, 3,3'-dithiodipropionic acid (DTDP) and 3-mercaptovaleric acid (3MV) were identified as two additional precursor carbon sources for in vivo biosynthesis of PTE in R. eutropha. Biosynthesis of copolymers of 3-hydroxybutyrate (3HB) and 3MP, which contributed 19-25% of cell dry matter, was compared referring to the different precursor substrates. Using DTDP as carbon source, which is probably cleaved into two molecules 3MP, yielded an about 2.3-fold higher molar 3MP content of the copolyester than TDP, which is probably cleaved into only one molecule 3MP. Furthermore, cultivation of R. eutropha in the presence of 3MV resulted in biosynthesis of copolymers consisting predominantly of 3HB with low amounts of 3MV and 3-hydroxyvalerate, each contributing less than 5 mol% of the constituents. In contrast, 4-mercaptobutyric acid could be not incorporated into PHAs, although - as documented in this study - five different strategies, various precursor substrates, R. eutropha and also a recombinant strain of Escherichia coli were employed. Therefore, this study not only extended the range of substrates suitable for PTE biosynthesis and also the range of PTE constituents in R. eutropha, it also demonstrates limits for PTE biosynthesis in this bacterium.     

Regulation Culture on Cytological，Biochemical and Physiological Characteristics of Somatic Carrot Em

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苹果斑点落叶病致病菌的鉴定及生物学特性研究

目的:明确l株分离于感病苹果株系群体的供试菌株AML0801的种类,探讨其生物学特性,为苹果抗病分子育种研究奠定基础.方法:通过形态特征描述,显微形态、超微结构观察等方法,从形态学上明确AML0801的分类.室内模拟侵染试验,了解AML0801致病性.ITS序列测定分析辅助验证供试菌株AML0801.结果:菌株AML0801的分生孢子、产孢表型等形态特征,符合苹果链格孢(Alternaria mali Roberts,A.mali)种的描述,ITS序列分析表明,AML0801与A.mali标准菌株同源性达99％.通过比较病原菌侵染试验证实AML0801具备一定的致病性.结论:结合供试菌株AML0801形态特征、ITS序列分析结果,以及室内模拟的致病性研究,可将供试菌株AML0801鉴定为苹果链格孢(A.mali).     

青枯病抗性不同的番茄品种根际拮抗菌拮抗能力差异研究

以植物基因型影响根际微生物的多样性为依据,通过平板拮抗试验,从番茄的感病品种和抗病品种、感病品种的严重发病植株和轻微发病植株根际筛选到36株抬抗菌,不同来源的抬抗菌群落具有不同的特征。严重发病的感病品种植株根际的拮抗菌群落多样性低,仅筛选到1个拮抗菌株,而轻微发病的感病品种和未发病的抗病品种植株根际筛选到的拮抗菌株数较多;抗病品种根际桔抗菌表现出强拮抗能力,而感病品种根际拮抗菌的拮抗能力较弱。测定植株根面和根颈处导管内青枯菌的种群数量、3株拮抗菌的防病效果,发现导管内青枯茵数量与防病效果基本一致,探讨了导管内青枯菌数量作为拮抗菌防病效果指标的可能性。     

Levels of Total Airborne Bacteria,Gram-Negative Bacteria,and Endotoxin According to Biosafety Levels in Korean Biosafety Laboratories

We assessed in this study the total airborne bacteria (TAB), gram-negative bacteria (GNB), and endotoxin (ET) concentrations in three Korean university laboratories and two hospital diagnostic laboratories categorized as biosafety level 1 (BL 1) or biosafety level 2 (BL 2). We also investigated the concentrations of TAB, GNB, and ET relative to the performance results of biosafety cabinets (BSCs). TAB and GNB were incubated, and ET was analyzed using the kinetic Limulus amebocyte lysate assay. A total of 246 (TAB = 95, GNB = 95, ET = 56) air samples were collected from nine laboratories. TAB, GNB, and ET concentrations were significantly higher in BL 1 laboratories and in laboratories that failed the BSC performance test compared to BL 2 laboratories and laboratories that passed the BSC performance test. Correlation coefficients were calculated to determine the relationships between TAB, GNB, and ET, and it was found that TAB significantly correlated with GNB (r = 0.35, p < .01) and ET (r = 0.29, p < .01). Careful attention needs to be paid to BL 1 laboratories, and BSCs should be managed on a regular basis to improve the air quality of university and hospital laboratories.     

Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H(2)-based ithoautotrophy and anaerobiosis

The self-transmissible megaplasmid pHG1 carries essential genetic information for the facultatively lithoautotrophic and facultatively anaerobic lifestyles of its host, the Gram-negative soil bacterium Ralstonia eutropha H16. We have determined the complete nucleotide sequence of pHG1. This megaplasmid is 452,156 bp in size and carries 429 potential genes. Groups of functionally related genes form loose clusters flanked by mobile elements. The largest functional group consists of lithoautotrophy-related genes. These include a set of 41 genes for the biosynthesis of the three previously identified hydrogenases and of a fourth, novel hydrogenase. Another large cluster carries the genetic information for denitrification. In addition to a dissimilatory nitrate reductase, both specific and global regulators were identified. Also located in the denitrification region is a set of genes for cytochrome c biosynthesis. Determinants for several enzymes involved in the mineralization of aromatic compounds were found. The genes for conjugative plasmid transfer predict that R.eutropha forms two types of pili. One of them is related to the type IV pili of pathogenic enterobacteria. pHG1 also carries an extensive "junkyard" region encompassing 17 remnants of mobile elements and 22 partial or intact genes for phage-type integrase. Among the mobile elements is a novel member of the IS5 family, in which the transposase gene is interrupted by a group II intron.     

Comparison of phbC genes cloned from Ralstonia eutropha and Alcaligenes latus for utilization in metabolic engineering of polyhydroxyalkanoate biosynthesis

Two cloned phbC genes, encoding polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha and from Alcaligenes latus, were transformed into a PHB-negative mutant of R. eutropha. The expression characteristics of both genes were compared for the biosyntheses of PHB and its copolymers. Each phbC gene had different characteristics not only in the biosyntheses of PHB, poly(3-hydroxybutyrate-3-hydroxy-valerate), and poly(3-hydroxybutyrate-4-hydroxybutyrate) but also in the resulting morphology of PHB granules.     

Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Synthesis of Anabiosis Autoinducers by Non-Spore-Forming Bacteria as a Mechanism Regulating Their Activity in Soil and Subsoil Sedimentary Rocks

Non-spore-forming bacteria of the genera Arthrobacterand Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d ₁extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d ₁preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis245 produced an effect on the collection strain Arthrobacter globiformisB-1112 and also on Micrococcus luteusand Bacillus cereus.The d ₁preparation from the permafrost isolate of Arthrobacterdiffered from the chemical analogue of this factor, 4-n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d ₁preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d ₁anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural long-term cooling.     

一株强神经毒力乙脑病毒株的生物学及其分子特征

研究一株神经毒力很强的乙型脑炎病毒株SA4株的生物学特性并进行基因组全序列分析,并找到其毒力强的分子基础。将SA4脑内接种昆明小鼠后每日观察发病和死亡鼠数并每日收取鼠脑,用空斑法检测接种后不同时间点的脑内病毒增殖滴度,同时以另一株乙脑病毒SA14按照相同方法进行平行实验作为对照。SA4株的全基因组测序序列结果与乙脑SA14株及世界各地共21株乙脑病毒的全基因组序列进行比较。结果显示,脑内接种SA4株的小鼠发病和死亡时间均比SA14株早1d,在相同时间点上SA4株的病毒滴度高0.5～1.0lgPFU/mL。SA4株与世界各地毒株基因同源性比对,核苷酸同源性为84.6%～99.0%,氨基酸同源性为95.2%～99.7%。与SA14相比,氨基酸序列存在17个位点的差异,分布于不同的区段,其中E区最多,为5个。证明SA4是一株在脑内具有较快繁殖力的神经毒力很强的毒株,其序列上17个氨基酸位点的替换很可能是其强神经毒力的分子基础。     

低温下壳聚糖处理对黄瓜幼苗生理生化特性的影响

50 mg/L的壳聚糖处理减轻了低温对黄瓜幼苗膜的伤害,低温(6℃)处理4 d后,幼苗膜透性和MDA含量明显低于对照,并且显著减缓了低温导致的光合速率、实际光化学效率(ФPS Ⅱ)、光化学猝灭系数(qP)和1光系数(F’v /Em’)的下降;同时,处理使抗氧化酶SOD、POD、CAT和APX活性提高,可溶性蛋白和脯胺酸含量增加.因此,壳聚糖处理增强了黄瓜幼苗的抗冷性,保护了膜系统,提高了活性氧清除能力,减轻了低温对光合机构的破坏.