Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

The catalytic properties and stability of d-xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg²⁺, Co²⁺ and Mn²⁺ but Ni²⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 mm Mg²⁺. The specific activity of the enzyme increased after treatment with 10 mm EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg²⁺ or Co²⁺, the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 mm Co²⁺ or 10 mm Mg²⁺. At 80°C, Co²⁺ is superior as a protector against thermal denaturation. At saturating concentrations of Mg²⁺ (35°C) the K_m-values of the EDTA-treated enzyme with respect to d-xylose and d-glucose were 2.8 and 149 mm and the dissociation constants of the enzyme-Mg²⁺ complex for xylitol and d-sorbitol were 0.455 and 4.47 mm, respectively.     

紫红链霉菌对钉螺酶组织化学的影响

为研究紫红链霉菌灭螺作用的机理,将钉螺分别浸泡于紫红链霉菌培养液(含菌4×106/ml)及去氯水、培养基中36h后,用酶组织化学方法显示各组钉螺肝脏、中枢神经节、头足部及鳃的Mg2 激活的三磷酸腺苷酶(Mg2 ATPase)、胆碱脂酶(ChE)、一氧化氮合酶(NOS)、乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)并观察其变化。结果显示:菌液浸泡组钉螺的Mg2 ATPase活性在肝脏、中枢神经节、头足部及鳃部均明显减弱或完全失活,LDH在中枢神经节、头足部也有一定程度减弱,ChE、NOS、SDH在肝脏、中枢神经节、头足部及鳃部与去氯水组无明显差异;培养基组与去氯水组钉螺相应部位的Mg2 ATPase、ChE、NOS、LDH、SDH活性一致。结果提示:紫红链霉菌的灭螺作用机理主要在于破坏钉螺体内Mg2 ATPase和LDH活性,使ATP生成和利用障碍,最终因能量缺乏而丧失生命功能直至死亡     

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

The ability of the NADPH oxidase of human neutrophil-derived cytoplasts to generate O2.-anions on the addition of phorbol 12-myristate 13-acetate is severely reduced in the presence of valinomycin and Zn2+ ions. The addition of NH4Cl or carbonyl cyanide m-chlorophenylhydrazone (K+ medium only) to these cytoplasts results in a restoration of O2.- generation. At an elevated pHo CCCP restores a greater rate of O2.- generation. Increasing the concentration of Zn2+ ions reduces the extent of the generation of O2.- on the addition of PMA. The restoration of O2.- generation by NH4Cl or CCCP requires the presence of valinomycin. The restoration of O2.- generation appears to be dependent on the movement of NH4+ ions or the anionic form of the uncoupler across the plasma membrane. The activity of the electrogenic NADPH oxidase of cytoplasts is limited by the movement of an ion to act as a compensator. The NADPH oxidase therefore exhibits respiratory control.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Nucleic Acid Homologies Among Species of Saccharomyces

Evolutionary divergence among species of the yeast genus Saccharomyces was estimated from measurements of deoxyribonucleic acid (DNA)/DNA and ribosomal ribonucleic acid (RNA)/DNA homology. Much diversity was found in the DNA base sequences with several species showing little or no homology to the three reference species, S. cerevisiae, S. lactis, and S. fragilis. These three reference species also showed little or no homology to each other. On the other hand the diversity among ribosomal RNA base sequences was small since most species showed a high degree of homology to the reference species. The arrangement of species based on ribosomal RNA homologies agrees in most cases with current taxonomic groupings. A yeast hybrid (S. fragilis x S. lactis) was shown to contain two nonhomologous genomes. A minimum genome size of 9.2 x 10(9) daltons for S. cerevisiae was calculated from the rate of DNA renaturation.     

Deoxyribonucleic Acid Homologies Among Some Pseudomonas Species

Phylogenetic relationships among a number of strains belonging to the genus Pseudomonas were explored by the use of in vitro deoxyribonucleic acid (DNA) hybridization. The fluorescent nomenspecies (P. fluorescens, P. putida, P. aeruginosa, P. cichorii, P. syringae, and related species), as well as the nonfluorescent species P. stutzeri, P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, were shown to belong to a single DNA homology complex which is isolated from other Pseudomonas species that have been studied [P. cepacia (= P. multivorans), P. caryophylli, P. marginata (= P. alliicola), P. pseudomallei, P. acidovorans, P. testosteroni, P. solanacearum, P. diminuta, P. facilis, P. delafieldii, P. saccharophila, P. palleronii]. A limited numerical analysis of the phenotypic properties of the examined strains supported, with some exceptions, their previous allocation to nomenspecies and biotypes. The internal structure and nomenclature of the "P. fluorescens homology complex" are discussed.     

Selective isolation of members of the Streptomyces violaceoruber clade from soil

Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms "Streptomyces coelicolor" A3(2) and "Streptomyces lividans" 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.     

水稻植物内生链霉菌中线型和环型质粒的检测

以广东番禺和五山地区水稻植株中分离到的内生链霉菌为对象,调查可能存在的内源质粒.利用脉冲电泳技术从8个菌株中检测到大小在60 kb～410 kb的线型质粒,其中4个菌株的线型质粒可能有保守的端粒复制基因.该结果与土壤链霉菌中检测到线型质粒和具有保守端粒复制基因的比例相似,表明水稻植物组织内部的独特环境不会造成链霉菌线型质粒的多样性分布产生大的变化.此外,从13个菌株中检测到6 kb～60 kb的环型质粒.     

Deoxyribonucleic Acid Homologies Among Species of the Genus Neisseria

Eleven aerobic species of Neisseria, a Mima sp., and a Herellea sp. were tested for deoxyribonucleic acid (DNA) homology in direct hybridization experiments. DNA labeled with either (14)C or (32)P was prepared from five species of Neisseria. Unlabeled DNA from the various microorganisms was immobilized on membrane filters, which, after pretreatment, were incubated with labeled DNA (4,000 counts per min per filter) for 14 hr at 67 C. The measure of relatedness was expressed as the relative percentage of direct binding compared to that obtained with homologous DNA. All serological types of N. meningitidis, including the newly proposed types, were homologous to the standard strain of N. meningitidis with one possible exception, type Z. The genus Neisseria is heterogeneous in nature, forming at least three distinct groups: first, N. meningitidis and N. gonorrhoeae; second, N. perflava, N. subflava, N. sicca, N. flavescens, and N. flava; third, N. catarrhalis and N. caviae. Mima and Herellea species show no significant homology with the Neisseria.     

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

利用Batch-ANIm快速分析无色杆菌属菌株间的亲缘关系

基因组平均核苷酸相似度(average nucleotide identity,ANI)已成为鉴定细菌种内关系的黄金方法,开发可用于快速分析大量基因组之间ANI值的生物信息学工具具有重要意义.本研究以广泛应用的ANI分析软件JSpecies为基础,采用Perl集成编写了能够根据设置的线程数、自动生成多个JSpecies配置文件以完成基因组序列载入与成对选择,快速完成大量基因组ANI计算与分析的工具Batch-ANIm(下载地址:http://www.microbialgenomic.com/Batch-ANIm.html).采用该工具对已测序的 109株无色杆菌属(Achromobacter)菌株进行ANIm(基于MUMmer算法)计算,共获得5 886个ANIm值.整体上,基于"100-ANIm"进化距离获得的聚类分析结果与核心基因组进化树比较一致,表明ANIm聚类分析可用于快速展示无色杆菌属菌株之间亲缘关系.很多无色杆菌属种内菌株之间的ANIm值小于95％,但种间ANIm值却大于95％,这表明从基因组水平上部分无色杆菌属菌株的分类学命名存在错误,特别是无色杆菌属的代表种A.xylosoxidans,命名为A.xylosoxidans的50个菌株,只有40个菌株真正属于A.xylosoxidans.同时,基于全基因组序列的ANI值比较可以将部分种名不确定的菌株分类学命名精确到种水平.     

利用Batch-ANIm快速分析无色杆菌属菌株间的亲缘关系

基因组平均核苷酸相似度(average nucleotide identity,ANI)已成为鉴定细菌种内关系的黄金方法,开发可用于快速分析大量基因组之间ANI值的生物信息学工具具有重要意义.本研究以广泛应用的ANI分析软件JSpecies为基础,采用Perl集成编写了能够根据设置的线程数、自动生成多个JSpecies配置文件以完成基因组序列载入与成对选择,快速完成大量基因组ANI计算与分析的工具Batch-ANIm(下载地址:http://www.microbialgenomic.com/Batch-ANIm.html).采用该工具对已测序的 109株无色杆菌属(Achromobacter)菌株进行ANIm(基于MUMmer算法)计算,共获得5 886个ANIm值.整体上,基于"100-ANIm"进化距离获得的聚类分析结果与核心基因组进化树比较一致,表明ANIm聚类分析可用于快速展示无色杆菌属菌株之间亲缘关系.很多无色杆菌属种内菌株之间的ANIm值小于95％,但种间ANIm值却大于95％,这表明从基因组水平上部分无色杆菌属菌株的分类学命名存在错误,特别是无色杆菌属的代表种A.xylosoxidans,命名为A.xylosoxidans的50个菌株,只有40个菌株真正属于A.xylosoxidans.同时,基于全基因组序列的ANI值比较可以将部分种名不确定的菌株分类学命名精确到种水平.     

三种植物与紫红链霉菌组合灭螺效果

探讨了不同植物与紫红链霉菌组合的灭螺效果,为研制高效低毒植物灭螺剂提供依据。将天名精（Carpesium abrotanoides）、苍耳子（Xanthium sibiricum）、青蒿（Artemisia carvifolia）3种植物水浸液分别与紫红链霉菌液（Streptomyces violaceoruber）组合后对钉螺进行浸杀对比实验,并比较了相同植物与紫红链霉菌液的复合颗粒灭螺剂的灭螺效果。结果表明：天名精、苍耳子与紫红链霉菌复合颗粒灭螺剂7 d钉螺死亡率分别比其1%混合液提高17.6%（P=0.019）和5.3%(P=0.362);青蒿与链霉菌复合颗粒灭螺剂7 d钉螺死亡率比其1%混合液降低6.7%(P=0.022);天名精、苍耳子和链霉菌复合颗粒灭螺剂较其混合液具有一定的增效作用。