栖热菌目、科、属、种*

阐述了栖热菌的发现和分类鉴定研究进程。现已发表有效种 1 4个 ,涉及 4个属 50多个菌株 ,未获有效公布种及未定种的菌株 50多个。形态、生理、G +C含量、DNA杂交、脂肪酸组成、 1 6SrRNA同源性和二级结构分析是栖热菌分类鉴定的主要方法。     

利用针对16S rDNA序列的限制性酶切分析鉴定栖热菌属

目的:建立一套酶切体系,用于鉴定栖热菌.方法:收集和整理栖热菌属16S rDNA序列信息,利用限制性内切酶对信息进行位点分析.结果:建立了一套鉴定栖热菌的酶切体系,通过模拟电泳对该体系进行验证.结论:该系统可有效地对已经公开发表但未鉴定到种的栖热菌属菌株进行种间分类.     

腾冲热海一株栖热菌裂解性噬菌体的分离及其特征

【目的】Thermus(栖热菌)属是嗜热细菌中比较古老的类群,本研究探索从腾冲热海热泉分离栖热菌噬菌体,并初步分析其特征。【方法】采用"双层平板法"从云南腾冲热海碱性热泉中分离纯化栖热菌噬菌体;对噬菌体及其宿主进行电镜形态观察,并进行噬菌体基因组限制性酶切片段多态性分析、噬菌体生理特征及蛋白组成分析。【结果】从腾冲热海热泉分离获得1株裂解性噬菌体,其宿主菌TC10通过16S rRNA基因序列分析鉴定为Thermus属菌株。此噬菌体为长尾型,头部直径67nm,尾管长837nm、宽10nm,最适感染温度为65℃-70℃,最适感染pH值为7.6,对氯仿不敏感,其形态与分离自俄罗斯勘察加半岛热泉的栖热菌噬菌体P23-45和P74-26有一定的差异,蛋白组成差异显著,为一株新的栖热菌噬菌体,命名为TTSP10(Tengchong Thermus Siphoviridae Bacteriophage)。     

一个产生L-乳酸脱氢酶的嗜热菌的新种——非解朊栖热菌

从广东省阳江、电白和韶关等地55—95℃的温泉中采集水样,经分离得到21株G-,不产生芽孢的高温菌。它们都能产生L—乳酸脱氢酶。根据表观特征,DNA的同源性和G十cmol％等特征可归人栖热菌属(Thermus)。但对葡萄糖不产酸和不液化明胶等表型特征不同于已发表的种。又根据该群内的DNA／DNA杂交结果,有高的同源性(大多在80％以上),而与已发表的种仅有40.5—70.7％的同源性。结果表明可成立一独立的类群,建议为栖热菌属的一个新种,因具不液化明胶的特性,命名为非解朊栖热菌(Thermus nonproteolyticus Cai ＆Yaagsp nov.)。以菌株HG—42为模式株,保藏于中国科学院微生物研究所。     

一株产异淀粉酶栖热菌的生长和产酶初步研究

异淀粉酶是一类在工业中有重要应用价值的淀粉酶,从云南梁河温泉水样中分离筛选到1株产异淀粉酶的菌株,初步鉴定为栖热菌(Thermus)。该菌在8~19 h为对数生长期,17 h生长最快,20 h后进入稳定期,产酶伴随菌体生长同时发生,进入稳定期后产酶迅速提高,39 h产酶量达最高,初始酶活达4.14 u/mL。     

从嗜热栖热菌中提取超氧物歧化酶的研究

嗜热栖热菌(Thermus thermophilus)ATCC 27634是一种适于75—80℃高温中生长的微生物。该菌超氧物歧化酶为一胞内酶,分子量为80000,由181个氨基酸组成。本试验对其发酵、破菌、Rnasel酶解、硫酸铵盐析、滤除小分子物质、上DE-32Ⅰ、DE-32Ⅱ柱分离等条件进行了一系列研究,提取到了热稳定性较商的Mn²⁺-SOD,比活力为3000u/mg,回收率达78.0%。     

Taq DNA耐热聚合酶在大肠杆菌中的克隆和高表达

从水生栖热菌(Thermus aquaticus)YT-1中分离得到的Taq DNA 聚合酶是一种广泛应用于PCR的耐热DNA聚合酶。由于天然菌株酶产量较低,培养条件要求严格,酶的纯化过程极为繁琐而使产品成本较高,因而促使人们构建适合于大规模生产的基因工程菌株。已有人分别通过不同的途径,使用不同的载体,成功地在大肠杆菌菌株中表达了Taq 耐热DNA聚合酶基因。我们通过与前人不同的途径,把这一基因克隆到大肠杆菌的载体质粒pJLA503上并使其得以高表达,为降低生产成本和进一步研究该酶的各种特性提供了有利条件。     

栖北散白蚁致病菌的分离鉴定与特性研究

[目的]了解散白蚁头部发红且死亡的原因,探究致病菌特性.[方法]从头部发红的散白蚁中分离菌株、培养并纯化,通过显微形态、革兰氏染色及16S rRNA基因序列分析鉴定菌株种类.进一步探究菌株产生红色素的条件并通过液相色谱和质谱联用等方法鉴定发酵产物.[结果]散白蚁鉴定为栖北散白蚁Reticulitermes speratus.从该白蚁头部分离得到一株产红色素的革兰氏阴性细菌RS.16S rDNA序列分析表明RS菌株为黏质沙雷氏菌Serratiamarcescens.RS菌株不同于其它大多黏质沙雷氏菌,在37℃仍然产生红色素.HPLC检测分析红色素为灵菌红素.[结论]本文首次报道从栖北散白蚁分离纯化一株产生灵菌红素的黏质沙雷氏RS菌,研究结果为灵菌红素的生产及散白蚁生物防治技术的研发提供了新的思路.     

栖热菌属高温菌RH99-02菌株产类胡萝卜素的研究

从腾冲热海分离到最高生长温度达80℃的一株嗜热非芽孢菌RH99-02菌株,经鉴定属Thermus属,对其所产类胡萝卜素进行了吸收光谱扫描及薄层层析分析。发酵研究表明振荡培养时的菌体生物量和色素产量上均高于静置培养,培养至50h菌体生物量和色素产量达到最大值,菌体色素产量为762μg/g干重。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

桔小实蝇对敌百虫抗性稳定性及再增长趋势

通过室内试验,研究了用LC₅₀及LC₉₀两种浓度敌百虫汰选14代建立的不同程度高抗性水平的桔小实蝇Bactrocera dorsalis抗性品系TrR₅₀和TrR₉₀在停止使用药剂后的抗性衰退规律,以及抗性衰退到原有水平的1/3时再用药汰选的抗性再增长规律。结果表明：两抗性品系对敌百虫抗性均不稳定,但抗性衰退速率不同,TrR₉₀完全隔离药剂4代后,抗性已衰退至原有水平的1/3,而TrR₅₀完全隔离药剂7代后,抗性才衰退至原有水平的1/3,但随后两品系抗性衰退均减缓,到19代后抗性均还处于低水平抗性阶段,无法衰退至敏感水平。通过对其抗性衰退趋势进行方程拟合,结果表明两品系抗性衰退均符合S型曲线模型。抗性再增长试验结果表明：桔小实蝇两抗性品系对敌百虫抗性再增长趋势不同,TrR₅₀继续使用药剂汰选,经过6代选育,抗性迅速上升,接近原有的抗性水平,随后保持平稳增长; 而TrR₉₀继续使用药剂汰选,经过9代选育,抗性才迅速上升,经过12代选育,抗性才接近原有的抗性水平。通过对两品系抗性再增长趋势进行方程拟合,结果表明两品系抗性再增长趋势不同,TrR₅₀品系抗性再增长趋势符合逻辑斯蒂模型,而TrR₉₀品系则符合二次曲线模型。     

Polyamine analysis for chemotaxonomy of thermophilic eubacteria: Polyamine distribution profiles within the orders Aquificales, Thermotogales, Thermodesulfobacteriales, Thermales, Thermoanaerobacteriales, Clostridiales and Bacillales

Cellular polyamines of 45 thermophilic and 8 related mesophilic eubacteria were investigated by HPLC and GC analyses for the thermophilic and chemotaxonomic significance of polyamine distribution profiles. Spermidine and a quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, were the major polyamine in Thermocrinis, Hydrogenobacter, Hydrogenobaculum, Aquifex, Persephonella, Sulfurihydrogenibium, Hydrogenothermus, Balnearium and Thermovibrio, located in the order Aquificales. Thermodesulfobacterium and Thermodesulfatator belonging to the order Thermodesulfobacteriales contained another quaternary penta-amine, N4-bis(aminopropyl)spermidine. In the order Thermotogales, Thermotoga contained spermidine, norspermidine, caldopentamine and homocaldopentamine. The latter two linear penta-amines were not found in Marinitoga and Petrotoga. In the order Thermales, Thermus and Marinithermus contained homospermidine, norspermine and the linear penta-amines. Meiothermus lacked penta-amines. Vulcanithermus contained linear penta-amines and hexa-amines but not homospermidine. Oceanithermus contained spermine alone. Within the order Thermoanaerobacteriales, the two quaternary branched penta-amines were found in Thermanaeromonas and Thermoanaerobacter. Caldanaerobacter contained N4-bis(aminopropyl)spermidine. Thermoanaerobacterium lacked penta-amines. Thermaerobacter of the order Clostridiales contained N4-bis(aminopropyl)spermidine and agmatine. Thermosyntropha, Thermanaerovibrio, Thermobrachium ( the order Clostridiales), Sulfobacillus, Alicyclobacillus, Anoxybacillus, Ureibacillus, Thermicanus ( the order Bacillales), Desulfotomaculum, Desulfitobacterium and Pelotomaculum (the family Peptococcaceae) ubiquitously contained spermine. Some thermophiles of Bacillales added linear and branched penta-amines.     

Effects of an improved biomechanical backpack strap design on load transfer to the shoulder soft tissues

The aim of the present study was to characterize shoulder strap structure and mechanical properties that may alleviate strains and stresses in the soft tissues of the shoulder. Utilizing a finite element model of the shoulder constructed from a single subject, we have quantified skin stresses exerted by backpack straps and the strains at the subclavian artery (SCA). For this end, standard shape straps with stiffness of 0.5, 1.2, and 5 MPa, were compared to the effects of optimized straps; a double-layered (soft outer layer and reinforced internal supporting layer) and newly-designed anatomically-shaped strap. Compared to the standard 0.5 MPa strap, the 5 MPa strap resulted in 4-times lower SCA strains and 2-times lower Trapezius stresses. The double-layered strap resulted in 40% and 50% reduction in SCA strains and skin stresses, respectively, with respect to the softer strap. The newly-designed anatomical strap exerted 4-times lower SCA strains and 50% lower skin stresses compared to the standard strap. This demonstrates a substantial improvement to the load carriage ergonomics when using a composite anatomical strap.     

Comparative study of nickel toxicity to growth and photosynthesis in nickel-resistant and -sensitive strains of Scenedesmus acutus f. alternans (Chlorophyceae)

Nickel (Ni) toxicity to growth and photosynthesis was studied in four strains of Scenedesmus acutus f. alternans. Effects of Ni dosage and duration of exposure on growth and photosynthesis were strain specific. Large differences in responses of both growth and photosynthesis to Ni were detected between three resistant strains (B4, Cu-Tol, and Ni-Tol) and one sensitive strain (UTEX 72). Growth of UTEX 72 was 18 times more sensitive to Ni than those of the three resistant strains. The order of Ni dosages (fmol Ni/pg cell dry weight) causing 50% inhibition (D₁₅₀) of growth rates in the four strains was Ni-Tol (10.5) > B4 (8.19) > Cu-Tol (4.60) > UTEX 72 (0.25). The effect of Ni dosage on photosynthetic rate as percentage of control corresponded to a saturation curve and was a strong function of duration of exposure. The DI₅₀s of photosynthetic rates were 3.5 times lower in UTEX 72 than in the three resistant strains, and in all four strains they decreased sharply with the increase in duration of exposure. The order of the four strains in DI₅₀s of photosynthetic rate was B4 (58.2) > Cu-Tol (38.0) > Ni-Tol (28.9) > UTEX 72 (8.24) for 6-h exposure and Ni-Tol (2.88) > Cu-Tol (1.30) > B4 (1.01) > UTEX 72 (0.15) for 24-h exposure. The DI₅₀s of photosynthetic rate for 6-h exposure were higher than those of growth rate in all four strains, and for 24-h exposure they were lower, except in UTEX 72. Thus, the relative Ni sensitivity of growth and photosynthesis of the four strains depends on the duration of exposure. The results of factorial analysis of variance suggested that Ni toxicity to photosynthesis is a consequence of a strong interaction among strain, Ni dosage, and duration of exposure.     

The role of nonhomologous end-joining components in telomere metabolism in Kluyveromyces lactis

The relationship between telomeres and nonhomologous end-joining (NHEJ) is paradoxical, as NHEJ proteins are part of the telomere cap, which serves to differentiate telomeres from DNA double-strand breaks. We explored these contradictory functions for NHEJ proteins by investigating their role in Kluyveromyces lactis telomere metabolism. The ter1-4LBsr allele of the TER1 gene resulted in the introduction of sequence altered telomeric repeats and subsequent telomere-telomere fusions (T-TFs). In this background, Lig4 and Ku80 were necessary for T-TFs to form. Nej1, essential for NHEJ at internal positions, was not. Hence, T-TF formation was mediated by an unusual NHEJ mechanism. Rad50 and mre11 strains exhibited stable short telomeres, suggesting that Rad50 and Mre11 were required for telomerase recruitment. Introduction of the ter1-4LBsr allele into these strains failed to result in telomere elongation as normally observed with the ter1-4LBsr allele. Thus, the role of Rad50 and Mre11 in the formation of T-TFs was unclear. Furthermore, rad50 and mre11 mutants had highly increased subtelomeric recombination rates, while ku80 and lig4 mutants displayed moderate increases. Ku80 mutant strains also contained extended single-stranded 3' telomeric overhangs. We concluded that NHEJ proteins have multiple roles at telomeres, mediating fusions of mutant telomeres and ensuring end protection of normal telomeres.     

Structure of shigella antigens. Heterogeneity of specific polysaccharides of 2 Shigella flexneri strains and 2 Sh. flexneri/Escherichia coli hybrids]

The S-specific polysaccharide from 2 Sh. flexneri wild strains (with serological var. X- and var. Y-specificity, respectively) and 2 Sh. flexneri E. coli hybrids (with the same specificities) can be separated by means of gel chromatography on Sephadex G-200 and G-50 into altogether 6 fractions per strain. Fraction G-200/1 (molecular weight greater than 10(6)D) represents a polymer consisting nearly exclusively of glucose and is present mainly in the two Y-type strains, much less in the two X-type strains. Fractions G-200/2 and G-200/3 (molecular weight approximately 10(5)D and approximately 2 - 10(4)D, respectively) seem to consist mainly of the S-specific side chains while fraction G-50/2 (molecular weight approximately 2000 D) presumably contains an SR-polysaccharide (core with one repeating unit.) Fraction G-50/3 (molecular weight approximately 100 D) contains the core polysaccharide and fraction G-50/4 splitting products (mainly KDO). No significant differences in chromatographical behaviour and quantitative composition could be found between the polysaccharides of the wild strains and the hybrid strains. Because of the well-known stability of the glucosaminyl linkages the sugar analysis was not only performed after acidic hydrolysis. In some cases the acid hydrolysate was reacted with HNO2 to cleave the glucosaminyl linkages. In most cases the values obtaines now were higher than those obtained directly.     

cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.     

Comparative activity of tobramycin and gentamicin against Pseudomonas,Proteus and Providencia species.

Tobramycin is a new antibiotic resembling gentamicin. We measured the minimal inhibitory concentrations of these two antibiotics against five bacterial species that cause hospital-acquired infections and are resistant to many presently available antibiotics. The organisms tested were 500 strains of Pseudomones aeruginosa, 100 strains of each of Proteus rettgeri and Pr. morganii, 50 strains of Pr. vulgaris and 250 strains of Providencia stuartii. Tobramycin was 2 to 4 times more active than gentamicin against Ps. aeruginosa; all except 6 of 70 strains resistant to 4 mug/ml of gentamicin were sensitive to 4 mug/ml of tobramycin. The two antibiotics showed a similar degree of activity against the other four species. Tobramycin promises to be of particular value in the treatment of Pseudomonas infections.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Antibiotic resistance of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> strains isolated from captive snakes

The minimum inhibitory concentrations (MICs) of 24 antibiotics were determined for 45 Stenotrophomonas maltophilia strains by the microdilution method at 37 and 30 °C (after 24 h and 48 h of incubation). The isolates were obtained from mouth swabs and pus of 116 captive snakes whereas the identical strains (based on PFGE) of the same origin were discarded. At 37 °C, the isolates showed a low frequency of resistance to levofloxacin (0 and 8.9 % of resistant strains after 24 and 48 h, MICs₅₀ 0.5 and 1 mg/L, MICs₉₀ 1 and 2 mg/L) and cotrimoxazole (2.2 % of resistant strains for 24 and 48 h, MICs₅₀ 4 mg/L for both time periods, MICs₉₀ 4 and 8). At 30 °C, the most effective drugs were also cotrimoxazole (2.2 and 6.7 %, MICs₅₀ 4 and 8, MICs₉₀ 8 and 32) and levofloxacin (8.9 and 46.7 %, MICs₅₀ 1 and 2, MICs₉₀ 2 and 4). The isolates were either identically or more susceptible to antibiotics than strains acquired from patients hospitalized at Olomouc University Hospital (the same region) with the exception of ciprofloxacin, cefoperazone, cefoperazone/sulbactam and ceftazidime.