恶臭假单胞菌SJTE-1中氧化17β-雌二醇的17β-羟甾类脱氢酶2及其转录调控因子AraC的鉴定

[目的]假单胞菌SJTE-1可高效转化17β-雌二醇,但其代谢机制尚不清楚。本文鉴定和表征了该菌株中参与雌二醇降解与调控过程的17β-羟甾类脱氢酶2(17β-HSD2)和转录调控因子AraC。[方法]我们通过荧光定量PCR分析了17β-hsd2和araC的转录水平;我们在大肠杆菌BL21(DE3)菌株中异源表达了17β-HSD2和AraC基因,并利用金属离子亲和层析法纯化获得了重组蛋白;我们体外表征了17β-HSD2的酶学性质,利用高效液相色谱鉴定了其产物;通过电泳迁移转移法和DNase酶I足迹试验,我们鉴定了重组蛋白AraC的结合能力与结合位点。[结果]17β-HSD2和AraC可被17β-雌二醇诱导表达;蛋白序列比对结果表明17β-HSD2含有短链脱氢酶/还原酶(SDR)和β-羟甾类脱氢酶的保守结构与残基。该酶以NAD+为辅助因子,在C17位点氧化17β-雌二醇,其Km值为0.082 mmol/L,Vmax值为56.26±0.02μmol/(min·mg);5 min内可转化97.4%以上的雌二醇。转录调控因子AraC可直接结合17β-hsd2基因启动子区的特异位点;雌二醇与雌酮可解除这一结合,启动17β-hsd2基因转录;过表达AraC蛋白可抑制17β-hsd2的转录。[结论]假单胞菌SJTE-1的17β-羟甾类脱氢酶2可高效催化17β-雌二醇转化,并受到转录因子AraC的直接调控。本工作可推进细菌的雌激素降解酶学机制与调控网络研究。     

Isolation and Characterization of a Mutant of Arthrobacter sp. Strain GLP-1 Which Utilizes the Herbicide Glyphosate as Its Sole Source of Phosphorus and Nitrogen

Arthrobacter sp. strain GLP-1, grown on glucose as a carbon source, utilizes the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus as well as its sole source of nitrogen. The mutant strain GLP-1/Nit-1 utilizes glyphosate as its sole source of nitrogen as well. In strain GLP-1, P_i was a potent competitive inhibitor of glyphosate uptake (K_i, 24 μM), while the affinity of P_i for the uptake system of strain GLP-1/Nit-1 was reduced by 2 orders of magnitude (K_i, 2.3 mM). It is concluded that the inability of strain GLP-1 to utilize glyphosate as a source of nitrogen is due to the stringent control of glyphosate uptake by excess phosphate released during the degradation of the herbicide.     

Multiplication of a DNA fragment in Streptomyces antibioticus--producer of oleandomycin

Two strains of Str. antibioticus producing oleandomycin were studied. Strain 326 was obtained from the initial laboratory strain and strain 1607 from strain 326 as a result of multistage selection aimed at increasing the antibiotic production capacity. Extrachromosomal ring DNA could be identified in strain 1607. The identified DNA was designated as eSA1 or extrachromosomal element of Streptomyces antibioticus 1. The molecular weight of this DNA is 21.3 Md. It is represented by 1 copy per chromosome. No eSA1 was isolated from strain 326 at CsCl-EtBr gradient. Hybridization studies according to Southern with the use as a probe of 32-eSA1 DNA showed that strain 326 contained 1 copy of eSA1 per chromosome in the integrated state. The hybridization studies, electron microscopy and analysis of the total DNA in CsC1-EtBr gradient showed that eSA1 in strain 1607 was tandemly multireplicated in the chromosome content. In the autonomous state its number was approximately equal to 1 copy per chromosome. The presence of eSA1 in strain 1607 in the autonomous state probably results from its segregation during homologous recombination due to tandem multireplication. The data are indicative of multiplication in strain 1607 of the chromosome fragment 23.3 Md in size. It is suggested that an increase in the oleandomycin production capacity of strain 1607 is associated with multiplication of the DNA fragment (eSA1) containing the genes determining production of the antibiotic.     

Isolation and characterization of a thermotolerant bacterium Ralstonia sp. strain PHS1 that degrades benzene, toluene, ethylbenzene, and o-xylene

A thermotolerant bacterium, designated as PHS1, was isolated from a hot spring in Pohang, Korea, on the basis of its ability to grow on benzene, toluene, ethylbenzene, and xylenes (BTEX) as a sole carbon source. Strain PHS1 is a gram-negative, rod-shaped aerobe and grows optimally at 42 degrees C and pH 7.2. According to 16 S rDNA analysis, strain PHS1 showed highest similarity to Ralstonia eutropha (previously named Alcaligenes eutrophus). Unlike its closest known Ralstonia species, however, strain PHS1 was able to utilize toluene, ethylbenzene, o-xylene, and both m- and o-cresol. The degradation of o-xylene by strain PHS1 is particularly important, since o-xylene is a compound of considerable environmental interest, owing to its recalcitrance; and very few microorganisms have been reported to utilize o-xylene as a sole carbon source. It was found that strain PHS1 transformed o-xylene to 2,3-dimethylphenol through direct oxygenation of the aromatic ring. The unique properties of strain PHS1, such as thermotolerance and the ability to degrade o-xylene, may have important implications for the treatment of BTEX-contaminated industrial effluents.     

HSV-1 HF株扩增子载体的构建及其在不同HSV血清型间的通用性研究

旨在构建HSV-1HF株的扩增子载体,研究其在不同血清型HSV辅助下的包装通用性。经酶切HF株的BAC-HSV-1,获得oriS和pac元件并测序。以pSilencer2.0-U6为骨架,以DsRed为报告基因构建HSV-1HF株的扩增子载体,利用脂质体2000转染扩增子载体至Vero细胞,分别应用HSV-1HF株和HSV-2HG52辅助HSV-1扩增子载体进行包装,待产生细胞病变效应后取上清,再次感染Vero细胞,观察Vero细胞内红色荧光蛋白表达情况。本研究首次构建了HSV-1HF株的扩增子载体,鉴定了HSV-1HF株oriS和pac元件,HSV-1HF株扩增子载体可以被HSV-1HF株和HSV-2HG52株包装并扩增。     

蜡状芽孢杆菌菌株Jp-A的分离鉴定及其降解苯酚特性

从某钢铁厂处理废水的活性污泥中驯化分离一株能高效降解苯酚的细菌（Jp-A）.通过形态观察、生理生化实验和16srRNA序列分析,初步鉴定Jp A为蜡状芽孢杆菌（Bacillus cereus）.在实验条件下,该菌在16、24和32 h内能将浓度分别为5、10和15 mmol·L^-1的苯酚完全降解,而30 mmol·L^-1的苯酚则完全抑制该菌的生长.该菌也能以甲苯、氯酚类和硝基酚类等芳香烃类物质作为唯一碳源和能源生长.双加氧酶检测表明,其通过间位途径开环裂解苯酚,该途径的关键酶邻苯二酚2,3 双加氧酶主要定位在细胞膜上,为诱导酶,补加葡萄糖能抑制该酶的产生.     

Genome Sequence of Pseudomonas aeruginosa Strain SJTD-1, a Bacterium Capable of Degrading Long-Chain Alkanes and Crude Oil

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.     

溃烂症眼斑拟石首鱼分离的灿烂弧菌的鉴定

摘要：【目的】从患溃烂症的眼斑拟石首鱼分离到优势菌株M-1,并进一步对该菌的系统发育学进行了分析。【方法】采用形态学、生理生化鉴定,结合16S rRNA和HSP60基因序列分析。【结果】16S rRNA基因同源性分析,菌株M-1与灿烂弧菌(AJ874361、AY046955)聚为一群;HSP60基因(groES)序列分析表明M-1与弧菌(EU653883、AY837440)聚为一个分支。【结论】菌株M-1属于灿烂弧菌(Vibrio splendidus)。     

超声波诱变选育乳链菌肽(Nisin)高产菌株

目的：从乳酸乳球菌中选育Nisin高产菌株。方法：利用超声波对乳酸乳球菌进行诱变,并用琼脂扩散法检测其效价。结果：获得一突变菌株S-1,该菌株的生物效价为343.53IU/mL,比原始菌株提高31.52%。结论：突变株S-1遗传性能稳定,为进一步菌种选育奠定基础。     

彭泽鲫卵源致病性水霉的鉴定及其生物学特性

从患病的彭泽鲫卵上分离3株丝状真菌,经人工感染试验证实其中1株丝状真菌JL1对彭泽鲫卵具有致病性,并进一步研究了其形态与生长特性,开展了ITS rDNA序列分析。实验结果表明,菌株JL1菌丝为透明管状结构,中间无横隔,分枝较少;游动孢子囊多数呈棒状,游动孢子呈多排排列,发育成熟后从孢子囊中释放出来,并迅速游离;藏卵器呈球形,与雄器同枝或异枝。菌株JL1的ITS rDNA序列与GenBank基因库中水霉属菌株自然聚类,同源性高达99%,与Saprolegnia sp.H(登录号:EF460351)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株JL1为水霉菌(Saprolegnia sp.)。此外,菌株JL1在5°C-30°C、pH 4-11范围内均能生长,最适生长温度和pH范围分别为25°C-30°C和6-9。同时菌株JL1对NaCl敏感,质量分数为2%的NaCl即可抑制其生长,可以作为该病防治的依据。     

In vitro and in vivo transferrable beta-lactam resistance due to a new plasmid-mediated oxyiminocephalosporinase from a clinical isolate of Proteus mirabilis

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.     

一株硅酸盐细菌的分离及解钾活性研究

目的利用硅酸盐细菌分离培养基,从昆明白泥山土壤样品中分离获得一株硅酸盐细菌——BN1。方法对分离获得的硅酸盐细菌——BM进行革兰染色、生理生化特征和16SrDNA测序分析,并对其解钾活性进行了初步研究。结果BN1初步鉴定为类芽胞杆菌属的菌株。结论该菌株——BN1对云母具有较强的解钾活性,为空白对照组的1．79倍。     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

尼古丁降解菌SCUEC1菌株的分离及其降解基因

【目的】分离并鉴定1株具有尼古丁降解能力的细菌,研究其尼古丁降解特性并对其降解基因进行分析,为尼古丁微生物降解提供基础。【方法】从烟草种植地土壤中分离1株具有尼古丁降解能力的细菌,通过16S r RNA基因和生理生化特性对该菌株进行鉴定,检测该菌株尼古丁降解率与生长量的关系,并进一步对该菌株进行尼古丁浓度耐受性测定,采用高通量测序技术对菌株进行全基因组测序,BLAST比对分析尼古丁降解相关基因。【结果】筛选到1株具有尼古丁降解能力的细菌,经鉴定命名为根癌土壤杆菌(Agrobacterium tumerficience)SCUEC1菌株,根癌土壤杆菌SCUEC1菌株尼古丁降解率可达到94.81%,该菌株在尼古丁浓度为0.50–5.00 g/L范围内生长良好且有较高的尼古丁降解能力。对根癌土壤杆菌SCUEC1菌株全基因组序列进行BLAST比对分析,推测该菌株的尼古丁降解代谢途径与中间苍白杆菌SYJ1菌株的尼古丁降解途径相似。【结论】本研究揭示了Agrobacterium tumerficienceSCUEC1菌株具备尼古丁降解特性,初步推测出尼古丁降解相关基因和降解代谢途径,为尼古丁微生物降解提供基础。     

拮抗菌SB1的鉴定及其抗菌物质的分析

对番茄根系菌株SB1的抗菌活性进行测定,结果表明该菌株对多种植物病原真菌、细菌具有明显的抑制作用,表现出广谱抗菌活性。通过菌体形态、生理生化反应及16SrDNA序列分析,鉴定菌株SB1为枯草芽孢杆菌内生亚种。以青枯雷尔氏菌为指示菌,测定了菌株SB1抗菌物质的理化性质及组成。结果表明,其抗菌物质表现出良好的热稳定性、水溶性和醇溶性,且对紫外线照射和蛋白酶K处理不敏感。高效液相色谱分析结果进一步显示菌株SB1的抗菌物质中含有抗菌肽Surfactin。     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

一株糖脂表面活性剂产生菌的筛选及干酪根降解

【目的】从油页岩环境中筛选可降解油页岩干酪根的产生物表面活性剂菌株。【方法】从抚顺油页岩矿废水样品中用血平板法初筛,排油圈法、乳化法和表面张力法复筛,获得产生物表面活性剂菌株。对目标菌株进行生理生化鉴定、16S r RNA基因序列和系统发育分析,用薄层色谱鉴定其发酵液表面活性成分,优化产表面活性剂的培养条件,初步考察其对油页岩干酪根的降解能力。【结果】筛选到一株产糖脂表面活性剂菌株B-1,初步鉴定为Pseudomonas sp.,该菌株有良好的排油和乳化能力以及较低的表面张力,可利用烷烃、不饱和脂肪酸和糖类作为碳源。在30-34°C范围内添加0.3%Na Cl的葡萄糖培养基(p H 7.0)中该菌生长旺盛,发酵液表面张力最低为27 m N/m。菌株B-1在添加一定量葡萄糖的无机盐培养基中作用30 d后对干酪根的降解率为2.85%,高于不添加葡萄糖无机盐培养基对照组的降解率(1.04%)。【结论】菌株B-1是一株性能良好的产糖脂表面活性剂细菌,有降解干酪根的潜力。     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that impaired citrate transport into the cell is a contributory factor to extracellular citrate accumulation by a strain of Candida guilliermondii

Candida guilliermondii IMK 1 is a mutant strain that accumulates concentrations of citric acid approximately seven times higher than does its parent strain, NRRL Y-448. In contrast to its parent, strain IMK 1 cannot use citrate as the sole carbon source, or assimilate citrate in the presence of glucose. Measurements of membrane ATPase activity show consistently lower values in the mutant strain than in its parent. It is suggested that failure to re-assimilate the excreted citrate is a major contributory factor in the extracellular accumulation of high citrate concentrations, and that this failure is related to a defect in the membrane structure. Correspondence to: N. A. Gutierrez