《微生物学杂志》2003年总目录

题目·研究报告·大肠杆菌murB和birA基因的转录通读现象单组分妥布霉素产生菌的快速筛选部分肝病患者和健康人血清中HBV基因型分析类胡萝卜素产生菌№5205的分类鉴定CpG ODNs对肿瘤细胞端粒酶活性及细胞周期和凋亡的影响     

黑暗链霉菌中tbmA基因的功能研究

PCR获得tbmA基因内部863 bp片段,构建基因阻断穿梭载体pSPU112-1,经接合转移导入Strepto-myces tenebrariusH6,筛选单交换阻断变株,并用Southern blot验证阻断变株的tbmA已经被破坏。经发酵产物分析,阻断变株不再合成氨甲酰妥布霉素,只合成安普霉素。首次从分子水平证明了tbmA只参与氨甲酰妥布霉素生物合成,而不参与安普霉素的生物合成。     

大肠埃希菌和肺炎克雷伯菌对三种氨基糖苷类抗生素的耐药性分析

【摘 要】 目的 评价庆大霉素、妥布霉素及阿米卡星三种氨基糖苷类抗生素（AGs）对大肠埃希菌和肺炎克雷伯菌的体外抗菌活性。方法 对902株大肠埃希菌和404株肺炎克雷伯菌,采用VITEK-2全自动微生物分析仪配套的AST-GN13药敏卡进行庆大霉素、妥布霉素及阿米卡星的体外药敏试验。结果 大肠埃希菌和肺炎克雷伯菌的产超广谱β-内酰胺酶（ESBLs）菌株检出率分别为40.8％和36.6％;产ESBLs的大肠埃希菌对庆大霉素、妥布霉素及阿米卡星的敏感率分别为42.4%、39.1%和96.5%,与非产ESBLs菌株比较,敏感率差异均有统计学意义（P<0.01）;产ESBLs的肺炎克雷伯菌对庆大霉素、妥布霉素及阿米卡星的敏感率分别为64.2%、62.8%和91.9%,与非产ESBLs菌株比较,敏感率差异均有统计学意义（P<0.01）;阿米卡星对产与非产ESBLs的大肠埃希菌和肺炎克雷伯菌均高度敏感,敏感率均在91%以上。结论 本地区大肠埃希菌和肺炎克雷伯菌产ESBLs菌株流行严重,ESBLs的产生可使大肠埃希菌和肺炎克雷伯菌对AGs的耐药情况加重,提示ESBLs和AGs引起的耐药可能存在一定的相关性。     

激光与抗性突变筛选技术相结合选育抗肿瘤抗生素博安霉素高产菌株

目的:获得博安霉素高产菌株,同时比较了铜蒸汽激光与妥布霉素抗性及二者复合诱变的选育效果。方法:采用铜蒸汽激光辐照30 min与妥布霉素100 r/m L抗性处理及其复合诱变选育博安霉素产生菌轮枝链霉菌(S.verticillus)B-31。结果:在复合诱变组中,获得一株高产突变株GB-160,经发酵罐应用后,发酵单位较出发菌株提高1.5倍,并且遗传性能稳定。结论:该方法能有效获得抗生素高产优质菌株。在医药生物工程中,具有较高的实用价值,为其它药物微生物选育提供借鉴。     

微生物比浊法测定妥布霉素地塞米松眼膏的效价

目的:分析微生物比浊法在测定妥布霉素地塞米松眼膏的效价。方法:分别使用三种方式测定妥布霉素地塞米松眼膏的含量,分别是微生物比浊法、管碟法以及高效相色谱法。结果:妥布霉素效价测定的线性区间在0.4至1.0U·mL-1之间,RDS为0.96%。结论:微生物比浊法的操作相对便捷,测量的结果也更为准确,能够广泛使用在产品的质量控制操作上。     

一种妥布拉霉素产生菌初筛方法的建立

从金黄色葡萄球菌１００１出发,选育到一株对妥布拉霉素和卡那霉素耐药而对阿泊拉霉素敏感的突变株１００１－１１。以金黄色葡萄球菌１００１－１１和对上述三种抗生素均敏感的枯草杆菌６３５０１为测定菌,建立了一种能同时检测妥布拉霉素产生菌菌落琼脂柱总效价和组分相对含量的初筛方法。     

服务行业健康人群携带沙门氏菌、志贺氏菌调查

为了解莱芜市饮食服务行业人员沙门氏菌、志贺氏菌携带情况 ,于 1998～ 2 0 0 0年对莱芜市饮食服务行业从业人员健康查体。用肛拭子法进行增菌分离培养鉴定 ,检出 1株格洛斯特鲁普沙门氏菌 (新型 ) ,8株福氏志贺Ⅱ型及鲍氏志贺氏Ⅱ型。用K B法做了药物敏感实验。 1株沙门氏菌、8株志贺氏菌均对妥布霉素、卡那霉素、丁氨卡那霉素高敏 ,对多粘菌素、青霉素、链霉素中度敏感 ,对红霉素低敏。     

妥布霉素产生菌激光育种的研究

本文首次报道铜蒸气激光选育妥布霉素产生菌—黑暗链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｔｅｎｅｂｒａｒｉｕｓ）的研究结果。在相同实验条件下,铜蒸气激光辐照黑暗链霉菌比其随后又以氯化锂复合处理的选育效果好。在铜蒸气激光辐照后,曾获得实验高产株,发酵单位比对照未辐照组提高７２％。     

309株铜绿假单胞菌的分布及耐药性

目的分析铜绿假单胞菌的耐药特点,为临床合理选药提供依据。方法对首都医科大学附属北京潞河医院2014年1月至12月分离的铜绿假单胞菌,采用全自动细菌鉴定仪,用微量稀释法进行药敏试验,并用WHONET 5.6软件对药敏结果进行统计分析。结果 309株铜绿假单胞菌对哌拉西林、哌拉西林/他唑巴坦、阿米卡星、妥布霉素的敏感性菌大于90.0%,对头孢吡肟、庆大霉素的敏感性分别为84.8%、84.5%,对亚胺培南、美罗培南的敏感率为71.5%、74.1%,对氨曲南的耐药率达78.0%;标本来源主要来自呼吸道标本,其次为分泌物标本、尿液标本;科室主要以呼吸科、神经外科、ICU为主。结论对铜绿假单胞菌感染应优先选择哌拉西林、哌拉西林/他唑巴坦、阿米卡星、妥布霉素等,临床应限制氨曲南的使用。     

产生妥布拉霉素的ER-16突变株的选育

以妥布拉霉素产生菌暗黑链霉菌AS 4.1098(410-Ⅱ)为出发菌株,经高温和亚硝基胍处理,获得6株无色突变株,对其中的W1028-M5用亚硝基胍及甲基磺酸乙酯继续处理,得到突变株E228,再经抗自身代谢终产物妥布拉霉素抗性株的选育,得到ER-16和ER-21等高产菌株。所产抗生素仅含两个组份,即氨甲酰妥布拉霉素和阿普拉霉素。不再含氨甲酰卡那霉素。测定的几项主要理化性质与出发菌株以及文献报道的数据完全一致。     

Engineering Streptomyces tenebrarius to synthesize single component of carbamoyl tobramycin

Aims: To engineer Streptomyces tenebrarius for producing carbamoyl tobramycin as a main component. Methods and Results: The aprH‐M gene fragment (apramycin biosynthetic gene from GenBank) in S. tenebrarius Tt49 was knocked out by genetic engineering to form S. tenebrarius T106 (△aprH‐M). Compared to the wild‐type strain, mutant strain T106 (△aprH‐M) no longer produced apramycin, while mainly synthesize carbamoyl tobramycin. TLC and HPLC‐MS analyses indicated that the mutant strain significantly increased the production of carbamoyl tobramycin. Conclusions: The metabolic flow for the apramycin and its analogues biosynthesis was blocked by disrupting the aprH‐M gene clusters. The aprH‐M gene clusters might be essential for the biosynthesis of apramycin. The mutant strain T106 mainly synthesized carbamoyl tobramycin. Significance and Impact of Study: The mutant T106 mainly produces carbamoyl tobramycin without synthesizing apramycin, which will reduce cost of postextraction from fermentation products. Therefore, it has good prospects for industrial application.     

Resistance to apramycin in Escherichia coli isolated from animals: detection of a novel aminoglycoside-modifying enzyme

The mechanisms of resistance to apramycin of five isolates of Escherichia coli from animals were investigated. Three isolates, which were resistant to all the aminoglycosides tested, did not transfer their resistance and did not produce aminoglycoside-modifying enzymes. The fourth isolate, which was resistant to apramycin, tobramycin, gentamicin, kanamycin and neomycin but not to amikacin, owed its resistance to production of the acetyltransferase AAC(3)IV. The gene specifying this enzyme was carried on a transposon, Tn800, on a plasmid designated R1535. The fifth isolate was resistant to apramycin, neomycin and kanamycin but not to gentamicin, tobramycin or amikacin. It produced an acetyltransferase that readily acetylated only apramycin, neomycin and paromomycin, a compound that is closely related to neomycin. Synthesis of this enzyme was specified by a chromosomal gene located near pyrD at about 20 min on the map of the E. coli K12 chromosome.     

Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7’-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.     

Fermentative hydrolysate of mycelial waste of aminoglycoside antibiotics production as component of culture media used in biosynthesis of tobramycin]

New nutrient media for cultivation of the tobramycin-producing organism were developed. As an additional source of nitrogen the media contain fermentative hydrolysate of the mycelial waste of manufacture of aminoglycoside antibiotics (tobramycin and apramycin). The use of the media provided a 20 to 50% decrease of consumption of soybean meal, an essential food raw material, and design of a low-waste technology for biosynthesis of tobramycin.     

链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.     

Identification and Functional Analysis of dTDP-Glucose-4,6-Dehydratase Gene and Its Linked Gene Cluster in an Aminoglycoside Antibiotics Producer of <Emphasis Type="Italic">Streptomyces tenebrarius</Emphasis> H6

Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B. Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S. tenebrarius H6 genomic DNA. With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated. DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF. In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S. griseus. Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv. citri str. 306 and glycosyltransferase from Pseudomonas putida KT2440. Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S. griseus. The function of the orfE gene was studied by targeted gene disruption. The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S. tenebrarius H6.     

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Method for selection of transposable DNA and characterization of a new insertion sequence, IS493, from Streptomyces lividans.

A method to select for transposable elements from Streptomyces spp. by using insertional inactivation of a repressor gene that functions in Escherichia coli was developed. Plasmid pCZA126, which can replicate in Streptomyces spp. or E. coli, contains a gene coding for the lambda cI857 repressor and a gene, under repressor control, coding for apramycin resistance. E. coli cells containing the plasmid are apramycin sensitive but become apramycin resistant if the cI857 repressor gene is disrupted. Plasmids propagated in Streptomyces spp. can be screened for transposable elements that have disrupted the cI857 gene by transforming E. coli cells to apramycin resistance. This method was used to isolate a new 1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2. IS493 duplicated host DNA at the target site, had inverted repeats at its ends, and contained two tandem open reading frames on each strand. IS493 was present in three copies in the same genomic locations in several S. lividans strains. Two of the copies appeared to be present in regions of similar DNA context that extended at least 11.5 kilobases. Several other Streptomyces spp. did not appear to contain copies of IS493.     

Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.