Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge
Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge
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DOI：
10.1007/s004380051191
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发表时间：
2000-04-01
期刊：
影响因子：
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通讯作者：
Selbitschka, W
中科院分区：
文献类型：
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作者：
Dröge, M;Pühler, A;Selbitschka, W
为了从活性污泥中发现的细菌群落中分离抗生素抗性质粒，将用绿色荧光蛋白作为识别标记标记的3-氯苯甲酸降解菌株假单胞菌B13的衍生物用作过滤杂交的受体。在含有3-氯苯甲酸盐作为唯一碳源和抗生素四环素、链霉素或壮观霉素的琼脂平板上选择接合子，并以每个受体10（-5）至10（-8）的频率回收接合子。共鉴定出12种不同的质粒，命名为pB 1-pB 12。它们的大小在41至69 kb之间，它们赋予宿主各种抗生素抗性模式。其中两个质粒pB 10和pB 11也介导了对无机汞的抗性。12个质粒中的7个被鉴定为广泛宿主范围的质粒，在过滤杂交中显示出极高的转移频率，范围为每个受体细胞10（-1）至10（-2）。12个质粒中的10个属于IncP不相容组，基于使用IncP组特异性PCR引物的复制子分型。PCR扩增产物的DNA测序进一步揭示了12个质粒中的8个属于IncP β亚组，而两个质粒被鉴定为IncP α质粒。IncP特异性PCR产物的分析揭示了IncP β质粒在DNA序列水平上的相当大的差异。为了表征IncP质粒的基因“负载”，克隆了限制性片段并建立了它们的DNA序列。这些片段编码的推定蛋白质的显着多样性进行了鉴定。除了转座酶和蛋白质参与抗生素耐药性，两个推定的DNA转化酶属于Din家族，甲基转移酶的I型限制/修饰系统，超氧化物歧化酶。鉴定了属于RND家族的推定外排系统的部分和未知功能的蛋白质。
In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncP beta subgroup, whereas two plasmids were identified as IncP alpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncP beta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase. parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.