Szczepanowski Rafael, Krahn Irene, Linke Burkhard, Goesmann Alexander, Pühler Alfred, Schlüter Andreas
Fakultät für Biologie, Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany.
Center for Biotechnology (CeBiTec), Bioinformatics Resource Facility, Universität Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany.
Microbiology (Reading). 2004 Nov;150(Pt 11):3613-3630. doi: 10.1099/mic.0.27317-0.
Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.
通过基于转化的方法，从一家污水处理厂的活性污泥细菌群落中分离出了10种赋予高水平红霉素抗性的不同抗生素抗性质粒。其中一种质粒，命名为pRSB101，介导对四环素、红霉素、罗红霉素、磺胺类药物、头孢菌素、壮观霉素、链霉素、甲氧苄啶、萘啶酸和低浓度诺氟沙星的抗性。质粒pRSB101已完成全序列测定和注释。其大小为47829 bp。pRSB101复制/分配（rep/par）模块与植物病原体柑桔溃疡病菌的pXAC33复制子之间存在保守的共线性。pRSB101的第二个主干模块编码一个具有与IncQ样质粒同源性的三Mob蛋白型移动（mob）系统。质粒pRSB101在IncP-1α质粒RP4的帮助下可移动，RP4提供反式转移功能。pRSB101上一个20 kb的抗性区域位于一个含整合子的Tn402样转座子内。1类整合子的可变区携带二氢叶酸还原酶基因dhfr1、壮观霉素/链霉素腺苷酸转移酶基因aadA2和一种迄今未知的安布勒A类超广谱β-内酰胺酶基因bla(TLA-2)。整合子特异性3'-片段（qacEDelta1-sul1-orf5Delta）与一个由mph(A)（大环内酯2'-磷酸转移酶I）、mrx（功能未知的疏水蛋白）和mphR(A)（调节蛋白）基因组成的大环内酯抗性操纵子相连。最后，在Tn402特异性转座酶基因tniA上游鉴定出一个带有四环素抗性基因tetA（四环素外排泵）和tetR的假定移动元件。pRSB101上的第二个“遗传负荷”区域包含四个不同的移动遗传元件、另一个属于新类别的整合子以及另外两个转座元件的足迹。在复制/分配模块和移动模块之间编码了一个由ATP结合盒（ABC）型ATP酶和通透酶以及RND家族的外排膜融合蛋白（MFP）组成的三方多药（MDR）转运蛋白。在从活性污泥细菌中分离出的其他8种红霉素抗性质粒上检测到了大环内酯抗性基因mph(A)、mrx和mphR(A)的同源物。从污水处理厂最终出水的质粒DNA制剂中也获得了pRSB101样repA扩增子，表明pRSB101样质粒随最终出水释放到环境中。
