Schlüter Andreas, Krahn Irene, Kollin Florian, Bönemann Gabriele, Stiens Michael, Szczepanowski Rafael, Schneiker Susanne, Pühler Alfred
Fakultät für Biologie, Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany.
Appl Environ Microbiol. 2007 Oct;73(20):6345-50. doi: 10.1128/AEM.01177-07. Epub 2007 Aug 3.
Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.
采用基于转化的方法，从污水处理厂活性污泥隔室中的细菌中分离出质粒pGNB1。这个60kb的质粒赋予对三苯甲烷染料结晶紫的抗性，并使其宿主细菌能够使结晶紫脱色。对pGNB1的部分测序表明，其骨架与先前测序的IncP-1β质粒非常相似。对该质粒的两个辅助区域进行了全序列测定，一个位于复制起始基因trfA的下游，另一个位于接合转移模块Tra和Trb之间。辅助区域L1包含一个与Tn5501相关的转座子和一个编码功能未知的Cupin 2保守桶状蛋白的基因。在辅助区域L2中鉴定出三苯甲烷还原酶基因tmr和一个被IS1071及另一个假定插入元件侧翼的截短的二氢硫辛酰胺脱氢酶基因。pGNB1的tmr基因亚克隆表明，该基因负责观察到的结晶紫抗性表型，并介导三苯甲烷染料结晶紫、孔雀石绿和碱性品红的脱色。质粒pGNB1及其相关表型可转移至α-变形菌苜蓿中华根瘤菌和γ-变形菌大肠杆菌。这是首次报道从污水处理厂细菌群落中分离出的一种携带三苯甲烷还原酶基因的IncP-1β接合性质粒。本文讨论了pGNB1编码的酶活性在三苯甲烷染料污染污水生物修复方面的应用。
