Boon N, Goris J, De Vos P, Verstraete W, Top E M
Laboratory of Microbial Ecology and Technology, Ghent University, B-9000 Ghent, Belgium.
Appl Environ Microbiol. 2001 Mar;67(3):1107-15. doi: 10.1128/AEM.67.3.1107-1115.2001.
We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.
我们研究了能够代谢苯胺和3-氯苯胺（3-CA）的五株细菌中质粒以及参与苯胺氧化脱氨作用的tdnQ基因的多样性。之前已描述并鉴定出三株菌，即睾丸酮丛毛单胞菌I2、食酸戴尔福特菌CA28和BN3.1。本研究中，菌株LME1和B8c分别从利谷隆处理过的土壤和污水处理厂中分离得到，二者均被鉴定为食酸戴尔福特菌。戴尔福特菌属和丛毛单胞菌属均属于丛毛单胞菌科。所有五株菌都拥有一个约100 kb的大质粒，但只有四株菌的质粒能够通过以苯胺或3-CA作为唯一碳源和/或氮源进行筛选而转移至受体菌株。质粒转移实验和Southern杂交表明，菌株I2的质粒负责苯胺的完全降解，但不负责3-CA的降解，而菌株LME1和B8c的质粒仅负责苯胺的氧化脱氨作用。几个从菌株CA28获得质粒的接合子克隆表现出不同的降解能力：所有接合子都能利用苯胺作为氮源，而只有部分接合子能够使3-CA脱氨。对于所有这四个质粒，发现Tn5271的IS1071插入序列位于一个1.4 kb的限制性片段上，该片段也能与tdnQ探针杂交。这一结果表明该插入序列元件参与了苯胺降解基因在环境中的传播。通过使用恶臭假单胞菌UCC22的tdnQ基因特异性引物，利用变性梯度凝胶电泳（DGGE）检测了这五株菌中PCR扩增片段的多样性。通过DGGE可区分出tdnQ片段的三个不同聚类。测序数据表明，I2、LME1、B8c和CA28的tdnQ序列密切相关，而BN3.1和恶臭假单胞菌UCC22的tdnQ序列与其他序列的相似度仅约为83%。Northern杂交表明，tdnQ基因仅在有苯胺存在时转录，而在只有3-CA存在时不转录。
