Zhong Wen-Hui, Sun Ming, He Guo-Qing, Feng Xiao-Shan, Yu Zi-Niu
College of Chemistry and Environmental Science, Nanjing Normal University, Nanjing 210097, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Mar;20(2):209-14.
2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
2,4-二氯苯酚是一种有毒且难生物降解的有机污染物。从几家2,4-二氯苯酚生产工厂排水区域采集的土壤样本中分离出一株能降解2,4-二氯苯酚的细菌菌株GT241-1，经鉴定为假单胞菌属。菌株GT241-1具有很强的2,4-二氯苯酚降解能力，在25 - 30摄氏度下，48小时内可分解90 mg/L的2,4-二氯苯酚中的91%，并且能够利用2,4-二氯苯酚、2,4-二氯苯氧乙酸（2,4-D）、苯甲酸盐和儿茶酚作为唯一碳源和能源。Southern杂交显示，菌株GT241-1的2,4-二氯苯酚羟化酶基因（dcpA）位于约10kb的EcoR I/Xba I片段上。回收该片段，连接到载体pUC19上，并转化到大肠杆菌DH5α中。通过斑点杂交从约1200个转化子中获得了一个目标转化子Z539。PCR和测序结果表明，完整的dcpA基因包含在pZ539的10kb EcoR I /Xba I片段中。该片段经HindmIII酶切后缩短至约2.4kb。将缩短后的片段亚克隆到载体pRSET-B中，得到转化子BS1-12。对亚克隆片段进行测序。测序结果表明，包含dcpA的亚克隆片段全长2389bp，编码区核苷酸跨度为276至2072位（1797bp），起始密码子为ATG，终止密码子为TAA。dcpA的序列分析以及由dcpA推导的氨基酸序列分析表明，它们与GenBank中登记的相关序列不同。亚克隆片段携带dcpA的启动子，这可从dcpA编码区上游长度为275bp这一事实推断出来，并通过2,4-二氯苯酚羟化酶活性测定结果进一步得到证实。检测了转化子Z539和BS1-12的2,4-二氯苯酚羟化酶活性，结果表明这些转化子具有2,4-二氯苯酚羟化酶活性。相比之下，这些转化子的活性低于菌株GT241-1。
