Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)
Characterization of a Second tfd Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in Ralstonia eutropha JMP134(pJP4)
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DOI：
10.1128/jb.182.15.4165-4172.2000
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发表时间：
2000-08
影响因子：
3.2
通讯作者：
Caroline M. Laemmli;J. Leveau;A. Zehnder;J. R. van der Meer
中科院分区：
文献类型：
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作者：
Caroline M. Laemmli;J. Leveau;A. Zehnder;J. R. van der Meer
在2，4-二氯苯氧基乙酸(2，4-D)降解质粒pJP4的tfdR和tfdK基因之间5.9kb的DNA区域内，我们鉴定了5个与pJP4上其他地方已存在的氯代辛酚和氯苯酚代谢基因(tfdCDEF和tfdB)有显著同源性的开放阅读框(ORF)。五个ORF的组织和分配如下：tfdDIICIIEIIFII和tfdBII(简称为fdII集群)，类比为tfdCDEF和tfdB(集群)。对从2，4-D培养的真核细菌JMP134中分离的mRNA进行的引物延伸分析表明，在该簇的第一个基因tfdDII的前面有一个单一的转录起始点，这表明tfdII基因是一个操纵子样的组织。通过在大肠杆菌中表达每个ORF，我们证实了tfdDII编码氯代牡丹酸环异构酶，tfdCII编码氯辛二酚1，2-双加氧酶，tfdEII编码双烯内酯水解酶，tfdFII编码马来酸乙酯还原酶，tfdBII编码氯苯酚羟基酶。斑点杂交结果表明，在2，4-D诱导下，TfdI和tfdII基因均有转录。因此，与tfdI簇相比，tfdII基因编码的功能似乎是多余的。TfdII基因没有从pJP4中消失的一个原因可能是需要保留2，4-D途径表达的调控基因tfdR和tfdS。
ABSTRACT Within the 5.9-kb DNA region between the tfdR andtfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows:tfdDIICIIEIIFIIand tfdBII (in short, thetfdII cluster), by analogy totfdCDEF and tfdB (thetfdI cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdDII, suggesting an operon-like organization for the tfdII genes. By expressing each ORF in Escherichia coli, we confirmed that tfdDII coded for a chloromuconate cycloisomerase, tfdCII coded for a chlorocatechol 1,2-dioxygenase, tfdEII coded for a dienelactone hydrolase, tfdFII coded for a maleylacetate reductase, and tfdBII coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that bothtfdI and tfdII genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfdII genes seem to be redundant with respect to those of the tfdI cluster. One reason why the tfdII genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR andtfdS.