Laemmli C M, Leveau J H, Zehnder A J, van der Meer J R
Swiss Federal Institute for Environmental Science and Technology and Swiss Federal Institute for Technology, CH-8600 Dübendorf, Switzerland.
J Bacteriol. 2000 Aug;182(15):4165-72. doi: 10.1128/JB.182.15.4165-4172.2000.
Within the 5.9-kb DNA region between the tfdR and tfdK 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: tfdD(II)C(II)E(II)F(II) and tfdB(II) (in short, the tfd(II) cluster), by analogy to tfdCDEF and tfdB (the tfd(I) 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, tfdD(II), suggesting an operon-like organization for the tfd(II) genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD(II) coded for a chloromuconate cycloisomerase, tfdC(II) coded for a chlorocatechol 1, 2-dioxygenase, tfdE(II) coded for a dienelactone hydrolase, tfdF(II) coded for a maleylacetate reductase, and tfdB(II) coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfd(I) and tfd(II) genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd(II) genes seem to be redundant with respect to those of the tfd(I) cluster. One reason why the tfd(II) genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS.
在来自嗜麦芽窄食单胞菌JMP134的2,4-二氯苯氧基乙酸（2,4-D）分解代谢质粒pJP4上，tfdR和tfdK基因之间的5.9千碱基DNA区域内，我们鉴定出五个开放阅读框（ORF），它们与pJP4上其他位置已存在的氯儿茶酚和氯酚代谢基因（tfdCDEF和tfdB）具有显著同源性。这五个ORF的组织和命名如下：tfdD(II)C(II)E(II)F(II)和tfdB(II)（简称为tfd(II)簇），类似于tfdCDEF和tfdB（tfd(I)簇）。对从以2,4-D培养的嗜麦芽窄食单胞菌JMP134中分离的mRNA进行引物延伸分析，在该簇的第一个基因tfdD(II)之前鉴定出一个单一的转录起始位点，这表明tfd(II)基因具有操纵子样的组织形式。通过在大肠杆菌中表达每个ORF，我们证实tfdD(II)编码氯粘康酸环异构酶，tfdC(II)编码氯儿茶酚1,2-双加氧酶，tfdE(II)编码二烯内酯水解酶，tfdF(II)编码马来酰乙酸还原酶，tfdB(II)编码氯酚羟化酶。从嗜麦芽窄食单胞菌JMP134中分离的mRNA的斑点杂交表明，tfd(I)和tfd(II)基因在2,4-D诱导下均被转录。因此，tfd(II)基因编码的功能相对于tfd(I)簇的功能似乎是冗余的。tfd(II)基因未从质粒pJP4上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。
