Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons
Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons
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DOI：
10.1128/aem.64.6.2086-2093.1998
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发表时间：
1998-06
影响因子：
4.4
通讯作者：
A. Hübner;C. E. Danganan;L. Xun;A. Chakrabarty;W. Hendrickson
中科院分区：
文献类型：
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作者：
A. Hübner;C. E. Danganan;L. Xun;A. Chakrabarty;W. Hendrickson
摘要 洋葱伯克霍尔德菌 AC1100 使用氯化芳香族化合物 2,4,5-三氯苯氧基乙酸 (2,4,5-T) 作为碳和能量的唯一来源。将途径中第一个中间体 2,4,5-三氯苯酚转化为 5-氯氢醌的酶已被纯化，由 58 和 22 kDa 的两个亚基组成，由 tftC 和 tftD 基因编码 (48)。从 58-kDa 多肽的 N 末端设计简并引物，并用于从 AC1100 基因组文库中分离含有 tftC 和 tftD 基因的克隆。 tftC和tftD的衍生氨基酸序列与皮氏伯克霍尔德氏菌的双组分单加氧酶HadA、大肠杆菌的HpaBC和肺炎克雷伯菌的HpaAH显示出显着的同源性。如 RNA 槽印迹和引物延伸分析所示，当 tftC 和 tftD 基因在 2,4,5-T 存在下生长时，它们的表达似乎被诱导。三组克隆的 tft 基因被用作探针来探索该通路的基因组组织。 B. 全染色体的脉冲场凝胶电泳分析。 cepacia AC1100 证明基因组由 4.0、2.7、0.53、0.34 和 0.15 Mbp 的 5 个复制子组成，分别指定为 I 至 V。 tft 基因位于较小的复制子上：tftAB 簇位于复制子 IV 上，tftEFGH 位于复制子 III 上，并且在复制子 III 和 IV 上都发现了 tftC 和 tftCD 操纵子的副本。当细胞在缺乏 2,4,5-T 的情况下生长时，基因会因染色体缺失和重排而频繁丢失，从而产生 2,4,5-T 阴性突变体。在一种突变体中，tftA 和 tftB 基因从一个复制子易位到另一个复制子，同时丢失 tftEFGH 和一个 tftCD 拷贝。
ABSTRACT Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftDgenes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences oftftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH ofKlebsiella pneumonia. Expression of the tftCand tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes ofB. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of thetftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, thetftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGHand one copy of tftCD.