Schools of Biological Sciences and Research Center for Biomolecules and Biosystems, Chung-Ang University, Seoul 156-756, Republic of Korea.
Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.
Microbiology (Reading). 2011 Oct;157(Pt 10):2891-2903. doi: 10.1099/mic.0.049387-0. Epub 2011 Jul 7.
Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36-39 kDa, and their structures were deduced to be dimeric. The K(m) values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.
萘降解菌 CJ2 是一株能够以 gentisate 途径代谢萘的细菌，最近的研究表明，在一个先前未被描述的萘降解基因簇内，该菌携带了第三份 gentisate 1,2-双加氧酶（GDO）的编码基因 nagI3。通过记录调控、转录和酶活性的模式，研究了该基因簇（尤其是 nagI3）在 CJ2 菌株萘代谢中的作用。对野生型细胞的转录分析表明，该第三簇是多顺反子的，并且 nagI3 以相对较高的水平表达。构建了所有三个 nagI 基因的单个缺失突变体，并评估了它们对 GDO 活性和细胞生长的影响。在三个缺失突变株中，CJ2ΔnagI3 的 GDO 活性严重降低，并且在芳香族底物上生长最慢。这些观察结果与 nagI3 可能防止萘降解菌 CJ2 细胞中积累有毒的 gentisate 的假说一致。从菌株 CJ2 中克隆了所有三个 nagI 基因到大肠杆菌中：nagI2 和 nagI3 基因成功过表达。GDO 的亚基质量约为 36-39 kDa，其结构被推断为二聚体。NagI2 和 NagI3 的 K(m)值分别为 31 和 10 µM，表明 NagI3 对 gentisate 的更高亲和力可能使野生型细胞免受 gentisate 的毒性。这些结果为解释为什么第三个基因簇，特别是 nagI3 基因，在 CJ2 菌株中很重要提供了线索。第三个基因簇中的基因组织与 Polaromonas sp. JS666 和 Leptothrix cholodnii SP-6 中的基因簇相匹配。虽然水平基因转移（HGT）是解释这种遗传模式的一个假设，但在祖先谱系内的基因复制同样有效。通过注意到 CJ2 菌株 nagI3 等位基因与 Polaromonas sp. JS666 和 L. cholodnii SP-6 中的同源物没有高度序列同一性，从而否定了 HGT 假说。
