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首页> 外文期刊>Applied Microbiology >Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1
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Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

机译：Nat样双加氧酶基因通过内部启动子在斯氏假单胞菌ZWLR2-1的2-氯硝基苯分解代谢基因簇中的组成性表达

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The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment.IMPORTANCE The release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory networks for the catabolism of synthetic compounds. The 2CNB utilizer Pseudomonas stutzeri ZWLR2-1 in this study has adapted itself to the new pollutant by evolving the always-inducible Nag-like dioxygenase into a constitutively expressed enzyme, and its expression has escaped the influence of salicylate. This may facilitate an understanding of how bacteria can rapidly adapt to the new synthetic compounds by evolving its expression system for key enzymes involved in the degradation of a xenobiotic.
机译：斯氏假单胞菌ZWLR2-1中编码2-氯硝基苯（2CNB）分解代谢途径的基因簇是Nag样双加氧酶（来自Ralstonia sp.U2菌株的萘双加氧酶NagAaAbAcAd家族的双加氧酶）基因簇和氯邻苯二酚的拼凑装配分解代谢簇。然而，通常存在于Nag样双加氧酶基因簇中的转录调节基因缺失，因此不清楚该簇如何表达。在这里研究了2CNB分解代谢簇的表达模式。结果表明，该表达是组成性的，而不是由其底物2CNB或水杨酸盐诱导的，而2CNB或水杨酸盐是Nag样双加氧酶家族中常见的表达诱导剂。逆转录-PCR表明存在至少一个包含所有2CNB降解结构基因的转录物。在基因簇中验证的三个启动子中，P1充当整个分解代谢操纵子的启动子，但内部启动子P2和P3也增强了下游基因的转录。 P3启动子，以前没有定义为启动子序列，是这三个启动子中最强的。它驱动了编码双加氧酶的cnbAcAd的表达，该酶催化2CNB分解代谢途径中的初始反应。生物信息学和突变分析表明，该P3启动子是通过复制18 bp的片段并引入额外的132 bp的片段而进化而来的。这些化学物质会生长。细菌必须克服的问题之一是发展用于表达相应分解代谢基因的调节装置。考虑到2CNB是仅从合成化学开始才存在的异种生物，它可能是研究合成化合物分解代谢调控网络中快速进化基础的分子机制的一个很好的例子。本研究中的2CNB利用者斯氏假单胞菌ZWLR2-1通过将始终可诱导的Nag样双加氧酶进化为组成型表达的酶，使其自身适应了新污染物，并且其表达已摆脱了水杨酸酯的影响。通过进化其针对异种生物降解的关键酶的表达系统，这可能有助于了解细菌如何快速适应新的合成化合物。

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