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Identification of genes induced under anaerobic benzene-oxidizing conditions in Dechloromonas aromatica strain RCB.

机译：鉴定厌氧苯氧化条件下诱导的芳香十氯单胞菌菌株RCB中的基因。

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摘要

Benzene (C6H6) is the simplest member of the aromatic hydrocarbon group of chemical compounds. Minute amounts of benzene are naturally released into the environment during volcanic eruptions and forest fires. This extremely stable aromatic compound is also an important industrial chemical and is an integral component of many petroleum products. In fact, benzene is amongst the top 20 in production volume for chemicals produced in the United States. Therefore, it is not surprising that the major reason for environmental contamination through benzene is by anthropogenic sources. Benzene is relatively soluble in water and migrates very quickly in the soil after its entry. The Environmental Protection Agency (EPA) has classified benzene as a Class A carcinogen.;Microorganisms play an integral role in the natural attenuation of benzene from the environment. Biodegradation of benzene by oxidation can occur under aerobic, anaerobic and microaerophilic conditions. Biooxidation of benzene under aerobic conditions is well-studied. However, oxygen is scarce in contaminated subsurface environments, and after the aerobic breakdown of benzene, oxygen is quickly depleted from the most heavily contaminated regions leading to the development of extensive anaerobic zones. As a result, there is increased focus on anaerobic benzene degradation as a potential bioremediation technique in anoxic subsurface environments.;In aerobic and microaerophilic environments, monooxygenase and dioxygenase enzyme systems have been established to be involved in the breakdown of the benzene ring. However, the genes and enzymes involved in anaerobic benzene oxidation pathway are still unknown. In the present study, Dechloromonas aromatica strain RCB, capable of benzene oxidation with nitrate as the electron acceptor, was used as a model system to investigate the initial steps of the anaerobic benzene oxidation pathway. Strain RCB is capable of completely mineralizing benzene to carbon dioxide in denitrifying conditions.;RNA-arbitrarily primed polymerase chain reaction (RAP-PCR), a differential gene expression technique used to randomly reverse-transcribe RNA into cDNA, was conducted to identify genes exclusively expressed during nitrate-dependent benzene oxidation. A total of seven genes were identified as differentially expressed in the presence of benzene using the RAP-PCR approach. Four differentially expressed genes were confirmed by a second method, semiquantitative reverse transcriptase PCR (RT-PCR). Microarray analysis was the second expression analysis technique conducted to identify genes expressed during benzene-oxidizing conditions. Based on fold induction and potential function, six genes were selected from the microarray data and their differential expression was confirmed by using semiquantitative RT-PCR. Interestingly, Daro1556, encoding a hypothetical protein, was identified by both RAP-PCR and microarray analysis. In order to verify the functions of the genes (selected from RAP-PCR and microarray analysis) in nitrate-dependent benzene oxidation, six deletion mutants were constructed in which the target gene was replaced by a tetracycline cassette. The correct insertion of the tetracycline cassette in the mutant genome was confirmed by PCR and Southern blotting.;Microarray results were further analyzed by using an unsupervised clustering approach, k-means. A couple of genes (Daro1358 and Daro1359) obtained from cluster analysis were also verified by semiquantitative RT-PCR. These two genes, part of the same operon, encode a two-component monooxygenase system, which is a member of the Rieske non-heme iron aromatic ring-hydroxylating oxygenase family of proteins. In the present investigation, for the first time, involvement of a monooxygenase system (Daro1358 and Daro1359) during benzene oxidation with nitrate reduction was observed. Based on the results obtained from k-means cluster analysis, a model was hypothesized for anaerobic benzene oxidation with nitrate as the electron acceptor in Dechloromonas aromatica strain RCB.
机译：苯（C6H6）是化合物芳烃基团中最简单的成员。在火山喷发和森林火灾期间，微量苯自然释放到环境中。这种极其稳定的芳族化合物也是重要的工业化学品，并且是许多石油产品的组成部分。实际上，苯是美国生产的化学品的前20大产量之一。因此，通过苯污染环境的主要原因是人为来源就不足为奇了。苯相对易溶于水，进入后在土壤中迁移非常快。环境保护署（EPA）已将苯分类为A类致癌物。微生物在环境中自然降解苯中起着不可或缺的作用。苯的氧化可在好氧，厌氧和微需氧条件下进行生物降解。苯在好氧条件下的生物氧化作用得到了很好的研究。但是，在受污染的地下环境中氧气稀缺，苯的好氧分解后，氧气从最受污染最严重的区域迅速消耗掉，从而形成了广阔的厌氧区。结果，人们越来越关注厌氧苯降解作为缺氧地下环境中潜在的生物修复技术。;在好氧和微需氧环境中，已经建立了单加氧酶和双加氧酶系统参与苯环的分解。然而，涉及厌氧苯氧化途径的基因和酶仍是未知的。在本研究中，以能够将硝酸盐作为电子受体的苯氧化的芳香族十氯单胞菌RCB用作模型系统，以研究厌氧苯氧化途径的初始步骤。 RCB菌株能够在反硝化条件下将苯完全矿化为二氧化碳。RNA随机引发的聚合酶链反应（RAP-PCR）是一种用于将RNA随机反转录为cDNA的差异基因表达技术，专门用于鉴定基因在硝酸盐依赖性苯氧化过程中表达。使用RAP-PCR方法，共鉴定出七个基因在苯存在下差异表达。通过第二种方法，即半定量逆转录酶PCR（RT-PCR），确认了四个差异表达的基因。微阵列分析是第二种表达分析技术，用于鉴定在苯氧化条件下表达的基因。基于折叠诱导和潜在功能，从微阵列数据中选择了六个基因，并通过半定量RT-PCR证实了它们的差异表达。有趣的是，通过RAP-PCR和微阵列分析均可鉴定出编码假定蛋白的Daro1556。为了验证该基因在硝酸盐依赖性苯氧化中的功能（选自RAP-PCR和微阵列分析），构建了六个缺失突变体，其中目标基因被四环素盒替代。通过PCR和Southern印迹证实四环素盒在突变基因组中的正确插入。；使用无监督聚类方法k-means进一步分析了微阵列结果。还通过半定量RT-PCR验证了从聚类分析获得的两个基因（Daro1358和Daro1359）。这两个基因是同一操纵子的一部分，编码一个双组分单加氧酶系统，该系统是Rieske非血红素铁芳香环羟化加氧酶家族的成员。在本研究中，首次观察到单加氧酶系统（Daro1358和Daro1359）在苯氧化过程中发生硝酸盐还原。根据从k均值聚类分析获得的结果，假设建立了一个模型，用于在芳香族十氯单胞菌RCB中以硝酸盐为电子受体的厌氧苯氧化。

著录项

  • 作者

    Gon, Rikhi.;

  • 作者单位

    Southern Illinois University at Carbondale.;

  • 授予单位 Southern Illinois University at Carbondale.;
  • 学科 Biology Molecular.;Biology Microbiology.;Biology Bioinformatics.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 164 p.
  • 总页数 164
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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