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首页> 美国卫生研究院文献>other >Unraveling RubisCO Form I and Form II Regulation in an Uncultured Organism from a Deep-Sea Hydrothermal Vent via Metagenomic and Mutagenesis Studies
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Unraveling RubisCO Form I and Form II Regulation in an Uncultured Organism from a Deep-Sea Hydrothermal Vent via Metagenomic and Mutagenesis Studies

机译：通过基因组和诱变研究从深海热液排放口中解开未培养生物中的RubisCO I型和II型法规。

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first major step of carbon fixation in the Calvin-Benson-Bassham (CBB) cycle. This autotrophic CO2 fixation cycle accounts for almost all the assimilated carbon on Earth. Due to the primary role that RubisCO plays in autotrophic carbon fixation, it is important to understand how its gene expression is regulated and the enzyme is activated. Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18′ S along the Mid-Atlantic Ridge. It is 13,046 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. In silico analyses uncovered several putative LysR binding sites and promoter structures. Functions of some of these DNA motifs were experimentally confirmed. For example, according to mobility shift assays LysR1’s binding ability to the intergenic region of lysR1 and cbbL appears to be intensified when CbbL or LysR2 are present. Binding of LysR2 upstream of cbbM appears to be intensified if CbbM is present. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR’s binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression. A model summarizes the findings and predicts putative interactions of the different proteins influencing RubisCO gene regulation and expression.
机译：核糖-1,5-二磷酸羧化酶/加氧酶（RubisCO）催化Calvin-Benson-Bassham（CBB）循环中碳固定的第一步。这种自养的二氧化碳固定循环几乎占了地球上所有被吸收的碳。由于RubisCO在自养碳固定中起主要作用，因此重要的是了解其基因表达如何被调节以及酶如何被激活。由于目前所有微生物大多数都不可培养，因此我们使用宏基因组学方法来鉴定与RubisCO表达相关的基因和酶。研究的宏基因组DNA片段源自中大西洋海脊8°18′S处的深海热液喷口场Nibelungen。它是13,046 bp，类似于Thiomicrospira crunogena的基因。该片段编码9个开放阅读框（ORF），其中包括两种类型的RubisCO：I型（CbbL / S）和II型（CbbM），两个LysR转录调节因子（LysR1和LysR2），两个von Willebrand因子A型（CbbO- m和CbbO-1），以及两个AAA + ATPase（CbbQ-m和CbbQ-1），预计将起RubisCO激活酶的作用。在计算机分析中发现了一些假定的LysR结合位点和启动子结构。这些DNA基序中的一些功能已通过实验得到确认。例如，根据迁移率变动分析，当存在CbbL或LysR2时，LysR1与lysR1和cbbL的基因间区域的结合能力似乎增强。如果存在CbbM，似乎会增强cbbM上游LysR2的结合。我们的研究表明CbbQ-m和CbbO-m激活CbbL，LysR1和LysR2蛋白促进CbbQ-m / CbbO-m表达。 CbbO-1似乎能激活CbbM，而CbbM本身似乎有助于增强LysR的结合能力，从而增强其自身的转录调控。此外，CbbM似乎会损害cbbL的表达。一个模型总结了这些发现，并预测了影响RubisCO基因调控和表达的不同蛋白质的推定相互作用。

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