Carboxysomes:The Role of Microcompartmentalization in Bacterial CO2 Fixation

所有的蓝藻和许多趋化自养细菌都含有多面体包涵体，称为羧酸体。这些微室完全由蛋白质组成，并充满1,5-二磷酸核酮糖羧化酶/加氧酶（RubisCO），被认为是碳浓缩机制（CCM）的一部分，有助于提高含有它们的生物体的二氧化碳固定效率。虽然羧基体对RubisCO的催化增强作用的分子机制尚不确定，但长期以来，CCM假说的原则是羧基体碳酸酐酶（CA）迅速将细胞质中的HCO3 -转化为RubisCO的底物CO2。该实验室最近的工作证实，羧基体外壳的一种蛋白质成分是一种新的CA，我们称之为EPSCOR-CA。它在羧酸体外壳中的定位表明，羧酸体的功能机制比标准CCM模型预测的更为复杂，可能表明微室在自养碳同化中的作用扩大。将结合生物化学和分子生物学方法来阐明构成其功能基础的羧基体的结构特征，进一步生物化学表征新发现的羧基体CA，并确定其与壳中其他蛋白质的相互作用。专性化学自养细菌Halothiobacillus neapolitanus将作为这些研究的模型系统，因为a)羧基体的均质制备可以从生物体中大量分离出来，b)其羧基体操纵子已经被克隆，表征并在大肠杆菌中表达，c)大量的羧基体突变体已经在H. neapolitanus中产生，d)细菌可以遗传操纵。e)在蓝藻和其他趋化自养生物物种中都观察到与假设的编码H. neapolitanus羧基体的操纵子同源的基因簇。考虑到含羧基体的生物对二氧化碳固定的巨大贡献及其代谢对全球碳循环的影响，所提出的工作的一个更广泛的含义将是阐明控制碳循环和影响天气和气候的最基本和最核心的生化过程之一。该PI将直接参与关键实验的进行，并将在研究的各个方面指导研究生和本科生的研究。参与这些项目的学生将获得生物化学和分子生物学研究技术的经验。

All cyanobacteria and many chemoautotrophic bacteria contain polyhedral inclusion bodies called carboxysomes. These microcompartments, which consist entirely of protein and are filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), are thought to be part of a carbon concentrating mechanism (CCM) that serves to enhance the CO2 fixation efficiency in organisms that contain them. Although the molecular mechanism by which the carboxysome provides a catalytic enhancement for RubisCO is not certain, it has long been a tenet of the CCM hypothesis that a carboxysomal carbonic anhydrase (CA) rapidly converts cytoplasmic HCO3 - into CO2, the substrate for RubisCO. Recent work from this laboratory established that a protein component of the carboxysomal shell is a novel CA that we have term EPSCOR-CA. Its localization in the carboxysomal shell suggests that carboxysomes function via a mechanism that is more complex than that predicted by the standard CCM model and might indicate an expanded role for the microcompartments in autotrophic carbon assimilation. Biochemical and molecular biological approaches will be combined to elucidate structural features of the carboxysome that form the basis for its function, further characterize the newly discovered carboxysomal CA biochemically and identify its interactions with other proteins in the shell. The obligate chemoautotrophic bacterium Halothiobacillus neapolitanus will serve as a model system for these studies since a) homogeneous preparations of carboxysomes can be isolated in large quantities from the organism, b) its carboxysome operon has been cloned, characterized and expressed in Escherichia coli, c) a substantial number of carboxysome mutants have been created in H. neapolitanus, d) the bacterium can be genetically manipulated, e) gene clusters homologous to the putative operon encoding H. neapolitanus carboxysomes have been observed in both cyanobacteria and other species of chemoautotrophs. Broader Impacts Given the large contribution of carboxysome-containing organisms towards CO2 fixation and the impact of their metabolism on the global carbon cycle, one broader implication of the work proposed will be the elucidation of one of the most basic and central biochemical processes that control the carbon cycle and influence weather and climate. The PI will be directly involved in conducting key experiments and will be the mentor for graduate and undergraduate student research in various aspects of the research. Students participating in these projects will gain experience in biochemical and molecular biological research techniques.