外膜孔蛋白在蓝细菌铁吸收中的作用机理研究

Cyanobacteria are prokaryotic organisms which show similar cellular characteristics of Gram-negative bacteria but perform oxygen-evolution photosynthesis. They are globally important primary producers and dominate in many iron-limiting oceans. It has been reported that global primary productivity is limited by iron deficiency, and the ways cyanobacteria take up iron efficiently to meet their high iron demand are still unclear. Our recent studies found that TonB-ExbB-ExbD dependent transport system plays an important role in inorganic iron uptake of cyanobacteria. But the mutant without all the four TonB-dependent outer membrane transporters could still survive and showed 40% of iron uptake rate of the wild type, which strongly suggested that other iron transport systems exist in cyanobacterial outer membrane. By mutant screening, it has been found that a porin mutant showed evidently slow growth in iron deficiency compared to the wild type. It has been further identified that the porin has a protein-protein interaction with TonB. Cyanobacterial porins probably play very important roles in iron uptake, but the detailed mechanisms need to be clarified. This project is going to identify the roles of the cyanobacterial porins in iron uptake and molecular mechanisms by the methods of gene knockout and complementation, protein location and protein-protein interaction, protein structural analysis, trace metal clean technique, and isotopic tracer method, etc.. The information derived from these studies will illustrate the unique iron metabolisms of cyanobacteria and the reason of their global distribution.

蓝细菌（俗称蓝藻）具有革兰氏阴性菌特征，但执行植物型光合作用，对全球初级生产力具有重要贡献。研究表明，海洋缺铁是限制全球初级生产力的重要因素，蓝细菌如何适应缺铁环境并在海洋中广泛分布是当前研究热点和难点。申请人前期研究发现，TonB依赖的主动转运系统对蓝细菌外膜铁吸收起重要作用，但将该系统的四个外膜转运蛋白全部敲除，并不会导致细胞致死或完全丧失铁吸收能力，这意味着其它外膜铁转运系统的存在。对模式蓝细菌Synechocystis sp. PCC6803进行突变株筛选发现，外膜孔蛋白突变株表现出低铁敏感表型。蛋白互作实验表明，孔蛋白与TonB具有蛋白相互作用。蓝细菌孔蛋白是否在长期进化过程中形成高效铁吸收能力亟待深入研究。本项目拟通过基因敲除与互补、缺铁生理表型、同位素铁吸收速率、蛋白铁结合能力及结构特征、转录调控及蛋白相互作用等分析手段，揭示孔蛋白在蓝细菌铁吸收过程中的作用及转运机制。

蓝藻（蓝细菌）是海洋重要的初级生产者，它们的生物量受到表层海水铁浓度不足的限制。但是蓝藻在长期的进化适应过程中，形成了高效的铁获取策略，为其广泛分布于广阔的缺铁海洋奠定了基础。前期研究表明，蓝藻的外膜上具有两套铁转运途径，一套为TonB依赖的主动转运系统，一套为被动扩散途径。本项目在上一个基金项目(31470171)”蓝细菌TonB-ExbB-ExbD依赖的铁吸收机制研究”的基础上，系统地研究了蓝藻外膜孔蛋白在铁转运中的作用，取得的主要创新性结论如下：（1）孔蛋白的编码基因存在于所有已测序的蓝藻基因组中，是蓝藻保守的离子和小分子转运蛋白，不同于TonB依赖的主动转运系统，主要分布于铁浓度多变的近海和淡水蓝藻种类；（2）不同孔蛋白对底物具有偏好性，在集胞藻等很多种类中具有铁锰偏好性的外膜孔蛋白，这可能是蓝藻在蛋白底物方面对缺铁环境的适应性进化策略之一；（3）外膜上的主动转运系统和被动扩散途径在蓝藻铁丰富和铁限制条件下具有互补调控作用，在铁相对充足时主要依赖被动扩散获取铁，主动转运系统不表达或低表达，而当铁浓度不足时，诱导表达主动铁转运系统。本研究揭示了蓝藻保守的外膜孔蛋白在铁获取中的作用，为从本质上认识铁限制对海洋初级生产者的生态学影响提供了科学依据。