Metabolic diversity in the chemoautotrophic Riftia pachyptila symbiont

在东太平洋的热液喷口区，巨大的深海管虫裂谷虫（Riftia pachyptila）与化学自养细菌以高度专门化的单特异性共生生活。虽然成虫没有消化系统，但它们的硫化物氧化内共生体可以从热液环境中固定二氧化碳，从而为宿主提供有机碳。反过来，细菌从蠕虫体内高而稳定的底物浓度中受益。最近对未培养的共生体的宏基因组进行了测序，为深入的基因组和代谢分析提供了基础。先前的全球蛋白质组学分析揭示了多种替代代谢途径，这些途径在同一宿主的共生体群体中同时表达，但似乎实现了冗余反应，或者甚至可能在相反的方向上起作用。这些结果有力地表明，在含有细菌的宿主组织（滋养体）内存在单个细菌亚群。在滋养体小叶内，细胞内共生体经历了一个分化过程：它们从小的、分裂的杆状体发展到小的球菌，最后发展到大的球菌。这些个体形态很可能与不同的代谢策略相吻合，并可能解释观察到的代谢多样性。拟议的项目将在三个子任务中解决这一假设：(I)形态型富集：为了验证细菌细胞周期阶段是否代表代谢不同的共生亚群，将通过梯度离心富集各自的形态型，并进行全面和比较的蛋白质组学分析。（II）蛋白定位：此外，参与潜在冗余或拮抗途径的关键酶将通过免疫组织化学定位于滋养体小叶切片。（III）能量限制：为了检查共生体对能量限制的反应，并确定个体亚群的限制特异性代谢模式之间的潜在差异，将进行比较蛋白质组学分析。富硫（富能量）滋养体组织的富形态组分和缺硫（缺能量）滋养体组织的富形态组分将进行比较。富硫和贫硫滋养体的全组织定量蛋白质组学分析将补充研究。

Along hydrothermal vent areas of the Eastern Pacific, the giant deep sea tube worm Riftia pachyptila lives in a highly specialized monospecific symbiosis with chemoautotrophic bacteria. While adult worms lack a digestive system, their sulfide-oxidizing endosymbionts fix CO2 from the hydrothermal environment and thus supply the host with organic carbon. In turn, the bacteria benefit from high and steady substrate concentrations inside the worm. The metagenome of the uncultured symbionts was recently sequenced and provides the basis for in-depth genomic and metabolic analyses. Previous global proteomic profiling revealed a variety of alternative metabolic pathways that were simultaneously expressed in the symbiont population of the same host, but which appear to fulfill redundant reactions, or which might even function in opposing directions. These results strongly suggest the existence of individual bacterial subpopulations inside the bacteria-containing host tissue, the trophosome. Within the trophosome lobules, the intracellular symbionts undergo a differentiation process: They develop from small, dividing rods to small cocci, and finally to large cocci. These individual morphotypes very likely coincide with different metabolic strategies and might explain the observed metabolic diversity. The proposed project will address this hypothesis in three subtasks: (I) Morphotype enrichment: To verify whether the bacterial cell cycle stages represent metabolically distinct symbiont subpopulations, the respective morphotypes will be enriched by gradient centrifugation and subjected to a comprehensive and comparative proteomic analysis. (II) Protein localization: Additionally, key enzymes involved in potentially redundant or antagonistic pathways will be localized in trophosome lobule sections by immunohistochemistry. (III) Energy limitation: To examine the symbionts response to energy limitation and to identify potential differences between limitation-specific metabolic patterns of the individual subpopulations, a comparative proteomic analysis will be performed. Enriched morphotype fractions from sulfur-rich (energy-rich) trophosome tissue and fractions from sulfur-depleted (energy-depleted) trophosomes will be compared. A whole-tissue quantitative proteomic analysis of sulfur-rich and of sulfur-depleted trophosome will complement the study.