The Subcellular Proteome of a Planctomycetes Bacterium Shows that Newly Evolved Proteins have Distinct Biochemical Characteristics

The Planctomycetes bacteria have unique cell architectures with heavily invaginated membranes as confirmed in this study by three-dimensional models reconstructed from FIB-SEM images of Tuwongella immobilis and Gemmata obscuriglobus. The subcellular proteome of T. immobilis was examined by differential solubilisation followed by LC-MS/MS analysis, which identified 1569 proteins in total. The Tris-soluble fraction contained mostly cytoplasmic proteins, while inner and outer membrane proteins were found in the Triton X-100 and SDS-soluble fractions, respectively. For comparison, the subcellular proteome of Escherichia coli was also examined using the same methodology. A notable difference in the overall fractionation pattern of the two species was a 5-fold higher number of cytoplasmic proteins in the SDS-soluble fraction in T. immobilis. One category of such proteins is represented by innovations in the Planctomycetes lineage, including unique sets of serine/threonine kinases and extracytoplasmic sigma factors with WD40 repeat domains for which no homologs are present in E. coli. Other such proteins are members of recently expanded protein families in which the newly evolved paralog with a new domain structure is recovered from the SDS-soluble fraction, while other paralogs may have similar domain structures and fractionation patterns as the single homolog in E. coli. The expanded protein families in T. immobilis include enzymes involved in replication-repair processes as well as in ribosomal and tRNA modification and degradation. These results show that paralogization and domain shuffling have yielded new proteins with distinct biochemical characteristics. Understanding the molecular intricacies of these adaptive changes might aid in the development of a model for the evolution of cellular complexity.

本研究通过对固定化土旺氏菌（Tuwongella immobilis）和隐球出芽菌（Gemmata obscuriglobus）的聚焦离子束扫描电镜（FIB-SEM）图像进行三维重构，证实浮霉菌门（Planctomycetes）细菌具有独特的细胞结构，其细胞膜存在大量内陷。本研究采用差异溶解法结合液相色谱-串联质谱（LC-MS/MS）分析，对固定化土旺氏菌的亚细胞蛋白质组进行了检测，共鉴定出1569种蛋白质。其中，三羟甲基氨基甲烷（Tris）可溶组分主要包含细胞质蛋白，而曲拉通X-100（Triton X-100）和十二烷基硫酸钠（SDS）可溶组分则分别富集了内膜蛋白与外膜蛋白。为便于对比，本研究采用相同实验方法对大肠杆菌（Escherichia coli）的亚细胞蛋白质组进行了检测。两个物种的整体分级分离模式存在显著差异：固定化土旺氏菌的SDS可溶组分中，细胞质蛋白的数量是大肠杆菌的5倍。这类差异蛋白可分为两类：一类属于浮霉菌门谱系特有的创新蛋白，包括独特的丝氨酸/苏氨酸激酶组以及带有WD40重复结构域的胞外σ因子，这类蛋白在大肠杆菌中无同源蛋白；另一类属于近期扩增的蛋白质家族成员，其中新演化出的旁系同源蛋白具有全新的结构域组成，可从SDS可溶组分中分离得到，而其余旁系同源蛋白的结构域组成与分级分离模式，则与大肠杆菌的单一同源蛋白相似。固定化土旺氏菌中扩增的蛋白质家族包括参与复制修复过程、核糖体及转运RNA（tRNA）修饰与降解的酶类。上述结果表明，旁系同源化与结构域洗牌过程催生了具有独特生化特性的新型蛋白质。阐明这些适应性变化的分子细节，或有助于构建细胞复杂性演化的理论模型。