Transcriptional landscape of the cell cycle in a model thermoacidophilic archaeon reveals similarities to eukaryotes

The cell cycle is a series of events that occur from the moment of cell birth to subsequent cell division. In eukaryotes, the cell growth, genome replication, genome segregation and cytokinesis, are strictly coordinated, defining discrete cell cycle phases, whereas in bacteria, these key processes may occur concurrently. Thermoacidophilic archaea in the genus Saccharolobus follow a defined cell cycle program, with the first pre-replicative growth (G1) phase, followed by the chromosome replication (S) phase, the second growth (G2) phase, and rapid genome segregation (M) and cytokinesis (D) phases. However, whether other processes, such as metabolism, catabolism, protein translation, antiviral defense, etc., also occur at specific cell cycle phases, like in eukaryotes, or are active throughout the cell cycle, like in bacteria, remains unclear. To address these questions, we synchronized the cultures of S. islandicus and performed an in-depth transcriptomic analysis of samples enriched in cells undergoing the M-G1, S and G2 phases. Differential gene expression and consensus gene co-expression network analyses provided a holistic view of the unfolding of the S. islandicus cell cycle. In addition to the core transcriptome network, which is expressed throughout the cell cycle, we show that diverse metabolic pathways, protein synthesis, cell motility and even antiviral defense systems, are expressed in a cell cycle dependent fashion. Our data also refines the understanding of the processes previously known to be linked to cell cycle, such as DNA replication. We show that most DNA replication genes are expressed prior to the S phase, during the M-G1, whereas expression of the major chromatin genes, and accordingly, chromatinization are concomitant with replication. A machine learning-based classifier allowed defining sets of signature genes characteristic of each of the analyzed cell cycle phases, emphasizing transcriptional stratification of the phases. Signature genes are more conserved across Thermoproteota than non-signature genes and their peak of expression, especially for the M-G1 and G2 specific genes, matches that of their homologs in yeast. Collectively, our data expose the complexity of the S. islandicus cell cycle and suggest that it more closely resembles the cell cycle of eukaryotes than previously appreciated. Cells were plated on solid media and 15 single colonies were inoculated in liquid media. forming 15 cultures that represent completely independent biological replicates. The 15 biological replicates were grown in 3 different batches (5 cultures per batch), each batch was grown and synchronized at a different time (5 cultures at a time).

细胞周期（cell cycle）是指从细胞诞生至后续细胞分裂所发生的一系列事件。在真核生物（eukaryotes）中，细胞生长、基因组复制、基因组分离与胞质分裂过程受到严格协调，由此定义出离散的细胞周期时相；而在细菌（bacteria）中，这些关键过程可同时发生。隶属于硫化叶菌属（Saccharolobus）的嗜热嗜酸古菌（Thermoacidophilic archaea）遵循一套明确的细胞周期程序：首先为复制前生长（G1）期，随后是染色体复制（S）期、第二次生长（G2）期，以及快速基因组分离（M）期与胞质分裂（D）期。不过，诸如代谢、分解代谢、蛋白质翻译、抗病毒防御等其他过程，究竟是如真核生物般在特定细胞周期时相发生，还是如细菌般在整个细胞周期中持续活跃，目前仍不明确。

为解答上述问题，我们对冰岛硫化叶菌（S. islandicus）的培养物进行了同步化处理，并对富集于M-G1、S及G2期细胞的样本开展了深入的转录组学分析。差异基因表达分析与共识基因共表达网络分析，全面呈现了冰岛硫化叶菌细胞周期的动态展开过程。除了在整个细胞周期中均有表达的核心转录组网络外，我们的研究显示，多样的代谢通路、蛋白质合成、细胞运动甚至抗病毒防御系统，均以细胞周期依赖的方式进行表达。我们的数据还深化了对已知与细胞周期相关过程的认知，例如DNA复制：绝大多数DNA复制基因在S期之前的M-G1期即已表达，而主要染色质基因的表达及其伴随的染色质组装则与复制过程同步。基于机器学习（machine learning）的分类器可用于定义各分析细胞周期时相的特征基因集，进一步凸显了各时相的转录分层特征。特征基因在热变形菌门（Thermoproteota）相较于非特征基因更为保守，且它们的表达峰值（尤其是M-G1期与G2期特异性基因）与酵母中其同源基因的表达峰值相吻合。

综上，我们的研究揭示了冰岛硫化叶菌细胞周期的复杂性，并表明其细胞周期相较于此前认知，与真核生物的细胞周期更为相似。

研究中将细胞接种于固体培养基，随后挑取15个单菌落接种至液体培养基，由此获得15份完全独立的生物学重复培养物。这15份生物学重复被分为3批培养（每批5份培养物），每批均在不同时间开展培养与同步化操作，且每批次单次同步化5份培养物。