A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea [RNA-seq]

Cell cycle regulation is of paramount importance for all forms of life. Here we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifested as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-folds) including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site in the promoters of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle. Overall design: Strains of Sis/pSeSD and Sis/pSeSD-aCcr1 were cultured in ATV medium under the conditions as described above. For transcriptomic analysis, culture was inoculated with an initial OD600 of 0.05. The cells were pelleted at 6,000 g for 10 min after 12 h of cultivation when the OD600 reached approximately 0.2. The pellet was resuspended in 1 ml PBS buffer. The cells were pelleted again and stored at -80°C. Total RNA was extracted using the Trizol reagent (Ambion, Austin, TX, USA). Total amounts and the integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Transcriptomic analysis was performed by Novogene (Beijing, China). About 3 µg of high-quality RNA per sample was used for the construction of RNA-Seq libraries. The libraries are sequenced by the Illumina NovaSeq 6000. Clean reads were aligned to the reference genome sequence of S. islandicus REY15A (31). The resulting data were then analysed by Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) analysis to reveal expression levels of all genes in the S. islandicus genome. Differential genome expression analysis (over-expression of aCcr1 versus empty vector) was performed using the DEGSeq R package. The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate padj<0.05 and |log2(foldchange)| > 0 were set as the threshold for significantly differential expression.