Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stephen D. Bell ( stedbell@indiana.edu ). The materials generated in this study are available from the Lead Contact with no restrictions.

Strains containing pSSRgD (empty vector) and pSSRgD-ClsN were grown to approximately OD 600 = 0.2 in TSVY + uracil [20 μg/ml]. D-(-)-arabinose was added to cultures to a final concentration of 0.2%. All induced samples were collected at four hours post-induction. Overexpression of ClsN was confirmed by western analysis.

S. islandicus REY15A (E235) was grown to early log phase and pelleted at room temperature. The cells were washed three times with 20 mM sucrose at room temperature and were then resuspended in 20 mM sucrose to an OD600 = 10-20. 300-700 ng of pSSRgD-based plasmid or 1 μg of the linearized oriC1 knockout construct were added to 50 μl of cells. The cells were then electroporated at 1.2 kV, 25 μF, 600 Ω. 900 μl of pre-warmed high salt buffer (22.7 mM ammonium sulfate, 2.9 mM potassium sulfate, 1.3 mM potassium chloride, 9.3 mM glycine) were added and the cells were transferred to sterile tubes and incubated at 78°C for 1 hour before plating on TSVY plates + uracil [20 μg/ml]. After incubation at 78°C for 7-14 days, isolated colonies were used to inoculate 1.5 mL TSVY + uracil [20 μg/ml]. pSSRgD transformants were confirmed by colony PCR and western blot detection of the overexpressed ClsN protein. ΔoriC1 transformants were confirmed by colony PCR and sequencing of the oriC1 locus.

The S. acidocaldarius wild-type strain DSM639 was grown in Brock’s medium () containing 0.2% sucrose and 0.1% tryptone, pH 3.2 at 78°C. For the MW001 and ΔfadR strains, uracil was added to 10 μg/ml (). The S. islandicus Δorc1-1 and Δorc1-1/Δorc1-3 strains were constructed previously from S. islandicus REY15A (E233S) () and were grown in TSVY medium (containing mineral salts, 0.2% sucrose, 0.1% tryptone, 0.05% yeast extract, and 1X vitamin solution) supplemented with 20 μg/ml of uracil. The S. islandicus agmatine auxotroph, E235, was grown with 50 μg/ml of agmatine (). Except for stationary-phase experiments, cells were collected during log phase (OD≈0.2). Stationary-phase cells of were collected after ODreached ∼1.0. To block transcription, 1 mg/ml of actinomycin D (dissolved in DMSO) was added to a log-phase culture of DSM639 to a final concentration of 15 μg/ml. For the temperature shift experiment, S. islandicus E233S was cultured in a 78°C water bath and shifted to a 65°C water bath for one hour. E. coli strains were grown in L-Broth (1% tryptone, 0.5% yeast estract 0.05%NaCl) supplemented with the appropriate antibiotics.

Method Details

Plasmid construction The gene encoding ClsN was amplified from S. acidocaldarius genomic DNA with the SacSMC_F and SacSMC_noSTOP_R primers and cloned into pET30 via NdeI and XhoI restriction sites to generate pET30-SacSMC. The gene encoding ClsN was amplified from S. islandicus genomic DNA with the SMC_F and SMCnoSTOP_R primers and cloned into pET33 via NcoI and XhoI restriction sites to generate pET33-SisSMC. Zheng et al., 2012 Zheng T.

Huang Q.

Zhang C.

Ni J.

She Q.

Shen Y. Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus. pSSRgD was generated by PCR amplification of the Sulfolobus tokodaii argD gene (ST1348) using ArgD_BstXI and ArgD_XmaI. Following digestion with BstXI and XmaI, the fragment was ligated into the XmaI and SacI sites of pSSR to replace the simvastatin resistance cassette in pSSR (). The gene encoding ClsN was amplified from S. islandicus REY15A, digested with FauI and SalI, and cloned in to the NdeI and SalI sited of pSSRgD. pCR-ArgD was generated by PCR amplification of the Sulfolobus tokodaii argD gene (ST1348) using StoArgD_FT7 StoArgD_RT3. The amplimer was assembled using the HiFi DNA Assembly kit (New England Biolabs) with the pCR-Script (Stratagene) backbone that had been amplified with primers reverse complementary to the T3 and T7 sequencing primers.

oriC1 Deletion Construct ORBs 2 and 3 (Origin Recognition Box) and intervening DNA were deleted from oriC1 in S. islandicus REY15A (E235) by gene deletion. The S. tokadaii argD gene was amplified from pCR-ArgD with OriC1-UP and oriC1-DO forward and reverse primers containing flanks that were homologous to 75 bp of sequence upstream and downstream of the deletion target.

Hi-C To crosslink DNA-DNA contacts, 20 mL of cell culture were mixed with (80-X) ml of ambient PBS buffer and X ml of 37% formaldehyde (Fisher Scientific). X was 5.4 for S. acidocaldarius (final formaldehyde concentration of 2%) and 10.8 for S. islandicus (final formaldehyde concentration of 4%). The reaction was incubated for 30 min at 25°C before quenching with glycine (at final concentrations of 125 mM for S. acidocaldarius and of 250 mM for S. islandicus) for 10 min at room temperature. The fixed cells were collected by centrifugation and washed twice with PBS buffer. The cell pellet was stored at −80°C until use. A frozen pellet of S. acidocaldarius was resuspended and diluted to an OD 600 of 4 in resuspension buffer (PBS buffer containing 1 mM EDTA), and 600 μL of the cell suspension were treated with proteinase K (at a final concentration of 24 μg/ml) for 15 min at 37°C to partially disrupt cell walls. The cells were collected by centrifugation and washed four times with resuspension buffer and once with 1 x NEBuffer 2. Centrifugation was done at 21,000 x g, 4°C for 5 min. A frozen pellet of S. islandicus was resuspended and diluted to an OD 600 of 4 in 1 x NEBuffer 2, and 600 μL of the cell suspension were centrifuged. Each cell pellet was resuspended in 75 μL of 1 x NEBuffer 2. 75 μL of the cell suspension were mixed with 8.33 μL of 10% SDS and heated at 65°C for 15 min. After cooling on ice for 90 s, 25 μL of cell lysate (corresponding to ∼4 × 108 cells) were mixed with 41.8 μL of 1 x NEBuffer 2, 3.2 μL of 10 x NEBuffer 2, 20 μL of 10% Triton X-100. Chromosomal DNA was digested by adding 10 μL of 100 U/μl HindIII (NEB) to the mixture and incubating the reaction for 4 h at 37°C. After cooling on ice, the reaction was mixed with 12 μL of dNTP mixture (0.67 mM dATP, 0.67 mM dGTP, and 0.67 mM dTTP in TE buffer), 20 μL of 0.4 mM biotin-14-dCTP (Thermo Fisher Scientific), 3.6 μL of 10 x NEBuffer 2, and 1 μL of 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB). After incubation for 30 min at 20°C, the reaction was quenched by adding 15.4 μL of 10% SDS and 2.8 μL of 0.5 M EDTA and incubating for 5 min at room temperature. For ligation, the reaction was mixed with 1426 μL of nuclease-free water, 200 μL of 10 x T4 DNA Ligase Reaction Buffer (NEB), 200 μL of 10% Triton X-100, and 20 μL of 400 U/μl T4 DNA ligase (NEB). The ligation reaction was incubated for 4 h at 16°C before adding 200 μL of 10% SDS, 100 μL of 0.5 M EDTA, and 10 μL of 20 mg/ml proteinase K. To reverse crosslinks, the reaction was incubated for 6 h at 65°C and then for at least 6 h at 37°C. The DNA was extracted twice with phenol:chloroform:isoamyl alcohol and isopropanol-precipitated together with 40 μg of glycogen. The purified DNA was dissolved in 40 μL of 1 x NEBuffer 2 containing 0.1 mg/ml RNase A and incubated for 30 min at 37°C. To remove biotin from unligated DNA, the solution was mixed with 48 μL of nuclease-free water, 6 μL of 10 x NEBuffer 2, 0.5 μL of 20 mg/ml BSA (Thermo Fisher Scientific, 5 μL of 2mM dGTP (in TE buffer), and 0.5 μL of 3 U/μl T4 DNA polymerase (NEB). After incubating for 4 h at 20°C, the DNA was extracted with phenol:chloroform:isoamyl alcohol and ethanol-precipitated. The DNA was dissolved in 100 μL of Buffer EB (QIAGEN) and sheared with a Bioruptor (Diagenode) at low power for 60 cycles (30 s on, 30 s off). 55.5 μL of the sheared DNA was used for library construction with NEBNext Ultra DNA Library Prep Kit for Illumina and NEBNex Multiplex Oligos for Illumina (NEB). First, end repair, adaptor ligation, and size selection were performed according to the manufacturer’s instructions. The purified DNA was subjected to biotin purification with Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). Before use, 10 μl/sample of beads were washed four times with B&W Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl) and resuspended in 135 μl/sample of B&W Buffer. 135 μL of the bead suspension was added to each DNA solution and rotated for 30 min at room temperature. The beads were washed three times with B&W Buffer followed by one wash with 1 x NEBuffer 2 without DTT. Each sample was resuspended in 15 μL of 0.1 x TE (pH 8.0) and used for PCR in a 50 μL reaction for 14 cycles. The PCR products were purified with AMPure XP Beads (Beckman Coulter) and paired-end sequenced (43 bp x 2) on the Illumina NextSeq platform at the Center for Genomics and Bioinformatics at Indiana University.

Hi-C of Non-crosslinked DNA Cells were harvested and resuspended in 600 μL of lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.42% SDS, 0.1 mg/ml RNase A). After incubation for 20 min at room temperature, the cell suspension was treated with 0.2 mg/ml of proteinase K for 1.5 h at 37°C. DNA was then extracted four times with phenol:chloroform:isoamyl alcohol, ethanol-precipitated, and dissolved in 1 x NEBuffer 2. 55 μL of the solution (containing approximately 25 μg of DNA) were mixed with 42 μL of 1 x NEBuffer 2 and 3 μL of 100 U/μl of HindIII (NEB). After incubation for 2 h at 37°C, the reaction was mixed with 6 μL of dNTP mixture (0.67 mM dATP, 0.67 mM dGTP, and 0.67 mM dTTP in TE buffer), 10 μL of 0.4 mM biotin-14-dCTP (Thermo Fisher Scientific), 2 μL of 10 x NEBuffer 2, and 1 μL of 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB). After incubation for 30 min at 20°C, the DNA was extracted with phenol:chloroform:isoamyl alcohol, ethanol-precipitated, and dissolved in 25 μL of 1 x T4 DNA Ligase Reaction Buffer (NEB). 20 μL of the solution was treated with 1 μL of 400 U/μl T4 DNA ligase (NEB) for 3h at 16°C. After heating for 20 min at 65°C, the reaction was mixed with 23 μL of nuclease-free water, 10 x T4 DNA Ligase Reaction Buffer (NEB), 20 mg/ml BSA (Thermo Fisher Scientific), 2.5 μL of 2 mM dGTP (in TE buffer), and 0.25 μL of 3 U/μl T4 DNA polymerase (NEB). After incubation for 4 h at 20°C, the DNA was extracted with phenol:chloroform:isoamyl alcohol, ethanol-precipitated, and dissolved in 22 μL of Buffer EB (Qiagen). 15 μL of the solution were diluted in 87 μL of Buffer EB and sheared with a Bioruptor (Diagnogen) at low power for 40 cycles (30 s on, 30 s off). After treatment with 0.5 μL of 10 mg/ml RNase A for 30 min at 37°C, the DNA was used for library preparation as described for the Hi-C experiment with crosslinking.

Generating Hi-C Contact Maps Servant et al., 2015 Servant N.

Varoquaux N.

Lajoie B.R.

Viara E.

Chen C.J.

Vert J.P.

Heard E.

Dekker J.

Barillot E. HiC-Pro: an optimized and flexible pipeline for Hi-C data processing. We mapped and processed raw Hi-C reads using HiC-Pro version 2.9.0 (). Reads were mapped to the genome of S. acidocaldarius DSM639 (GenBank ID: CP000077.1 ) or S. islandicus REY15A (GenBank ID: CP002425.1 ) with default parameters. The start positions of the genomes were modified as explained in the next paragraph. For the overexpression experiment of ClsN, reads were also mapped to the sequence of pSSRgD-ClsN. Invalid reads pairs including dangling ends, self-circle ligation, and duplicates were discarded. Remaining valid read were used to generate raw Hi-C matrices (see Table S1 ). The genome was binned at 15-kb for S. acidocaldarius and 30-kb for S. islandicus. When combining biological replicates, valid read pairs from them were pooled before generating raw Hi-C matrices. The intra-bin ligations were discarded by setting the values on the corresponding diagonal to zero. The data were iteratively corrected with the MAX_ITER parameter of 500. The obtained matrices were normalized so that the sum of interaction scores is equal to 1 for each row and column. In our preliminary analysis of S. acidocaldarius, we noticed that interaction score was extraordinarily high between the first bin and the last bin. These bins are adjacent on the circular chromosome of S. acidocaldarius but not are neighbors based on the genomic coordinates. HiC-Pro failed to discard self-ligation junctions of the restriction fragment spanning the boundary of the two bins, as it was originally developed to analyze Hi-C data from eukaryotic linear chromosomes. To solve this problem, we re-defined the genomic coordinates of each Sulfolobus species so that they started from the first base of the first restriction site in the original definition (S. acidocaldarius DSM639: 1,011 bp from the start, S. islandicus REY15A: 966 bp from the start). These modified genomes were used to map reads and generate Hi-C matrices. The obtained data were visualized and analyzed with R software ( http://www.R-project.org ).

Identification of Compartments Lieberman-Aiden et al., 2009 Lieberman-Aiden E.

van Berkum N.L.

Williams L.

Imakaev M.

Ragoczy T.

Telling A.

Amit I.

Lajoie B.R.

Sabo P.J.

Dorschner M.O.

et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. 2 ratio of interaction score versus expected value (genome-wide average of interaction scores for the corresponding genomic distance) for each pair of bins. Obtained matrices were converted into Pearson correlation matrices, in which the entry a i,j represents the Pearson correlation coefficient between the ith row and the jth column of the corresponding observed/expected matrices. Obtained Pearson correlation matrices were then used for principal component analysis. We defined the sign of the first principal component such that the value is positively correlated with RNA expression level. The first principal component value was used as compartment index. Generation of Pearson correlation matrices and principal component analysis were done with HiTC ( Servant et al., 2012 Servant N.

Lajoie B.R.

Nora E.P.

Giorgetti L.

Chen C.J.

Heard E.

Dekker J.

Barillot E. HiTC: exploration of high-throughput ‘C’ experiments. Compartment index was calculated according to previous work (). First, distance-normalized Hi-C matrices were generated by calculating the logratio of interaction score versus expected value (genome-wide average of interaction scores for the corresponding genomic distance) for each pair of bins. Obtained matrices were converted into Pearson correlation matrices, in which the entry arepresents the Pearson correlation coefficient between the ith row and the jth column of the corresponding observed/expected matrices. Obtained Pearson correlation matrices were then used for principal component analysis. We defined the sign of the first principal component such that the value is positively correlated with RNA expression level. The first principal component value was used as compartment index. Generation of Pearson correlation matrices and principal component analysis were done with HiTC ().

3D Model of Chromosome Structure Lesne et al., 2014 Lesne A.

Riposo J.

Roger P.

Cournac A.

Mozziconacci J. 3D genome reconstruction from chromosomal contacts. Humphrey et al., 1996 Humphrey W.

Dalke A.

Schulten K. VMD: visual molecular dynamics. We converted Hi-C contact matrices into 3D models of chromosome structure using ShRec3D (). This algorithm assumes that the spatial distance between two loci are inversely proportional to the interaction score. All the 3D structures were rendered with VMD ().

Quantification of Compartment Strength To quantify compartment strength, every genomic bin was assigned to one of the two compartments according to compartment index. Based on this assignment, every bin pairs were classified as either of “A-A,” “B-B,” and “A-B.” For each of the three groups, the sum of observed interaction scores and the sum of expected interaction scores was calculated. Before this calculation, DNA contacts within 45 kb (S. acidocaldarius) or 60 kb (S. islandicus) were discarded, because such short-range interactions constitute a large part of genomic contacts in cells but reflect compartment strength very little. The log 2 ratio of the observed sum versus the expected sum was used to estimate compartment strength.

Directional Preference of Hi-C contacts Le et al., 2013 Le T.B.

Imakaev M.V.

Mirny L.A.

Laub M.T. High-resolution mapping of the spatial organization of a bacterial chromosome. 2 values of the upstream and downstream vectors by paired t test. Directional preference was defined as t-value of the test. Directional preference was calculated according to a previous study (). To calculate the directional preference for a given bin, we collected Hi-C interaction scores between the bin and bins located either downstream or upstream within the distance of 90 kb or 300 kb. We then compared logvalues of the upstream and downstream vectors by paired t test. Directional preference was defined as t-value of the test.

Purification of Recombinant ClsN S. acidocaldarius and S. islandicus ClsN proteins were expressed with C-terminal His 6 -tags in E. coli Rosetta (DE3) cells. Cultures were grown in LB at 37°C to an OD 600 = 0.6-0.8 and induced with 1 mM IPTG for 3 hours at 37°C. Cells were lysed by French press in buffer (50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole, 10% glycerol, 5 mM β-mercaptoethanol, pH 7.5) containing Roche Mini-Complete Protease Inhibitors. The soluble lysate was heat treated for 20 minutes at 65°C, and the heat-stable fraction was purified first over Ni-NTA agarose (QIAGEN) and then over a HiTrap Heparin column (GE Healthcare). Proteins were further purified over a HiLoad 26/600 Superdex 200 column (GE Healthcare) in 50 mM sodium phosphate, 150 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, pH 7.5.

Affinity Purification of Antibodies 1 mg of purified, recombinant S. acidocaldarius ClsN protein was coupled to a 1 mL HiTrap NHS-activated HP column (GE Healthcare) in standard coupling buffer (200 mM sodium carbonate, 500 mM NaCl, pH 8.3) according to the manufacturer’s protocol (GE Healthcare). Rabbit anti-ClsN polyclonal serum was diluted 1:10 in 1X TBS, passed through a 0.45 μm filter, and recirculated through the affinity column at < 1 ml/minute for 45 minutes at room temperature. The column was washed with 1X TBS, TBS-T, and then eluted with 100 mM glycine, pH 2.5 into Tris, pH 8.5, 100 mM final molarity. Fractions were spotted onto Immobilon-P membrane (Millipore) and those containing antibody were detected with anti-rabbit HRP secondary antibody.

ChIP-Seq Samson et al. (2013) Samson R.Y.

Xu Y.

Gadelha C.

Stone T.A.

Faqiri J.N.

Li D.

Qin N.

Pu F.

Liang Y.X.

She Q.

Bell S.D. Specificity and function of archaeal DNA replication initiator proteins. Chromatin immunoprecipitation (ChIP) was performed as described in. Cultures of Sulfolobus were crosslinked with 1% formaldehyde for 20 minutes. After quenching with 125 mM glycine, the cells were pelleted and washed with 1X PBS. The pellets were resuspended in TBS-TT (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, 0.1% Triton X-100, pH 7.5) and sonicated using a Diagenode Bioruptor to generate DNA fragments ranging from 200-1000 bp. Extract was then clarified by centrifugation. 10 μg of extract (based on protein concentration) were used in each 100 μl ChIP reaction. Samples were rotated for 2-3 hours with 3 μl of antiserum or 75 μl of purified anti-S. acidocaldarius ClsN antibodies at 4°C. 25 μl of a 50% slurry of protein A Sepharose were then added and the samples were rotated for another hour at 4°C. Each ChIP reaction was then washed five times at room temperature with TBS-TT, once with TBS-TT containing 500 mM NaCl, and once with TBS-TT containing 0.5% Tween-20 and 0.5% Triton X-100. Protein-DNA complexes were eluted from the protein A Sepharose in 20 mM Tris, 10 mM EDTA, 0.5% SDS, pH 7.8 at 65°C for 30 minutes. Crosslinking was reversed and protein was digested by incubating the samples with 10 ng/μl Proteinase K for 6 hours at 65°C followed by 10 hours at 37°C. The samples were extracted with phenol/chloroform/isoamyl alcohol first, then chloroform alone and the DNA was precipitated in 100% ethanol containing 20 μg of glycogen. After washing with 70% ethanol and air drying, the DNA was resuspended in 50 μl TE buffer. ChIP reactions were performed in triplicate and pooled. 50 μl of ChIP reactions and 100 ng of input DNA were used to construct DNA libraries using Illumina TruSeq ChIP Library Preparation Kits. The manufacturer’s instructions were followed, except DNA size selection was performed using a 0.6 X ratio of AMPure XP beads rather than purification by agarose gel. DNA quality, size and quantity were verified using a High Sensitivity DNA chip on an Agilent Technologies 2100 Bioanalyzer and with a Qubit dsDNA High Sensitivity Assay kit on a Qubit 2.0 Fluorometer (Invitrogen). Libraries were sequenced using the Illumina HiSeq platform at the Wellcome Trust Center for Human Genetics, Oxford, UK or the Illumina NextSeq platform at the Center for Genomics and Bioinformatics at Indiana University. Langmead and Salzberg, 2012 Langmead B.

Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Reads from single or multiple biological replicates were mapped to the genome of S. acidocaldarius DSM639 or S. islandicus REY15A using Bowtie 2 version 2.3.2 with default parameters (). Multi-mapping reads were retained for downstream analyses. For a plasmid-containing strain of S. islandicus, reads were separately mapped to the genome or the corresponding plasmid. To calculate protein enrichment on the chromosome, ChIP-seq coverage of the region of interest was divided by input coverage of the region after normalizing the total number of reads mapped to the chromosome. To calculate protein enrichment on the plasmid, numbers of reads mapped to the chromosome and the plasmid were summed and used for read number normalization. Protein enrichment in protein-coding genes was calculated according to RefSeq annotation downloaded from UCSC Archaeal Genome Browser ( http://archaea.ucsc.edu/index.html ). Ramírez et al., 2016 Ramírez F.

Ryan D.P.

Grüning B.

Bhardwaj V.

Kilpert F.

Richter A.S.

Heyne S.

Dündar F.

Manke T. deepTools2: a next generation web server for deep-sequencing data analysis. Metagene analyses on RefSeq protein-coding genes were performed using functions implemented in deepTools version 3.0.0 (). After mapping, ChIP reads and input reads were compared using bamCompare (parameters: -bs 10,–scaleFactorsMethod readCount). The output file was processed using computeMatrix and plotProfile. Zhang et al., 2008 Zhang Y.

Liu T.

Meyer C.A.

Eeckhoute J.

Johnson D.S.

Bernstein B.E.

Nusbaum C.

Myers R.M.

Brown M.

Li W.

Liu X.S. Model-based analysis of ChIP-Seq (MACS). Haug-Baltzell et al., 2017 Haug-Baltzell A.

Stephens S.A.

Davey S.

Scheidegger C.E.

Lyons E. SynMap2 and SynMap3D: web-based whole-genome synteny browsers. Pooled paired-end reads were used for peak calling with MACS2 () using the option -m 1 50. Overlap of broad ClsN peaks with conserved gene cluster was examined using SynMap2 ().

Marker Frequency Analysis (MFA-Seq) MFA was performed by NGS sequencing on the Illumina NextSeq at the Center for genome Biology at Indiana University. DNA was isolated from exponentially growing (OD600 nm = 0.2) S. islandicus ΔoriC1 cells and, as reference, from a stationary phase population confirmed by flow cytometry to be exclusively G2 cells. ∼28 million reads were obtained for both samples. Read counts were grouped in 1 kilobase bins and the count for exponentially growing cells divided by the stationary phase values. The midpoint of the lowest trough was set arbitrarily to a value of 1.

RNA-seq Samson et al., 2008 Samson R.Y.

Obita T.

Freund S.M.

Williams R.L.

Bell S.D. A role for the ESCRT system in cell division in archaea. RNA was extracted from S. acidocaldarius and S. islandicus using the method described previously (). After treatment with DNase, the RNA was ethanol precipitated and dissolved in DEPC-treated water. Without ribosomal RNA depletion, the RNA samples were used for preparing strand-specific libraries with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to the protocol for use with purified mRNA or rRNA-depleted RNA in the manufacturer’s manual. RNA fragmentation was carried out based on RIN values determined by TapeStation (Agilent) at the Center for Genomics and Bioinformatics at Indiana University. The libraries were paired-end sequenced (43 bp x 2) on the Illumina NextSeq at the Center for Genomics and Bioinformatics at Indiana University. Langmead and Salzberg, 2012 Langmead B.

Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Ramírez et al., 2016 Ramírez F.

Ryan D.P.

Grüning B.

Bhardwaj V.

Kilpert F.

Richter A.S.

Heyne S.

Dündar F.

Manke T. deepTools2: a next generation web server for deep-sequencing data analysis. Patro et al., 2017 Patro R.

Duggal G.

Love M.I.

Irizarry R.A.

Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Reads from biological replicates were pooled and mapped to both strands using Bowtie 2 version 2.3.2 (parameter:–maxins 1000) (). After removal of multi-mapping reads, the remaining reads were processed using bamCoverage implemented in deepTools version 3.0.0 () to calculate Reads Per Kilobase region per Million mapped reads (RPKM) for genomic bins of fixed size. To calculate Transcripts Per Million (TPM), reads from each biological replicate were mapped to RefSeq protein-coding genes in a strand-specific manner using Salmon version 0.8.2 (). The average of the TPM values from biological replicates was used for downstream analyses.

Probe Preparation for DNA FISH DNA fragments of 6.1-6.3 kb were amplified by PCR from S. acidocaldarius genomic DNA and cloned into pCR-XL-2-TOPO using TOPO XL-2 Complete PCR Cloning Kit (Thermo Fisher Scientific). The primers used for amplification are as follows. P1F and P1R, P2F and P2R, P3F and P3R, P4F and P4R and P5F and P5R. Fluorescently-labeled FISH probes were generated from these plasmids using FISH Tag DNA Multicolor Kit (Thermo Fisher Scientific). For each probe, 1 μg of plasmid DNA was used and the final product was dissolved in 12 μL of nuclease-free water.

DNA FISH Robinson et al., 2007 Robinson N.P.

Blood K.A.

McCallum S.A.

Edwards P.A.

Bell S.D. Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus. 8 cells were collected by centrifugation at room temperature. The cell pellet was resuspended in PBS containing 1.5% paraformaldehyde and incubated at 25°C for 30 min. After centrifugation, the cell pellet was permeabilized by resuspension in 0.2% Triton X-100 and 0.04% SDS at 25°C for 20 min. After washing once with PBS, the cells were resuspended in PBS containing 0.1 mg/ml of RNase A and incubated at 25°C for 30 min. The cells were washed once with PBS and then once with pre-hybridization buffer (2 x SSC, 36.4 mM phosphate buffer [pH 6.9], 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 60% formamide, 50 μg/ml BSA). The cells were resuspended in 40 μL of hybridization buffer (1.15 μL of a probe labeled with Alexa Fluor 488, 1.15 μL of a probe labeled with Alexa Fluor 647, 2 x SSC, 36.4 mM phosphate buffer [pH 6.9], 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 60% formamide, 50 μg/ml BSA). The suspension was heated at 60°C for 10 min and then incubated at 30°C overnight. After adding 200 μL of wash buffer (2 x SSC, 60% formamide, prewarmed to 30°C), the cells were pelleted and washed twice with wash buffer, once with 2 x SSC, once with 1 x SSC, and then once with PBS. The cell pellet was resuspended in 120-200 μL of PBS, and 10 μL of the suspension were spread onto a polyD-lysine-coated coverslip. After partially air-dried, the coverslip was rinsed twice with PBS and inverted onto a slide spotted with 13 μL of VECTASHIELD (Vector Laboratories) containing 1.5 ng/μl of DAPI. The coverslip was sealed with nail polish. DNA FISH was performed as described previously () with modifications. Approximately 5 × 10cells were collected by centrifugation at room temperature. The cell pellet was resuspended in PBS containing 1.5% paraformaldehyde and incubated at 25°C for 30 min. After centrifugation, the cell pellet was permeabilized by resuspension in 0.2% Triton X-100 and 0.04% SDS at 25°C for 20 min. After washing once with PBS, the cells were resuspended in PBS containing 0.1 mg/ml of RNase A and incubated at 25°C for 30 min. The cells were washed once with PBS and then once with pre-hybridization buffer (2 x SSC, 36.4 mM phosphate buffer [pH 6.9], 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 60% formamide, 50 μg/ml BSA). The cells were resuspended in 40 μL of hybridization buffer (1.15 μL of a probe labeled with Alexa Fluor 488, 1.15 μL of a probe labeled with Alexa Fluor 647, 2 x SSC, 36.4 mM phosphate buffer [pH 6.9], 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 60% formamide, 50 μg/ml BSA). The suspension was heated at 60°C for 10 min and then incubated at 30°C overnight. After adding 200 μL of wash buffer (2 x SSC, 60% formamide, prewarmed to 30°C), the cells were pelleted and washed twice with wash buffer, once with 2 x SSC, once with 1 x SSC, and then once with PBS. The cell pellet was resuspended in 120-200 μL of PBS, and 10 μL of the suspension were spread onto a polyD-lysine-coated coverslip. After partially air-dried, the coverslip was rinsed twice with PBS and inverted onto a slide spotted with 13 μL of VECTASHIELD (Vector Laboratories) containing 1.5 ng/μl of DAPI. The coverslip was sealed with nail polish.

Microscopy Schober et al., 2008 Schober H.

Kalck V.

Vega-Palas M.A.

Van Houwe G.

Sage D.

Unser M.

Gartenberg M.R.

Gasser S.M. Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast. DNA FISH samples were imaged using a Metemorph-driven Olympus BX-61, with a Z stack of 21 planes (0.1 μm step size) collected for each channel. 3D distances between fluorescent foci were quantified using the ImageJ plugin SpotDistance () with a visual inspection. Between 161 and 333 cells were analyzed per probe pair, as indicated in Figure 2 C.

Chromatin Fractionation Cells were harvested from Sulfolobus cultures at OD 600 = 0.2 for exponential samples and OD 600 = 1.0 for stationary samples. Pelleted cells were resuspended in 100 μl/0.1 OD 600 unit/ml of chromatin extraction buffer (25 mM HEPES, 15 mM MgCl 2 , 100 mM NaCl, 400 mM sorbitol, 0.5% Triton X-100, pH 7.5) and incubated on ice for 10 minutes. Extracts were centrifuged for 20 minutes at 14,000 g, 4°C. The soluble fraction was transferred to a new tube and the pelleted chromatin fraction was resuspended in a volume of chromatin extraction buffer equivalent to the soluble fraction. The chromatin fraction was then sonicated three times for 30 s each.

Flow Cytometry Sulfolobus cells were fixed with 72% ethanol, stained with SYTOX Green (Invitrogen) and DNA contents were analyzed with an LSRII flow cytometer in the Flow Cytometry Facility at Indiana University.