The procedure employed to delete genes from the T. kodakarensis genome permits a statistical definition of essentiality (Hileman and Santangelo, 2012). Plasmids are constructed and used to transform a parental strain (here T. kodakarensis TS559) so that the target locus is flanked by two sets of direct repeats. Spontaneous recombination in this intermediate strain between one set of the repeats results in the markerless deletion of the target locus, whereas an equally-probable recombination between the second set of repeats regenerates the parental strain. When only the parental strain is recovered, after screening >30 isolates generated from at least two independently-constructed intermediate strains, the target locus is defined operationally as essential for T. kodakarensis viability under our laboratory conditions.

TK1901-TK1902-TK1903 (encoding Cdc6, DNA polymerase D small and large subunits, respectively) form an operon (Jager et al., 2014) and essentiality has been previously established for TK1902 and TK1903 (Figure 1; Cubonova et al., 2013). Surprisingly, this was not true for TK1901, the only gene in T. kodakarensis that encodes a recognizable Cdc6 homolog. The design of the plasmid constructed to delete TK1901 ensured retention of the upstream promotor and so continued expression of TK1902-TK1903, and avoided deletion of any sequences in the adjacent ~900 bp region predicted to contain the origin of replication based on homology with the origin region in Pyrococcus furiosus, a related member of the Thermococcales (Figure 1; Farkas et al., 2011; Cossu et al., 2015).

The presence of the TK1901 deletion was confirmed by diagnostic PCR and Southern blotting in two independent isolates (Figure 1). Amplicon sequencing confirmed that the 1,248 bp deletion extended precisely from the ATG-start codon to TGA-stop codon of TK1901, and this was subsequently re-confirmed by deep-sequencing (see below) of the entire genome of one isolate, designated T. kodakarensis Δcdc6. This isolate was phenotypically similar to the parental T. kodakarensis TS559 strain: cultures grew at similar rates and reached the same final cell densities (Figure 1) and T. kodakarensis Δcdc6 was genetically competent and supported the autonomous replication of pTN1-based plasmids (Santangelo et al., 2008).

Regions adjacent to an origin(s) are over-represented in growing cells, and marker frequency analyses comparing the number of sequencing reads across the genome has been used to identify replication origin(s) in many archaeal genomes (Andersson et al., 2010; Hawkins et al., 2013; Pelve et al., 2013). Genomic DNA was therefore isolated, fragmented, and deep-sequenced from growing and stationary phase cultures of T. kodakarensis TS559 and Δcdc6. The sequences obtained confirmed the deletion of TK1901 but, in repeated experiments, all regions of the genome were equally represented in the DNA reads from both growing and stationary phase cells of both T. kodakarensis TS559 and Δcdc6 (Figure 2 and Supplementary Figure 1). Given this unanticipated result, to provide confidence in the laboratory and computational procedures, the experiments were repeated with genomic DNAs from Escherichia coli MG1655 and P. furiosus strain JFW02(Farkas et al., 2012), species with established origins of replication. Quantification of the whole genome sequencing (WGS) reads clearly and correctly identified the origin loci established in the genomes of E. coli and P. furiosus (Supplementary Figure 2; Burland et al., 1993; Farkas et al., 2012).

Using the same markerless-genome modification techniques, the presumed origin sequences (Ojha and Swati, 2010) were easily deleted in T. kodakarensis Δcdc6 (Figure 1). All the bioinformatically identified mini-origin recognition boxes (mini-ORBs; green) and one full ORB (blue) were deleted while retaining the promoters (red), transcription start sites (arrows), and translation start sites (yellow) for TK1902-1903, and for TK1900 (Figure 1). Both amplicon- and whole genome deep-sequencing confirmed the precision of the deletion. A representative isolate, designated T. kodakarensis Δcdc6 Δori, was phenotypically indistinguishable from T. kodakarensis TS559 and Δcdc6. All three strains grew at the same rate, achieved the same final cell densities, were genetically competent and supported plasmid replication. Quantification of WGS reads also failed to identify any preferred origin(s) sequences and indicated that replication was initiated at many sites around the genome of T. kodakarensis Δcdc6 Δori (Figure 2).

Although, the WGS reads did not identify origins of replication, with >2,000x genome coverage, they did identify spontaneous recombination events at two locations in subpopulations (<10%) of T. kodakarensis TS559 cells (Figure 3). The recombinations inverted an ~150 kbp region or excised ~100 kbp, and these events resulted in small spikes and dips in the marker frequency analyses at sites previously established to contain vestigial prophage TKV2 and TKV3 genomic sequences (Figure 3; Fukui et al., 2005; Tagashira et al., 2013). Only a small number of sequences were obtained that extended across the sites of recombination, (Figure 3) but these were more prevalent in DNAs isolated from growing than from stationary phase cells. Based on PCR amplicons, these recombination events also occurred in T. kodakarensis Δcdc6 and Δcdc6 Δori. As deletion of TKV3 severely hinders growth (Tagashira et al., 2013) and several presumably essential genes are within the deleted sequences, these recombination events are likely lethal and prevent the effected cells from contributing to continued culture growth.

T. kodakarensis KOD1 has been the focus of continuous research since its isolation in 1994 (Morikawa et al., 1994). It seemed possible therefore that the cdc6- and origin-independent replication of T. kodakarensis TS559 might result from mutations acquired and selected during laboratory culture. The genome sequence determined here for T. kodakarensis TS559 was therefore compared with that published for T. kodakarensis KOD1 (Fukui et al., 2005). All the changes known to have been intentionally introduced to generate T. kodakarensis TS559 from T. kodakarensis KOD1 were present, and although there were no large genome rearrangements, an additional 35 single nucleotide differences were identified. Some of these changes are within open reading frames, but none would be predicted to radically change DNA replication or recombination (Table 1).

T. kodakarensis is oligoploid (7–19 genomes/cell; Spaans et al., 2015) and this is consistent with the use of RDR. Nutrient-stress, the absence of defined DNA segregation strategies, and the potential for continued cell division without DNA replication may occasionally result in T. kodakarensis cells with only one genome. Such cells would be unable to restart growth by RDR initiation but could do so if a cdc6-oriC system of replication initiation was also available. To evaluate whether retention of cdc6 or presumptive origin sequences promotes long-term viability, aliquots were taken from stationary phase cultures maintained at 85°C for extended periods without nutrient addition, and assayed for viability. Extended (several months) incubation at elevated temperatures in nutrient poor conditions was predicted to deplete energy reserves, introduce stress into the genome, and potentially reduce ploidy as genomes were consumed to provide nutrients. Cells in cultures of T. kodakarensis TS559 and Δcdc6, strains with the presumed origin (oriC) region, were viable for ~40 days longer than cells in cultures of T. kodakarensis Δori (Figure 4). Retention of Cdc6, however, did not influence long-term viability.

RadA was required for growth—presumably by RDR initiation—of the origin-deleted halophilic H. volcanii strains but non-essential in the parental, origins-containing strain (Hawkins et al., 2013). Despite repeated attempts with different transforming DNA strategies, we were unable to generate T. kodakarensis strains with RadA (TK1899) or RadB (TK2231) deleted, regardless of the retention or absence of cdc6 and/or origin sequences.

T. kodakarensis strains were grown in artificial seawater (ASW) supplemented with 5 g/L of both yeast extract and tryptone (YT) and 2 g/L of sulfur (S°) or 5 g/L sodium pyruvate (Pyr) at 85°C. P. furiosus strain JFW02 (Farkas et al., 2012) was grown at 95°C as described using maltose as a carbon source (Adams et al., 2001). E. coli strain MG1655 was grown in Luria-Bertani (LB) broth at 37°C. The growth of cultures was measured by increases in optical density at 600 nm (OD₆₀₀). T. kodakarensis cultures were harvested at an OD₆₀₀ of 0.2 (early exponential) and 0.6 (late-exponential) while the stationary phase cells were harvested at an OD₆₀₀ of ~1.0. In Figure 4, four independent cultures of each T. kodakarensis strain were maintained at 85°C in sealed vessels with no additions to the cultures over ~180 days. Loss of culture viability was defined as the inability of aliquots, sampled multiple times over 3 days, to initiate culture growth when inoculated into fresh medium. The inability of 9 or more individual aliquots, removed from cultures over 3 consecutive days, to support outgrowth confirmed that these cultures had lost all viable CFUs.

Standard procedures (Hileman and Santangelo, 2012) were used to construct plasmids, pOSUTK1901B and pJG4 respectively, that were used to delete TK1901 or TK1901 plus the origin sequences from T. kodakarensis TS559. In the resulting strains, T. kodakarensis Δcdc6 and Δcdc6 Δori, the Cdc6 encoding sequence [TK1901; 1,248 bp] was deleted but the promoter for the TK1901-TK1903 operon was retained to sure expression of TK1902-1903. Similarly, in T. kodakarensis Δcdc6 Δori, the putative origin (640 bp) with one origin recognition box (ORB) and three mini-ORBs were deleted, but the promoters for TK1901-1903 and TK1900 were retained (Figure 1). Use of the same procedures, and plasmids designed to precisely delete the TK1899 (RadA) and TK2231 (RadB) sequences, did not generate viable strains with the desired deletions.

Genomic DNA was isolated from all strains as described (Santangelo et al., 2007). Cells pelleted from cultures at the designated OD₆₀₀ were resuspended in 10% (w/v) sucrose, 20 mM Tris-HCl pH 8.0, and 5 mM EDTA. SDS (2% final concentration) and proteinase K (0.25 mg/ml) were added to the resulting lysate and the mixture was incubated for 1 h at 55°C. NaCl (1 M final concentration) was then added, the mixture was chilled, centrifuged, and an equal volume of isopropanol was added to the clarified supernatant. The precipitated nucleic acids were pelleted, resuspended with 10 mM Tris-HCl pH 8.0, 50 μg of RNase A added and incubation continued at 37°C for 30 min. The DNA remaining was further purified by repeated phenol/chloroform/isoamyl alcohol (25:24:1) extraction and an alcohol precipitation.

The procedure used has been previously described (Cubonova et al., 2013). In Figure 1, TK1901 (Probe 1, pink) is only detectable in strain TS559 whereas origin sequences (Probe 3, blue) are detectable in strains TS559 and Δcdc6, but not strain Δcdc6 Δori; probe 3 highlights a smaller product in Δcdc6 that reflects deletion of TK1901. Probe 2 (orange) detected the sequences encoding TK1902 in all samples and the fragment lengths identified are appropriate for the corresponding strains.

Sequencing libraries were prepared using TruSeq DNA library preparation kits (Illumina, San Diego, CA) and were subjected to WGS (pair-ended, 2 × 150 bp per read; 1 × 125 bp reads for E. coli) on an Illumina Hi-Seq 2000 platform (University of Colorado Denver Genomics and Microarray Core Facility). Individual genome coverages ranged from 3,300× to 7,800×.

The reference genome for Thermococcus kodakarensis KOD1 (https://www.ncbi.nlm.nih.gov/nuccore/NC_006624.1) was downloaded from Genbank and manually edited to account for the laboratory manipulations made in the lineage leading to T. kodakarensis TS559. The reference genomes for P. furiosus COM1 and Escherichia coli MG1655 were downloaded from Genbank here (https://www.ncbi.nlm.nih.gov/nuccore/CP003685 and https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3, respectively).

The T. kodakarensis TS559 and KOD1 genomes were aligned and differences identified by using Universal Genotyper (GATK version v2.1-8; Van der Auwera et al., 2013) and MUMmer (version 3.1; Kurtz et al., 2004). Low quality sequences, regions with <5x coverage and a small number of variants identified in DNA from only growing or stationary phase cells of the same isolate were not included. The coordinates of the RefSeq GFF file were updated to account for identified insertions and deletions, and the resulting variants annotated using snpEff (version 4.0e; Cingolani et al., 2012).

An index was built for each reference genome with bowtie2-build (version 2.2.9; Langmead and Salzberg, 2012) using default settings. Illumina adaptor sequences and low quality bases (quality score <10) were trimmed from the 3′-end of each read using cutadapt (version 1.11; Martin, 2011) with reads discarded if more than half the bases were trimmed. The filtered reads were aligned to the reference genome using bowtie2 (version 2.2.9; Langmead and Salzberg, 2012) selecting the best alignment for each read. The alignment statistics are reported in Supplementary Table 1. Reads that did not align as proper pairs were treated as single end reads. For copy number estimation, each reference genome was binned into 1 kb windows with a 500 bp sliding overlap between windows using bedtools (version 2.17.0; Quinlan and Hall, 2010). For each sample, the coverage for each 1 kb was calculated as the number of sequenced bases that overlap with that window. The %G+C content of each window was calculated, and corrections made for potential bias in library preparation and sequencing due to GC content. The average coverage for each GC bin was plotted against the GC content and smoothed by a Lowess regression model. A correction factor, calculated by dividing the global mean coverage by the fitted model, was applied to each sample. Source code available at http://github.com/dpastling/plethora.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Certain commercial equipment, instruments, or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified is necessarily the best available for the purpose.