Genome sequencing of a genetically tractable Pyrococcus furiosus strain reveals a highly dynamic genome

Abstract

The model archaeon Pyrococcus furiosus grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPR-associated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The P. furiosus genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.

Fig 1

Dot plot of the alignment of the P. furiosus NCBI reference sequence (NC_003413) on the x axis and the P. furiosus COM1 strain on the y axis indicates high overall synteny with two major inversions. Inverted segments in COM1 synonymous with the circular diagram in Fig. 2 are block A (positions 1495214 to 1295163), block E (positions 1909827 to 1495215), and block C (positions 199132 to 162245). The small segment (positions 210431 to 211213) is a result of an IS element insertion between PF0401 and PF0402 in COM1.

Fig 2

COM1 genome organization (left) compared to the P. furiosus reference sequence (NC_003413, right). Black tick marks on the outside of the circular diagrams represent full-length IS elements in both genomes, with two additional IS elements located at the boundaries on the COM1 genome between blocks A and D and blocks B and E. The block boundary between A and E indicates the start/end of the reference sequence and is just upstream of the origin of replication (ori) in both genomes. PF numbers associated with each block are ordered in the 5′-to-3′ direction of each arrow: A (red), PF0001 to PF0189; B (yellow), PF0190 to PF0347; C (green), PF0349 to PF0388; D (blue), PF0390 to PF1603′; E (purple), PF1603′ to PF2065. PF1603 is disrupted by an IS element in the COM1 genome.

Fig 3

Maps of IS element insertions in both coding and potential regulatory regions of the COM1 genome compared to the NCBI reference sequence. The riboflavin synthase subunit alpha (PF0061) gene (A) and the region upstream of the cold-induced protein A (PF0190) (B) in COM1 are disrupted by IS elements. IS elements are represented by white arrows and are flanked by direct repeat sequences (boxed). A single copy of the direct repeat sequence is located at the same positions on the reference sequences, and the nucleotide sequence alignments are identical for the two genomic regions minus the IS element insertion and additional direct repeats. In the case of the riboflavin synthase gene, at the protein level the COM1 sequence has a premature stop codon (∼16 bp into the IS element).