Genome Sequencing of Sulfolobus sp. A20 from Costa Rica and Comparative Analyses of the Putative Pathways of Carbon, Nitrogen, and Sulfur Metabolism in Various Sulfolobus Strains

Abstract

The genome of Sulfolobus sp. A20 isolated from a hot spring in Costa Rica was sequenced. This circular genome of the strain is 2,688,317 bp in size and 34.8% in G+C content, and contains 2591 open reading frames (ORFs). Strain A20 shares ~95.6% identity at the 16S rRNA gene sequence level and <30% DNA-DNA hybridization (DDH) values with the most closely related known Sulfolobus species (i.e., Sulfolobus islandicus and Sulfolobus solfataricus), suggesting that it represents a novel Sulfolobus species. Comparison of the genome of strain A20 with those of the type strains of S. solfataricus, Sulfolobus acidocaldarius, S. islandicus, and Sulfolobus tokodaii, which were isolated from geographically separated areas, identified 1801 genes conserved among all Sulfolobus species analyzed (core genes). Comparative genome analyses show that central carbon metabolism in Sulfolobus is highly conserved, and enzymes involved in the Entner-Doudoroff pathway, the tricarboxylic acid cycle and the CO₂ fixation pathways are predominantly encoded by the core genes. All Sulfolobus species encode genes required for the conversion of ammonium into glutamate/glutamine. Some Sulfolobus strains have gained the ability to utilize additional nitrogen source such as nitrate (i.e., S. islandicus strain REY15A, LAL14/1, M14.25, and M16.27) or urea (i.e., S. islandicus HEV10/4, S. tokodaii strain7, and S. metallicus DSM 6482). The strategies for sulfur metabolism are most diverse and least understood. S. tokodaii encodes sulfur oxygenase/reductase (SOR), whereas both S. islandicus and S. solfataricus contain genes for sulfur reductase (SRE). However, neither SOR nor SRE genes exist in the genome of strain A20, raising the possibility that an unknown pathway for the utilization of elemental sulfur may be present in the strain. The ability of Sulfolobus to utilize nitrate or sulfur is encoded by a gene cluster flanked by IS elements or their remnants. These clusters appear to have become fixed at a specific genomic site in some strains and lost in other strains during the course of evolution. The versatility in nitrogen and sulfur metabolism may represent adaptation of Sulfolobus to thriving in different habitats.

Keywords: Sulfolobus; carbon metabolism; comparative genomics; genome sequencing; nitrogen metabolism; strain A20; sulfur metabolism.

Figure 1

A transmission electron micrograph showing the morphology of Sulfolobus sp. A20.

Figure 2

Phylogenetic tree of genome-sequenced Sulfolobus strains based on the 16S rRNA gene sequences. Metallosphaera sedula DSM 5348 is used as the outgroup. Numbers denote the bootstrap percentages obtained with 1000 replicates.

Figure 3

Venn diagram of the conservation of protein-coding ORFs of the genome-sequenced type strains of Sulfolobus species. The overlaps between the ellipses show the gene groups shared by different strains with the number of shared gene groups indicated. The number of genes for each strain in a section of the diagram is shown by an underlined number in italics. Each underlined number in italics in the middle of the diagram indicates the number of core genes for a strain analyzed.

Figure 4

The 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle and the dicarboxylate/ 4-hydroxybutyrate (DC/HB) cycle in Sulfolobus. Homologs of the enzymes in the two pathways in strain A20 are indicated by ORF numbers for the strain: ① acetyl-CoA caroxylase, ② malonyl-CoA reductase (NADPH), ③ malonate semialdehyde reductase (NADPH), ④ 3-hydroxypropionate-CoA ligase (AMP-forming), ⑤ 3-hydroxypropionyl-CoA dehydratase, ⑥ acrryloyl-CoA reductase (NADPH), ⑦ propionyl-CoA carboxylase, ⑧ methylmalonyl-CoA epimerase, ⑨ methylmalonyl-CoA mutase, ⑩ succinyl-CoA reductase, ⑪ succinic semialdehyde reductse (NADPH), ⑫ 4-hydroxybutyrate-CoA ligase (AMP forming), ⑬ 4-hydroxybutyryl-CoA dehydratase, ⑭ crotonyl-CoA hydratase, ⑮ (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺), ⑯ acetoacetyl-CoA beta-ketothiolase, ⑰ pyruvate synthase, ⑱ pyruvate:water dikinase, ⑲ PEP carboxylase, ⑳ malate dehydrogenase (NAD), ㉑ fumarate hydratase, ㉒ fumarate reductase, ㉓ succinyl-CoA synthetase (ADP-forming).