The complete genome of hyperthermophile Methanopyrus kandleri AV19 and monophyly of archaeal methanogens

Abstract

We have determined the complete 1,694,969-nt sequence of the GC-rich genome of Methanopyrus kandleri by using a whole direct genome sequencing approach. This approach is based on unlinking of genomic DNA with the ThermoFidelase version of M. kandleri topoisomerase V and cycle sequencing directed by 2'-modified oligonucleotides (Fimers). Sequencing redundancy (3.3x) was sufficient to assemble the genome with less than one error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1,692 protein-coding genes and 39 genes for structural RNAs were identified. M. kandleri proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to the high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that M. kandleri belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons indicate that, in both trees constructed using concatenated alignments of ribosomal proteins and trees based on gene content, M. kandleri consistently groups with other archaeal methanogens. M. kandleri shares the set of genes implicated in methanogenesis and, in part, its operon organization with Methanococcus jannaschii and Methanothermobacter thermoautotrophicum. These findings indicate that archaeal methanogens are monophyletic. A distinctive feature of M. kandleri is the paucity of proteins involved in signaling and regulation of gene expression. Also, M. kandleri appears to have fewer genes acquired via lateral transfer than other archaea. These features might reflect the extreme habitat of this organism.

Figure 1

The gene repertoires of M. kandleri and other archaeal methanogens. (A) Conserved and unique proteins in M. kandleri. (B) A Venn diagram of the shared and unique portions of the proteomes of archaeal methanogens. The number of COGs that are unique for the three methanogens is shown in parentheses in the central section. The number of COGs that include a given archaeal species and two or more bacteria, but no other archaea, is shown in brackets.

Figure 2

Genome trees of archaea. (A) Gene content tree. (B) Tree based on conserved gene pairs. Solid lines show terminal branches and internal branches with >90% bootstrap support, and dotted lines show internal branches with <90% support. The three methanogen species are highlighted by red, green, and blue. The bacterial species included as an outgroup are shown in magenta. Species abbreviations: Euryarchaeota: A. fulgidus (Arcfu), M. thermoautotrophicum (Metth), M. jannaschii (Metja), M. kandleri (Metka), Pyrococcus horikoshii (Pyrho), Pyrococcus abyssi (Pyrab), Thermoplasma volcanium (Thevo), Thermoplasma acidophilum (Theac), Halobacterium sp. (Halsp); Crenarchaeota: Aeropyrum pernix (Aper), Sulfolobus solfataricus (Sulso); bacteria: Thermotoga maritima (Thema), Deinococcus radiodurans (Deira), Escherichia coli (Escco).

Figure 3

The ML phylogenetic tree of archaea constructed by using concatenated alignments of ribosomal proteins. Thick solid lines indicate internal branches with Resampling of Estimated Log-Likelihoods bootstrap support >95%, thin solid lines indicate branches with 90–95% bootstrap support, and dotted lines indicate branches with <70% support. Distances are indicated in substitutions per site. Methanogens are highlighted. Species name abbreviations are as in Fig. 2.

Figure 4

Predicted functional systems and general metabolic pathways of M. kandleri compared with the counterparts in other archaeal methanogens. Specific enzymatic reactions predicted in a given species but not in the other two are color-coded blue (M. kandleri), green (M. thermoautotrophicus), and red (M. jannaschii). Gene identifiers are shown only for M. kandleri. The methanogenesis pathway is shown by magenta arrows. Reactions for which no candidate enzyme was confidently predicted are shown by dashed arrows. Final biosynthetic products are shown as follows: light blue for amino acids, dark yellow for nucleotides, brown for sugars, pink for cofactors. MF, methanofuran; FMF, formyl-methanofuran; H4MPT, tetrahydromethanopterin; CoM, coenzyme M.