The novel extremely acidophilic, cell-wall-deficient archaeon Cuniculiplasma divulgatum gen. nov., sp. nov. represents a new family, Cuniculiplasmataceae fam. nov., of the order Thermoplasmatales

Abstract

Two novel cell-wall-less, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains were isolated from acidic streamers formed on the surfaces of copper-ore-containing sulfidic deposits in south-west Spain and North Wales, UK. Cells of the strains varied from 0.1 to 2 μm in size and were pleomorphic, with a tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0-1.2 and 37-40 °C, with the optimal substrates for growth being beef extract (3 g l- 1) for strain S5T and beef extract with tryptone (3 and 1 g l- 1, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and small relative amounts of intact and core diether lipids were present. Strains S5T and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. The DNA G+C content was 37.3 mol% in strain S5T and 37.16 mol% for strain PM4. A similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales, with 73.9-86.3 % identity with sequences from members of the order with validly published names. The average nucleotide identity between genomes of the strains determined in silico was 98.75 %, suggesting, together with the 16S rRNA gene-based phylogenetic analysis, that the strains belong to the same species. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. are proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T and PM4 ( = JCM 30641 = VKM B-2940). The type strain of Cuniculiplasma divulgatum is S5T ( = JCM 30642T = VKM B-2941T).

Fig. 1

Electron micrographs of cells of strain PM4 (4 days of growth). (a) Tight cluster of cells, showing general pleomorphism. Occasionally, cells are spherical (upper inset) or appear Y-shaped (lower inset). Appendices can be seen as fimbriae-like structures (see upper inset; fi). Arrowheads indicate direction of shadow casting. (b) Ultrathin sections show the rather obvious cell pleomorphism and, in detail (inset), it is seen that only a unit membrane surrounds the cytoplasm. The cytoplasm is rather dense but often contains electron-translucent areas of different sizes, which in general represent voids (*). Ghost-like remnants of cells (#) are present.

Fig. 2

Ultrastructure of strain S5^T. (a) Cluster of cells, which appear rather pleomorphic when prepared as whole-mount cells and PtC-shadow-contrasted (arrowhead, direction of shadow-cast; arrow, ring-shaped cell). When seen in ultrathin sections (b, c), cells are surrounded by a single unit membrane (cm) and in general show a densely packed cytoplasm without distinct inclusions.

Fig. 3

Lipid composition of strain S5^T. The column on the left displays the relative abundances of the major lipid groups. The horizontal bars show the relative contributions of individual lipid structures to the major groups. 1G, Monoglycosidic; 1G-PG, monoglycosidic phosphatidylglycerol; 2G, diglycosidic; 3G, triglycosidic; 4G, tetraglycosidic; AR, archaeol; G-AR, glycosidic AR; GDD, glycerol dibiphytanyl diether; GDGT, glycerol dibiphytanyl glycerol tetraether; GDGT-0 to -4, GDGT with zero to four cyclopentane moieties; G-OH, glycosidic-hydroxylated; GTGT, glycerol trialkyl glycerol tetraether; H-GDGT, H-shaped GDGT; IP, intact polar; Me-GDGT, methylated GDGT; MK, menaquinone; OH, core hydroxylated; P-AR, phospho-AR; PG, phosphatidylglycerol; TPQ, thermoplasmaquinone; unsAR, unsaturated AR.

Fig. 4

Lipid composition of strain PM4. See Fig. 3 for details.

Fig. 5

16S rRNA gene-based maximum-likelihood tree showing the phylogenetic positions of strains S5^T and PM4 and type strains of all members of the order Thermoplasmatales with validly published names; the analysis involved 11 nucleotide sequences. There were a total of 1323 positions in the final dataset. Methanomassiliicoccus luminyensis B10^T (GenBank accession no. HQ896499.1) was used as an outgroup (not shown). Bar, 5 substitutions per 100 nucleotide positions.