Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a unique, intimate association of two archaea

Abstract

Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.

FIG. 1.

Growth of the coculture in the fermentor. In the preculture, around 30% of the I. hospitalis cells were occupied by N. equitans cells (two to four per Ignicoccus cell). (A) Growth curves. I. hospitalis cells, •; total N. equitans cells, shaded ×; attached N. equitans cells, plain shaded square; free N. equitans cells, unshaded ×. (B) Numbers of N. equitans cells (ranging from 0 to 10) attached to cells of I. hospitalis are represented by no shading (n = 0) increasing to the darkest shading (n = 10). The insert is an enlarged view of the cell concentrations within the first 5 h after inoculation.

FIG. 2.

Epifluorescence microphotographs of the coculture at different growth phases after staining with BacLight. Dead cells stained red, and living cells stained green. (A) At 3.25 h after inoculation, exponential growth phase. (B) At 7.5 h after inoculation, transition into the stationary phase. (C) At 10 h after inoculation, stationary phase. (D) At 23 h after inoculation, stationary phase.

FIG. 3.

Growth of the coculture in the fermentor. In the preculture, nearly all I. hospitalis cells were occupied by at least 10 N. equitans cells. (A) Growth curves. I. hospitalis cells, •; total N. equitans cells, shaded ×; attached N. equitans cells, plain shaded square; free N. equitans cells, unshaded ×. (B) Numbers of N. equitans cells (ranging from 0 to 10) attached to cells of I. hospitalis are represented by no shading (n = 0) increasing to the darkest shading (n = 10). The insert is an enlarged view of the cell concentrations within the first 10 h after inoculation.

FIG. 4.

Transmission electron micrographs of ultrathin sections of I. hospitalis and N. equitans following cryoprocessing as described in Materials and Methods. I.ho., I. hospitalis cell; CM, cytoplasmic membrane; OM, outer membrane; Pp, periplasm; PV, periplasmic vesicles; N.eq., N. equitans cell. White arrows point to the contact site where the I. hospitalis outer membrane is in close contact with the cytoplasmic membrane. Black arrows, fibrous material in the gap between the two cells. Bars, 1 μm.