TEXT

Top Abstract Text References

It has been shown previously that strains of extremely halophilic archaea of the euryarchaeotal genera Halobacterium and Haloferax produce toxic bacteriocin-like proteins, termed halocins, possibly for competition with related sensitive strains (5, 8, 9, 13, 15). Here we present evidence for the production of similar specific proteinaceous toxins by strains of the extremely thermophilic crenarchaeote Sulfolobus.

Strains and cell growth. The strains of Sulfolobus sp. described in this communication were isolated from samples taken from solfataric fields throughout Iceland. The methods for sampling and enrichment were similar to those described previously (16). The minimal medium (4), used either in liquid form or in Gelrite (Kelco, San Diego, Calif.) gels, was supplemented with 2 g of tryptone (Difco, Detroit, Mich.) per liter and adjusted to pH 3.2 with sulfuric acid. More than 400 isolates were obtained from heterotrophic enrichment cultures via single colonies. All these strains belonged to one species provisionally named "Sulfolobus islandicus" (16).

Demonstration of sulfolobicin production. The strains were screened for the inhibition of the growth of Sulfolobus solfataricus P1 (DSM 1616) by a "spot-on-lawn" procedure. Two microliters each of exponentially growing cultures of 420 different "S. islandicus" strains was spotted onto 1.5-ml soft layers of 0.2% Gelrite routinely seeded with about 6 × 10⁷ cells of S. solfataricus and laid over 0.8% Gelrite supporting gels, as described by Zillig et al. (16). The spots of 41 cultures were surrounded by sharp-edged, nearly clear zones of growth inhibition (halos) with an area of about 0.8 cm² after incubation at 80°C for 48 h. The size of the halo did not depend on the incubation time. The inhibitory agent was not infectious and therefore not a virus. The effect rather appeared to be caused by an inhibitory substance resembling a bacteriocin (1, 6), which we thus called sulfolobicin, according to standard terminology.

Soluble sulfolobicin is not excreted into the culture medium. In cell-free culture supernatants, sulfolobicin activity could be detected only after about a 100-fold concentration, e.g., by precipitation with ammonium sulfate at 30% saturation, or with polyethylene glycol 6000 (105 g/liter) and NaCl (58 g/liter) (overnight at 4°C), or by centrifugation for 5 h at 50,000 rpm in a 55 Ti rotor (Beckman). For estimation of the activity, 2 µl of twofold serial dilutions of samples in 20 mM Tris-acetate, pH 6, was applied to standard lawns of S. solfataricus. The highest dilution producing recognizable inhibition was considered to contain 1 arbitrary unit (AU) of sulfolobicin. Maximal extracellular sulfolobicin activity was detected when the cells entered the stationary phase. The total extracellular activity of a 500-ml culture was about 5 × 10³ AU. An approximately 30-times-higher amount of the toxin could be purified from the cells of a 500-ml culture following the procedure described below.

View larger version (110K): [in this window] [in a new window]   FIG. 1.   (A) Electron micrograph of cell-derived vesicles with which extracellular sulfolobicin activity was associated. Vesicles were negatively stained with 2% uranyl acetate. Bar, 150 nm. (B) Electron micrograph of a fragment of the surface layer of a vesicle, stained with 2% uranyl acetate, and its diffraction pattern showing a clear reflex of the second order at (11 nm)1. Bar, 20 nm.

Purification procedure. For the extraction and purification of sulfolobicin, cultures of the producer cells were grown to the late stationary phase. The cells were collected, suspended in buffer A (20 mM Tris-acetate, pH 6), and disrupted by sonication (Branson sonifier fitted with a macro tip; 7 min). Residual unbroken cells were removed by centrifugation at 3,000 rpm in a Minifuge 2 (Heraeus). The cell ghosts were collected by high-speed centrifugation (30 min at 39,000 rpm in a Beckman SW41 rotor). No sulfolobicin activity was present in the supernatant. The ghosts were washed twice in buffer A and then subjected to extraction with either 6 M urea, 1 M NaCl, 0.1% Triton X-100 (all in buffer A), diethylether, or trichlormethan or the mixture trichlormethan-methanol-water (65:25:4) or n-butanol-acetic acid-water (80:20:20). Only Triton X-100 extraction was able to release the sulfolobicin from the ghosts.

Chemical nature. To elucidate the chemical nature of the sulfolobicin, the purified preparation was treated with -amylase, - and -glucosidases, lipase, phospholipase C, lipoprotein lipase, pronase E, proteinase K, and trypsin (all from Sigma and used as recommended by the manufacturer). The assay mixtures containing 0.1 mg of the enzyme tested per ml and 20 AU of the sulfolobicin per µl were incubated for 3 h at 37°C. The activity was determined by the spot-on-lawn test in comparison with a corresponding control without enzyme. No decrease of sulfolobicin activity was detected after treatment with glycolytic or lipolytic enzymes. Incubation with all proteolytic enzymes tested led to the complete loss of sulfolobicin activity, indicating that an intact protein is required for activity.

Molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 0.7-mm gels as described by Schägger and von Jagow (14) was used to estimate the molecular mass of the sulfolobicin. Since no protein band was visible on a Coomassie blue-stained gel with 100 AU of sulfolobicin purified as described above (Fig. 2A, lane 2), the sulfolobicin band was detected via its activity. A Coomassie blue-stained gel with 100 AU of sulfolobicin and molecular mass standards was washed in distilled water for 6 h and laid over a soft layer seeded with S. solfataricus. A zone of growth inhibition was observed after development of the lawn at 80°C for 48 h (Fig. 2B, lane 2). The molecular mass of the sulfolobicin was estimated from its mobility to be approximately 20 kDa. To directly visualize the sulfolobicin band by Coomassie blue staining, we had to apply about 10⁵ AU of the toxin (Fig. 2A, lane 1). The sulfolobicin from isolated S-layer-coated membrane vesicles had the same molecular mass as that solubilized and purified from cell membranes.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified sulfolobicin. (A) Coomassie blue-stained gel. (B) A portion of the Coomassie blue-stained gel containing lanes 2 and 3 laid onto an indicator lawn. Lane 1, 105 AU of sulfolobicin; lanes 2, 100 AU of sulfolobicin; lanes 3, protein markers with molecular masses of 39.2, 26.6, and 20.1 kDa. The arrows indicate the clearing of the lawn at the position of sulfolobicin (B) and a Coomassie blue-stained protein band with the same mobility (A).

Concentration dependence of archaeocidal effect. Addition of sulfolobicin (100 AU/ml) to an S. solfataricus culture at an optical density at 600 nm of 0.25 caused a decrease in the number of CFU to about 50% in 20 min, whereas the optical density remained constant (data not shown). Thus, the effect of the toxin is archaeocidal rather than archaeolytic. The decrease of the fraction of viable cells as a function of the sulfolobicin concentration is shown in Fig. 3.

View larger version (10K): [in this window] [in a new window]   FIG. 3.   Survival of S. solfataricus in the presence of sulfolobicin. Different amounts of sulfolobicin were added to 25 ml of growing cultures of S. solfataricus containing about 5 × 106 cells/ml. After 24 h of growth, the samples were plated for the detection of CFU. CFU(0) was determined before addition of sulfolobicin.

Plasmids of sulfolobicin-producing strains. Some of the sulfolobicin-producing strains of "S. islandicus," e.g., HEN2/2, contained conjugative plasmids (11). The production of and the resistance to sulfolobicin were, however, not transferred to transcipients by the DNAs of these plasmids (D. Prangishvili and W. Zillig, unpublished results). The results indicate that the genes for sulfolobicin production and immunity might be located on the chromosomes of the producer cells.

Perspectives. Although sulfolobicin shares key characteristics of bacteriocins, such as the proteinaceous nature, the killing mode of action, and the narrow range of activity directed primarily against closely related strains (6), it is in some respects different. In contrast to many bacteriocins, sulfolobicin is apparently not released from the producer cells in soluble form in liquid medium but remains bound to the membranes of the cells or of cell-derived S-layer-coated membrane vesicles. These vesicles resemble recently described enzyme-containing killer vesicles produced by different gram-negative bacteria (2).