The archaeocins produced by Sulfolobus are entirely different from halocins, since their activity is predominantly associated with the cells and not the supernatant (Prangishvili et al. 2000). Prangishvili et al. (2000) were the first to isolate and characterize these proteinaceous toxins, which they called "sulfolobicins", in keeping with bacteriocin nomenclature. Provisionally, the producer strain has been named "Sulfolobus islandicus". Screening for sulfolobicin activity involves spotting samples of exponentially growing "S. islandicus" cells onto lawns of the sensitive strain S. solfataricus P1. Following incubation, nearly clear zones with sharp borders are generated, the size of the zone of inhibition being inversely proportional to the concentration of sensitive cells in the lawn. To date, the spectrum of sulfolobicin activity appears to be restricted to other members of the sulfolobales: the sulfolobicin inhibited S. solfataricus P1, S. shibatae B12, and six non-producing strains of "S. islandicus". Activity appears to be archaeocidal but not archaeolytic. It does not inhibit S. acidocaldarius DSM639, nor does purified sulfolobicin from strain HEN2/2 inhibit Hbt. salinarum R1 or Escherichia coli (Prangishvili et al. 2000).
Unlike halocins, sulfolobicins are not secreted into the culture medium in any significant quantity, and classical inducing agents (UV light, temperature and pH shifts, and exposure to sensitive cells) used to increase secretion have not been successful (Prangishvili et al. 2000). Analysis of sulfolobicin activity in a 500 ml culture revealed that 30 times more activity can be purified from the cell pellet than from the culture supernatant. To visualize activity in culture supernatants, the supernatant from stationary phase cultures had to be concentrated 100-fold, either by precipitation or centrifugation, before any activity was detected when spotted onto a lawn of sensitive cells.
Extracellular activity is associated with spherical particles 90 to 180 nm in diameter. These particles are present in a ratio of 1:100 cells, and are also produced by strains that do not make sulfolobicin. When purified using CsCl density gradient centrifugation, these particles form a discrete band with a density of approximately 1.29 g/ml. Electron micrographs of this material revealed an inner core with a surrounding layer having a periodicity of 22 nm, the same as the lattice constant of the Sulfolobus S-layer (Prangishvili et al. 2000).
Purification of sulfolobicin involves harvesting cells from late stationary phase, sonicating them, collecting the resultant cell ghosts by high-speed centrifugation, and releasing the sulfolobicin with Triton X-100. Activity elutes in the range of 30 to 40 kDa on size exclusion chromatography, in contrast to 20 kDa on SDS-PAGE. These data suggest that this archaeocin may aggregate (Prangishvili et al. 2000). Activity of purified sulfolobicin remains stable after 6 months at 4°C or 5 days at 85 °C. Enzymatic treatment with a-amylase, a- and P-glucosidases, phospholipase C, and lipoprotein lipase had no effect on activity. However, treatment with pronase E, proteinase K, and trypsin completely destroyed activity, indicating activity is associated with a proteinaceous component (Prangishvili et al. 2000).
Sulfolobicins exhibit some classical bacteriocin characteristics, as they are proteinaceous and are directed against strains that are closely related to the producer. Although some of the producer strains contain conjugative plas-mids, neither sulfolobicin production nor immunity can be transferred to non-producer strains, suggesting that the genes for these traits may be located on the chromosome. Although evidence suggests that sulfolobicins remain bound to cells or associated with S-layer-coated vesicles, it does not exclude the possibility that an undetectable amount of sulfolobicin may leak out from cells or vesicles into the surrounding medium. Indeed, such a scenario could account for the generation of large zones of inhibition on solid medium where the concentration of free sulfolobicin would remain more localized and high. This phenomenon also is seen with cell-bound bacteri-ocins (Prangishvili et al. 2000).
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