Screening for Sulfolobales, their Plasmids and their Viruses in Icelandic Solfataras

Abstract

We have sampled acidic springs, water and mud holes of 14 major solfataric fields of Iceland in order to isolate both heterotrophic and autotrophic members of the order Sulfolobales and to find affiliated genetic elements e.g. plasmids and viruses. The diverstiy of 120 isolates was analysed by comparing DNA restriction fragment patterns. The 44 heterotrophic isolates belonged to only two types, the solfataricus (S) type and the islandicus (I) type as judged by the restriction patterns of their DNAs. None of the heterotrophic isolates was able to grow autotrophically. The more than 70 autotrophic isolates belonged to only three types, the Desulfurolobus (D) type, the closely related A type and, rarely, the B type. None of the autotrophic isolates was able to grow heterotrophically. Primary heterotrophic colonies often gave rise to obligately autotrophic isolates when submitted to autotrophic selection, probably because they constituted synthrophic associatons. Inversely, primary autotrophic colonies sometimes yielded obligately heterotrophic isolates when submitted to heterotrophic selection. Cell-free filtrates of the field samples precipitated with PEG 6000 yielded different types of viruslike particles as visualized by electron microscopy. Some of these were probably Thermoproteus viruses. No infectious or even lytic virus was obtained from these samples. Four different multicopy plasmids, three, pRN1, pRN2 and pHE7 from heterotrophic hosts and one, pDL10, originally found in Desulfurolobus ambivalens DSM 3772, occurring in all but two autotrophs of the D type, were characterized and used for developing cloning vectors. All 18 representatives of the heterotrophic S type and none of the I type were lysogenic for the virus SSV1 originally discovered in S. shibatae . Different lysogens exhibited different ratios of free circular and linearly integrated viral DNA. The I type isolate KVEM10H1 multiplied SSV 1 but did not integrate its genome into the chromosome. One heterotrophic I type and one autotrophic D type isolate carried doublestranded DNA viruses, SIRV and DAFV. SIRV is a stiff rod, 0.95 μm long and 0.026 μm in diameter, consisting of 33 kbpairs of linear double stranded DNA, a strongly basic DNA binding protein and terminal tentacles involved in attachment to thin filaments, most probably pili. It lacks a membrane or hydrophobic coat and represents a novel virus tpye. The flexible filamentous DAFV, 2.3 μm long and 0.027 μm in diameter, containing 56 kbpairs of linear double-stranded DNA, appears to be enwrapped in a membrane. It resembles representatives of the lipotrixviridae, most closely the virus TTV2.