Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae.

The DNA sequence of the Sulfolobus shibatae virus SSV1 is the first complete sequence of an archaebacterial virus genome. The viral DNA is a closed double-stranded DNA circle of 15465 bp. The features of the sequence, the positions of all 11 transcripts, the three characterized proteins, and the open reading frames are described.

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.

Gene expression in archaebacteria: physical mapping of constitutive and UV-inducible transcripts from the Sulfolobus virus-like particle SSV1.

The transcription of the genome of the UV-inducible Sulfolobus virus-like particle SSV1 was studied. Eight different transcripts could be distinguished by Northern analysis that were present in uninduced cells and the coordinately increased in amount after UV induction of SSV1. Using single-stranded DNA probes from different parts of the genome, the approximate map positions of these RNAs and the directions of transcription were determined. In two cases, terminator read-through resulted in the formation of more than one RNA species from a single 5' end and therefore the eight different RNAs corresponded to only five different transcriptional starts. Two RNAs sharing a common 5' end encode SSV1 structural proteins. The 5' end of these transcripts was determined by S1 nuclease analysis. About 20 nucleotides upstream of the transcriptional start of these RNAs, there is an AT-rich region resembling putative promoter sequences which have been found at a similar distance 5' to the genes encoding stable RNAs in Thermoproteus. In addition to the eight constitutive transcripts, a UV-inducible RNA of 0.3 kb was mapped on the SSV1 genome. In contrast to all other RNAs, it was not detectable in uninduced cells and it is expressed shortly before the amplification and packaging of the SSV1 genome commences.

Plasmid-related anaerobic autotrophy of the novel archaebacterium Sulfolobus ambivalens.

Three different species of the genus Sulfolobus, S. acidocaldarius, S. solfataricus (= Caldariella) and S. brierleyi, have been distinguished by the conditions required for optimal growth, by the component patterns of their DNA-dependent RNA polymerases and by DNA sequence data. Many isolates of these species are able to grow chemolithoautotrophically using CO2 as the sole carbon source and the oxidation of S(0) with O2 yielding sulphuric acid, as the energy source, though a few others grow only heterotrophically. We show here that a strain of a novel Sulfolobus species, S. ambivalens, is alternatively able to live by an anaerobic mode of chemolithoautotrophy, also using CO2 as the sole carbon source, but using reduction of S(0) with H2, yielding H2S as the energy source. This mode of growth is correlated with the amplification of a plasmid, pSL10.

SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12.

Sulfolobus acidocaldarius, strain B12, which harbours a double-stranded DNA species both as a plasmid and in a linear form, which is integrated at a specific site of the chromosome, produces virus-like particles upon u.v. irradiation. These particles contain the same circular DNA and a number of coat proteins and are probably surrounded by a lipid membrane. They are lemon shaped, 100 x 60 nm in size and carry tail structures at one pole. The host cell recovers and remains lysogenic after virus production. Though a large fraction of liberated particles is found attached to structures derived from the cells, neither adsorption nor infection of a number of Sulfolobus isolates has so far been observed.

Genome organization and transcription in archaebacteria.

The genome organization of the archaebacteria is investigated in three model systems: a) rRNA genes of various archaebacteria, b) a plasmid of 15.6 kb from Sulfolobus acidocaldarius which exists in free or integrated form, c) the 59 kb genome of phage phi H of Halobacterium halobium as a model for the unusual structural variability of DNA in this organism. Several variants of this phage have been isolated, their genomes differ by several insertions, a deletion, and an inversion. The frequent inversion and circularization of a 12 kb segment of DNA appears to be linked to the presence of two copies of an IS element at its flanks. DNA-dependent RNA polymerases have been isolated from a large number of archaebacteria including representatives of 4 families of the novel order Thermoproteales . As shown by immunological methods, they are closely related to those of eukaryotes. Two different types of RNA polymerase exist in the two main branches of the archaebacteria. The role of one component of the enzyme of Thermoplasma acidophilum was elucidated using an in vitro transcription system.

A plasmid in the archaebacterium Sulfolobus acidocaldarius.

A plasmid of mol. wt. approximately 9 x 10 has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.

Altered transcriptional termination in a rifampicin-resistant mutant of Escherichia coli which inhibits the growth of bacteriophage T7.

A spontaneous rifampicin-resistant mutant of E. coli K12, RpoB26, which inhibits the growth of bacteriophage T7 has been isolated. The mutation is an RNA polymerase mutation; it also restores the wild-type effect of polar mutations in a rho-deficient strain, probably by restoring transcriptional termination. The efficiency of plating (e.o.p.) of wild-type T7, and of some early region deletion and point mutants of T7 tested, is reduced on RpoB26 by a factor of 10(-4). However, some deletion mutants are inhibited more severely (up to 10(-7) on RpoB26. We argue that these differences may reflect variations in the frequency of transcriptional termination before gene 1, an essential gene which codes for the T7 RNA polymerase (Summers and Siegel 1970; Chamberlin et al. 1970). We also present data which suggest that the product of a late T7 gene plays a role, by some interaction with the product of gene 1, in the inhibition of T7 in RpoB26. We suggest that different levels of expression of gene 1 may lead to different degrees of inhibition of T7 strains in RpoB26.

A possible role in transcription for the single-stranded DNA binding protein of bacteriophage T7.

A spontaneous rifampicin-resistant mutant of E. coli, RpoB26, which inhibits the growth of bacteriophage T7, has been described in the accompanying paper (Schwarz et al.). The rifr mutation appears to increase the rate of transcriptional termination in a rho-deficient strain. T7 mutants with the ability to grow (Gor+) on the Rifr mutant were isolated, and some of their properties were investigated. One of these Gor+ mutants has a small deletion, located between nucleotides 9694 and 9820 of the T7 DNA sequence (Dunn and Studier 1980), which affects the size of the T7 single-stranded DNA binding protein (ssDBP), the product of gene 2.5 (p 2.5) (Dunn and Studier 1980). The Gor+ phenotype was also mapped to this region by genetic methods. Gor+ is recessive to the wild-type (Gor-) phenotype. This suggests that the T7 ssDBP may normally increase the frequency of transcriptional termination in the early region by binding to single-stranded nucleic acid configurations, and so affecting molecular conformations involved in the termination process. Excessive termination in RpoB26 could therefore be compensated for by alterations of the ssDBP.