Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication

Abstract

pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5 end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.Whereas major studies on viral systems in Bacteria and Eukarya have shed sufficient light on basic and regulated gene expression in these organisms, very little regarding the elucidation of the mechanisms of viral gene expression and regulation in hyperthermophilic Archaea has been addressed. A few archaeal viruses have been investigated in depth at molecular and physiological levels (23,25,48,53). These viruses are known to be highly unusual in terms of morphology and genome structure/sequence (38,39,40,41,45). A survey of the extrachromosomal elements in the hyperthermophilic crenarchaeon Sulfolobus (64, 65) has revealed the existence of many new viruses, with the Fuselloviridae being the most common family among the Sulfolobales to date (37). Sulfolobus spindleshaped virus 1 (SSV1) is the best-studied member of this family, and it behaves as a temperate virus both in Sulfolobus shibatae and in the nonnatural but related Sulfolobus solfataricus host (53); infection, integration of DNA into the host chromosome, and the production of virions cause apparently no phenotype change but do cause a significant growth retardation of the host cells, which can be visualized as turbid plaques on plated lawns of indicator host cells around propagation foci (53, 65).Transcription studies conducted on SSV1 have pointed out that the copy number of the episomal DNA as well as the virus titer remain essentially constant in the unirradiated host (48). DNA replication increases after induction by UV or other DNA-damaging agents and seems to be mediated by transcription at the promoter T ind (48,53). Furthermore, the structural genes of SSV1 are constitutively and coordinately transcribed in nonirradiated cells, and the amount of these...

The transcription programme of the protein-primed halovirus SH1

“…The thermophilic archaeal virus SSV1 shows a similar behaviour, with many genes being transcribed as operons from single 59 start sites, but with terminator read-through resulting in the formation of several RNA species (Fröls et al, 2007;Reiter et al, 1987). The archaeal satellite virus pSSVx also utilizes transcriptional read-through of terminators (Contursi et al, 2007).…”

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

“…T4 polynucleotide kinase (Fermentas Life Sciences) was used to label 5= ends of single-stranded oligonucleotides T lys -rv (5=-AAGTTCTTCAATGCGTCTTCTGATT-3=), T ind -fw (5=-TCTGAGCTACTAATACTGCTTGAAT-3=), and 16S-rv (5=-CTCTCCT ACTCGGGTGGAGCAAC) with radioactive [␥- 32 P]ATP, following the manufacturer's instructions. Purification of radiolabeled oligonucleotides was achieved by gel filtration chromatography using Illustra Nick columns (Amersham Biosciences), and hybridizations with single-stranded DNA probes were carried out as described elsewhere (33). Probes were eventually removed by boiling in 0.1% (wt/vol) sodium dodecyl sulfate (SDS) for 10 min Western blot analysis.…”

Archaeal Extrachromosomal Genetic Elements

“…In the early growth phase, pSSVx is present at a low copy number in the host, and only three genes, i.e., plrA and genes for ctRNA1 and ctRNA2, are expressed at high levels. Genes encoding ctRNA1 and ctRNA2, identified by Northern hybridization, are transcribed in a direction opposite that of copG (ORF51) and ORF91 (121). Therefore, the two RNAs are complementary to the copG and ORF91 mRNAs, respectively.…”

“…Like SSV2, it exhibits a pattern of inducible replication during host growth (120). Due largely to the genomic simplicity of pSSVx, gene expression in this genetic element has been investigated in detail (121,122). There are 11 genes in pSSVx.…”