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.