H J Monstein1, C D de la Cour, J Jonasson. 1. Molecular Biology Laboratory-LMO, Division of Clinical Microbiology, University Hospital, Linköping, Sweden.
Abstract
BACKGROUND: Previous studies have revealed that extensive nonrandom fragmentation of ribosomal RNA occurs during conversion of Helicobacter pylori to the coccoid form. The 16S rRNA fragmentation has been characterised in some detail. The aim of the present study was to define corresponding cleavage-sites in the 3'-half of the 23S rRNA molecule. MATERIALS AND METHODS: Northern blot analysis using 23S rRNA specific antisense riboprobes and a 5'-end-labelled oligonucleotide probe was used to analyse the 23S rRNA fragmentation pattern in coccoid H. pylori type strain CCUG 17874T and H. pylori 26695, for which the genome has been sequenced. A double-stranded cDNA-dependent (ds-cDNA) primer-extension analysis technique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the peptidyl-transferase centre was used to determine cleavage sites at the nucleotide level. RESULTS: We report here the mapping of putative cleavage sites within domains IV and V, enclosing the peptidyl transferase centre, in the 3'-half of the 23S rRNA molecule. Three cleavage sites were located in domain IV. Two other cleavage sites were located in the peptidyl transferase centre, and one presumptive multiple-break site between helices 77 and 78 in domain V. The DNA motifs were different from the postulated A + U rich single-strand cleavage sites recognised by RNase E, which has been implicated in rRNA degradation in Escherichia coli. CONCLUSIONS: The present analysis suggests that a hitherto unknown mechanism is responsible for the nonrandom fragmentation of rRNA in coccoid H. pylori, which may have important consequences for the growth, and survival of the bacterium.
BACKGROUND: Previous studies have revealed that extensive nonrandom fragmentation of ribosomal RNA occurs during conversion of Helicobacter pylori to the coccoid form. The 16S rRNA fragmentation has been characterised in some detail. The aim of the present study was to define corresponding cleavage-sites in the 3'-half of the 23S rRNA molecule. MATERIALS AND METHODS: Northern blot analysis using 23S rRNA specific antisense riboprobes and a 5'-end-labelled oligonucleotide probe was used to analyse the 23S rRNA fragmentation pattern in coccoid H. pylori type strain CCUG 17874T and H. pylori 26695, for which the genome has been sequenced. A double-stranded cDNA-dependent (ds-cDNA) primer-extension analysis technique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the peptidyl-transferase centre was used to determine cleavage sites at the nucleotide level. RESULTS: We report here the mapping of putative cleavage sites within domains IV and V, enclosing the peptidyl transferase centre, in the 3'-half of the 23S rRNA molecule. Three cleavage sites were located in domain IV. Two other cleavage sites were located in the peptidyl transferase centre, and one presumptive multiple-break site between helices 77 and 78 in domain V. The DNA motifs were different from the postulated A + U rich single-strand cleavage sites recognised by RNase E, which has been implicated in rRNA degradation in Escherichia coli. CONCLUSIONS: The present analysis suggests that a hitherto unknown mechanism is responsible for the nonrandom fragmentation of rRNA in coccoid H. pylori, which may have important consequences for the growth, and survival of the bacterium.