Probing 23S ribosomal RNA cleavage sites in coccoid Helicobacter pylori

Background. Previous studies have revealed that extensive nonrandom fragmen tation 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 antis ense riboprobes and a 5'-end-labelled oligonucleotide probe was used to ana lyse the 23S rRNA fragmentation pattern in coccoid E-I. pylori type strain CCUG 17874(T) and H. pylori 26695, for which the genome has been sequenced. A double-stranded cDNA-dependent (ds-cDNA) primer-extension analysis techn ique using 23S rRNA ds-cDNA and a primer targeting the vicinity of the pept idyltransferase centre was used to determine cleavage sites at the nucleoti de level. Results. We report here the mapping of putative cleavage sites within domai ns IV and V, enclosing the peptidyl transferase centre, in the 3'-half of t he 23S rRNA molecule. Three cleavage sites were located in domain IV. Two o ther cleavage sites were located in the peptidyl transferase centre, and on e presumptive multiple-break site between helices 77 and 78 in domain V. Th e DNA motifs were different from the postulated A + U rich single-strand cl eavage sites recognised by RNase E, which has been implicated in rRNA degra dation in Escherichia coli. Conclusions. The present analysis suggests that a hitherto unknown mechanis m is responsible for the nonrandom fragmentation of rRNA in coccoid H. pylo ri, which may have important consequences for the growth, and survival of t he bacterium.