ISOLATION AND CHARACTERIZATION OF CAULOBACTER CRESCENTUS RIBOSOMAL RNA OPERONS

Abstract

Ribosomal RNA genes of Caulobacter crescentus were isolated from a hybrid phage library, subcloned into the plasmid pBR 325, and the organization of the rRNA genes was determined. It was demonstrated that C. crescentus has two rRNA operons. A low copy number of ribosomal RNA operons is observed in other slow growing bacterial species, while multiple copies are observed in species capable of rapid cell growth.;The DNA sequence of the 5' and 3' ends of the 16S rRNA gene and the 5' end of the 23S gene from C. crescentus were determined and then compared to the comparable regions of the E. coli gene and other species. This analysis revealed that C. crescentus forms a new category among the established phylogenetic subclasses. Sequence analysis of the 16S-23S intergenic spacer region demonstrated the presence of tRNA('ile) and tRNA('ala) genes. The same two tRNAs have been found in the intergenic spacer regions of rRNA operons from several bacteria and eukaryote plastids.;In vitro transcription, RNA processing, and DNA sequence analysis of the regions 5' and 3' to the 16S structural gene have identified possible transcriptional promoter sites and RNase III processing sites. The regions where transcription is initiated contain Pribnow sequences similar to those seen at E. coli transcription promoters. A transcript was shown to initiate in vitro and in vivo near the 3' end of the 16S gene. This transcript includes an open reading frame from which a polypeptide could be synthesized that is similar in length but different in amino acid composition to polypeptides predicted from the identical region within the 16S rRNA structural gene of several other organisms. The hydrophilic/hydrophobic nature of these polypeptides is well conserved between the species despite the amino acid sequence heterogeneity. A role for this putative polypeptide in regulating transcription of the entire operon or, perhaps, transcription from this internal promoter is proposed.;Finally, a new technique for mapping genes on the C. crescentus chromosome using suicide plasmids containing a drug-resistance marker is described.