Cloning and Expression in Escherichia coli of Proteus vulgaris Genes for 16S Ribosomal RNA

SUMMARY: In contrast to the established systems of plasmid-coded homologous ribosomal DNA (rDNA) cistrons in Escherichia coli little is known about the fate of heterologous rRNA. In order to study expression of foreign rDNA, rRNA cistrons from Proteus vulgaris were cloned in phage vector Charon 35, subcloned in pBR322 and transformed in E. coli. The inserts of two clones (pPM2 and pPM14) were characterized by restriction analysis and Southern hybridization. Each of them harboured a complete rrn cistron. The location of rRNA genes of clone pPM2 was also verified by R-loop analysis. The 5’ flanking region of the 16S rRNA of pPM2 was sequenced and compared to the E. coli counterparts. High-level homologies exist in the functional parts of this region, e.g. promoters, box A and RNAase III recognition site. The copy number of pPM2 and pPM14 was estimated to be 8 and 10, respectively. Clones showed a markedly reduced growth rate (generation time about 57 to 70 min) as compared to the non-transformed cells (generation time 40 min). rDNA cistrons of P. vulgaris were properly expressed and the transcripts are processed as demonstrated by the presence of 16S rRNA from P. vulgaris in both ribosomes and 30S ribosomal subunits isolated from the transformed E. coli cells. The fraction of heterologous rRNA in ribosomes was about 25%.