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

Abstract

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 pPM 24) 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 I11 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%.

Comparative sequence analyses reveal frequent occurrence of short segments containing an abnormally high number of non-random base variations in bacterial rRNA genes

“…This view is supported by reports that an organism may have distinct types of rRNA genes with high levels of base variations throughout the entire gene (Gunderson et al, 1987 ;Mylvaganam & Dennis, 1992 ;Carranza et al, 1996 ;Wang et al, 1997 ;. Niebel et al (1987) demonstrated that the rRNA cistrons from Proteus vulgaris were expressed and the products correctly processed and assembled into ribosomes when transformed into E. coli. Recently, Asai et al (1999) reported a complete exchange of rRNA genes between different bacterial species and replacement of a 23S rRNA gene segment of E. coli by the corresponding region of yeast.…”

Expression of the chromosome-coded rRNA genes of Proteus vulgaris in Escherichia coli.

“…Transfer of an rrn operon from tgt11 to chromosome About 70% of the recombinant phages contained the 7.5 kb rrn operon from P. vulgaris originating from plasmid pPM14 (10). Compared with non-lysogenized E. coli Y l089, EcoRI restricted chromosomal DNA from various lysogenized clones contained two additional bands of size 6.5 and 1 kb, respectively, which hybridize with P. vulgaris 16S rRNA, indicating the successful integration of the recombinant phage into the chromosome.…”

“…16S rRNA was isolated from 30S ribosomal subunits and separated on a 2.8% (w/v) PAGE gel. Individual spots of the fingerprints could be assigned to origins from either homologous E. coli or heterologous P. vulgaris 16S rRNA using information obtained from fingerprints of mixed 16S rRNAs analyzed previously (10). The amount of heterologous 16S rRNA was estimated by scintillation counting of selected isolated oligonucleotides (16) occurring in a single copy only in the 16S rRNA primary structure.…”

“…Raibaud, Institut Pasteur, France. E. coli Y1088 and Y1089 (6) were obtained from Promega Biotec, Madison, Wisconsin; t gt 11 (17) was purchased from Promega Biotec; Proteus vulgaris IFAM 1731 and the clones pPM2 and pPM14 (10) were from the culture collection of the Institute fur Allgemeine Mikrobiologie, Kiel. Cloning in phage Agtll.…”

“…Transformation of these plasmids in Escherichia coli lead to the formation of hybrid ribosomes in the corresponding clones (10) to which P, vulgaris 16S rRNA contributed about 25%. As demonstrated by in vitro transcription experiments (9) heterologous promoters are as efficiently recognized as the homologous promoters.…”