An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria

Abstract

Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

Figure 1

(A) Deletion mutations introduced into the chromosome. Open and filled rectangles are rRNA and tRNA genes, respectively. Deleted regions are shown by broken lines. cat⁺ and lacZ⁺ indicate the deleted regions are replaced with the coding regions of these genes. The rRNA promoters (P1P2) and terminators (ter) also are shown. The crosses in the 16S and 23S rRNA genes indicate the positions of the Spc and erythromycin resistance mutations, respectively. ▴ indicates the position of an intervening sequence in the 23S rRNA gene of S. typhimurium (St) or P. vulgaris (Pv). The four DNA fragments with tRNA genes cloned in pTRNA65 are shown below the rRNA operons. (B) pTRNA plasmids. Relative positions and orientation of the tac promoter, tRNA genes and the 5S rRNA gene are indicated by arrows. The rRNA transcription terminators and the truncated 16S and 23S rRNA genes also are shown. Truncation of the genes is indicated by a prime. The plasmids are not drawn to scale. (C) An rRNA plasmid carrying the wt rrnC operon. Filled and open boxes indicate stable RNA genes and their flanking sequences, respectively. The size of the BamHI–PstI fragment containing the rrnC operon is 8.4 kb.

Figure 2

The pedigree of rrn-deletion strains. Inactivated rRNA operons are indicated by capital letters derived from their specific operon names (for example, A for rrnA). When the inactivation was carried out by a deletion/insertion mutation, a capital letter is followed by a lowercase letter c or z representing the inserted gene cat⁺ or lacZ⁺, respectively (for example, Ac for rrnA∷cat⁺). pTRNA, pHK-rrnC⁺, and pSTL102 contain Spc, Km, and Ap resistance markers, respectively. All rRNA operons cloned in the plasmids shown contain the gene for tRNA Glu-2.

Figure 3

Autoradiograms of Southern blot hybridization showing the inactivation of chromosomal rRNA operons. DNA was analyzed as described in ref. . The number of operons inactivated and strain numbers are shown on the top. The rRNA operon carried by each fragment is shown on the left. (A) Inactivation of the 16S rRNA genes. The SalI–SmaI fragment (probe I, Fig. 1A) of the 16S rRNA gene was labeled with ³²P and used for hybridization. TA520, 531, and 542 contain an rRNA plasmid, pHK-rrnC⁺ and thus give an additional band (8.4 kb) carrying the plasmid-borne rrnC operon (see Fig. 1C) just above the rrnD band (8.1 kb). (B) Inactivation of the 23S rRNA genes. Probe I was removed from the membrane shown in A, and the cellular DNA was rehybridized with ³²P-labeled probe II carrying the DNA sequence between the HpaI and the SalI sites in the 23S rRNA gene (Fig. 1A). TA520, 531, and 542 again gave a plasmid-derived band. The ΔrrnG∷lacZ⁺ construct contains the HpaI–SalI region of the gene (Fig. 1A). Therefore, in TA447, 488, 516, 520, and 531 in which the rrnG operon was inactivated with this construct, the rrnG⁺-containing band (15.5 kb) disappeared and a new band with the expected size (11.6 kb, indicated by an arrow) appeared just above the rrnE band (11.2 kb). This band disappeared in TA542 in which ΔrrnG∷lacZ⁺ was replaced with ΔrrnG∷cat⁺.

Figure 4

Expression of homogeneous rRNA in Δ7 prrn strains. 16S rRNA molecules in the rrn⁺ strain and in Δ7 prrn strains carrying either wt (pHK-rrnC⁺) or mutant (pSTL102) rRNA plasmid were analyzed by primer extension. DNA primers annealed to 16S rRNA molecules were extended in the presence of one dideoxynucleotide (ddATP) and three deoxynucleotides (dGTP, dCTP, and dTTP). Because mutant 16S rRNA produced from pSTL102 contains a C to U change, primers hybridized to wt and mutant 16S molecules are extended by four nucleotides and one nucleotide, respectively.

Figure 5

Expression of foreign rRNA in Δ7 prrn strains. (A) Primer extension analysis. 23S rRNA molecules in the rrn⁺ strain and in Δ7 prrn strains carrying either E. coli (pSTL102) or S. typhimurium (pSt1-Km) rRNA plasmid were analyzed by primer extension as described in Fig. 4. The primer used in this experiment hybridizes to a common sequence in E. coli and S. typhimurium 23S rRNAs but gives different extension products because of a sequence difference between the two molecules. (B) Fragmentation of 23S rRNA. Total cellular RNA prepared from the rrn⁺ strain and Δ7 prrn strains carrying plasmids with E. coli (Ec, pSTL102), S. typhimurium (St, pSt1-Km), or P. vulgaris (Pv, pPM2) rRNA is shown. Fragmented 23S rRNA molecules are indicated by a prime.