Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons

Abstract

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

FIG. 1

The common structure of the rRNA operons in E. coli. Open and filled rectangles represent rRNA (16S, 23S, and 5S) and tRNA genes, respectively. rrnB, rrnC, rrnE, and rrnG contain the spacer tRNA gene for Glu-2, and the other operons (rrnA, rrnD, and rrnH) contain the spacer tRNA genes for Ile-1 and Ala-1B (25). Distal tRNA genes are encoded by only three operons: rrnC contains the tRNA genes for Asp-1 and Trp, and rrnD and rrnH contain the tRNA genes for Thr-1 and Asp-1, respectively. The figure also indicates the relative positions of promoters (P1 P2), terminators (ter), and relevant restriction sites.

FIG. 2

The basic strategy for allele exchange. Thick and thin lines represent chromosomal and plasmid sequences, respectively. The hatched rectangles indicate the 16S and 23S rRNA genes. The 5S rRNA and tRNA genes are not shown. Stippled and open rectangles represent the ampicillin and chloramphenicol resistance genes, respectively, and closed rectangles the sacB-Km^r cassette. ori indicates the relative position of the ColE1-type replication origin. Broken lines indicate possible crossover sites for a successful allele exchange. In panels B, C, and D, only a part of the chromosome is shown. See Fig. 1 for definitions of the other symbols.

FIG. 3

The pedigree of rrn deletion strains. Inactivated rRNA operons are indicated by uppercase letters derived from their specific operon names (for example, A for rrnA). When the inactivation was carried out by a deletion-insertion mutation, an uppercase letter is followed by a lowercase c or z, representing the inserted gene cat⁺ or lacZ⁺, respectively (for example, Ac for rrnA::cat⁺). See Table 1 for precise genotypes. polA mutant strains are resistant to tetracycline since these mutations are linked to Tn10. pTRNA and pHK-rrnC⁺ contain spectinomycin (Spc^r) and Km^r markers, respectively.

FIG. 4

Microscopic examination. Cells from exponential-phase cultures were stained with BacLight (Molecular Probes Inc., Eugene, Oreg.) and analyzed by fluorescence microscopy.

FIG. 5

Physiological effects of rrn inactivation. (A) Relative growth rates, rRNA/protein ratios, and ribosome efficiencies. Growth rates (doublings per hour) were determined by monitoring the turbidity of each culture with a Klett-Summerson photoelectric colorimeter and are presented relative to the rrn⁺ strain values. The maximum standard error of growth rate measurements was 0.07. The actual growth rate of the rrn⁺ strain, TA563, was 2.0 doublings/h. rRNA/protein ratios were determined from the data presented in panel B. Ribosome efficiencies were calculated from growth rates and rRNA/protein ratios as described by Bremer and Dennis (5). (B) Relative total RNA/total protein and tRNA/rRNA ratios. The total RNA/total protein and tRNA/rRNA ratios in each total RNA sample were determined as described previously (1). These parameters were normalized to the total amount of RNA in the rrn⁺ strain. Closed and open bars represent the amounts of rRNA and tRNA, respectively. The maximum standard error of RNA measurements was 0.05. Note that the Δ6 strain contains only the tRNA plasmid while the Δ7 strain contains both the tRNA and rRNA plasmids.