Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli

Abstract

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.

Figure 1

(a) Recombineering, the strategy applied for in frame deletion of E. coli chromosomal genes. Chromosomal gene orf is replaced by a drug-resistance PCR cassette during recombination utilizing short (39–40nt) homologies (shown with striped boxes). Open arrows indicate position of checking primers used to verify gene replacements by PCR. (b) Gene essentiality assay. Nonessential gene such as rplY can readily be replaced with a drug-resistance cassette using recombineering (see scheme, plate and gel on the left column). In the case of rplY knockout, a few normally growing rplY<>cat/rplY+ diploids are seen among thousands of growth-impaired rplY<>cat knockouts on the LB-Cm plate. Only cat insert-related PCR product is synthesized when rplY configuration is tested in rplY<>cat Cm^R recombinants. At the same time, only gene orf<>cat orf/gene orf+ gene partial diploids are viable when essential gene such as rplE or rplR is replaced (right column: scheme, plate and gels). In these cases only rare gene<>cat/gene+ diploids survive on the selective LB-Cm plate. Agarose gels below the plate show that in the case of rplE and rplR gene replacements both, cat-insert and wild type gene-related PCR products are synthesized.

Figure 2

(a) The E. coli spc operon with rplE and rplR genes. The hairpin needed for feedback regulation of the operon is indicated with an asterisk. (b) The replacement of rplE orf with cat was made either in the absence or presence of an rplE-expressing plasmid. Only rplE<>cat/rplE+ diploids survive in the absence of the plasmid (left). The rplE<>cat haploids survive if cells carry an L5-expressing plasmid, showing that rplE is indeed essential while regulatory hairpin is not (right). (c) Agarose gel showing the configuration of rplE in viable Cm^R recombinants in the presence (+) or absence (−) of an rplE-expressing plasmid, as analyzed by PCR of rplE chromosomal region.

Figure 3

Growth of wild type W3110 (WT) and L25-defective KNB800 (ΔL25) strains in the presence of plasmids providing expression of E. coli rplY for L25 protein (pKAB101), B. subtilis gene ctc for CTC protein (pKAB110) and 5’-terminal portion of ctc encoding for N-terminal fragment (NfrCTC) of CTC protein (pKAB104). WT + control plasmid (1), ΔL25 + control plasmid (2), ΔL25 + L25-expressing plasmid (3), ΔL25 + NfrCTC-expressing plasmid (4), ΔL25 + CTC-expressing plasmid (5). Growth medium was supplemented with kanamycin. Incubation at 37°C for 16 hours.

Figure 4

Protein-synthesizing activity of wild type (●) and ΔL25 (▲) cells determined by standard β-galactosidase assay. The β-galactosidase activity is normalized to the cell mass (OD₆₀₀).

Figure 5

(a) A sucrose gradient analysis of ribosomes from L25-defective (ΔL25, left panel) and wild type (WT, right panel) cells. Crude cell lysates were fractionated by centrifugation on 15–30% sucrose gradients. (b) Two-dimensional polyacrylamide gel analysis of protein content of L25-defective (left panel) and wild type (right panel) ribosomes.

Figure 6

Poly(U)-dependent polyphenylalanine synthesis by wild type (●) and L25-defective (▲) ribosomes.

Figure 7

Time course of GFP (a) and luciferase (b) mRNA translation in E. coli cell-free translation system containing wild type (WT, ●) and L25-defective (ΔL25, ▲) ribosomes. In case of GFP synthesis, an aliquot was taken at 30 min time point from the wild type and L25-defective systems and analyzed by SDS-PAGE with subsequent autoradiography (right panel of section (a)).