Systematic Chromosomal Deletion of Bacterial Ribosomal Protein Genes

Abstract

Detailed studies of ribosomal proteins, essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual ribosomal protein genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, 9 ribosomal proteins (L15, L21, L24, L27, L29, L30, L34, S9 and S17) were found nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli ribosomal protein genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function, and may ultimately allow systematic substitution of ribosomal proteins with RNA.

Eukaryotes first: how could that be?

“…There is a general conundrum here: any claim as to the antiquity and homology of similar structures and processes in diverse lineages must invoke selection to maintain them, and yet-if invoked-this same selection should be adequate to create them. It is only if there are many equally good ways to satisfy such selection (high neutral specificity) that similarity implies homology, and we know too little about this for complex systems such as the ribosome, even yet [59].…”

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

“…To test the effects of our S4 mutants on ribosome assembly in vivo, we asked whether they complement a partial rpsD chromosomal deletion (Shoji et al 2011). In strain DH10β, deletion of the initial 176 amino acids encoded by rpsD (S4) is covered by an arabinose-inducible plasmid expressing WT E. coli S4.…”

“…Interestingly, these strains readily accumulated mutations in the S4 coding sequence, which were usually C-terminal truncations. These S4 mutations may be favored because they compensate the streptomycin-resistant rpsL (S12) allele in the parental strain (Shoji et al 2011). Because of this, we sequenced rpsD DNA from all bacterial cultures used in subsequent experiments, both before and after the experiment was completed.…”

“…Standard cloning methods were used to create plasmids pTrc-WT, pTrc-Δ41, pTrc-Y47A, pTrc-L51A, pTrc-D95A, and pTrc-R200A from plasmid pTrc-S16 (Shoji et al 2011). pTrc plasmids confer ampicillin resistance (100 μg/mL) and are IPTG-inducible (0.75 mM) and were transformed into chemically competent ΔS4 DH10β cells carrying a helper plasmid, pCDDSara-S4, to allow for strain maintenance (Shoji et al 2011).…”

“…pTrc plasmids confer ampicillin resistance (100 μg/mL) and are IPTG-inducible (0.75 mM) and were transformed into chemically competent ΔS4 DH10β cells carrying a helper plasmid, pCDDSara-S4, to allow for strain maintenance (Shoji et al 2011). Strains carrying pCDDSara are streptomycin (50 μg/mL)-resistant, arabinose (0.2% weight/volume)-inducible, and sucrose-sensitive (5% weight/volume).…”