Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), 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 RP 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, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP 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 RPs with RNA.     

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.     

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m²G966 and m⁵C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m²G966 or m⁵C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.     

A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase

The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)₆-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (His)₆-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (k_cat/K_M = 0.3 s^− 1 M^− 1) was ∼ 25-fold more efficient than its ancestor but > 10⁷-fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli

The TOPRIM domain found in many nucleotidyl transferases contains a DxD motif involved in magnesium ion coordination for catalysis. Medium- to high-copy-number plasmid clones of Yersinia pestis topoisomerase I (YpTOP) with Asp-to-Asn substitution at the first aspartate residue (D117N) of this motif could not be generated in Escherichia coli without second-site mutation even when expression was under the control of the tightly regulated BAD promoter and suppressed by 2% glucose in the medium. Arabinose induction of a single-copy YpTOP-D117N mutant gene integrated into the chromosome resulted in ∼ 10⁵-fold of cell killing in 2.5 h. Attempt to induce expression of the corresponding E. coli topoisomerase I mutant (EcTOP-D111N) encoded on a high-copy-number plasmid resulted in either loss of viability or reversion of the clone to wild type. High-copy-number plasmid clones of YpTOP-D119N and EcTOP-D113N with the Asn substitution at the second Asp of the TOPRIM motif could be stably maintained, but overexpression also decreased cell viability significantly. The Asp-to-Asn substitutions at these TOPRIM residues can selectively decrease Mg²⁺ binding affinity with minimal disruption of the active-site geometry, leading to trapping of the covalent complex with cleaved DNA and causing bacterial cell death. The extreme sensitivity of the first TOPRIM position suggested that this might be a useful site for binding of small molecules that could act as topoisomerase poisons.     

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.     

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.