Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB. Received: 15 July 1998 / Accepted: 13 October 1998     

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu.

Summary The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86, 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.     

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB.     

Gene expression of chloroplast translation elongation factor Tu during maize chloroplast biogenesis

We have examined the expression of a maize nucleartuf gene(tufA) coding for the chloroplast translation elongation factor EF-Tu. Southern analysis revealed that the maize chloroplast EF-Tu was encoded by at least two distinct genes in the nuclear genome. In order to know the effect of light on the expression of thetufA gene during maize chloroplast biogenesis, we have analyzed the steady-state level of thetufA mRNAs by Northern analysis. The steady-state level of thetufA mRNAs was similar in both continuous light- and dark-grown seedlings. The level of thetufA mRNAs also maintained at relatively same level during light-induced greening of etiolated seedlings and all examined developmental stages. These results indicate that the gene expression of the maize chloroplast EF-Tu is rarely light-regulated at it’s mRNA level during chloroplast biogenesis.     

Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The mitochondrial elongation factor EF-Tu (tufM) in rice (Oryza sativa L.) was isolated and characterized. The rice tufM cDNA clone contained 1,726 nucleotides and coded for a 453 amino acid protein including a putative mitochondrial transit peptide of 64 amino acid residues. This coding region was composed of 12 exons and 11 introns. The deduced amino acid sequence showed 62% and 88% identities with rice chloroplast EF-Tu (tufA) and Arabidopsis mitochondrial EF-Tu, respectively. As previously observed for the rice tufA gene, the tufM gene is likely present as one copy in rice. The mitochondrial EF-Tu gene was differentially expressed during flower development, and the other translational EF-Tu genes (chloroplast EF-Tu and cytosolic EF-1 alpha) were also distinctly expressed in a temporal manner. Phylogenetic analysis of the rice tufM gene showed that the mitochondrial tufA homologue of Reclinomonas was more closely related to the mitochondrial tufM genes of flowering plants than fungal and other mitochondrial tuf genes. In addition, the tufM encoded an N-terminal extension showing significant similarity to that of rps14 (or sdhB), which is also a nuclear-encoded rice mitochondrial gene.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

Rates of growth, ribosome synthesis and elongation factor synthesis in a tufA defective strain of E. coli

Summary A tufA defective strain of E. coli was isolated which by a single deletion event acquired a tufA-lacZ fusion gene and lost the normal functional tufA gene (see accompanying paper). A correlation between the growth rate and the rate of ribosome synthesis showed that the average rate of protein synthesis was decreased to about 50% in the tufA defective strain whereas the number of EF-Tu molecules per ribosome was about 80% compared to a normal strain. The results indicate that tufB gene expression was preferentially stimulated in the tufA defective strain but the increased EF-TuB synthesis was not sufficient to make up for the loss of normal EF-TuA synthesis. Introduction of a plasmid that carries a complete tufA gene and the preceeding fusA gene but not the str-promotor into the tufA defective strain did not alleviate the slow growth or low rate of EF-Tu synthesis showing that the high rate of EF-TuA synthesis compared to the other proteins in the str operon is not augmented by a strong second promotor for the tufA gene. The tufA-lacZ fusion which takes the place of the normal tufA gene was expressed at a high rate and the -galactosidase activity increased with the growth rate as expected.     

A comparison of the activities of the products of the two genes for elongation factor Tu

Summary The two species of Elongation Factor Tu coded for by the tufA and tufB genes were synthesized in UV-irradiated E coli infected by transducing phages bearing the separate genes. Both proteins interact similarly with EFTs, GDP, and phe-tRNA. Although the phe-tRNA·EFTu·GMP·PNP complex containing the tufA gene product binds some-what more tightly to ribosomes, both proteins promote the complete process of binding phe tRNA to ribosomes at similar rates.     

Cloning of an EcoRI fragment carrying E. coli tufA gene.

Summary EcoRI fragments of the transducing phage fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Ap^r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA·DNA hybridization of RNA transcribed from phage rif ^d18 carrying tufB with DNA from pTUAl.     

A technique for targeted mutagenesis of the EF-Tu chromosomal gene by M13-mediated gene replacement

A generally applicable system for targeted mutagenesis of a chromosomal sequence is described. The Escherichia coli tufA gene was mutated using a recombinant M13mp9 phage vector carrying a tuf gene. Integration via crossing over with the chromosomal tufA target gene produced an M13 lysogen. These lysogens were screened for resistance to kirromycin. The M13 phage carrying tufA mutations were efficiently retrieved by a genetic procedure. Genetic mapping was performed with the M13 vectors. The same recombinant M13 phage was used for mutagenesis, lysogen formation, gene replacement, retrieval, mapping and sequencing of kirromycin mutants. Three different mutations yielding resistance to kirromycin were found: two of these have previously been found and characterised, while the third mutation, Gly316 Asp, is a new mutant. We also report the identification of a fourth kirromycin-resistant mutant, Gln124 Lys.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

Growth and translation elongation rate are sensitive to the concentration of EF-Tu

We have used quantitative immunoblotting to estimate the amount of EF-Tu in a variety of S. typhimurium strains with wild-type, mutant, insertionally inactivated or plasmid-borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild-type strain, at moderate to fast growth rates, our data show that EF-Tu makes up 8–9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild-type EF-Tu level, irrespective of which tuf gene remains active, or whether that gene is wild-type or a kirromycin-resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction in elongation rate relative to the level of EF-Tu we calculate that in glucose minimal medium the in vivo saturation level of wild-type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non-viable on minimal medium when the level of EF-Tu is reduced.     

Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rRNA and S7 mRNA

Summary A 6.5 kb region from the genome of the cyanobacterium, Anacystis nidulans 6301 was cloned using the tobacco chloroplast gene for ribosomal protein S12 as a probe. Sequence analysis revealed the presence of genes for ribosomal proteins S12 and S6 and elongation factors EF-G and EF-Tu in this DNA region. The arrangement is rps12 (124 codons)-167 bp spacer-rps7 (156 codons)-77 bp spacer-fus (694 codons)-26 bp spacer-tufA (409 codons), which is similar to that of the Escherichia coli str operon. The deduced amino acid sequences of the A. nidulans S12 and EF-Tu show high homology (72%–82%) with the E. coli and chloroplast counterparts while those of the A. nidulans S7 and EF-G give low homology (51%–59%). Striking structural homology was found between the potential S7 binding region of 16S rRNA and the beginning of S7 mRNA, suggesting that feedback regulation of rps7 expression operates in A. nidulans.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.     

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii

Summary Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifty, nifU, nifS and nifV mutants were Nif^– (failed to grow on molybdenum) while nifM mutants were Nif^–, Vnf^– and Anf^–. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf^– Anf–. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.