Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2′-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37→Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T→C:G transversion mutation in a mutT^– mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.     

A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae

A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with ~76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused ~14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2′-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 × 10⁻³ μM⁻¹ s⁻¹, which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.     

Analysis of a chemotaxis operon in Rhizobium meliloti*

Genes controlling chemotaxis towards L-amino acids and d -mannitol in Rhizobium meliloti have been identified by Tn5 insertions that lead to chemotaxis-deficient mutants. The tagged genes span an 8.7 kbp region that has been sequenced. These genes are part of a large operon containing three novel open reading frames, orf1, orf2 and orf9, and six familiar chemotaxis (che) genes, cheY1-cheA-cheW-cheR-cheB-cheY2, that have been assigned by their similarity to known Escherichia coli genes. The second copy of cheY may be part of a second signalling chain; orf1 and orf2 encode sequence motifs that resemble the signalling domain of E. coli MCPs (methyl-accepting chemotaxis proteins), while the product of orf9 may contain a transmembrane domain. No protein methylation has been observed in Rhizobium meliloti in response to l -amino acids. However, the presence of cheR (methyltransferase gene) and cheB (methyl-esterase gene) suggested that MCPs are likely components of the chemotactic response in R. meliloti. Therefore, it is postulated that two chemotaxis pathways are functional in R meliloti: one responds to l -amino acids via ORF1-ORF2, whereas the other (probably responding to specific plant exudates) acts via MCP-like receptors, and both interact with the central components CheW-CheA-CheY1 and/or CheY2.     

Selection against hypermutability in Escherichia coli during long term evolution

Summary A population of a mutT strain of E. coli was maintained in a chemostat for 2,200 generations. Afterwards the rate, of mutation to resistance to three antibiotics was determined by the Luria-Delbrück fluctuation test. It was found that the strain had a distinctly reduced mutability after the long-term cultivation compared with the original strain. Nevertheless the mutability was still much higher than that of a wild-type strain. After transduction of the mutT gene into another genetic backgroud the transductants showed the same mutability as the original strain indicating that the mutT allele itself had not changed. Our results support the hypothesis that under new environmental conditions mutator strains have an advantage due to their more efficient production of beneficial mutations. After optimal adaptation there is selection against high mutation rates due to the increased mutational load in the mutator population.     

Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent Insertion element, IS 186

Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study we report the sequence of a gene encoding a second nucleoside transport system, nupC (in addition to nupG.) An open reading frame (ORF) of 1200bp was identified that codes for a hydrophobic polypeptide of 43 560 Da and an NupC fusion protein was shown to be membrane associated. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homology to several membrane-associated proteins, including LacY and Cyd. Analysis of the nupC promoter region revealed the presence of at least two putative CRP-binding sites, centred at–40bp and–89bp, which probably flank a CytR-binding site. In addition, an adjacent IS186 element was identified and found to reside within a putative terminator structure, downstream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.     

Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport

Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.     

An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK

Summary A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Antimutagens against mutT.

Summary InE. coli strain 91P containing the mutator genemutT ^-, caffeine, spermine and quinacrine, but not guanosine, act as antimutagens, reducing the frequencies of mutation from ara^-Ara.     

Characterization of a mitochondrially targeted single-stranded DNA-binding protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.     

A point mutation in the core subunit gene of yeast mitochondrial RNA polymerase is suppressed by a high level of specificity factor MTF1

Analysis of a transcribed region in the mitochondrial genome of Oenothera revealed an open reading frame (ORF) of 577 codons (orf577) that is also conserved in carrot, here encoding a protein of 579 amino acids (orf579). RNA editing alters the mRNA sequence of orf577 in Oenothera with 46 C to U transitions, many of which improve sequence similarity with the homologous Marchantia gene orf509. The deduced polypeptides show significant similarity with the ccll-encoded protein involved in cytochrome c biogenesis in the photosynthetic bacterium Rhodobacter capsulatus. A highly conserved domain is also found in plastid ORFs, suggesting that these bacterial, chloroplast and mitochondrial genes encode polypeptides with analogous functions in assembly and maturation of cytochromes c.     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

Point mutations in two conserved glycine residues within the integral membrane protein FhuB affect iron(II) hydroxamate transport

Summary A region of substantial homology, comprising 32 amino acids around a highly conserved glycine residue, is located near the C-terminal ends of the hydrophobic Fhu, Fec, Fep, Fat, and Btu transport proteins involved in the uptake of ferrisiderophores and vitamin B₁₂ into Escherichia coli and Vibrio anguillarum. Furthermore, a region similar in location and sequence containing an invariant glycine at an equivalent position was identified in the hydrophobic component of all other periplasmic binding protein-dependent (PBT) systems. In the FhuB protein, which is twice the size of the other PBT-related inner membrane proteins and which displays an internal homology, two conserved glycine residues are present. Alteration of Gly at positions 226 and 559 to Ala, Val, or Glu reduced iron(III) hydroxamate uptake, suggesting that this homologous region may play a general role in the mechanism of PBT-dependent transport.