Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.     

A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures.

The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy. Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da. Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E. coli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis, E. coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T. scotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P. haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.     

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.     

2'-Versus 3'-OH specificity in tRNA aminoacylation. Further support for the "secondary cognition" proposal.

Purified Escherichia coli tRNAAla and tRNALys were each converted to modified species terminating in 2'- and 3'-deoxyadenosine. The modified species were tested as substrates for activation by their cognate aminoacyl-tRNA synthetases and for misacylation with phenylalanine by yeast phenylalanyl-tRNA synthetase. E. coli alanyl- and lysyl-tRNA synthetases normally aminoacylate their cognate tRNA's exclusively on the 3'-OH group, while yeast phenylalanyl-tRNA synthetase utilizes only the 2' position on its own tRNA. Therefore, the finding that the phenylalanyl-tRNA synthetase activated only those modified tRNAAla and tRNALys species terminating in 3'-deoxyadenosine indicated that the position of aminoacylation in this case was specified entirely by the enzyme, an observation relevant to the more general problem of the reason(s) for using a particular site for aminoacylation and maintaining positional specificity during evolution. Initial velocity studies were carried out using E. coli tRNAAla and both alanyl- and phenylalanyl-tRNA synthetases. As noted in other cases, activation of the modified and unmodified tRNA's had essentially the same associated Km values, but in each case the Vmax determined for the modified tRNA was smaller.     

Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method

A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes. In the E. coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues. The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues. A possible correlation of the observed effects with thermostability is discussed.     

Immunochemical comparison of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase among the Enterobacteriaceae.

The bifunctional enzyme of the tryptophan operon, phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase (PRAI-InGPS;EC 4.1.1.48), was characterized by an immunochemical study of six representative members of the Enterobacteriaceae: Escherichia coli, Salmonella typhimurium, Enterobacter aerogenes, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris. PRAI-InGPS was purified from E. coli, and antisera were prepared in rabbits. These antisera were utilized in quantitative microcomplement fixation allowing for a comparison of the overall antigenic surface structure of the various homologous enzymes. These data showed E. coli PRAI-InGPS and S. marcescens and E. carotovora PRAI-InGPS (taken as a group) to have an index of dissimilarity of approximately 10, whereas the other organisms had values intermediate. In addition, antiserum to E. coli tryptophan synthetase beta2 subunit was used in microcomplement fixation to extend the previous comparison of this subunit (Rocha, Crawford, and Mills, 1972) to E. carotovora and P. vulgaris. Indexes of dissimilarity for E. coli compared to P. vulgaris of E. carotovora were 1.0 and 1.7, respectively. Agar immunodiffusion using PRAI-Ingps antisera showed significant cross-reaction among E. coli, E. aerogenes, S. typhimurium, and P. vulgaris whereas the enzymes from S. marcescens and E. carotovora cross-reacted to a lesser extent, with the latter reaction being quite weak. Comparative enzyme neutralization using E. coli PRAI-InGPS antisera showed significant cross-reactions among the enzymes in that all were neutralized at least 25%. The data taken together indicate that the trpC gene products in the Enterobacteriaceae are a homologous group of proteins, that the genetic divergene of the trpC gene is basically the same as the trpA gene, and that both are less conserved than the trpB gene. Furthermore, the PRAI-InGPS, enzyme active site appears to represent a more evolutionarily conserved region of the protein. These findings indicate that, with respect to PRAI-InGPS, similarity to E. coli among the organisms examined is in the following order: (E. aerogenes, S. typhimurium, P. vulgaris) greater than (S. marcescens, E. carotovora).     

Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.     

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.     

Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli.     

Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.     

Express method of isolation of mammalian phenylalanine-tRNA-synthetase and preparation of monoclonal antibodies against this enzyme

A rapid and efficient procedure for isolating homogeneous beef liver phenylalanyl-tRNA synthetase (EC.6.1.1) was developed that enables to purify the enzyme 5000 fold and to achieve the activity of 8 e.a.u. per mg of protein. The molecular mass of the native enzyme was estimated to be 260 kDa, for alpha subunit - 59 kDa, and for beta - 72 kDa. Two cellular clones were derived by means of hybridization of immunised splenocytes with myeloma cells. They secrete monoclonal antibodies, designated P6 and P1 2, that bind to human placental and bovine liver phenylalanyl-tRNA synthetases but not to the same enzymes from E. coli and T. thermophilus. P6 and P1 2 antibodies do not affect the aminoacylation capacity of human or bovine phenylalanyl-tRNA synthetases. By immunoblotting, it was shown that P6 antibodies recognize the alpha subunit of the enzyme.     

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.     

Purification and properties of T4 phage thymidylate synthetase produced by the cloned gene in an amplification vector

We have introduced the T4 thymidylate synthetase gene, resident in a 2.7-kilobase EcoRI restriction fragment, into an amplification plasmid, pKC30. By regulating expression of this gene from the phage lambda pL promoter within pKC30 in a thyA host containing a temperature-sensitive lambda repressor, the T4 synthetase could be amplified about 200-fold over that after T4 infection. At this stage, a 20-fold purification was required to obtain homogeneous enzyme, mainly by an affinity column procedure. The purified plasmid-amplified T4 synthetase appeared to be identical with the T2 phage synthetase purified from phage-infected Escherichia coli in molecular weight, amino end group analysis, and immunochemical reactivity. The individual nature of the phage and host proteins was revealed by the fact that neither the T2 nor the T4 enzyme reacted with antibody to the E. coli synthetase, nor did antibody to the phage enzymes react with the E. coli synthetase. These differences were corroborated by DNA hybridization experiments, which revealed the absence of apparent homology between the T4 and E. coli synthetase genes. The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.     

Regulation of isocitrate dehydrogenase by phosphorylation in Escherichia coli K-12 and a simple method for determining the amount of inactive phosphoenzyme.

In several Escherichia coli K-12 strains grown on a limiting concentration of glucose, isocitrate dehydrogenase (IDH) was inactivated about 90% after cessation of growth upon exhaustion of the glucose. Such inactivation has been previously observed in several E. coli strains but not in E. coli K-12 (unless acetate was added to the bacterial culture when growth ceased). IDH was inactivated 75 to 80% in all E. coli K-12 strains we examined during growth on acetate. The inactivation involved phosphorylation of the enzyme and is considered to be a regulatory mechanism facilitating metabolite flow along the glyoxylate shunt. Phospho-IDH interacted with antibodies to enzymatically active IDH. We have devised a method, based on this immunological cross-reaction, for determining the proportions of active and inactive (phospho-) IDH in cell extracts.     

Characteristics of the B subunit of a thermolabile Escherichia coli enterotoxin produced by the A-B+ E. coli strain

The preparation and identification of B subunit of thermolabile enterotoxin produced by A-B+ gene-containing strain are described. The E. coli strain studied is shown to produce protein identical in its molecular properties and antigenic specificity to B subunit obtained from the whole thermolabile enterotoxin. Partial antigenic affinity between B subunits of thermolabile enterotoxin obtained from different sources and B subunit from cholera enterotoxin has been established in immunochemical studies. Electrophoretic and immunochemical analysis has confirmed the absence of A-subunit admixtures in B-subunit preparation obtained from /A-B+/E. coli strain.     

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

Modification of the alpha-subunit of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA]

L-Phenylalanyl-tRNA synthetase from E. coli MRE-600 (EC 6.1.1.20) was alkylated with N-chlorambucilyl-[14C] phenylalanyl-tRNA. After removal of the affinity reagent tRNA moiety bp alkaline hydrolysis of the ester bond between the N-chlorambucilyl-phenylalanyl residue and the 3'-end of tRNA, The enzyme was dissociated into subunits in the presence of SDS. Separation of the subunits was performed by SDS electrophoresis. The bulk of the radioactivity of the N-chlorambucilyl-[14C] phenylalanyl residue was found at the position of the alpha-subunit of the enzyme. The results obtained are consistent with a specific binding of the phenylalanyl-tRNA analog to the alpha-subunit of the enzyme followed by covalent binding of the N-chlorambucilyl-phenylalanyl moiety to the protein.