Isolation and characterization of acetylornithine delta-transaminase of wild-type Escherichia coli W. Comparison with arginine-inducible acetylornithine delta-transaminase.

The enzyme that catalyzes the reversible conversion of N-acetylglutamic γ-semialdehyde and l-glutamate to α-N-acetyl-l-ornithine and α-ketoglutarate, acetylornithine δ-transaminase, has been isolated in homogeneous form and crystallized from both the wild-type and the arginine-inducible strains of Escherichia coli W. The molecular weight of the wild-type transaminase is 119,000 while the molecular weight of the arginine-inducible enzyme is 61,000. However, the arginine-inducible acetylornithine δ-transaminase is not a breakdown product of the wild-type, arginine-repressible transaminase. Analysis of crude extracts of the wild-type and arginine-inducible strains by varying the acrylamide concentration in polyacrylamide disc gel electrophoresis showed that arginine-inducible and wild-type transaminases differed in ionic charge. Immunochemical analysis of the two transaminases showed that neither enzyme would cross-react with antibodies prepared against its counterpart. Treatment of the two enzymes with sodium dodecyl sulfate, followed by disc gel electrophoresis revealed that both transaminases were composed of 31,000-dalton subunits. Tryptic digestion of the two transaminases showed that nearly identical peptides were present. The overall data suggest that the wild-type and inducible transaminases were products of two different structural genes. The two transaminases have different molecular weights, ionic charges, and antigenic determinants, but both are composed of similar molecular weight subunits and show a high degree of similarity in amino acid content and peptide composition.     

Inducible and repressible acetylornithine delta-transaminase in Escherichia coli: different proteins

Two acetylornithine δ-transaminases which have different physical and kinetic properties have been isolated from a mutant of E. coli W. Sephadex gel filtration has shown the molecular weight of one transaminase to be approximately 119,000; the second transaminase has a molecular weight of about 61,000. The two transaminases can be separated by ammonium sulfate fractionation. The K_m values of the smaller and larger molecular-weight species for N^α-acetylornithine are 3.1 mm and 1.3 mm, respectively. The K_m for α-ketoglutarate is 1.1 mm for both enzymes. The presence of arginine in the growth medium represses the synthesis of the 119,000 molecular-weight transaminase and induces the synthesis of the 61,000 molecular-weight species.     

Cloning the Escherichia coli K-12 argD gene specifying acetylornithine delta-transaminase

The argD gene of Escherichia coli was shown to be present in plasmids pLC2-28 and pLC3-11 of the collection of Clarke and Carbon [Cell 9 (1976) 91-99]. The gene was cloned into pBR322 as a 6.3-kb BamHI fragment. Enzyme determination showed that the cloned DNA contains the structural gene for acetylornithine delta-transaminase. The argD DNA was used as a probe in hybridization experiments which indicated that the argM gene resides in a duplicated portion of E. coli DNA that is highly similar to the argD region.     

Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase

Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced. The deduced amino acid sequences show substantial similarity. Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous. However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S. cerevisiae ACOAT transaminates ornithine about as efficiently as E. coli does. The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.     

Derepression Kinetics of Ornithine Transcarbamylase in Escherichia coli

A mathematical model for the derepression of ornithine transcarbamylase (OTC) in Escherichia coli strain W was derived from a set of 14 assumptions concerning the arginine regulon. The model assumes that active repressor for the arginine regulon is unstable and is only formed when the level of arginyl-tRNA is in excess of the level necessary to maintain protein synthesis for a given cell doubling time. The presence of active repressor was assumed to inhibit the synthesis of messenger RNA coding for the synthesis of the enzymes of the arginine biosynthetic pathway. Numerical estimates of the model's parameters were made and, by simulation on a digital computer, the model was shown to fit kinetic data for derepression of OTC in E. coli W cells in minimal medium growing in flask culture with a doubling time of 60 min and growing in a chemostat with a generation time of 460 min for an assumed OTC-specific mRNA half-life (t_1/2) of 9 min. The model was also shown to predict the increase in the size of bursts of OTC synthesis elicited by addition of arginine to cultures of derepressing E. coli cells with the increase in the delay time before arginine addition. Approximate analytical solutions to the model were obtained for the early phase of derepression and for repression of OTC. These were used to derive graphical methods for determining t_1/2 from repression and derepression transient changes in the OTC level.     

Mutational evidence for identity of penicillin-binding protein 5 in Escherichia coli with the major D-alanine carboxypeptidase IA activity.

The defect in D-alanine carboxypeptidase IA activity in the dacA11191 mutant of Escherichia coli was correlated with a defect in the release of penicillin G from penicillin-binding protein 5. The results suggest that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity of the wild type and that the mutation results in a defect in the deacylation step catalyzed by this enzyme.     

MutL associates with Escherichia coli RecA and inhibits its ATPase activity

Different DNA repair systems are known to cooperate to deal with DNA damage. However, the regulatory role of the cross-talk between these pathways is unclear. Here, we have shown that MutL, an essential component of mismatch repair, is a RecA-interacting protein, and that its highly conserved N-terminal domain is sufficient for this interaction. Surface plasmon resonance and capillary electrophoresis analyses revealed that MutL has little effect on RecA-ssDNA filament formation, but dose down-regulate the ATPase activity of RecA. Our findings identify a new role for MutL, and suggest its regulatory role in homologous recombination.     

Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis.

Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

The identity of Escherichia coli bacterioferritin and cytochrome b1.

Two different procedures were used to prepare pure samples of 'cytochrome b1' (column chromatography) and 'bacterioferritin' (immunoprecipitation) from Escherichia coli K-12 strain CA265. Both were crystallized, and X-ray-crystallographic data were compared with those from the bacterioferritin of E. coli strain W3300 used as a standard. We conclude that 'cytochrome b1' and 'bacterioferritin' are identical.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

ld-Carboxypeptidase activity in Escherichia coli

The activities of the LD-carboxypeptidases of Escherichia coli K 12 and of a mutant strain 155 with reduced activities were studied with the aid of ether treated cells. Evidence was obtained that was consistent with the suggestion that in both strains two LD-carboxypeptidase activities are present. Activity I degrades the nucleotide activated precursor UDP-MurNAc-tetrapeptide and activity II splits off D-alanine residues from position 4 of the peptide subunits in the nascent murein. In the mutant strain activity I is reduced 10fold compared with strain K 12, whereas activity II is not affected. The two activities could be distinguished with regard to their sensitivity to D-amino acids and the beta-lactam antibiotic thienamycin.     

Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli

This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU). A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing methods. These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites. These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.