The molecular basis of leucine auxotrophy of quinone-treated Escherichia coli. Active site-directed modification of leucyl-tRNA synthetase by 6-amino-7-chloro-5,8-dioxoquinoline.

Leucyl-tRNA synthetase from Escherichia coli is rapidly inactivated by 6-amino-7-chloro-5,8-dioxoquinoline (quinone), a model substance for cytostatic quinones. Loss of activity follows pseudo-first order kinetics. The quinone masks essential--SH groups that are reactive with N-ethylmaleimide. Specific protection of the enzyme by leucine provides evidence for active site-directed modification. Half-maximal protection is found at a concentration of 150 micron which is identical with the dissociation constant of the enzyme.substrate complex. The competitive inhibitor leucinol also protects the enzyme from inactivation by the quinone. MgATP enhances the protective effect of leucinol about 250-fold, thus substantiating recently published findings on synergistic coupling of ligands to aminoacyl-tRNA synthetases. The results support the assumption that the bacteriostatic quinone directly interferes with leucyl-tRNA synthetase in growing cells. Active-site-directed inhibition of the enzyme could adequately explain the phenotypically observed auxotrophy for leucine of quinone-treated E. coli.     

Growth inhibition of Escherichia coli by E colicin plasmids

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.     

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum

Ketopantoate reductase catalyzes the second step of the pantothenate pathway after ketoisovalerate, common intermediate in valine, leucine and pantothenate biosynthesis. We show here that the Corynebacterium glutamicum ilvC gene is able to complement a ketopantoate reductase deficient Escherichia coli mutant. Thus ilvC, encoding acetohydroxyacid isomeroreductase, involved in the common pathway for branched-chained amino acids, also exhibits ketopantoate reductase activity. Enzymatic activity was confirmed by biochemical analysis in C. glutamicum. Furthermore, inactivation of ilvC in C. glutamicum leads to auxotrophy for pantothenate, indicating that ilvC is the only ketopantoate reductase- encoding gene in C. glutamicum.     

The effect of intracellular ppGpp levels on glutamate and lysine overproduction in Escherichia coli

Although the enhancement of amino-acid synthesis by guanosine-3',5'-tetraphosphate (ppGpp) is well known, the effect of intracellular ppGpp levels on amino-acid overproduction in Escherichia coli has not been investigated. In this study, we demonstrate that overexpression of the relA gene, encoding ppGpp synthetase, increases the accumulation of amino acids, such as glutamate and lysine, in amino-acid-overproducing strains of E. coli. Elevation of intracellular ppGpp levels due to depletion of required amino acids also enhances glutamate overproduction. Moreover, the extent of overproduction is highly dependent on the intracellular ppGpp level. These results demonstrate that amino-acid overproduction in E. coli is closely connected to amino-acid auxotrophy via the accumulation of ppGpp.     

Carboxymethyl cellulose decomposition by intestinal bacteria of cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.     

Carboxymethyl Cellulose Decomposition by Intestinal Bacteria of Cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Die Bildung höherer Alkohole bei Aminosäuremangelmutanten von Saccharomyces cerevisiae

1. The influence of varying amounts of amino acids on the uptake of threonine, isoleucine, valine and leucine and their degradation to higher alcohols was investigated using a mutant strain of Saccharomyces cerevisiae, mating type a, genetic markers ade2, hom2, thr4, ilv2, leu1.
2. The cell mass is increased by increasing concentrations of threonine, isoleucine, valine and leucine, the latter two resulting in a higher dry weight. The amino acids are completely utilised at low concentrations. At higher contents up to 20% of the amino acids remain in the medium. The uptake of threonine, isoleucine, valine and leucine depends on the relative amounts of the concentrations of these amino acids in the medium. A greater amount of an amino acid is taken up if its concentration is comparatively higher than those of the other amino acids. There is a competition between the amino acids for the uptake into the cells.

Higher amounts of intracellular isoleucine and leucine are converted to 2-and 3-methylbutanol when compared with the degradation of valine and threonine to isobutanol and n-propanol-1, isoleucine and leucine up to 90%, valine up to 24% and threonine up to 20%. There is a competition between the four amino acids for their degradation to the corresponding higher alcohols. This behaviour confirms the earlier assumption of a degradation of the four amino acids by unspecific enzymes.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.