A role for a pyridoxne derivative in the multivalent repression of the isoleucine and valine biosynthetic enzymes

Starvation of a pdx mutant of Escherichia coli strain B in the presence of repressing levels of isoleucine, valine and leucine leads to a derepression of the normally repressible ilv genes. The derepression of the ilvA gene under these conditions results in the accumulation of apothreonine deaminase. Addition of pyridoxine leads to a sudden increase in threonine deaminase activity, and to restoration of repression. The pyridoxine component needed for the repression signal is probably not threonine deaminase but, more likely, some transient (“immature”) form of the enzyme.     

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

Summary A set of dilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing dilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO ⁺, while the ilvA538 allele is trans-recessive to ilvA ⁺. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.     

An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation.     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Gene relA function in the expression of amino acid operons. I. Effect of the allelic state of gene relA on phenotypic manifestations of auxotrophic threonine and isoleucine mutations in Escherichia coli K-12]

The substitution of the relA gene mutant allele with wild type allele of this gene in strictly auxotrophic strain of Escherichia coli K-12 GT25, carrying thr B1007 mitation, results in the appearance of the partial dependence of the bacterial growth upon threonine. On the other hand, the introduction of relA mutation into genome of incomplete threonine auxotroph, which was isolated as pseudorevertant from the strict threonine auxotroph CP78, recovered the strict dependency of the growth on the presence of threonine in the medium. The introduction of relA mutation into genome of partial isoleucine auxotroph, carrying a mutation in ilvA gene, reduces the residual activity of threonine deaminase under the conditions of derepression and results in the appearance of strict dependency of bacterial growth on the presence of isoleucine. These data indicate that operons, which control the biosynthesis of threonine and isoleucine, are positively regulated by the product of relA gene. The possibility of using leaky mutations, which lead to incomplete block of these amino acids synthesis, for testing allelic state of relA gene is discussed.     

Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli.

Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon.     

Evaluation of the<Emphasis Type="Italic"> Escherichia coli</Emphasis> threonine deaminase gene as a selectable marker for plant transformation

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to -ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.Abbreviations ELISA enzyme-linked immunosorbent assay - Ile isoleucine - OMT l-O-methylthreonine - nptII neomycin phosphotransferase II - TD threonine deaminase     

Regulation of the formation of isoleucyl-tRNA-synthetase and the level of isoleucine biosynthetic enzymes in E. coli K12

Summary During derepression of threonine deaminase and acetolactate synthetase due to valine deficiency—initiated by -aminobutyric acid limited growth of E. coli K12 or by limited valine supply to an ilv/leu auxotroph of E. coli K12—no alteration of the specific activity of isoleucyl-tRNA-synthetase occurs. Leucine limited growth of the auxotroph, leading to an even higher derepression of the isoleucine biosynthetic enzymes, also does not affect the specific activity of isoleucyl-tRNA-synthetase. However, under growth conditions where the same degree of derepression of threonine deaminase is due to isoleucine deficiency, as in E. coli K12B or two valine resistant mutants thereof grown in the presence of valine, or in the auxotroph during growth-limiting isoleucine supply, a specific two- to three-fold derepression of the isoleucyl-tRNA-synthetase takes place. But there is no strict correlation between the degree of derepression of threonine deaminase due to isoleucine deficiency and the degree of derepression of isoleucyl-tRNA-synthetase, as especially shown in case of the valine resistant mutant Val R4 and Val R5 grown in the presence of valine.These results demonstrate that the rate of formation of isoleucyl-tRNA-synthetase and of threonine deaminase are not regulated by the same molecular devices and that a certain degree of isoleucine deficiency is a prerequisite for a derepression of isoleucyl-tRNA-synthetase.     

Genetic analysis of uxuR and exuR genes: evidence for ExuR and UxuR monomer repressors interactions

Summary The uxuAB operon is under the dual control of uxuR- and exuR-encoded repressors whereas the exu regulon genes are regulated by the sole ExuR repressor. Mutations affecting the two exuR and uxuR regulatory genes were selected to investigate the relationship between the two repressors. The isolation of exuR and uxuR negative dominant mutations on a multicopy plasmid indicated that the active form of the two repressors was multimeric.The introduction of a uxuR negative dominant allele into a wild-type strain resulted in a significant increase in exu gene expression. This unexpected effect may have been the consequence of the formation of hybrid repressor molecules. This protein must be composed of native ExuR⁺ subunits aggregated with altered UxuR subunits. The same interference was observed for the exuR negative dominant allele on uxu gene derepression. The hypothesis given here implies that the two regions of the ExuR and UxuR repressors involved in the subunit aggregation present enough homologies to allow the formation of hybrid repressor molecules.     

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.     

Complementation Between Different Mutations in the ilvA Gene of Escherichia coli K-12

An ilvA mutation carried by a ?80i(lambda)dilv transducing phage complemented some ilvA mutations and did not complement others. Complementation was accompanied by appearance of threonine deaminase activity in vivo. These results divided the ilvA mutations into two sets which formerly appeared to define two cistrons.     

Positive and negative regulation of expression of the l-sorbose (sor) operon by SorC in Klebsiella pneumoniae

Summary In Klebsiella pneumoniae the gene products involved in the degradation of the ketose l-sorbose are encoded in the sor operon. It comprises, besides structural genes for uptake and catabolism, a promoter-proximal gene sorC, encoding a protein SorC of M_r 40 kDa, for which no enzymatic function has been detected. All sor genes are coordinately expressed and inducible by l-sorbose. Polar insertions and frameshift mutations in sorC cause a pleiotropic negative effect on the expression of all other sor genes. This defect is complemented in trans by the wild-type sorC ⁺ allele for frameshift mutations, but not for polar insertions. A single promoter for all sor genes, for which SorC is the activator, thus seems to be located in front of sorC. The repressor activity of SorC was demonstrated by complementation of constitutive sorC alleles with a sorC ⁺ allele leading to inducible expression of all sor genes, including sorC, which, as visualized by the use of a series of lacZ fusions, thus autoregulates its expression, both as an activator and a repressor.     

Genetics of carbon catabolite repression in Saccharomyces cerevisiae: genes involved in the derepression process

Summary A recessive mutant cat1-1, wild type CAT1, was isolated in Saccharomyces cerevisiae. It did not grow on glycrrol nor ferment maltose even with fully constitutive, glucose resistant maltase synthesis. It prevented derepression of isocitrate lyase, fructose-1,6-diphosphatase and maltase in a constitutive but glucose sensitive maltase mutant. Derepression of malate dehydrogenase was retarded and slowed down. Sucrose fermentation and invertase synthesis was not affected. Respiration was normal. From this mutant, two reverse mutants were isolated. One was recessive, acted as a suppressor of cat1-1 and was called cat2-1, wild type CAT2; the other was dominant and allelic to CAT1 and designated CAT1-2 ^d. CAT1-2 ^d and cat2-1 caused an earlier derepression of enzymes studied but did not affect the repressed nor the fully derepressed enzyme levels. CAT1-2 ^d and cat2-1 did not show any additive effects. It is proposed that carbon catabolite repression acts in two ways. The direct way represses synthesis of sensitive enzymes, during growth on repressing carbon sources whereas the other way regulates the derepression process. After alleviation of carbon catabolite repression, gene CAT1 becomes active and prevents the activity of CAT2 which functions as a repressor of sensitive enzyme synthesis. The CAT2 gene product has to be eliminated before derepression can actually occur. The time required for this causes a delay in derepression after the depletion of a repressible carbon source. cat1-1 cannot block CAT2 activity and therefore, derepression is blocked. cat2-1 is inactive and derepression can start after carbon catabolite repression has ceased. CAT1-2 ^d is permanently active as a repressor of CAT2 and eliminates the delay in derepression.     

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichia coli K-12

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.     

Restoration of enzyme activity by recessive missense suppressors in the fungus Coprinus.

Summary The acu-1 locus in Coprinus is the structural gene for acetyl-CoA synthetase. Five suppressor gene mutations, which suppress the acu-1,34 missense allele, were induced by mutagen treatment. All five suppressors were shown to have properties expected for tRNA structural gene mutations: they are recessive, they show a gene dosage effect in any doubly heterozygous combination of two sup ⁺ mutations and they are allele specific in action.Crosses between suppressed mutants established that at least four suppressor loci were represented. Doubly suppressed mutants derived from these crosses were used to show that the gene dosage effect is maintained when two sup ⁺ mutations are in cis as well as trans combinations in the two nuclei of the basidiomycete dikaryon.Extracts of the unsuppressed acu-1.34 mutant contained less than 2% of wild type acetyl-CoA synthetase activity whereas extracts of four of the five suppressor strains showed activities ranging from 28 to 37% of wild type. Only a slight increase in activity was detected in the fifth suppressor strain but this was associated with a temperature sensitive sup ⁺ phenotype. All five sup ⁺ mutations restored the ability of the acu-1.34 mutant to induce isocitrate lyase, an enzyme which, under the conditions of growth used, can only be induced when acetyl-CoA synthetase activity is present. Thus all five suppressors act to restore normal acu-1 protein function.     

Na+-Dependent Transport of Amino Acids and Its Significance for Growth of a Lysine-producing Bacterium

A lysine-producing mutant Brevibacterium flavum HUT 8052, a threonine plus methionine (or threonine plus homoserine) auxotroph, grew rapidly as nearly as the wild strain in a medium supplemented with NaCl (60 µg/ml), threonine (100 µg/ml), and methionine (33.3 µg/ml). With NaCl concentrations less than 20 µg/ml, the mutant grew little or very slowly, The peculiar growth behavior of the mutant including the above phenomenon could be reasonably explained by the finding of Na⁺-dependent amino acids transport and the feedback inhibition of homoserine dehydrogenase by threonine in the bacterium.

The threonine transport was stimulated by Na⁺ and Li⁺. though the latter being less effective. The transport of threonine was inhibited by serine. The uptake of serine, isoleucine, leucine and valine was also stimulated by Na⁺     

Repressor cleavage as a prophage induction mechanism: hypersensitivity of a mutant lambda cI protein to recA-mediated proteolysis

λcIind^s prophage is hypersensitive to derepression by ultraviolet-irradiation. We have utilized this mutant to test current models for prophage derepression. We find that cIind^s repressor is cleaved by RecA protein in vivo at lower ultraviolet doses and with more rapid kinetics than cI⁺ repressor, and that induction of the recA8 gene or other LexA-repressed genes is not required for cleavage. Our results support the concept that RecA-directed proteolysis is the primary mechanism for prophage derepression.     

Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant.

A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases. Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine. The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine. In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme. Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine. Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition. Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain. Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase. Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression. The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.