The effect of an ilvA Mu phage insertion on ilv gene expression in Escherichia coli K-12

Physiological analysis of an E. coli K-12 strain carrying a Mu phage integrated into the ilvA structural gene shows that there is a polar affect on ilvD gene expression, whereas, the ilvE gene maintains a normal multivalent regulation response. It was also demonstrated that the ilvC and ilvB genes can be derepressed and repressed in response to ilv multivalent control. These experiments demonstrate that the ilvE structural gene can be regulated independently from the ilvA and ilvD structural genes and that the ilvC structural gene does not require the complete ilvA gene product (threonine deaminase) for its induction.     

A New Map Location for the ilvB Locus of ESCHERICHIA COLI

We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.     

Regulation of cyclic AMP of the ilvB-encoded biosynthetic acetohydroxy acid synthase in Escherichia coli K-12

Summary The biosynthetic acetohydroxy acid synthase activities of E. coli K12 are encoded by three genetic loci namely, ilvB (acetohydroxy acid synthase I), ilvG (acetohydroxy acid synthase II) and ilvHI (acetohydroxy acid synthase III). The previously reported involvement of cyclic AMP in the regulation of the biosynthetic acetohydroxy acid synthase isozymes in E. coli K-12 was found to be due to the effect of this nucleotide on the expression of ilvB. Cyclic AMP had no effect on acetohydroxy acid synthase activity in strains lacking wild-type ilvB activity but containing the remaining isozymes. Very little activity of acetohydroxy acid synthase coded for by ilvB was found when ppGpp and cyclic AMP were severely limited. Addition of cyclic AMP under these conditions increased ilvB expression 24-fold. The data suggest that in addition to multivalent repression and ppGpp, cyclic AMP plays a major role in the regulation of the ilvB biosynthetic operon.     

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.     

Enhancing the carbon flux and NADPH supply to increase L-isoleucine production in Corynebacterium glutamicum

Our previous work has shown that L-isoleucine production in Corynebacterium glutamicum IWJ001 could be increased by overexpressing ilvA1 encoding a feedback-resistant threonine dehydratase, ilvBN1 encoding a feedback-resistant acetohydroxy acid synthase, lrp encoding the global regulator Lrp, brnFE encoding the two-component export system BrnFE, or ppnk1 encoding NAD kinase. The main purpose of this study is to further increase the L-isoleucine production in C. glutamicum IWJ001 by overexpressing the above genes in various combinations. Several C. glutamicum strains IWJ001/pDXW-8-ppnk1-lrp-brnFE, IWJ001/pDXW-8-ilvBN1-ilvA1-lrp-brnFE, IWJ001/pDXW-8-ilvBN1-ilvA1-ppnk1, and IWJ001/pDXW-8-ppnk1-ilvBN1-ilvA1-lrp-brnFE were constructed, and L-isoleucine production and activities of several key enzymes in these strains were analyzed. Compared with the control strain IWJ001/pDXW-8, L-isoleucine production increased in all of the four strains. IWJ001/pDXW-8-ilvBN1-ilvA1-ppnk1 showed the highest L-isoleucine production and produced 32.3 g/L L-isoleucine in 72 h fed batch fermentation. The results indicate that L-isoleucine production in C. glutamicum could be increased by enhancing the carbon flux and NADPH supply in the biosynthetic pathway.     

The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus

The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207–1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M_r of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.     

Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications.     

Role of Acetohydroxy Acid Isomeroreductase in Biosynthesis of Pantothenic Acid in Salmonella typhimurium

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of α-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound α-ketopantoate (K_m = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which α-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of α-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with α-ketopantoate or β-alanine. The mutations that conferred pantoate auxotrophy (designated panE) to these isolates appeared to be clustered, but were not linked to panB or panC. All panE strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of ilvC⁺ derivatives of panE ilvC8 strains. These strains required either α-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced, panE ilvC⁺ strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate. panE ilvC⁺ strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some S. typhimurium ilvG mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A. panE ilvC⁺ strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the α-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels. panE ilvC⁺ strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to ilvC by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.     

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes.     

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.     

Deregulation of acetohydroxy-acid synthase: Loss of allosteric inhibition conferred by mutations in the catalytic subunit

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, ΔQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ß-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, ΔQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.     

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

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     

Cloning and expression of the ilvB gene of Escherichia coli K-12

Summary A plasmid containing theilvB operon, which codes for acetohydroxy acid synthase I ofEscherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and F'ilvB4 treated with endonucleaseSalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of theilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of theilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functionalilvB promoter and control region. The DNA from this plasmid was used as a probe to show that the rate of synthesis ofilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.     

Genetic Studies of Leucine Biosynthesis in Bacillus subtilis

The mutations in a series of leucine auxotrophs isolated after treatment with nitrosoguanidine, ultraviolet light, and ICR-191 have been mapped between ilvC and pheA on the Bacillus subtilis chromosome. A fine structure map of the region was constructed by transformation. Analysis of several strains by assaying levels of their leucine bioysnthetic enzymes has shown that the region encodes three enzymes. The order of the genes with respect to the biosynthetic steps catalyzed by the gene products is 1–3–2.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

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.