Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

Summary We studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO ⁺ G ⁺ ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when grown with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO ⁺ strain. The valine sensitivity of E. coli K-12 is a consequence of this regulatory pattern.     

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.     

Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12

Summary A strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants.The strain carrying the ilv0603 mutation is resistant to valine inhibition (Val^r) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Val^r acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Val^r acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Val^r peak is missing in a strain carrying an ilvG (amber) mutation.     

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.     

Analysing overexpression of l-valine biosynthesis genes in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum

l-Valine biosynthesis was analysed by comparing different plasmids in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum strains in order to achieve an optimal production strain. The plasmids contained different combinations of the genes ilvBNCDE encoding for the l-valine forming pathway. It was shown that overexpression of the ilvBN genes encoding acetolactate synthase is obligatory for efficient pyruvate conversion and to prevent l-alanine as a by-product. In contrast to earlier studies, overexpression of ilvE encoding transaminase B is favourable in pyruvate-dehydrogenase-negative strains. Its amplification enhanced l-valine formation and avoided extra- and intracellular accumulation of ketoisovalerate.     

Regulation of isoleucine-valine biosynthesis in an ilvDAC deletion strain of Escherichia coli K-12

Studies of isogenic strains of Escherichia coli K-12 with and without the ilvDAC115 deletion described by Kiritani showed that a strain carrying this lesion does not have the ilvA, ilvD or ilvC structural genes but has normal multivalent regulation of the ilvB and ilvE structural genes. It was also shown that the regulatory locus (ilvO) for the ilvADE operon (defined by Ramakrishnan and Adelberg) affects the expression of the ilvB and ilvC structural genes and is located outside of the ilvDAC115 deletion. These experiments demonstrate that there is multivalent control of at least two ilv biosynthetic enzymes in the absence of the ilvA gene product.     

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.     

Chromatographic detection of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12.

Two valine-sensitive acetohydroxy acid synthase activities were separable from $\text{Escherichia}\text{coli}$ K-12 cells by virtue of their different affinities for DEAE-cellulose eluted with a KC1 gradient. These activities appeared to be independent from a valine-resistant cryptic component expressed only in $\text{ilvO}$ regulatory mutants. The properties of the first and second activity were coincident to those of extracts of $\text{ilvB}$ and $\text{ilvHI}$ mutants, respectively. These data prove that the $\text{ilvB}$ and $\text{ilvHI}$ gene products exist in the cell as physically distinct acetohydroxy acid synthase isoenzymes.     

Construction of the new <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 MG 1655 novel strain with improved growth characteristics for application in metabolic engineering

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The “polar effect” of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val^S phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph ^wt from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying λattL-Cm^R-λattR and transduced with phage P1 into MG1655; later, the Cm^R marker was removed with the use of λXis/Int recombinase. Parallel to this procedure, a spontaneous Val^R mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of L-Val (50 μg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val^R strain had been generated in the region of the identified ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val^R phenotype in the obtained strain, was transferred to MG1655-rph ^wt using cotransduction, by analogy to the transfer of rph ^wt. Evaluation of rates of aerobically growing cells (μ, hour-1) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655,MG1655-rph ^wt, and MG1655-(rph ^wt, ilvG-15), respectively.     

Hyper-recombination in uvrD mutants of Escherichia coli K-12

Summary A mutant strain of E. coli which was isolated initially because of its strong hyper-recombination phenotype was shown to carry a lesion in uvrD. The presence of this mutation, designated uvrD210, increased the frequency of recombination between chromosomal duplications in F-prime repliconant cells and reduced linkage between closely linked markers in crosses with Hfr donors. A comparable hyper-rec phenotype was demonstrated in strains carrying other alleles of uvrD previously referred to as mutU4, uvr502 and recL152. The recombination activity of a uvrD210 strain was abolished by mutation of recA but the mutator activity associated with this allele proved to be independent of recA. It is suggested that uvrD mutations reduce the fidelity of DNA replication and that the accumulation of lesions in the newly synthesized strand provides additional sites for initiating recombination.     

Evidence for mutations in the structural gene for homocitrate synthase in Saccharomycopsis lipolytica.

Summary Eight strains devoid of homocitrate synthase activity were found among lysine requiring mutants of the yeast Saccharomycopsis lipolytica. Genetic analysis of these strains showed that they were all affected at the same locus LYS 1. Three lines of evidence suggest that this locus defines a structural gene for homocitrate synthase. First, the mutations show various degrees of intragenic complementation; it could be shown in some cases that the hybrid enzyme formed in vivo displayed modified properties in vitro. Second, reversion of some of these mutations can result in a modified enzyme (desensitized). Third, a feedback mutant of homocitrate synthase was directly isolated from the wild type strain, and shown to carry a single mutation at or near LYS 1.We also present here the first attempts at genetic fine mapping in Saccharomycopsis lipolytica.Abbreviations used lys lysine - arg arginine - ade adenine - ura uracile - TDL 4,5-transdehydrolysine - Sm Saccharomycopsis - KR kilorads Part of a thesis submitted by C.G. to the Université de Paris VI, Paris, France     

Yeast LEU5 is a PET-like gene that is not essential for leucine biosynthesis

Summary Alpha-IPM synthase catalyzes the first committed step in leucine biosynthesis in the yeast S. cerevisiae. LEU4 is known to encode this enzyme activity. A second gene, LEU5, has been proposed to encode a second enzyme with this activity.We cloned LEU5 and genetically defined the locus. LEU5 maps to chromosome VIII and is tightly linked to CEN8.Five different mutations in LEU5 were analyzed: a sitedirected deletion and a disruption, as well as three distinct mutations produced by chemical mutagenesis. In a leu4 background, each leu5 mutation causes a Leu — phenotype; in a LEU4 background, none of the mutations alters the Leu+ phenotype. This shows that LEU5 is not essential for leucine biosynthesis. In either a leu4 or LEU4 background, each leu5 mutation causes a glycerol — phenotype. This operationally defines LEU5 as a PET gene.Two distinct suppressors of the Pet — phenotype of leu5 strains have been isolated. These suppressors revert the Pet — phenotype of each of four mutant leu5 alleles that were tested. Suppression occurs regardless of the allele at LEU4. Moreover, the suppressors co-revert the Leu — phenotype for each of the four leu5 mutations that is combined with a leu4 allele. This establishes the presence of a gene other than LEU5 that encodes a second alpha-IPM synthase. Further analysis provided no evidence for synthase activity that is encoded by LEU5.Abbreviation EMS ethylmethane sulfonate - IPM isopropylmalate - NPD nonparental ditype - PD parental ditype - TT tetratype     

Fluoroquinolone Resistance Linked to Both gyrA and parC Mutations in the Quinolone Resistance–Determining Region of Shigella dysenteriae Type 1

We examined the quinolone resistance–determining region (QRDR) of gyrA, gyrB, and parC of recently isolated fluoroquinolone-resistant S. dysenteriae type 1 strains from south Asia and compared data with fluoroquinolone-susceptible strains associated with previous epidemics of 1978, 1984, and 1994. In fluoroquinolone-resistant strains, double mutations (Ser⁸³ → Leu, Asp⁸⁷ → Asn or Gly) and a single mutation (Ser⁸⁰ → Ile) were detected in the QRDRs of gyrA and parC, respectively.     

Increased and decreased sensitivity to carbon catabolite repression of enzymes of acetate metabolism in mutants of Aspergillus nidulans.

Summary The creA204, creB15 and creC27 mutations have been shown to cause carbon catabolite derepression of acetyl CoA synthase and isocitrate lyase in Aspergillus nidulans. A recessive mutation, cre-34, which is linked to the creC gene, results in these enzymes being more sensitive than cre or wildtype strains to catabolite repression. The acetamidase levels of strains containing cre mutations have been investigated and provide support for the hypothesis that an acetate metabolite, rather than acetamide, induces this enzyme.     

Isolation and expression analysis of two yeast regulatory genes involved in the derepression of glucose-repressible enzymes

Summary Yeast strains carrying one of the two regulatory mutations cat1 and cat3 are defectve in derepression of several glucose-repressible enzymes that are necessary for utilizing non-fermentable carbon sources. Hence, these strains fail to grow on ethanol, glycerol or acetate. The synthesis of isocitrate lyase, malate synthase, malate dehydrogenase and fructose-1,6-bisphosphatase is strongly affected in cat1 and cat3 strains. Genes CAT1 and CAT3 have been isolated by complementation of the cognate, mutations after transformation with an episomal plasmid gene library. The restriction map of CAT1 proved its allelism to the earlier isolated SNF1 gene. Both genes appear to exist as single-copy genes per haploid genome as indicated by Southern hybridization. Northern analysis has shown that the 1.35 kb CAT3 mRNA is constitutively expressed, independent of the carbon source in the medium. Derepression studies with CAT3 transformants using a multi-copy plasmid showed over-expression of glyoxylate cycle enzymes. This result would be consistent with a direct effector function for the CAT3 gene product.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.