Maurilio De Felice

The acetolactate synthase isoenzymes of Escherichia coli K-12

SummaryStrains 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.

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

SummaryWe 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.

Regulation of Isoleucine and Valine Biosynthesis

Publisher Summary This chapter discusses the regulation of isoleucine and valine biosynthesis. Isoleucine and valine biosynthesis is a model system for the study of mechanisms by which the cell coordinates different parts of its total metabolism. Threonine deaminase is the first enzyme in isoleucine biosynthesis. A different threonine deaminase, termed catabolic, appears only in anaerobiosis and in the absence of glucose. A third threonine deaminase, present in Escherichia coli K-12 and Salmonella Typhimurium , deaminates only D-threonine, and its presence explains why ilvA mutants of these organisms grow upon addition of D-threonine to the medium. The second step in the pathway is common to isoleucine and valine biosynthesis and is catalyzed by acetolactate synthase activity. It involves the conversion of either two molecules of pyruvate to form α-acetolactate, or one molecule of pyruvate and one molecule of α-ketobutyrate to form α-aceto-α-hydroxybutyrate. The acetolactate synthase isoenzymes have some relation to lysine biosynthesis. The third step in isoleucine biosynthesis and the second in valine biosynthesis are catalyzed by the enzyme isomeroreductase. The last intermediates in the pathway for isoleucine and valine biosynthesis are the keto acids from which the corresponding amino acids are synthesized by transamination.

The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.

Metabolic interlock between the acetolactate synthase isoenzymes and lysine biosynthesis in Escherichia coli K-12

SummarySome of the strains containing mutations in the genes for the acetolactate synthase isoenzymes are temperature sensitive (ts). Suppression of the acetolactate synthase defect due to one of these mutations suppresses also the ts phenotype; moreover, a genetic cross shows that the two phenotypes cannot be dissociated.The ts phenotype is accompanied by a decreased efficiency of transduction with Pl phage. Observations at the light microscope show formation of abnormal cells. Under specific conditions diaminopimelate stimulates growth and restores normal transduction efficiency. The rate of diaminopimelate formed and excreted by non-growing cells decreases when an acetolactate synthase mutation is present.We give evidence that the ts phenotype is due to an increased formation of lysine from diaminopimelate; this causes a starvation for the latter and therefore cell wall abnormalities. In fact, even at the permissive temperature, the lysine pool is 8x increased in a strain with an acetolactate synthase defect, while a slight decrease in the diaminopimelate pool is observed. Moreover, introduction into a ts strain of a mutation in lysA (the gene coding for diaminopimelate decarboxylase) cures the ts phenotype. Finally among the temperature resistant revertants we found some lysine auxotrophs.

Biochemical evidence for multiple forms of acetohydroxy acid synthase in Spirulina platensis

Two isoforms of acetohydroxy acid synthase (AHAS), the first enzyme of the branched-chain amino acids biosynthetic pathway, were detected in cell-free extracts of the cyanobacterium Spirulina platensis and separated both by ion-exchange chromatography and by hydrophobic interaction. Several biochemical properties of the two putative isozymes were analysed and it was found that they differ for pH optimum, FAD requirement for both activity and stability, and for heat lability. The results were partially confirmed with the characterization of the enzyme extracted from a recombinant Escherichia coli strain transformed with one subcloned S. platensis coli strain transformed with one subcloned S. platensis AHAS gene. The approximate molecular mass of both AHAS activities, estimated by gel filtration, indicates that they are distinct isozymes and not different oligomeric species or aggregates of identical subunits.

Molecular cloning and expression of Spirulina platensis acetohydroxy acid synthase genes in Escherichia coli

The coding sequence for Spirulina platensis acetohydroxy acid synthase (AHAS, EC 4.1.3.18) is shown to be contained within a 4.2 Kb ClaI fragment (ilvX) that has been cloned from a recombinant lambda library. This fragment was able to complement a suitable mutant of Escherichia coli when inserted into the ClaI site of plasmid pAT153 in either orientation, demonstrating that transcription of ilvX originated within the cloned fragment. The probe used for hybridization experiments was the corresponding gene from Anabaena sp. PCC7120. The same probe allowed us to identify a second putative gene encoding AHAS in the S. platensis genomic library.

On the permeability of biological membranes

Abstract Studies of the permeability of biological membranes to the amino acids isoleucine, leucine, and valine are very active in several laboratories. These studies show that this is a very useful model system for the understanding of cellular permeability. p]In this paper, we describe the different transport systems for isoleucine, leucine, and valine present in the microorganism Escherichia coli K-12. Each transport system is defined by its kinetic constants (Km and Vmax) for the substrate(s), by a set of uptake inhibitors, and by the characterization of mutant strains lacking a specific transport system. p]E. coli K-12 contains: li](a) an oligopeptide transport system, functioning with a wide range of oligopeptides and absent in strains carrying the opp-1 mutation. 1. (b) a dipeptide transport system, showing little specificity and absent in a strain carrying the dpp-1 mutation. 2. (c) a “very high affinity” transport system (also called LIV-1) for isoleucine, leucine, valine, methionine, threonine, and alanine. This system is repressible and probably requires a protein that can be isolated in pure form. 3. (d) a “leucine-specific”, high affinity transport system, requiring a protein that also can be isolated in pure form. 4. (e) two “high affinity” transport systems. One of them, the “high affinity-1”, catalyzes the transport of isoleucine, leucine, and valine and requires the activity of a protein coded by the brnQ gene. The other, “high affinity-2”, catalyzes the transport of isoleucine, leucine, and valine and requires the activity of a protein coded by the brnS gene. Both of them also require the activity of a protein coded by the brnR gene. These transport systems are called in other laboratories the LIV-II transport system. 5. (f) three “low affinity” transport systems, each one specific for isoleucine, leucine, or valine. A mutation, brnT8, in the isoleucine low affinity transport system, located at 61 min on the map of E. coli K-12, abolishes all residual isoleucine permeability in a strain lacking the other transport systems. This result shows that the isoleucine low affinity uptake is not a diffusion-controlled process and suggests that the systems described in this paper constitute all the transport systems for isoleucine, leucine, and valine uptake in E. coli K-12.

Chromatographic detection of the acetohydroxy acid synthase isoenzymes of Escherichiacoli K-12

Two valine-sensitive acetohydroxy acid synthase activities were separable from Escherichiacoli 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 ilvO regulatory mutants. The properties of the first and second activity were coincident to those of extracts of ilvB and ilvHI mutants, respectively. These data prove that the ilvB and ilvHI gene products exist in the cell as physically distinct acetohydroxy acid synthase isoenzymes.

Acetohydroxy acid synthase isoenzymes of Escherichia coli K-12: A trans-acting regulatory locus for ilvHI gene expression

SummaryWe isolated an Escherichia coli K-12 regulatory mutation affecting the acetohydroxy acid synthase III isoenzyme. This mutation was found to lie outside the structural genes ilvHI for this isoenzyme and was designated lrs-1. A strain carrying this mutation was found to be altered in the leucine-mediated control of ilvHI mRNA and acetohydroxy acid synthase III synthesis observed in the isogenic lrs+ strain. These alterations appeared to be a consequence of a reduced intracellular concentration of a single one of five tRNALeu isoaccepting species.