Evolution of aromatic biosynthesis and fine-tuned phylogenetic positioning of Azomonas,Azotobacter and rRNA group I pseudomonads

The evolutionary history of biochemical pathways can be determined in microbial groupings for which phylogenetic trees have been established. This has been demonstrated best in Superfamily B, an assemblage of rRNA homology groups containing lineages that lead to genera such as Escherichia and other enteric microbes, Pseudomonas (Group I), Xanthomonas, Oceanospirillum, and Acinetobacter. The rRNA homology group that defines Group I pseudomonads also includes Azomonas and Azotobacter, but particular dendrogram points of evolutionary divergence for these genera within Superfamily B have not been established. Phylogenetic relationships at such intergeneric levels can be deduced by analysis of aromaticpathway enzyme arrangement and regulation in selected groupings where dynamic evolutionary changes have occurred. A case in point is illustrated by Axomonas insignis, Azotobacter paspali, and Azotobacter vinelandii — a grouping that appears to be homogeneous with respect to the evolutionary state of the aromatic pathway. The conclusion that this phylogenetic cluster diverges from an ancestor common to pseudomonad subgroup Ia (rather than to subgroup Ib) is based upon the absence of chorismate mutase-F and arogenate dehydratase, enzymes making up a twostep pathway of phenylalanine biosynthesis that is absent in subgroup Ia, but present in subgroup Ib. Of further interest, Azomonas insignis and Azotobacter sp. were found to comprise a distinctive and recently evolved sublineage, differing from subgroup Ia species in their loss of a regulatory isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (ADHP synthase-trp) that is subject to feedback inhibition by l-tryptophan. DAHP synthase-trp is an ancient character state of Superfamily B that has been retained during the evolutionary history of most members of this Superfamily.Abbreviation DAHP 3-Deoxy-d-arabino-heptulosonate 7-phosphate     

Clues fromXanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

Summary The recent placement of major Gramnegative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading toEscherichia coli, Pseudomonas aeruginosa, andAcinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping,Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping.X. campestris is likeP. aeruginosa (and unlikeE. coli) in its possession of dual flow routes to bothl-phenylalanine andl-tyrosine from prephenate. Like all other members of Superfamily B,X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that ofP. aeruginosa inX. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-d-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., inE. coli), as two isozymes (e.g., inP. aeruginosa), or as one enzyme (inX. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus,E. coli has gained an isozyme, whereasX. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme ofX. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. InX. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway inX. campestris is thus far unique in nature.     

Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps. sp. ATCC 11299a)

The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either l-tyrosine or l-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by l-tyrosine with 50% inhibition of activity being achieved at 4.0 M effector. DAHP synthase-phe is inhibited 50% by 40 M l-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol. This two-isozyme pattern of DAHP synthase has not been described previously, although it may prove to be widespread.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - E4P d-erythrose-4-phosphate - PEP phosphoenolpyruvate - DTT dithiothreitol - BSA fraction V bovine serum albumin     

The stable phylogenetic distribution of the recently evolvedl-phenylalanine-inhibited isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in enteric bacteria

Escherichia coli and some other enteric bacteria possess three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, each of which is inhibited by one aromatic amino acid. Thel-phenylalanine-sensitive isozyme of DAHP synthase has evolved most recently since it is absent in all other members of the Gram-negative cluster that contains enteric bacteria as a subcluster. A comprehensive survey of enteric genera was carried out to determine whether the newly evolved isozyme is a stable, conserved trait. The results obtained show that all the genera of the contemporaryEnterobacteriaceae family possess the recently evolved phenylalanine-sensitive isozyme in addition to the tyrosine-and tryptophansensitive isozymes of DAHP synthase. However, physiological manipulation was usually necessary to derepress the tryptophan-sensitive DAHP synthase in order to demonstrate its presence.Florida Experiment Station Journal Series No. 9603.     

The phylogenetic origin of the bifunctional tyrosine-pathway protein in the enteric lineage of bacteria

Because bifunctional enzymes are distinctive and highly conserved products of relatively infrequent gene-fusion events, they are particularly useful markers to identify clusters of organisms at different hierarchical levels of a phylogenetic tree. Within the subdivision of gram-negative bacteria known as superfamily B, there are two distinctive types of tyrosine-pathway dehydrogenases: (1) a broad- specificity dehydrogenase (recently termed cyclohexadienyl dehydrogenase [CDH]) that can utilize either prephenate or L-arogenate as alternative substrates and (2) a bifunctional CDH that also posseses chorismate mutase activity. (T-proteins). The bifunctional T-protein, thought to be encoded by fused ancestral genes for chorismate mutase and CDH, was found to be present in enteric bacteria (Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Erwinia, Serratia, Morganella, Cedecea, Kluyvera, Hafnia, Edwardsiella, Yersinia, and Proteus) and in Aeromonas and Alteromonas. Outside of the latter "enteric lineage," the T-protein is absent in other major superfamily-B genera, such as Pseudomonas (rRNA homology group I), Xanthomonas, Acinetobacter, and Oceanospirillum. Hence, the T-protein must have evolved after the divergence of the enteric and Oceanospirillum lineages. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase-phe, an early-pathway isozyme sensitive to feedback inhibition by L- phenylalanine, has been found in each member of the enteric lineage examined. The absence of both the T-protein and DAHP synthase-phe elsewhere in superfamily B indicates the emergence of these character states at approximately the same evolutionary time.      

Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.     

Evolutionary implications of features of aromatic amino acid biosynthesis in the genus Acinetobacter

Key enzymes of aromatic amino acid biosynthesis were examined in the genus Acinetobacter. Members of this genus belong to a suprafamilial assemblage of Gram-negative bacteria (denoted Superfamily B) for which a phylogenetic tree based upon oligonucleotide cataloging of 16S rRNA exists. Since the Acinetobacter lineage diverged at an early evolutionary time from other lineages within Superfamily B, an examination of aromatic biosynthesis in members of this genus has supplied improtant clues for the deduction of major evolutionary events leading to the contemporary aromatic pathways that now exist within Superfamily B. Together with Escherichia coli, Pseudomonas aeruginosa and Xanthomonas campestris, four well-spaced lineages have now been studied in comprehensive detail with respect to comparative enzymological features of aromatic amino acid biosynthesis. A. calcoaceticus and A. lwoffii both possess two chorismate mutase isozymes: one a monofunctional isozyme (chorismate mutase-F), and the other (chorismate mutase-P) a component of a bifunctional P-protein (chorismate mutase-prephenate dehydratase). While both P-protein activities were feedback inhibited by l-phenylalanine, the chorismate mutase-P activity was additionally inhibited by prephenate. Likewise, chorismate mutase-F was product inhibited by prephenate. Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase were detected. The major isozyme (>95%) was sensitive to feedback inhibition by l-tyrosine, whereas the minor isozyme was apparently insensitive to allosteric control. Prephenate dehydrogenase and arogenate dehydrogenase activities were both detected, but could not be chromatographically resolved. Available evidence favors the existence of a single dehydrogenase enzyme, exhibiting substrate ambiguity for prephenate andl-arogenate. Dehydrogenase activity with either of the latter substrates was specific for NADP⁺, NAD⁺ being ineffective. Consideration of the phylogeny of Superfamily-B organisms suggests that the stem ancestor of the Superfamily possessed a single dehydrogenase enzyme having ambiguity for both substrate and pyridine nucleotide cofactor. Since all other members of Superfamily B have NAD⁺-specific dehydrogenases, specialization for NADP⁺ must have occurred following the point of Acinetobacter divergence, leading to the dichotomy seen in present-day Superfamily-B organisms.     

Phylogenetic distribution of components of the overflow pathway tol-phenylalanine within the enteric lineage of bacteria

The enteric lineage of prokaryotes (traditional enteric bacteria,Aeromonas, andAlteromonas) encompasses closely related genera that share many common character states of aromatic amino acid biosynthesis. For example, they uniformly employ the tightly regulated bifunctional P-protein (chorismate mutase: prephenate dehydratase) to forml-phenylalanine via phenylpyruvate. A second, unregulated pathway to phenylalanine, originally termed the overflow pathway inPseudomonas aeruginosa, consists of a monofunctional chorismate mutase (CM-F) and a cyclohexadienyl dehydratase. The evolution of the overflow pathway has been dynamic in the enteric lineage.Serratia marcescens, Erwinia herbicola, Erwinia amylovora, and several otherErwinia species possess an intact pathway.Salmonella, Klebsiella, andErwinia carotovora possess an incomplete overflow pathway, whileEscherichia, Proteus, Aeromonas, andAlteromonas lack it altogether.     

Stimulation of L-asparaginase production inEscherichia coli by organic and amino acids

The effect of 18 amino acids and 7 organic acids on the production ofl-asparaginase EC-2 by a strain ofEscherichia coli in a chemically defined medium was investigated under moderate aeration. All the amino acids and some of the organic acids stimulated the enzyme production. The specific activity without stimulants was about 0.16 nkat per mg dry weight, with stimulants it lay between 1 and 6 nkat per mg dry weight but withl-leucine andl-methionine the values were 12 nkat and 17 nkat per mg, respectively. When two organic or amino acids were added simultaneously at concentrations that were suboptimal for stimulation, the stimulating effects were cumulative in most cases. When cells were grown under conditions approaching anaerobiosis, the specific activity reached, even in the absence of stimulants, values as high as 5 nkat per mg; under these conditions, a further substantial increase in specific activity was only caused byl-leucine andl-methionine. Stimulating effects ofdl-lactate and of some amino acids were also found in other strains ofEscherichia coli. The ability to grow on a medium withl-asparagine as the sole source of both nitrogen and carbon was found in two strains; growth took place even when there was no measurable activity ofl-asparaginase EC-2.     

Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy.

The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Acinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enzymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirillum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only Escherichia coli and its close relatives.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Restriction enzyme analysis of the chloroplast and nuclear 45s ribosomal DNA ofAllium sectionsCepa andPhyllodolon (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of molecular characters. Restriction enzyme analysis of the chloroplast DNA (cpDNA) of the bulb onion (Allium cepa), Japanese bunching onion (A. fistulosum), wildAllium species in sect.Cepa andPhyllodolon, and the outgroupsA. ampeloprasum andA. tuberosum detected 39 polymorphisms.Allium cepa andA. vavilovii were identical for all characters. Cladistic analysis generated three most-parsimoniousWagner trees of 44 steps differing only in a zero-length branch.Allium fistulosum andA. altaicum (sect.Phyllodolon) comprised a monophyletic lineage separated from theA. cepa andA. vavilovii of sect.Cepa. The unresolved node was composed ofA. galanthum, A. roylei, and the lineage containingA. cepa, A. vavilovii, A. fistulosum, andA. altaicum. The clade containingA. altaicum, A. cepa, A. fistulosum, A. galanthum, A. roylei, andA. vavilovii remained resolved for strict consensus ofWagner trees of 48 steps or less.Allium pskemense andA. oschaninii were increasingly distant.Allium oschaninii has been proposed as the progenitor of the bulb onion, but was more closely related to the common progenitor of all species in sect.Cepa andPhyllodolon. Phylogenies estimated from cpDNA characters usingDollo parsimony resulted in a single most-parsimonious tree of 46 steps and agreed with phylogenies based onWagner parsimony. Polymorphic restriction enzyme sites in the 45s ribosomal DNA were not used to estimate phylogenies because of uncertain homologies, but are useful for identifying interspecific hybrids. The maternal phylogenies estimated in this study help to distinguish wildAllium species closely related to the bulb onion. Although not in agreement with classifications based on morphology, the phylogenies closely reflected crossability among species in sect.Cepa andPhyllodolon.     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

Influence des parasites oophages sur la régulation des populations deCassida deflorata Suff

Summary The layings ofCassida deflorata Suffr. are parasited in southern France byMymaridae (Anaphoidea sp. andFulmekiella ovata soyka),Trichogrammatidae (Monorthochaeta nigra Blood.) andEulophidae (Foersterella flavipes Foerst. andTetrastichus rhosaces Walk.). In the Alpes-Maritimes, two successive generations ofMymaridae andTrichogrammatidae seem to attack the layings ofC. deflorata. In Corsica, parasitism byMymaridae andF. flavipes is very frequent and may be the essential regulating factor of the populations ofCassida. In the other regions that have been studied, although the large number ofCassida imagos appearing in spring show that mortality during the aestivation and hibernation is not the primary factor, it does not seem that the limitation of the populations is essentially caused by the activity of oophagous insects.      

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Nouvelles espèces de Torymides paléarctiques (Hym. Torymidae)

Summary In this paper four new palearctic species ofTorymidae are described:Monodontomerus gladiatus andTorymus favardi from South of France,Glyphomerus signifer from Austria andPseudotorymus amethystinus from Algeria.Torymus favardi is a parasite of bothDryocosmus australis mayr andAndricus singulus (mayr), whereasGlyphomerus signifer has been bred from galls ofDiplolepis spinosissimae (gir.).      

Heterostyly in species ofNarcissus (Amaryllidaceae) andHugonia (Linaceae) and other disputed cases

The hypothesis ofHenriques andFernandes that several Iberian species ofNarcissus (Amaryllidaceae) are tristylous is reconsidered. Contrary to the opinion ofBateman and most subsequent authors, we believe that the available evidence indicates that some populations ofN. triandrus andN. fernandesii, at least, are tristylous; other populations ofN. triandrus are distylous.Hugonia cf.penicillanthemum (Linaceae) from new Caledonia is distylous, but it remains possible that other species ofHugonia are tristylous. The disputed occurrence of heterostyly in S. African species ofBauhinia (Leguminosae),Cleome (Capparaceae) andAneilema (Commelinaceae), and inAgelaea (Connaraceae) is discussed.     

Breeding ofl-threonine hyper-producer ofEscherichia coli W

Summary l-Threonine hyper-producing mutants were obtained fromEscherichia coli W strain KY-8366, by reducingl-threonine degradation activity and enhancingl-threonine biosynthetic activity. Anl-threonine degradation reaction test using resting cells of KY-8366 suggested that the main pathway ofl-threonine degradation by KY-8366 is via glycine. A strain with reducedl-threonine degradation activity was obtained among those mutants that could not utilizel-threonine as sole nitrogen source. Rifampicin-resistant mutants andl-lysine plus methionine-insentitive mutants were isolated. These mutants showed enhanced aspartokinase levels and accumulated morel-threonine than the parental strains. Mutant H-4290 accumulated 58 g/l ofl-threonine.     

Influence of temperature and relative humidity on the development ofAmblyseius alstoniae (Acari: Phytoseiidae)

Reproduction and development ofAmblyseius alstoniae Gupta is significantly affected by temperature and relative humidity (rh), optimum being a temperature of 25°C andrh of 70%. At this combination, the viability of eggs, fecundity and daily egg production were higher, oviposition period and longevity were of longer duration, and mortality was minimal. Increase or decrease in these temperature andrh levels leads to a drop in fecundity, longevity and oviposition period and an increase in mortality. Longevity of females is always greater than that of males, irrespective of temperature andrh. Female: male ratio decreases with increase in temperature, irrespective ofrh.