Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Prolonged environmental stress via a two step process selects mutants of Escherichia,Salmonella and Pseudomonas that grow at 54°C

A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48°C with nalidixic acid selected mutants (T48) able to grow at 48°C. A prolonged incubation at 54°C of the T48 mutants selected mutants (T54) able to grow at 54°C. These mutants were susceptible to the same bacteriophages as the original mesophilic strains. Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48°C or 54°C or on a minimal agar with glucose at 37°C. The T48 alleles mapped in the gyrA region of E. coli or S. typhimurium chromosome. In S. typhimurium the T54 alleles, which permit growth at 54°C, were shown by cotransductional analysis to be linked to gyrA.     

The value of mutants unable to carry out photorespiration

Manipulation of the CO₂ concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO₂ fixation and amino acid metabolism are highlighted.Abbreviations GS cytoplasmic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - PFR Photon fluence rate - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SGAT serine:glyoxylate aminotransferase     

Amino acid metabolism in euryhaline bivalves: regulation of glycine accumulation in ribbed mussel gills

Glycine levels in isolated ribbed mussel (Modiolus demissus) gill tissue increased slightly and decreased markedly when incubated at high and low salinities, respectively. Low levels of the enzymes involved in the biosynthesis of serine from triose phosphate intermediates, the serine hydroxymethyltransferase, and serine dehydrase were detected in gill tissue homogenates. Experiments using gill tissue incubated with (U-14C)-glycine and (U-14C)-serine indicated interconversion between serine and glycine and transfer of label to alanine, asparate, glutamate, CO2, organic acids, and protein. Glyoxylate was metabolized more slowly than glycine and was probably converted to glycine for catabolism. Studies using (1-14C)-glycine and (2-14C)-glycine with isolated gill tissue and mitochondria indicated that the mitochondrial glycine cleavage enzyme was the major route of glycine catabolism. Metabolic controls activating or inhibiting the glycine cleavage enzyme regulate tissue glycine accumulation and catabolism during hypersalinity or hyposalinity stress.     

The glycine cleavage system: Composition,reaction mechanism,and physiological significance

Summary The glycine cleavage system catalyzes the following reversible reaction: Glycine + THF + NAD⁺ 5,10-methylene-THF + + CO₂ + NH₃ + NADHReversibility of the overall reaction was established through the studies with the enzymes prepared from liver mitochondria of rat and cock and from extracts ofArthrobacter globiformis grown on glycine. The glycine cleavage system is composed from four protein components. The four proteins were revealed to exist originally as an enzyme complex in the liver mitochondria. Partial reactions of glycine cleavage and glycine synthesis were studied in detail with partially purified individual protein components. Particularly a protein-bound intermediate of glycine metabolism could be isolated and its nature and role were clarified. A tentative scheme was presented to explain the whole process of the reversible glycine cleavage.The glycine cleavage system was shown to represent the major pathway of catabolism of both glycine and serine in vertebrates, including mammals, birds, reptiles, amphibians, and fishes. Serine catabolism in these animals proceeds mainly by way of the cleavage of serine to form methylene-THF and glycine rather than deamination by serine dehydratase. In ureotelic and ammonotelic animals methylene-THF formed from the -carbon of glycine as well as the-carbon of serine could be further oxidized to CO₂ in either the mitochondria or the soluble tissue fractions, while in uricotelic animals methylene-THF could hardly be oxidized to CO₂ and instead, was utilized mostly for purine synthesis. Glycine synthesis by the glycine cleavage system did not appear to have appreciable physiological significance in animals.Abbreviation THF dl,l-tetrahydrofolic acid. an invited article     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO₂ concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with ¹⁴C showed that the barley mutants incorporated [¹⁴C]formate and [2-¹⁴C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway. Received: 14 August 1998 / Accepted: 30 September 1998     

Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis

Summary The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). We have isolated, mapped and characterized a large number of Salmonella typhimurium mutants auxotrophic for ALA. These mutants carry defects in either one of two genes, both required for ALA synthesis. The previously identified hemA gene maps at 35 min, and the hemL gene maps at 5 min on the S. typhimurium genetic map. Mutants in hemA and hemL are defective for aerobic and anaerobic respiration, and appear to be oxygen sensitive. The Hem^– phenotype of hemL mutants is less severe than that of hemA mutants. Although hemA and hemL mutants are deficient in heme synthesis, genetic tests indicate that they still synthesize two minor products of the heme pathway, siroheme and cobalamin (vitamin B₁₂), under anaerobic conditions. In contrast, hemB, hemC and cysG mutants, blocked after ALA synthesis, make neither siroheme nor vitamin B₁₂. Double mutants defective in both hemA and hemL also make siroheme. We suggest that hemA and hemL are required for one route of ALA synthesis and that a second, minor route of ALA synthesis may operate in S. typhimurium; this second pathway would be independent of the hemA and hemL functions.Abbreviations Amp ampicillin - Cam chloramphenicol - Kan kanamycin - Tet tetracycline - Str streptomycin - X-gal 5-bromo-4-chloro-3-indolyl--d-galactoside - DES diethyl sulfate     

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Regulation of tryptophan metabolism in Coprinus cinereus: Isolation and characterisation of mutants resistant to 5-fluoroindole

171 mutations conferring resistance to the indole analogue 5-fluoroindole (5 FI) were isolated in the filamentous basidiomycete fungus Coprinus cinereus. 5 FI is thought to be toxic because it is converted intracellularly to 5-fluorotryptophan (5 FT) which feedback inhibits the first enzyme of the tryptophan biosynthetic pathway, anthranilate synthase. Mutations were assigned to five loci, iar-1-iar-5 on the basis of functional analyses and mapping experiments. iar-5 mutations mapped in the anthranilate synthase structural gene and gave rise to an enzyme feedback resistant to tryptophan and its analogue. Mutants at other loci had regulatory changes. iar-1 and iar-3 mutants had elevated levels of two pathway enzymes measured (anthranilate synthase and tryptophan synthase) and were cross resistant to analogues of other aromatic amino acids suggesting that the entire aromatic pathway was derepressed. iar-3 mutants were unable to degrade metabolically derived typtophan to anthranilic acid unlike iar-1 mutants which excreted high levels of anthranilic acid. iar-2 mutants appeared to have a constitutive degradative pathway. iar-4 mutants had a blocked degradative pathway and unusual levels of tryptophan pathway enzymes.Abbreviations 5 FI 5-fluoroindole - 5 FT 5-fluorotryptophan - pFP para-fluorophenylalanine - mFT meta-fluoro-tyrosine     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Induction and regulation of cellulase synthesis in Trichoderma pseudokoningii mutants EA3-867 and N2-78

Two mutants, EA₃-867 and N₂-78, with high cellulase yields were obtained from wild strains of Trichoderma pseudokoningii Rifai, 1096 and Mo₃, respectively, by mutagenic treatments with a linear accelerator, ⁶⁰Co, u.v., nitrosoguanidine (NTG) and diethylsulphate (DTS). The mutants grew slowly to produce small colonies on agar plates with synthetic medium. On agar plates of peptone-yeast extract, the small colonies were as large as those of wild strains. The cellulase activities of these mutants in Koji extracts, shake flask culture filtrates, and enzyme preparations were markedly higher than those of their parents. The mutant N₂-78 reached quite high cellulase activity level when cultured for 60 h in shake flasks in a simple medium containing milled straw, wheat bran, mineral salts plus waste glucose molasses. The cellulase saccharifying activities on CMC, filter paper and cotton, were 255, 8.2 and 13.4 mg glucose/ml enzyme, respectively, or 11, 4.3 and 6 times more than those of its parent Mo₃.The cellulase synthesis of EA₃-867 and N₂-78 was strongly induced by sophorose, isolated from pods of Sophora japonica L., and was inhibited by glucose, sugar phosphates, glycerol and organic acids. We conclude that cellulase synthesis of the mutants is regulated by catabolite repression as well as by induction. The increase in cellulase production by both mutants results from changes in the regulatory systems for cellulase synthesis, i.e. the mutants showed higher sensitivity to inducer and lower susceptibility to catabolite repression than did the wild types.A cellulase preparation of Trichoderma pseudokoningii Rifai N₂-78 induced by sophorose was fractionated by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography, selective inactivation and polyacrylamide gel electrophoresis. The components C₁(exo-β1,4-glucanase), C_x(endo-β1,4-glucanase) and β-glucosidase were separated, and their molecular weights were estimated to be 67 000, 62 000 and 42 000 respectively. The homogeneity of C₁ was verified by polyacrylamide gel electrophoresis, immunoelectrophoresis and ultracentrifugal analysis. It is a glycoprotein and is rich in glycine, aspartic acid, threonine, serine and glutamic acid. The C₁ showed a strong synergistic action with C_x in the degradation of cotton, Avicel and Walseth cellulose.A poly(A)-RNA, induced by sophorose in N₂-78 mycelium, was isolated by oligo(dT)-cellulose affinity chromatography.     

Proline over-production results in enhanced osmotolerance in Salmonella typhimurium

Summary Mutants of S. typhimurium with enhanced osmotolerance were isolated. These mutants were obtained as strains which over-produced proline due to regulatory mutations affecting proline biosynthesis. The mutations are located on FproBA and upon transfer to other S. typhimurium strains, they confer enhanced osmotolerance on the recipients. The osmotolerant mutants not only have higher intracellular proline levels than the osmosensitive parental strain, but the proline levels in the osmotolerant mutants are regulated such that they increase in response to osmotic stress. Possible reasons why elevated proline levels lead to enhanced osmotolerance are discussed.     

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Four xylanases belonging to glycoside hydrolase family 10—Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulomonas fimi Cex (CF)—were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric β-1,4-linked xylopyranose as a precipitate during reaction with α-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X₂ as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.     

Isolation of stable aroA mutants of Salmonella typhi Ty2: properties and preliminary characterisation in mice

Summary Derivatives of the Salmonella typhi strain Ty2 carrying stable mutations in the aroA gene were isolated. The mutations were generated by transducing an aroA::Tn10 marker into Ty2 and selecting for derivatives which were tetracyline sensitive and dependent on aromatic compounds for growth. Isolates that did not revert to aromatic compound independence at a detectable frequency were obtained. An S. typhimurium derived aroA specific DNA probe was used to demonstrate the presence of DNA rearrangements in the aroA region of the chromosome of some of the S. typhi aroA mutants. Most of these isolates still expressed Vi antigen. Aromatic compound dependent mutants of S. typhi were less virulent in mice than S. typhi Ty2 following intraperitoneal challenge with bacteria suspended in mucin. Mice immunised with one of these mutants, named WBL85-1, were protected against a potentially lethal challenge of S. typhi Ty2.     

Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana

Summary. Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses. Received December 9, 1999 Accepted February 2, 2000     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W₁₀BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.Dedicated to Prof. Dr. A. Trebst on the occasion of his 65th birthday     

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.