A metabolic prototype for eliminating tryptophan from the genetic code.

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli.

BACKGROUND: To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. RESULTS: To overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate. CONCLUSIONS: This approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium.

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Ca(2+)/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids.

This study describes a novel mode of activation for the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca(2+)/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca(2+)/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca(2+)/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca(2+)/calmodulin action by binding to the calmodulin-binding site.

Overexpression, purification and characterization of Dictyostelium calcineurin A.

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Primary structure, expression and developmental regulation of a Dictyostelium calcineurin A homologue.

cDNA clones for the catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) from Dictyostelium discoideum were isolated by functional screening of a lambda gt11 lysogen expression library with labeled Dictyostelium CaM. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shown to display similar enzymatic properties as the enzyme from other sources. On Western blots specific antibodies against the protein recognized a band of approximately 80 kDa that migrated with an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed.

On the role of calcium during chemotactic signalling and differentiation of the cellular slime mould Dictyostelium discoideum.

Transient cytosolic calcium elevations are required for chemotaxis and differentiation of Dictyostelium discoideum since Ca2+ chelating buffers introduced into the cells by scrape loading inhibited motility as well as orientation in a Ca2+ specific manner. Ca2+ changes are provided either by intrinsic cytosolic Ca2+ oscillations that can be determined as periodic Ca2+ efflux, or by receptor-mediated Ca2+ liberation from the InsP3-sensitive store and Ca2+ influx. Cytosolic Ca2+ homeostasis as well as oscillations seem to be regulated by two different Ca2+ stores, the acidosomes and the InsP3-sensitive store, both of which are dependent on Ca2+ pumps and V-type H+ ATPases. Ca2+ transients are sensed by calmodulin-binding proteins. The latter have been detected in Dictyostelium by 125I-calmodulin labeling. A calmodulin-dependent protein phosphatase, calcineurin A, was cloned, sequenced, purified and characterized biochemically. Overproduction of calcineurin A as well as antisense constructs will help to the elucidation of its function in signal transduction. Surprisingly, protein synthesis is also controlled by Ca2+/calmodulin. An integral ribosomal protein of the 60S subunit, L19, proved to be a calmodulin-binding protein and calmodulin antagonists of different classes, inhibited in vitro translation of Dictyostelium and wheat germ extracts.

The catalytic subunit of cAMP-dependent protein kinase from Ascaris suum. The cloning and structure of a novel subtype of protein kinase A.

A complete cDNA clone encoding the catalytic subunit of cAMP-dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 amino acids is 48% identical with the sequence of mouse C alpha subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N-terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N-terminal region and an acidic amino acid near the C-terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N-terminal and C-terminal parts of the protein. There is strong evidence that a single gene encodes cAMP-dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X-ray structure of the mammalian enzyme suggest a high degree of conservation in the three-dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952-957] and the mammalian enzyme.

Cytosolic nucleoside diphosphate kinase associated with the translation apparatus may provide GTP for protein synthesis.

Elongation of nascent polypeptides in a Dictyostelium discoideum in vitro translation system did not require the addition of ATP and GTP when creatine phosphate and creatine phosphokinase were present. However, depletion of the exogenous energy supply completely abolished incorporation of amino acids. Addition of dTTP, a nucleoside triphosphate that can be utilized by nucleoside diphosphate kinase (NDP kinase) to phosphorylate endogenous ADP and GDP, partially restored protein synthesis. Dictyostelium ribosomes were found to contain NDP kinase activity that could not be released by 1 M KCl. Thermal denaturation studies, specific inhibition with antibodies, and Western blotting identify the activity as cytosolic NDP kinase. These data support the idea that GTP can be fed into the translation machinery efficiently by NDP kinase associated with active ribosomes.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt.

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

Nucleotide sequence of the gene for ribosomal protein S17 from Dictyostelium discoideum.

The nucleotide sequence of the gene for the Dictyostelium homologue of eukaryotic ribosomal protein S17 has been assembled from cDNA and genomic DNA clones. The predicted primary structure of the S17 protein displays a similar level of sequence identity with its counterparts from higher eukaryotes (53%) as other Dictyostelium ribosomal proteins. Although Dictyostelium genes usually are organized in a rather simple manner, the rps17 gene harbors two introns. One of them, located immediately 3' from the ATG initiator codon, appears to be ubiquitously conserved in eukaryotic rps17 genes.

Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

Elongation in a Dictyostelium in vitro translation system is affected by calmodulin antagonists.

We have previously shown that the Dictyostelium discoideum ribosomal protein L19 specifically binds Ca2+/calmodulin [Sonneman et al. (1991) J. Biol. Chem. 266, 23091-23096]. To investigate the role of calmodulin in the regulation of protein synthesis, we have now established an in vitro protein synthesizing system from Dictyostelium cells which can elongate polypeptide chains with high efficiency. Various calmodulin antagonists affected translation in this system. The inhibitory effects of the antagonists could be partially reversed by addition of calmodulin. A monoclonal antibody against D. discoideum calmodulin also specifically inhibited protein synthesis. Similar effects of calmodulin antagonists were found in a standard wheat germ in vitro translation system.

Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme.

The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined. The cpo codes for a protein of 278 amino acids (aa). The mature enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304. Expression studies showed that the cpo from P. pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans. Based on the overproduction of the CPO in E. coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P. pyrrocinia. Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S. aureofaciens ATCC10762 revealed an identity of 38%.

Serine/threonine protein phosphatases in Dictyostelium discoideum: no evidence for type I activity.

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.

A 138-kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity.

A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide.

A ribosomal calmodulin-binding protein from Dictyostelium.

Using 125I-calmodulin as a probe, we have recently identified specific Ca2+/calmodulin-binding proteins in cell extracts from the cellular slime mold, Dictyostelium discoideum: a major 22-kDa activity, a soluble 78/80-kDa protein, and several membrane-associated high Mr proteins (Winckler, T., Dammann, H., and Mutzel, R. (1991) Res. Microbiol. 142, 509-519). cDNA clones for at least two of these proteins have been isolated by ligand screening of a lambda gt11 prophage expression library. Antibodies directed against the lacZ-cDNA-encoded fusion protein from one of the clones recognized a single 22-kDa component in D. discoideum extracts which comigrated with the endogenous 22-kDa calmodulin-binding protein. The cDNA-derived nucleotide sequence predicts a protein of Mr 21,659 with 56% sequence identity (69% homology) with rat ribosomal protein L19. The endogenous 22-kDa calmodulin-binding activity was associated with ribosomes. It was found to be an integral constituent of the large ribosomal subunit, since it cosedimented with 60 S ribosomal subunits in sucrose density gradients in the presence of 0.5 M NH4Cl. Our observations point to a physiological role for calmodulin in the Ca2+ regulation of eukaryotic protein synthesis. Support for this comes from recent studies showing inhibition of protein synthesis by calmodulin antagonists in Ehrlich ascites tumor cells (Kumar, R. V., Panniers, R., Wolfman, A., and Henshaw, E.C. (1991) Eur. J. Biochem. 195, 313-319).

Ca2+/calmodulin-binding proteins in Dictyostelium discoideum.

We have initiated a systematic study of Ca2+/calmodulin-regulated enzymes in the cellular slime mold Dictyostelium discoideum. Using 125I-labelled D. discoideum calmodulin (CaM) as a functional probe, several Ca2+/CaM-binding proteins were detected in crude cell lysates. Proteins with apparent molecular weights of 22 kDa and 78-80 kDa, respectively, were found in the soluble fraction. In addition, membrane-bound high molecular weight CaM-binding proteins were identified. Binding of CaM to all of the proteins required the presence of Ca2+ ions and competed efficiently with nonradioactive CaM from both Dictyostelium and bovine brain. The CaM antagonists melittin, W-7 and R24571 inhibited CaM binding. With a functional cloning approach, we previously obtained cDNA clones by screening a lambda gt11 lysogen expression library; in this paper, we report the analysis of CaM-binding activity by one of the recombinant cDNA clones in Escherichia coli. When rabbit antiserum was raised against it, the antiserum recognized a 78-80-kDa protein in Dictyostelium extracts which comigrated on SDS-polyacrylamide gels with 78-80-kDa CaM-binding activity.