Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties nikkomycin Cz, and nikkomycins Cx and Cz, respectively. The nikV mutants formed nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized nikkomycins I, J, X, and Z, but amounts of nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.     

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase–dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins C_x and C_z. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins L_x and L_z, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine; the putative activities of each of their products are discussed. Received: 1 February 1999 / Accepted: 29 April 1999     

Functional characterization of orf6 and orf9 genes involved in the biosynthesis of L-oleandrose from Streptomyces antibioticus Tü99

Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and heterologously expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is in support of proving their functions. Furthermore biosynthetic pathway 5,5-gem-dimethyl-6-deoxyglucose (TDP-Lnoviose) has been proposed.     

Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée)

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.     

Molecular characterization of the zerknüllt region of the Antennapedia complex of D. subobscura

We have characterized at the molecular level the zerknüllt (zen) region of the Drosophila subobscura Antennapedia complex. The sequence comparison between D. subobscura and D. melanogaster shows an irregular distribution of the conserved and diverged regions, with the homeobox and a putative activating domain completely conserved. Comparisons of the promoter sequence and pattern of expression of the gene during development suggest that the regulation of zen has been conserved during evolution. The conservation of zen expression in a subpopulation of the polar cells indicates the existence of an important role in such cells. We describe a transitory segmented pattern of expression of zen in both species, suggesting the existence of interactions with a pair rule gene. Some indirect clues indicate that the z2 gene might be absent from the D. subobscura genome. A chromosome walk initiated to reach the proboscipedia gene of D. subobscura reveals that the distance between pb and zen is at least four times the one described for D. melanogaster and for D. pseudoobscura. Finally, we present cytological evidence showing that the ANT-C is inverted in D. subobscura as compared to D. melanogaster.     

Molecular cloning and functional expression of two key carotene synthetic genes derived from Blakeslea trispora into E. coli for increased β-carotene production

Blakeslea trispora is used commercially to produce β-carotene. Isopentenyl pyrophosphate isomerase (IPI) and geranylgeranyl pyrophosphate synthase (GGPS) are key enzymes in the biosynthesis of carotenoids. The cDNAs of genes ipi and carG were cloned from the fungus and expressed in Escherichia coli. Greater GGPS activity was needed in the engineered E. coli when IPP activity was increased. The introduction of GGPS and IPI increased the β-carotene content in E. coli from 0.5 to 0.95?mg/g dry wt.     

Molecular cloning and characterization of two pathogenesis-related β-1,3-glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium scitamineum

Key message

Two β-1,3-glucanase genes from sugarcane were cloned and characterized. They were all located in apoplast and involves in different expression patterns in biotic and abiotic stress.

Abstract

Smut caused by Sporisorium scitamineum is a serious disease in the sugarcane industry. β-1,3-Glucanase, a typical pathogenesis-related protein, has been shown to express during plant–pathogen interaction and involves in sugarcane defense response. In this study, β-1,3-glucanase enzyme activity in the resistant variety increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1 (GenBank Accession No. KC848050) and ScGluD1 (GenBank Accession No. KC848051) were cloned and characterized. Phylogenetic analysis suggested that ScGluA1 and ScGluD1 clustered within subfamily A and subfamily D, respectively. Subcellular localization analysis demonstrated that both gene products were targeted to apoplast. Escherichia coli Rosetta (DE3) cells expressing ScGluA1 and ScGluD1 showed varying degrees of tolerance to NaCl, CdCl₂, PEG, CuCl₂ and ZnSO₄. Q-PCR analysis showed up-regulation of ScGluA1 and slight down-regulation of ScGluD1 in response to S. scitamineum infection. It suggested that ScGluA1 may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1 was inhibited. The gene expression patterns of ScGluA1 and ScGluD1, in response to abiotic stresses, were similar to sugarcane response against smut infection. Together, β-1,3-glucanase may function in sugarcane defense mechanism for S. scitamineum. The positive responses of ScGluA1 and the negative responses of ScGluD1 to biotic and abiotic stresses indicate they play different roles in interaction between sugarcane and biotic or abiotic stresses.     

Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37 °C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.     

Contribution of the allelicMtz 3 andMtz 4 allotype genes to the formation of individual rabbit serum α2-macroglobulin molecules

The contribution of the allelicMtz ³ andMtz ⁴ genes to the formation of individual rabbit serum α₂-macroglobulin (α₂M) molecules was examined by precipitation of α₂M from rabbits of known genotype with antiallotype antisera. The α₂M was isolated fromz ³z³ andz ⁴z⁴ homozygous andz ³z⁴ heterozygous rabbits, iodinated with I¹²⁵ and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α₂M or α₂M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α₂M. Nearly all α₂M molecules have z3 or z4 antigenic determinants; approximately 50% of α₂M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60% of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known allelic blood protein systems of rabbits, allelic exclusion does not occur in α₂M molecules of heterozygotes; rather, hybrid molecules are formed. Presented in part at the Fifty-fourth and Fifty-fifth Annual Meetings of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 12–17, 1970, and Chicago, Illinois, April 12–17, 1971. This investigation was supported in part by U.S. Public Health Service Grants AI-09241 and AI-07043. B.H.B. performed this investigation in partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College; he is supported by a postdoctoral fellowship from the Schweppe Foundation. K.L.K. is the recipient of U.S. Public Health Service Research Career Development Award AI-28687.     

Inactivation of the tricarboxylic acid cycle aconitase gene from Streptomyces viridochromogenes Tü494 impairs morphological and physiological differentiation

The tricarboxylic acid (TCA) cycle aconitase gene acnA from Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation.     

Nucleosides LXXXIII. Synthetic studies on nucleoside antibiotics. 11. Synthesis of methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside and -hexopyranosiduronic acid (derivatives related to the carbohydrate moiety of gougerotin)

Methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside (17) and its uronic acid (19) were synthesized via a series of reactions starting from 1,2:5,6-di-O-isopropylidene-3-O-tosyl-α-D-glucofuranose. A method suitable for the large scale preparation of 3,4-dideoxy- 1,2:5,6-di-O-isopropylidene-α-D-erythro-hex-3-enofuranose(2) was devised.     

Molecular cloning of the self-incompatibility genes S1 and S3 from almond (Prunus dulcis cv. Ferragnès)

Almond (Prunus dulcis) displays gametophytic self-incompatibility. In the work reported here, we cloned two novel S-RNase genes from almond cultivar Ferragnès (genotype S1S3) using PCR. The S1-RNase gene has the same coding region as the Sb gene cloned from almond cultivated in the USA; however, their introns are different in sequence. S1 was cloned and sequenced from six different cultivars originating in Europe. The full-length of the S3-RNase gene was cloned using two primers corresponding to the start and stop codons contexts. Two introns are present in the S3 gene, unique among the S-RNase genes. Sequence-specific PCR was performed to confirm that the two cloned genes co-segregate with the S-locus using progenies of a controlled cross between Tuono (S1Sf) and Ferragnès (S1S3). Based on the structural differences of S- and S-like RNase genes, we discuss the evolutionary relationship between the two groups of RNase genes. Received: 18 February 2001 / Accepted: 26 June 2001