Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

The role of sulphur in detoxication of cadmium in young sugar beet plants

In young sugar beet plants cadmium suppressed the activity of nitrate reductase, glutamine synthetase and glutamate dehydrogenase, whereas sulphur exhibited a protective role towards activity of these enzymes, except of glutamine synthetase. Protein synthesis was suppressed in the absence of S in nutrient medium; the lowest level was at 10^-3 M Cd²⁺. Chloroplast pigment contents were increased by S while Cd²⁺, even in the lowest concentration, (10⁻⁵ M) showed a repressive effect. The highest concentrations of Cd²⁺ (10−3 M) caused a decrease in dry mass, whereas S induced its increase. Nitrate content was increased in the presence of Cd²⁺ and decreased by increased concentration of S. Acknowledgement: The authors acknowledge financial support of the Ministry for Science and Technology of Serbia. The paper was presented at 9th Congress of the Federation of European Societies of Plant Physiology, Brno, Czech Republic, 3–8 July 1994.     

Korkormicins,novel depsipeptide antitumor antibiotics fromMicromonospora sp C39500: Fermentation,precursor directed biosynthesis and biological activities

Micromonospora sp C39500, isolated in our laboratory from a soil sample, produced a complex of seven novel depsipeptide antitumor antibiotics, designated korkormicins. The major component of the complex, korkormicin A, has a MW of 1452 and a molecular formula of C₆₆H₈₄N₁₆O₂₂. Korkormicin A exhibits potentin vivo antitumor activity against P388 leukemia and M109 lung carcinoma implanted intraperitoneally (ip) in mice, with effective doses of 0.05–0.20 mg kg^–1 injection^–1, for five or three ip injections, respectively. It is also active against Gram-positive bacteria but inactive against Gram-negative bacteria. The production of korkormicin A was enhanced by 3-fold when 0.1%l-valine was added to the production culture at 48h. A titer of 401.0 g ml^–1 was achieved in the fermenter culture supplemented with 0.1%l-valine.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .