Cloning and in vitro characterization of dTDP-6-deoxy-l-talose biosynthetic genes from Kitasatospora kifunensis featuring the dTDP-6-deoxy-l-lyxo-4-hexulose reductase that synthesizes dTDP-6-deoxy-l-talose

Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA_Kkf, RmlB_Kkf, RmlC_Kkf, and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, α-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using ¹H and ¹³C NMR spectroscopy. K. kifunensistal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.     

The correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities : The role of hydrogen bonding and conformational changes

2-methyl-1,4-naphtoquinone 1 (vitamin K₃, menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E_1/2) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E_A). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E_A) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.     

The site of inhibition of mitochondrial electron transfer by coenzyme Q analogues.

The effects of 33 quinone derivatives on mitochondrial electron transfer in yeast were examined. Twenty-two of the compounds were also tested for their effects on the growth of yeast cells. Four strong inhibitors of electron transfer were identified: 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole, 7-ω-cyclohexyloctyl-6-hydroxy-5,8-quinolinequinone, 7-n-hexadecyl-mercapto-6-hydroxy-5, 8-quinolinequinone, and 3-n-dodecylmercapto-2-hydroxy-1, 4-naphthoquinone. They inhibit the growth of yeast with ethanol as an energy source, but not when glucose is the energy source. The NADH oxidase activity of isolated mitochondria is 50% inhibited by these quinone derivatives at about 10^?8m, or 0.5 μmol/g mitochondrial protein; 1000-fold higher concentrations do not affect electron transfer from NADH or succinate to coenzyme Q₂. The effects of the inhibitors on cytochrome spectra indicate that they block electron transfer between cytochromes b and c₁. A possible antagonism between these compounds and coenzyme Q at a site between cytochromes b and C₁ is discussed in terms of Mitchell's “protonmotive Q cycle” hypothesis (Mitchell, P. (1976) J. Theor. Biol. 62, 327–367). 6-β-naphthylmercapto-5-chloro-2,3-dimethoxy-1,4-benzoquinone inhibits electron transfer between succinate and coenzyme Q₂ or phenazine methosulfate, suggesting a site in the succinate-coenzyme Q reductase complex with a different quinone specificity from that of the site in the cytochrome bc₁ complex. Seven of the quinone derivatives inhibit growth on both glucose and ethanol media, indicating that their effect is not the result of inhibition of respiration.     

Direct ESI-MS analysis of O-acyl glycosylated cardiolipins from the thermophilic bacterium Alicyclobacillus acidoterrestris

We used direct ESI-MS analysis to identify derivatives of cardiolipin molecular species (i.e. O-acyl glycosylated cardiolipins) from the thermophilic bacterium Alicyclobacillus acidoterrestris. We used triple-quadrupole type mass spectrometer for analysis of this complex lipid and enzymatic hydrolysis and ¹H and ¹³C NMR for the identification of these cardiolipin derivatives. These techniques enabled us to identify and quantify the specific molecular species profiles of derivatives of cardiolipin directly from lipid extracts of the bacterium including the identification of the sugar moiety as α-d-mannose and all five acyls including their positional isomers.     

Biosynthesis of antimalarial lignans from Holostylis reniformis

Holostylis reniformis biosynthesizes 8-8′ linked lignans without 9,9′-oxygenation. To elucidate the biosynthetic pathways to these lignans, the reputed precursors [U-¹⁴C]phenylalanine, [9-³H₁]coniferyl alcohol, and [9-³H₁]isoeugenol were administered to roots of the plant, which led to the incorporation of ³H and ¹⁴C into ten 2,7′ linked-lignans (aryltetralone lignans) and two 7,7′-epoxylignans (furan lignans). These administration experiments demonstrated that the lignans were propenylphenol-derived and that H. reniformis can exhibit regioselective control over radical-radical coupling (via isoeugenol radicals). Regiospecific control over propenylphenol-derived lignan biosynthesis was observed, together with diastereoselective control of C2-C7′ bond formation for the aryltetralone lignans (7′R). These experiments provide evidence that isoeugenol is a biosynthetic intermediate to the aryltetralone and furan lignans.     

Reactive derivative of adenosine-5′-triphosphate formed by irradiation of ATP γ-p-azidoanilide

UV and ¹H NMR spectra changes demonstrate that p-azidoanilides of ATP, ITP, pyrophosphate after irradiation at 313 nm, transform to similar reactive intermediates. The latter react readily with morpholine, iso-propylamine, tert-butylamine, imidazole, mercaptoethanol the reactivity being qualitatively correlated with nucleophilicity. By analogy with the transformation of p-azidoanilide, it is concluded that the reactive intermediate is the respective derivative of quinone diimine. Photoaffinity labelling of proteins with p-azidoanilide derivatives may proceed as the reaction of the nucleophilic centers of proteins with this quinone diimine derivative, rather than as a direct attack by a nitrene biradical.     

Tubby-tagged balancers for the Drosophila X and second chromosomes

We generated FM7a and CyO balancer chromosomes bearing a Tubby¹ (Tb¹) dominant transgene. Flies heterozygous for these FM7a and CyO derivatives exhibit a phenotype undistinguishable from that elicited by the Tb¹ mutation associated with the TM6B balancer. We tested two of these Tb-bearing balancers (FM7-TbA and CyO-TbA) for more than 30 generations and found that the Tb¹ transgene they carry is stable. Thus, these new Tb-tagged balancers are particularly useful for balancing lethal mutations and distinguish homozygous mutant larvae from their heterozygous siblings.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Biosynthesis and enantioselectivity in the production of the lilac compounds in Actinidia arguta flowers

Biosynthesis of the lilac alcohols and alcohol epoxides from linalool in ‘Hortgem Tahi’ kiwifruit (Actinidiaarguta) flowers was investigated by incubating flowers with rac-linalool, rac-[4,4,10,10,10-²H₅]linalool, (R)-8-hydroxylinalool and (R)-8-oxolinalool. All substrates were incorporated into the lilac alcohols although the (R)-configured compounds are not normally present in flowers. Biosynthesis of the lilac alcohol epoxides from rac-1,2-epoxy[4,4,10,10,10-²H₅]linalool and rac-[4′,4′, 8′, 8′,8′-²H₅]lilac aldehyde epoxide, rather than the lilac alcohols, was examined. Both substrates were non-enantioselectively converted to the lilac alcohol epoxides, suggesting two biosynthetic pathways for these compounds, contrary to previous reports. Their ability to process unnatural substrates indicates that A.arguta flowers have a greater biosynthetic capability than is suggested by their phytochemical composition. Linalool, the lilac compounds, and their biosynthetic intermediates were measured in the pistils, stamen, petals and sepals to determine if localisation in different organs contributed to only (S)-linalool being processed to the lilac compounds. Both linalool enantiomers were present in all organs, while most (97%) of the lilac compounds, and their precursors, were found in the petals. (S)-Linalool was not depleted from the flower petals, with respect to (R)-linalool, during the time of maximum production of the metabolites of (S)-linalool.     

Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis

A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.     

Biosynthesis of wyosine derivatives in tRNAPhe of Archaea: role of a remarkable bifunctional tRNAPhe:m1G/imG2 methyltransferase

The presence of tricyclic wyosine derivatives 3′-adjacent to anticodon is a hallmark of tRNA^Phe in eukaryotes and archaea. In yeast, formation of wybutosine (yW) results from five enzymes acting in a strict sequential order. In archaea, the intermediate compound imG-14 (4-demethylwyosine) is a target of three different enzymes, leading to the formation of distinct wyosine derivatives (yW-86, imG, and imG2). We focus here on a peculiar methyltransferase (aTrm5a) that catalyzes two distinct reactions: N¹-methylation of guanosine and C⁷-methylation of imG-14, whose function is to allow the production of isowyosine (imG2), an intermediate of the 7-methylwyosine (mimG) biosynthetic pathway. Based on the formation of mesomeric forms of imG-14, a rationale for such dual enzymatic activities is proposed. This bifunctional tRNA:m¹G/imG2 methyltransferase, acting on two chemically distinct guanosine derivatives located at the same position of tRNA^Phe, is unique to certain archaea and has no homologs in eukaryotes. This enzyme here referred to as Taw22, probably played an important role in the emergence of the multistep biosynthetic pathway of wyosine derivatives in archaea and eukaryotes.     

Oceanicella actignis gen. nov., sp. nov., a halophilic slightly thermophilic member of the Alphaproteobacteria

Two isolates, designated PRQ-67^T and PRQ-68, with an optimum growth temperature of about 50 °C, growth range in medium containing between 1 and 9% NaCl and an optimum pH for growth between 7.5 and 8.0, were recovered from a shallow marine hot spring on a beach, Praia do Fogo, at Ribeira Quente, on the Island of São Miguel in the Azores. Comparisons of 16S rRNA gene sequences show these strains to be most closely related (93.1–94.7% similarity) to species of the genus Amaricoccus, within the family Rhodobacteraceae. Strains are non-pigmented and form non-motile pleomorphic cells that stain Gram-negative, are aerobic, oxidase and catalase positive. The major fatty acids are C_18:1ω7c and C_18:1ω7c 11-methyl. Ubiquinone 10 is the major respiratory quinone. Major polar lipids are phosphatidylcholine, phosphatidylglycerol and one aminolipid. Based on 16S rRNA gene sequence analysis, physiological and biochemical characteristics we describe a new species of a novel genus represented by strain PRQ-67^T (=DSM 22673^T = LMG 25334^T) for which we propose the name Oceanicella actignis.     

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-¹⁴C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K_m values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-l-methionine as a substrate, respectively.     

Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with ¹³C-labelled precursors in an ¹⁸O₂ atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1 → O-2 → O-5. In addition, the incubation of W. thyrsiflora root cultures with ¹³C-labelled lachnanthocarpone established a direct biosynthetic precursor–product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.     

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond,proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6^T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6^T and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso ω9c (10.7%), and 16:1 ω7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6^T could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6^T (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.     

The Biosynthesis of the Pyrenocines in Cultures of Pyrenochaeta terrestris

[¹⁴C]Acetate, [¹⁴C]formate, and methyl[¹⁴C]methionine all serve as precursors of pyrenocines A, B, and C when added to cultures of Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, the pathogen responsible for disease known as pink root of onion (Allium cepa L.). This information supports the hypothesis that these metabolites are methyl-substituted polyketides in origin. Pyrenocine A arises from acetate via uncharacterized intermediates and is subsequently converted to pyrenocine B. The biosynthetic role of pyrenocine C remains uncertain.     

The biosynthetic origin of oxygen functions in phenylphenalenones of Anigozanthos preissii inferred from NMR- and HRMS-based isotopologue analysis

The biosynthetic origin of 9-phenylphenalenones and the sequence according to which their oxygen functionalities are introduced were studied using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). ¹³C-labelled precursors were administered to root cultures of Anigozanthos preissii, which were simultaneously incubated in an atmosphere of ¹⁸O₂. Two major phenylphenalenones, anigorufone and hydroxyanigorufone, were isolated and analyzed by spectroscopic methods. Incorporation of ¹³C-labelled precursors from the culture medium and ¹⁸O from the atmosphere was detected. O-Methylation with ¹³C-diazomethane was used to attach ¹³C-labels to each hydroxyl and thereby dramatically enhance the sensitivity with which NMR spectroscopy can detect ¹⁸O by means of isotope-induced shifts of ¹³C signals. The isotopologue patterns inferred from NMR and HRESIMS analyses indicated that the hydroxyl group at C-2 of 9-phenylphenalenones had been introduced on the stage of a linear diarylheptanoid. The oxygen atoms of the carbonyl and lateral aryl ring originated from the hydroxyl group of the 4-coumaroyl moiety, which was incorporated as a unit.     

Gaiella occulta gen. nov., sp. nov., a novel representative of a deep branching phylogenetic lineage within the class Actinobacteria and proposal of Gaiellaceae fam. nov. and Gaiellales ord. nov

Two isolates, with an optimum growth temperature of about 35-37 °C and an optimum pH for growth between 6.5 and 7.5, were recovered from a deep mineral water aquifer in Portugal. Strains form rod-shaped cells and were non-motile. These strains were non-pigmented, strictly aerobic, catalase and oxidase positive. Strains F2-233^T and F2-223 assimilated carbohydrates, organic acids and amino acids. Major fatty acids were novel iso internally branched such as 17:0 iso 10-methyl, 17:0 iso and 15:0 iso 8-methyl. The peptidoglycan contained meso-diaminopimelic acid and menaquinone MK-7 was the major respiratory quinone. Analysis of the 16S rRNA gene shows the strains to cluster with species of the genera Thermoleophilum, Patulibacter, Conexibacter and Solirubrobacter to which they have pairwise sequence similarity in the range 87-88%. Based on 16S rRNA gene sequence analysis, physiological and biochemical characteristics we describe a new species of a novel genus represented by strain F2-233^T (=CECT 7815^T = LMG 26412^T) for which we propose the name Gaiella occulta gen. nov., sp. nov. We also propose that this organism represents a novel family named Gaiellaceae fam. nov. of a novel order named Gaiellales ord. nov.