Nocardia Sp Research Articles

Microbial transformation of three bufadienolides by Nocardia sp. and some insight for the cytotoxic structure–activity relationship (SAR)

Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan’su. Biotransformation of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl 15β-hydroxyl bufotalin, via an unprecedented 14β,15β-epoxy ring cleavage and a regio-selective acetoxylation. This product showed significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure–activity relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR research.

Development of a Rapid Normal-Phase LC-Positive Ion APCI-MS Method for Simultaneous Detection and Quantitation of Cholesterol, Androst-4-ene-3,17-dione, and Androsta-1,4-diene-3,17-dione

This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4-diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M+H]+ ions are taken into consideration for quantitation of AD and ADD, and [M-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation (r2 > or = 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 microg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as Nocardia sp. These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.

Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid

Aldehyde oxidoreductase (carboxylic acid reductase) catalyzes the Mg 2+, ATP and NADPH dependent reduction of carboxylic acids to their corresponding aldehydes. The identification of the gene from Nocardia sp. NRRL 5646 and its expression in E. coli BL21-CodonPlus ®(DE3)-RP/pHAT305 provided an avenue to develop a biocatalyst for reduction of carboxylic acids. In addition to aromatic acids, the recombinant carboxylic acid reductase also accepts several aliphatic mono, di and tri carboxylic acids as substrates. A recently identified Nocardia sp., phosphopantetheinyl transferase gene ( npt) enhanced the activity of carboxylic acid reductase. Coexpression of car and npt in E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.83 resulted in a purified recombinant carboxylic acid reductase with improved specific activity of 2.2 U/mg protein. The utility of the recombinant carboxylic acid reductase as a biocatalyst has been demonstrated using vanillic acid as substrate. E. coli BL21-CodonPlus ®(DE3)-RP/pHAT305 expressing Car reduced 50% of vanillic acid to vanillin in 10 h. E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.83 resting cells expressing Car and Npt reduced 90% of vanillic acid to vanillin in 6 h. Enhanced, in vivo cofactor NADPH regeneration by glucose dehydrogenase ( gdh) was accomplished using E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.85, that carried car, npt, and gdh. Resting cell reactions using E. coli BL21-CodonPlus ®(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin efficiently reduced 5 g/L of vanillic and benzoic acids within 2 h.

Nocardia sp. vanillic acid decarboxylase

Vanillic acid is an abundant renewable chemical useful for the biocatalytic synthesis of aromatics. The bacterium, Nocardia sp. NRRL 5646 efficiently catalyzes the decarboxylation of vanillic acid to the phenolic product, guaiacol. For purification and characterization of vanillic acid decarboxylase (VAD), a simple, rapid enzyme assay was devised to couple the vanillic acid decarboxylase reaction with horseradish peroxidase/H 2O 2. Guaiacol formed in the decarboxylation reaction is oxidized by peroxidase to tetraguaiacol, a chromophoric substance, which is measurable at 470 nm. Soluble, VAD activity was inducible in cells grown with vanillic acid. VAD was purified 185-fold by a combination of DEAE cellulose, phenylsepharose and sephacryl S-200 chromatographies. SDS-PAGE gave an estimated molecular mass of 46 ± 0.8 kDa while active native VAD was 92 kDa by gel chromatography. VAD required no cofactors, displayed optima at pH 7.0 and 30 °C, and a K m of 0.8 mM for vanillic acid. The N-terminal amino acid sequence was determined to be AEYTLPDLDYDYSALEPHIS. 4-Hydroxybenzoic acid, vanillic acid, and syringic acid were excellent substrates, while VAD showed no activity for isovanillic acid, ferulic acid, p-coumaric acid, 2-hydroxybenzoic acid and 3-hydroxybenzoic acid. Purified VAD required a phenolic functional group para to the carboxylic acid moiety in the best substrates. The vanillic acid decarboxylation reaction is non-oxidative in nature. Conduct of the decarboxylation process in deuterium oxide results in incorporation of one deuterium atom para to the phenolic hydroxyl group of guaiacol. Vanillic acid decarboxylation likely involves enzymatic tautomerization to a vinylogous β-keto acid that spontaneously decarboxylates to guaiacol.

Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.

Novel bioactive bacterial metabolites from a marine derived bacterium Nocardia Sp. Merv 21695

Novel bioactive bacterial metabolites from a marine derived bacterium Nocardia Sp. Merv 21695

217 POSTER Circulating levels of ligand as a potential biomarker for optimal dosing of targeted antibody drugs to the epidermal growth factor receptor

This chapter provides an overview of antitumor antibiotics. The actinomycins are a group of antibiotics possessing a quinoid-containing phenoxazine chromophore(3-amino-l,8-dimethyl-2-phenoxazone-4,5-dicarboxylic acid) and differ in the amino acid components on two symmetrically substituted polypeptide side chains attached through the carboxylic group. These side chains are necessary for antitumor activity. The new group of ansamycin antibiotics, ansamitocins, having potent antitumor activity was isolated from a fermentation broth of Nocardia sp. No. C-1500s by Higashide et al. Structure of ansamitocin was found to be similar to maytansine and related maytansinoids obtained from plant sources by Kupchan et al. It is very important that the discovery of a microorganism producing ansamitocin opened the way for production of new antitumor ansa macrolides by fermentation, because maytansinoides are only harvested from tropical plants, Maytenus serrata and M. buchananii, and their contents in the plants are extremely low.

Biodegradation of Aliphatic and Aromatic Hydrocarbons by Nocardia sp. H17-1

We investigated the biodegradation of hydrocarbon components by Nocardia sp. H17-1 and the catabolic genes involved in the degradation pathways of both aliphatic and aromatic hydrocarbons. After 6 days of incubation, the aliphatic and aromatic fractions separated from Arabian light oil were degraded 99.0 ± 0.1% and 23.8 ± 0.8%, respectively. Detection of the catabolic genes involved in the hydrocarbon degradation indicated that H17-1 possessed the alkB genes for n-alkane biodegradation and catA gene for catechol 1,2-dioxygenase. However, H17-1 had neither the C23O gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity. The investigation of the genes involved in the biodegradation of hydrocarbons supported the low degradation activity of H17-1 on the aromatic fractions.

Replication and inheritance ofNocardiaplasmid pC1

Nocardia sp. C-14-1, isolated from acrylic fiber wastewater, can degrade long-chain alkanes and succinonitrile efficiently. Here we report the characterization of an indigenous plasmid pC1. The overall nucleotide sequence of pC1 consisted of 5841 bp. The five ORFs, encoding a DNA recombinase, replication protein (Rep(pC1)) and three proteins of unknown function, were predicted on pC1. The Rep(pC1) displayed its homology with the Rep of Rhodococcus large plasmid p33701, suggesting a theta type of replication. An Escherichia coli plasmid (containing the single rep(pC1) gene) propagated autonomously in low copy number in Nocardia or Rhodococcus, suggesting that rep(pC1) was an essential gene for plasmid replication. The plasmid (containing the single rep(pC1) gene) presented as inheritance unstable hints that other pC1 loci were required for the stable inheritance of plasmids. By comparison of the plasmid-borne Rep proteins, we classify Rhodococcus or Nocardia plasmids into four groups.

Nocardiosis and kidney transplantation: case report in a recently transplanted patient

The patient is a 47-year-old white woman who was on the hemodialysis from 1999 to 2002, when she received a cadaveric renal transplant (deceased donor). The immediate postoperative immunosupression consisted of oral tacrolimus, predinisone and mycofenolato mofetil (MMF), and her medications at that time of her admission were tacrolimus (5mg 12/12 h), MMF (1000mg 12-12 h), and predinisone (10mg/ day). After 8 weeks, the patient went to the hospital and she was admitted presenting fever (37,9ºC), cough, malaise and vomiting. The chest radiography revealed a mass in the left superior lobe, which was initially treated with levofloxacin associated to ceftriaxone. There was partial improvement of the cough and total remission of the fever. The patient was discharged after 3 weeks of treatment in stable condition, with negative blood and bronchial cultures. After ten days, she returned to the hospital with relapsed symptoms and a subcutaneous purulent collection was detected in her left leg, and the culture of the drained material evidenced a filamentous microorganism, identified as Nocardia sp, later specified as Nocardia asteroides. Treatment with sulfametoxazole-trimetroprin 800mg t.i.d was initiated, and after five days, the patient was pyretic and treatment was kept for six months.

Nocardimicins A, B, C, D, E, and F, Siderophores with Muscarinic M3 Receptor Inhibiting Activity from Nocardia sp. TP-A0674

In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites, the extract of Nocardia sp. TP-A0674 was found to be highly active. Bioassay-guided fractionation of it led to the isolation of six new siderophores, nocardimicins A (1), B (2), C (3), D (4), E (5), and F (6), as active principles. Their chemical structures were determined by spectroscopic and degradation analysis. Of these congeners, nocardimicin B (2) inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor most potently with a Ki value of 0.13 microM. Compound 2 showed more selective activity to M3 and M4 receptors than other subtypes.

Novel biotransformation of pentacyclic triterpenoid acids by Nocardia sp. NRRL 5646

Six pentacyclic triterpene acids, ursolic acid, oleanolic acid, betulinic acid, 23-hydroxybetulinic acid, glycyrrhetinic acid, and senegenin, were metabolized by the microbe Nocardia sp. NRRL 5646 to selectively furnish their corresponding 28-methyl esters. Notably, ursolic acid ( 1 ) was converted to oleanolic acid methyl ester ( 4 ) via two intermediates, oleanolic acid ( 2 ), and ursolic acid methyl ester ( 3 ), which are formed by participation of ‘retro-biosynthetic’ methyl migration from C-19 to C-20. Senegenin ( 11 ) was selectively converted to a nortriterpene methyl ester, senegenic acid methyl ester ( 12 ), via an unprecedented C–C bond cleavage. The stereochemical assignments of compounds 11 and 12 were made unambiguously for the first time using 2D NMR spectroscopy.

Effects of Crude Oil, Oil Components, and Bioremediation on Plant Growth

The phytotoxic effects of crude oil and oil components on the growth of red beans (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated. In addition, the beneficial effects of bioremediation with the oil-degrading microorganism, Nocardia sp. H17-1, on corn and red bean growth in oil-contaminated soil was also determined. It was found that crude oil-contaminated soil (10,000 mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0–1000 mg/kg of aliphatic hydrocarbons such as decane (C10) and eicosane (C20). Phytotoxicity was observed in soils contaminated with 10–1000 mg/kg of the poly aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH-contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree.

Corneal infections after implantation of intracorneal ring segments.

To report risk factors, clinical course, and outcome in patients with infectious keratitis following implantation of intracorneal ring segments (ICRS). The records of 8 patients with culture-proven infectious keratitis after ICRS (Ferrara or Intacs) implantation were retrospectively reviewed. Age, gender, corneal findings, ocular abnormalities, the condition that led to ICRS implantation, immediate prior use of a contact lens, elapsed time between implantation and the onset of symptoms, previous medications, and systemic disorders were noted. Culture-positive infectious keratitis developed in 7 eyes of 7 patients (2 men and 5 women) with a mean age of 35 years who underwent Ferrara implantation for the treatment of keratoconus and in a 29-year-old man who underwent Intacs implantation for correction of low myopia. Contact lens use, diabetes, and trauma were factors possibly associated with the risk of infection in three cases. Microorganisms, identified in all cases, included Staphylococcus aureus, Streptococcus viridans, Streptococcus pneumoniae, Pseudomonas sp, Nocardia sp, Klebsiella sp, and Paecylomices sp. Onset of symptoms of infection varied from less than 1 week to 22 months postoperatively, depending on the infecting organism. Infectious keratitis following ICRS implantation is a sight-threatening complication for which early recognition and rapid institution of appropriate treatment may result in a better visual outcome.

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563 Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9 bp upstream of the translational start site. The 3 ′ end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5 kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS–PAGE migration of the subunit.

Transvalencin A, a Thiazolidine Zinc Complex Antibiotic Produced by a Clinical Isolate of Nocardia transvalensis: I.Taxonomy, Fermentation, Isolation and Biological Activities

A new thiazolidine-type antibiotic with zinc in its structure, designated transvalencin A, was isolated from Nocardia sp. IFM 10065, a clinical isolate from a patient with actinomycotic mycetoma. The strain was identified as Nocardia transvalensis based on its morphological, phenotypic and phylogenetic characteristics. Transvalencin A showed antimicrobial activity against fungi such as Trichophyton mentagrophytes and Cryptococcus neoformans. The antibiotic is also active against Gram-positive bacteria such as Micrococcus luteus. We observed higher activity for fungi in an acidic medium than in a neutral medium.

Nocathiacins (I), New Thiazolyl Peptide Antibiotics from Nocardia sp. Part 2. Isolation, Characterization, and Structure Determination.

Nocathiacins (I), New Thiazolyl Peptide Antibiotics from Nocardia sp. Part 2. Isolation, Characterization, and Structure Determination.

Nocathiacins, New Thiazolyl Peptide Antibiotics from Nocardia sp. Part 1. Taxonomy, Fermentation and Biological Activities.

Nocathiacins, New Thiazolyl Peptide Antibiotics from Nocardia sp. Part 1. Taxonomy, Fermentation and Biological Activities.

A survey of filamentous organisms at the deer Island treatment plant

The Deer Island Treatment Plant (DITP) treating both domestic and industrial wastewater, has a peak flow capacity of up to 1270 million gallons per day (MGD) (3342 m3 min−1). The DITP contains a Pilot Plant, which consists of two identical pure oxygen activated sludge treatment trains, each with a maximum capacity of 1 MGD (2.63 m3 min−1) to simulate the maximum flow of the full size facility. This study documents the community of filamentous organisms living in the activated sludge under various operational conditions in the Pilot Plant. Sixteen types of filamentous organisms were identified. The major filamentous organisms present in the Pilot Plant were Type 1701, Type 0041, Sphaerotilus natans, Microthrix parvicella, and Nocardia sp. Nocardia sp. was quite dominant in the early stages of the experimental period during high ambient temperatures (summer), indicating that temperature is one determining factor in the distribution of Nocardia sp. Thiothrix sp. was very sensitive to the dissolved oxygen (DO) level, with low DO values favoring the growth of the organisms. Microbiological observations made during the start‐up and stable operation of the full‐scale secondary treatment plant (maximum capacity 780 MGD) are also reported. No dominant filamentous species existed during the start‐up period. Type 0803 and Type 1701, indicators of low oxygen level for the applied food to microorganism ratio, confirmed the low DO conditions of activated sludge tanks.

Ubiquinone Q9 (I) from a Marine Isolate of an Actinobacterium Nocardia sp.

ChemInformVolume 34, Issue 19 Natural Products Ubiquinone Q9 (I) from a Marine Isolate of an Actinobacterium Nocardia sp. T. A. Kuznetsova, T. A. Kuznetsova Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorA. S. Dmitrenok, A. S. Dmitrenok Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorM. P. Sobolevskaya, M. P. Sobolevskaya Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorL. S. Shevchenko, L. S. Shevchenko Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorV. V. Mikhailov, V. V. Mikhailov Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this author T. A. Kuznetsova, T. A. Kuznetsova Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorA. S. Dmitrenok, A. S. Dmitrenok Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorM. P. Sobolevskaya, M. P. Sobolevskaya Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorL. S. Shevchenko, L. S. Shevchenko Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this authorV. V. Mikhailov, V. V. Mikhailov Pac. Inst. Bio-Org. Chem., Russ. Acad. Sci., Vladivostok 690022, RussiaSearch for more papers by this author First published: 14 May 2003 https://doi.org/10.1002/chin.200319199Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume34, Issue19May 13, 2003 RelatedInformation