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Two strains identified as Nocardia caishijiensis (SORS 64b) and Pseudonocardia carboxydivorans (AGLS 2) were isolated as endophytes from Sonchus oleraceus and Ageratum conyzoides respectively. The analysis of their extracts revealed them to be strongly bioactive. The N. caishijiensis extract gave an LC50 of 570 μg/ml(-1) in the brine shrimp cytotoxicity assay and an EC50 of 0.552 μg/ml(-1) in the DPPH antioxidant assay. Antimicrobial activity was observed against Methicillin resistant Staphlococcus aureus (MRSA) and Escherichia coli ATCC 25922 (14 mm), Klebsiella pneumoniae ATCC 706003 (13 mm), S. aureus ATCC 25923 (11 mm) and Candida tropicalis (20 mm). For the extract of P. carboxydivorans the EC50 was 0.670 μg/ml(-1) and it was observed to be more bioactive against Bacillus subtilis DSM 10 ATCC 6051 (21 mm), C. tropicalis (20 mm), S. aureus ATCC 25923 (17 mm), MRSA (17 mm), E. coli K12 (W1130) (16 mm) and Chlorella vulgaris (10 mm). The genotoxicity testing revealed a 20 mm zone of inhibition against the polA mutant strain E. coli K-12 AB 3027 suggesting damage to the DNA and polA genes. The TLC and bioautography screening revealed a diversity of active bands of medium polar and nonpolar compounds. Metabolite analysis by HPLC-DAD via UV/vis spectral screening suggested the possibility of stenothricin and bagremycin A in the mycelium extract of N. caishijiensis respectively. In the broth and mycelium extract of P. carboxydivorans borrelidin was suggested along with α-pyrone. The HPLC-MS revealed bioactive long chained amide derivatives such as 7-Octadecenamide, 9, 12 octadecandienamide. This study reports the rare actinomycetes N. caishijiensis and P. carboxydivorans as endophytes and evaluates their bioactive metabolites., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

This addendum to "Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach " includes characteristics of the phages NOC1, NOC2 and NOC3 not discussed in the previous paper. The phage adsorption and host interaction properties, their sensitivity to pH and temperature are inferred. NOC2 is seen to be more temperature resistant while others are not. All the phages show pH sensitivity. There is a variance observed in the behavior of these phages. Also, applicability of the phage based system to large scale reactors is studied and discussed here.

Nocardithiocin is a thiopeptide compound isolated from the opportunistic pathogen Nocardia pseudobrasiliensis. It shows a strong activity against acid-fast bacteria and is also active against rifampicin-resistant Mycobacterium tuberculosis. Here, we report the identification of the nocardithiocin gene cluster in N. pseudobrasiliensis IFM 0761 based on conserved thiopeptide biosynthesis gene sequence and the whole genome sequence. The predicted gene cluster was confirmed by gene disruption and complementation. As expected, strains containing the disrupted gene did not produce nocardithiocin while gene complementation restored nocardithiocin production in these strains. The predicted cluster was further analyzed using RNA-seq which showed that the nocardithiocin gene cluster contains 12 genes within a 15.2-kb region. This finding will promote the improvement of nocardithiocin productivity and its derivatives production.

Aim: To study the synthesis of phytohormones (auxins, cytokinins, abscisic acid) under cultivation of Nocardia vaccinii IMV B-7405 (surfactants producer) in media containing different carbon sources (glycerol, refined sunflower oil, as well as waste oil after frying potatoes and meat)., Methods: Phytohormones were extracted from supernatants of culture liquid (before or after surfactant separation) by ethylacetate (auxins, abscisic acid) and n-butanol (cytokinins), concentrated and purified by thin-layer chromatography, then quantitative determination was performed using a scanning Sorbfil spectrodensitometer., Results: While growing in medium with refined oil IMV B-7405 strain synthesized 1.8 ± 0.09 g/l extracellular surfactant, also maximum amount of auxins (245-770 µ/l) and cytokinins (134-348 µl). Cultivation of N. vaccini LMV B-7405 on waste oil was accompanied by decreasing amount of phytohormones to 23-84 µ/l (auxins) and 16-90 µ/l (cytokinins) and increasing surfactant concentration to 2.3-2.6 g/l. The level of abscisic acid synthesis was practically not dependent on the nature of growth substrate, was substantially lower than that of auxins and cytokinins and ranged from 2 to 12 µ/l., Conclusions: Obtained data demonstrate the possibility of using oil-containing industrial waste for the simultaneous synthesis of both surfactants and phytohormones, and indicate the need for studies of the effect of producer cultivation conditions on the biological properties of the target products of microbial synthesis.

Key enzymes of glucose metabolism were detected in the cells of surfactants producer Nocardia vaccinii IMV B-7405 grown on this substrate. It has been established that glucose catabolism is performed through gluconate (FAD(+)-dependent glucose dehydrogenase activity 698 ± 35 nmol x min(-1) x mg(-1) of protein). Oxidation of gluconate to 6-phosphogluconate is catalised by gluconokinase (178 ± 9 nmol x min(-1) x mg(-1) of protein). 6-Phosphogluconate was involved into pentose phosphate cycle by constitutive NADP(+)-dependent 6-phosphogluconate dehydrogenase (activity 357 ± 17 nmol x min(-1) x mg(-1) of protein). The data obtained serve as the basis for theoretical calculations of optimal molar ratio of concentrations of energetically nonequivalent substrates for intensifying the surfactants synthesis on their mixture.

Aromatic amines are important industrial products having in their molecular structure one or more aromatic rings. These are used as precursors for the synthesis of dyes, adhesives, pesticides, rubber, fertilizers and surfactants. The aromatic amines are common constituents of industrial effluents, generated mostly by the degradation of azo dyes. Several of them are a threat to human health because they can by toxic, allergenic, mutagenic or carcinogenic. The most common are benzenesulfonic amines, such as 4-ABS (4-aminobenzene sulfonic acid) and naphthalene sulfonic amines, such as 4-ANS (4-amino naphthalene sulfonic acid). Sometimes, the mixtures of toxic compounds are more toxic or inhibitory than the individual compounds, even for microorganisms capable of degrading them. Therefore, the aim of this study was to evaluate the degradation of the mixture 4-ANS plus 4-ABS by a bacterial community immobilized in fragments of volcanic stone, using a packed bed continuous reactor. In this reactor, the amines loading rates were varied from 5.5 up to 69 mg L(-1) h(-1). The removal of the amines was determined by high-performance liquid chromatography and chemical oxygen demand. With this information, we have studied the substrate inhibition of the removal rate of the aromatic amines during the degradation of the mixture of sulfonated aromatic amines by the immobilized microorganisms. Experimental results were fitted to parabolic, hyperbolic and linear inhibition models. The model that best characterizes the inhibition of the specific degradation rate in the biofilm reactor was a parabolic model with values of RXM=58.15±7.95 mg (10(9) cells h)(-1), Ks=0.73±0.31 mg L(-1), Sm=89.14±5.43 mg L(-1) and the exponent m=5. From the microbial community obtained, six cultivable bacterial strains were isolated and identified by sequencing their 16S rDNA genes. The strains belong to the genera Variovorax, Pseudomonas, Bacillus, Arthrobacter, Nocardioides and Microbacterium. This microbial consortium could use the mixture of aromatic amines as sources of carbon, nitrogen, energy and sulfur., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The possibility of replacing glucose and pure glycerol in mixed substrates for surtace-active substances (SAS, biosurfactants) biosynthesis of Nocardia vaccinii IMB B-7405 on molasses (sugar production waste) and crude glycerol (by-product of biodiesel production) was established. It was established that the increasing concentration of crude glycerol to 6% in mixture with 1.0% molasses was accompanied by increase of amount of SAS synthesized more than twice, and the increasing content of molasses to 3.0% in mixture with 1.0% crude glycerol--by some decrease in the level of surfactant as compared to that in a medium containing 1.0% monosubstrates. It was shown that the increasing concentration of sodium nitrate to 2-fold in medium cultivation of N. vaccinii IMB B-7405 allowed to increase to 7.0% content of grude glycerol in mixture with 1.0% molasses. Under such conditions of cultivation concentration of exocellular SAS synthesized was 7,5 g/l, that to 1,3 fold higher than in basic medium with a lower content of nitrogen source.

The objective of this study was to isolate and identify the chemical structure of a biosurfactant produced by Nocardia farcinica strain BN26 isolated from soil, and evaluate its in vitro antitumor activity on a panel of human cancer cell lines. Strain BN26 was found to produce glycolipid biosurfactant on n-hexadecane as the sole carbon source. The biosurfactant was purified using medium-pressure liquid chromatography and characterized as trehalose lipid tetraester (THL) by nuclear magnetic resonance spectroscopy and mass spectrometry. Subsequently, the cytotoxic effects of THL on cancer cell lines BV-173, KE-37 (SKW-3), HL-60, HL-60/DOX, and JMSU-1 were evaluated by MTT assay. It was shown that THL exerted concentration-dependent antiproliferative activity against the human tumor cell lines and mediated cell death by the induction of partial oligonucleosomal DNA fragmentation. These findings suggest that THL could be of potential to apply in biomedicine as a therapeutic agent.

It has been established that in cells of Nocardia vaccinii IMB B-7405 (surfactant producer) glucose catabolism is performed through pentose phosphate cycle as well as through gluconate (activity of NAD+-dependent glucose-6-phosphate dehydrogenase and FAD+-dependent glucose dehydrogenase 835 ± 41 and 698 ± 35 nmol.min-1.mg-1 of protein respectively). 6-Phosphogluconate formed in the gluconokinase reaction is involved in the pentose phosphate cycle (activity of constitutive NADP+-dependent 6-phosphogluconate dehydrogenase 357 ± 17 nmol.min-1.mg-1 of protein). Glycerol catabolism to dihydroxyacetonephosphate (the intermediate of glycolysis) may be performed in two ways: through glycerol-3-phosphate (glycerol kinase activity 244 ± 12 nmol.min-1.mg-1 of protein) and through dihydroxyacetone. Replenishment of the C4-dicarboxylic acids pool in N. vaccinii IMV B-7405 grown on glucose and glycerol occurs in the phosphoenolpyruvate(PEP)carboxylase reaction (714-803 nmol.min-1.mg-1 of protein). 2-Oxoglutarate was involved in tricarboxylic acid cycle by alternate pathway with the participation of 2-oxoglutarate synthase. The observed activity of both key enzymes of gluconeogenesis (PEP-carboxykinase and PEP-synthase), trehalose phosphate synthase and NADP+-dependent glutamate dehydrogenase confirmed the ability of IMV B-7405 strain to the synthesis of surface active glycoand aminolipids, respectively.

The effect of copper cations (0.01-1.0 mM) and surface-active agents (surfactants) of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Alc-5017 and Nocardia vaccinii IMV B-7405 in the form of culture liquid on the destruction of oil in water (3.0-6.0 g/L) and soil (20 g/kg), including in the presence of Cd2+ and Pb2+ (0.01-0.5 mM), was investigated. It was shown that the degree of oil degradation in water and soil after 20 days in the presence of low concentrations of Cu2+ (0.01-0.05 mM) and culture liquid of strains IMV B-7241, IMV Ac-5017, and IMV B-7405 was 15 - 25% higher than without copper cations. The activating effect of Cu2+ on the decomposition of complex oil and Cd2+ and Pb2+ pollution was established: after treatment with surfactant of A. calcoacelicus IMV B-7241 and R. erythropolis IMV Ac-5017 destruction of oil in water and soil was 85-95%, and after removal of the copper cations decreased to 45-70%. Intensification of oil destruction in the presence of copper cations may be due to their stimulating effect on the activity of alkane hydroxylases as in surfactant-producing strains, and natural (autochthonous) oxidizing microbiota.

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.

The role of the outer-membrane channel from a mycolic acid containing Gram-positive bacteria Nocardia farcinica, which forms a hydrophilic pathway across the cell wall, was characterized. Single channel electrophysiology measurements and liposome swelling assays revealed the permeation of hydrophilic solutes including sugars, amino acids and antibiotics. The cation selective N. farcinica channel exhibited strong interaction with the positively charged antibiotics; amikacin and kanamycin, and surprisingly also with the negatively charged ertapenem. Voltage dependent kinetics of amikacin and kanamycin interactions were studied to distinguish binding from translocation. Moreover, the importance of charged residues inside the channel was investigated using mutational studies that revealed rate limiting interactions during the permeation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The bacterial strains of the genus Nocardia were used for the bioconversion of fumaric acid to L-malic acid. The ability of the bacterial strain Nocardia sp. CCM 4837/A to produce L-malic acid from fumaric acid was investigated under various conditions. The optimal temperature for the bioconversion was approximately 37 °C, and the optimal pH was around 8.0. The addition of an inductor (fumarate salt) to the fermentation medium was necessary to enhance enzyme activity. The presence of detergent Triton X-100 (0.02-0.1 %) in the reaction mixture rapidly increased the enzyme activity of fumarase. The specific fumarase activity of intact cells Nocardia sp. CCM 4837/A increased from 2.8 to 75 U/mg after optimising the experimental conditions described here. Pretreatment of the Nocardia cells with malonate was not necessary because succinate was not detected as a by-product under our experimental conditions.

CYP154C5 from Nocardia farcinica is a bacterial cytochrome P450 monooxygenase active on steroid molecules. The enzyme has recently been shown to exhibit exclusive regioselectivity and stereoselectivity in the conversion of various pregnans and androstans, yielding 16α-hydroxylated steroid products. This makes the enzyme an attractive candidate for industrial application in steroid hormone synthesis. Here, crystal structures of CYP154C5 in complex with four different steroid molecules were solved at resolutions of up to 1.9 Å. These are the first reported P450 structures from the CYP154 family in complex with a substrate. The active site of CYP154C5 forms a flattened hydrophobic channel with two opposing polar regions, perfectly resembling the size and polarity distribution of the steroids and thus resulting in highly specific steroid binding with Kd values in the range 10-100 nM. Key enzyme-substrate interactions were identified that accounted for the exclusive regioselectivity and stereoselectivity of the enzyme. Additionally, comparison of the four CYP154C5-steroid structures revealed distinct structural differences, explaining the observed variations in kinetic data obtained for this P450 with the steroids pregnenolone, dehydroepiandrosterone, progesterone, androstenedione, testosterone and nandrolone. This will facilitate the generation of variants with improved activity or altered selectivity in the future by means of protein engineering.

The production of surfactants by Nocardia vaccinii IMV B-7405 in glycerol -and hydrocarbon-containing medium after addition Cd(3+) Pb(2+) and Cu(2+) was investigated. It was established that the introduction of 0.1 mM Cu(2+) in the exponential growth phase of IMV B-7405 strain or simultaneous addition of Cu(2+) (0.1 mM) and Cd(2+) (0.3 mM), Cu(2+) (0.1 mM) and Pb(2+) (0.3 mM) in stationary phase was accompanied by the increase of conditional concentration of the surfactant (by 53 and 20-26%, respectively) compared with indexes in the medium without metals cations. It was established that the surfactants of N. vaccinii IMV B-7405 possessed protective functions from heavy metals influence. After surfactants elimination the survival of cells of strain IMV B-7405 in the presence of Cu(2+) (1.5-2.5 mM), Cd(2+) or Pb(2+) (0.1-0.3 mM) decreased a few times (to 5-45%). The inhibition action of Cu(2+) on alkane hydroxylase activity (the first enzyme of hydrocarbon catabolism) and stimulation--on phosphoenolpyruvate synthetase (enzyme of surface-active glycolipids biosynthesis) in MB B-7405 have been established.

The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid-AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to L-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of L-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37 × 10(-3) g U(-1) h(-1) was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m(3) stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the L-(+)-tartaric acid industrial production.

The synthesis of surfactants by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 on industrial waste (food and oil-processing industry, production of biodiesel) was investigated. The possibility of replacing the expensive substrates (n-hexadecane and ethanol) by industrial waste (oil and fat industry, fried sunflower oil, glycerol, liquid paraffin) for the surfactant biosynthesis was established. The conditional concentration of surfactants was maximal on oil containing substrates and exceeded those on n-hexadecane and ethanol 2-3 times. The highest rates of surfactants synthesis were observed on fried sunflower oil with the use of inoculum grown on carbohydrate substrates (glucose, molasses). It was established that the addition of glucose (0.1%) was accompanied by 2-4-fold intensification of surfactants synthesis by R. erythropolis IMV Ac-5017 and N. vaccinii IMV B-7405 on fried sunflower oil (2%).

Bacteria affiliated in the genus Nocardia are aerobic and Gram-positive actinomycetes that are widely found in aquatic and terrestrial habitats. As occasional pathogens, several of them cause infection diseases called 'nocardiosis' affecting lungs, central nervous system, cutaneous tissues and others. In addition, members of the genus Nocardia exhibit an enormous metabolic versatility. On one side, many secondary metabolites have been isolated from members of this genus that exhibit various biological activities such as antimicrobial, antitumor, antioxidative and immunosuppressive activities. On the other side, many species are capable of degrading or converting aliphatic and aromatic toxic hydrocarbons, natural or synthetic polymers, and other widespread environmental pollutants. Because of these valuable properties and the application potential, Nocardia species have attracted much interest in academia and industry in recent years. A solid basis of genetic tools including a set of shuttle vectors and an efficient electroporation method for further genetic and metabolic engineering studies has been established to conduct efficient research. Associated with the increasing data of nocardial genome sequences, the functional diversity of Nocardia will be much faster and better understood., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The environmental pollutant 4-sec-butylphenol (4-sec-BP) which possesses estrogenic properties was transformed by the aerobic Gram-positive bacteria Mycobacterium neoaurum and Nocardia cyriacigeorgica into three main products (P1-P3) which were isolated and structurally characterized in detail. Two of them were products of a process resembling anaerobic metabolism of alkylphenols based on modifications of the alkyl side chain of 4-sec-BP. The first product (P1) was identified as 4-(2-hydroxy-1-methylpropyl)-phenol. The second product P2 was isolated as a mixture of at least four structures which could be identified as I 4-sec-butylidenecyclohexa-2,5-dienone; II 4-(1-methylenepropyl)-phenol; III 4-(1-methylpropenyl)-phenol; and IV 4-(1-methylallyl)-phenol. In contrast to P1 and P2, the third product (P3) resulted from a modification of the hydroxyl group of 4-sec-BP. This product was only formed by M. neoaurum and was identified as the glucoside conjugate 4-sec-butylphenol-α-D-glucopyranoside. Since in general, fungi synthesize sugar conjugates to detoxify hazardous pollutants, the formation of this conjugate is a peculiarity of M. neoaurum. Thus, altogether, six products were formed from 4-sec-BP and different transformation pathways are introduced. The hydroxylating and glucosylating capacity of the characterized bacteria open up applications in environmental protection.

The authors studied the effect of components of crude glycerol (potassium and sodium salts, ethanol, methanol) - the by-products of biodiesel production on formation of surfactants (surface-active substances, SAS) by Nocardia vaccinii K-8, as well as possibility to intensify the SAS synthesis by the strain K-8 on crude glycerol in the presence ofbiosynthesis precursors (glucose, sun-flower oil, organic substances). It has been established that the introduction of potassium (sodium) chloride in concentration 2.5 % and ethanol (methanol) in concentration 0.3 % into the medium with refined glycerol (1 %) was accompanied by the 14-1.7-fold increase of conditional SAS concentration as compared with indices on the medium without adding salts and alcohols. Under cultivation conditions of strain K-8 on the medium with crude glycerol the conditional SAS concentration was 3-fold higher than on the medium with refined substrate. Introduction of glucose (0.05 %), sun-flower oil (0.05 %), fumarate and citrate (0.1 %) during the stationary growth phase of N. vaccinii K-8 into the medium with crude glycerol (2.2%) was accompanied by the increase in the amount of synthesized SAS h by 17-44 % compared with cultivation of bacteria on the medium without precursors.

The effect of surface-active substances (SAS's) of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017, and Nocardia vaccinii K-8 on phytopathogenic bacteria has been studied. It was shown that the survival of cells (10(5)-10(7) in a milliliter) of the Pseudomonas and Xanthomonas phytopathogenic bacteria was found to be 0-33% after treatment with SAS preparations of the IMV Ac-5017 and IMV B-7241 strains for 2 h (0.15-0.4 mg/mL). In the presence of N. vaccinii K-8 SAS preparations (0.085-0.85 mg/mL), the number of cells of the majority of the studied phytopathogenic bacteria decreased by 95-100%. These data show prospects for using microbial SAS's for the construction of ecologically friendly drugs for regulating the number of phytopathogenic bacteria.

Nocardia brasiliensis is an important etiologic agent of mycetoma. These bacteria live as a saprobe in soil or organic material and enter the tissue via minor trauma. Mycetoma is characterized by tumefaction and the production of fistula and abscesses, with no spontaneous cure. By using mass sequencing, we determined the complete genomic nucleotide sequence of the bacteria. According to our data, the genome is a circular chromosome 9,436,348-bp long with 68% G+C content that encodes 8,414 proteins. We observed orthologs for virulence factors, a higher number of genes involved in lipid biosynthesis and catabolism, and gene clusters for the synthesis of bioactive compounds, such as antibiotics, terpenes, and polyketides. An in silico analysis of the sequence supports the conclusion that the bacteria acquired diverse genes by horizontal transfer from other soil bacteria, even from eukaryotic organisms. The genome composition reflects the evolution of bacteria via the acquisition of a large amount of DNA, which allows it to survive in new ecological niches, including humans.

During screening of marine actinomycetes for anti-mycobacterial activity, a new phenoxazine derivative (1) was isolated, along with 6-phenazinediol (2), 6-methoxy-1-phenazinol (3), nocardamin (4), and 3-pyridinecarboxylic acid (5), from a culture of Nocardiopsis sp. 236 collected from the west Pacific. The chemical structure of 1 was established on the basis of 1D-, 2D-NMR, and HRQ-TOF MS data. All compounds were evaluated for their anti-mycobacterial activity in vitro, and only compounds 2 and 3 exhibited weak activity.

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH(2)) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH(2), indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of gram-negative bacteria. The monobactams are of renewed interest due to emerging gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-L-phenylglycine (L-pHPG) into positions 1, 3, and 5 and L-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is L-pHPG-L-Arg-D-pHPG-L-Ser-L-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the L-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis.

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.

Nocardia nova SH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 × 10(7) CFU per μg of plasmid DNA was achieved. By applying this optimized protocol to N. nova SH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an α-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, β-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP.

A marine actinomycete, designated strain BM-17, was isolated from a sediment sample collected in the Arctic Ocean. The strain was identified as Nocardia dassonvillei based on morphological, cultural, physiological, biochemical characteristics, along with the cell wall analysis and 16S rDNA gene sequence analysis. A new secondary metabolite (1), N-(2-hydroxyphenyl)-2-phenazinamine (NHP), and six known antibiotics (2-7) have been isolated from the saline culture broth of the stain by sequentially purification over macroporous resin D101, silica gel, Sephadex LH-20 column chromatography and preparative HPLC after the stain was incubated in soy bean media at 28°C for 7 days. The chemical structures of the compounds were elucidated on the basis of spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. The new compound showed significant antifungal activity against Candida albicans, with a MIC of 64 μg/ml and high cancer cell cytotoxicity against HepG2, A549, HCT-116 and COC1 cells., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Nocardia brasiliensis is a Gram-positive facultative intracellular bacterium frequently isolated from human actinomycetoma. However, the pathogenesis of this infection remains unknown. Here, we used a model of bacterial delipidation with benzine to investigate the role of N. brasiliensis cell wall-associated lipids in experimental actinomycetoma. Delipidation of N. brasiliensis with benzine resulted in complete abolition of actinomycetoma without affecting bacterial viability. Chemical analyses revealed that trehalose dimycolate and an unidentified hydrophobic compound were the principal compounds extracted from N. brasiliensis with benzine. By electron microscopy, the extracted lipids were found to be located in the outermost membrane layer of the N. brasiliensis cell wall. They also appeared to confer acid-fastness. In vitro, the extractable lipids from the N. brasiliensis cell wall induced the production of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and CCL-2 in macrophages. The N. brasiliensis cell wall extractable lipids inhibited important macrophage microbicidal effects, such as tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, phagocytosis, bacterial killing, and major histocompatibility complex class II (MHC-II) expression in response to gamma interferon (IFN-γ). In dendritic cells (DCs), N. brasiliensis cell wall-associated extractable lipids suppressed MHC-II, CD80, and CD40 expression while inducing tumor growth factor β (TGF-β) production. Immunization with delipidated N. brasiliensis induced partial protection preventing actinomycetoma. These findings suggest that N. brasiliensis cell wall-associated lipids are important for actinomycetoma development by inducing inflammation and modulating the responses of macrophages and DCs to N. brasiliensis.

This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

Nargenicin A1 is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin A1 was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15 mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin A1 was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin A1 production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin A1 in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.

17α-Methyltestosterone (MT), a synthetic anabolic androgenic steroid, is widely used in aquafarming for the production of an all male fish population such as Nile tilapia. This study isolated, identified and characterized MT-degrading bacteria in the sediment and water from a masculinizing pond of Nile tilapia fry. Based on the phylogeny, physiological properties and cell morphology, the three isolated MT-degrading bacteria were related closely to Rhodococcus equi, Nocardioides aromaticivorans, and Nocardioides nitrophenolicus. Growth of the three isolated strains was found to be inhibited for MT concentrations in the range of 1.0-10mg/L. The inhibition of cell growth was found to be modeled using the Haldane's substrate inhibition model. The kinetic constants ranged from 0.13 to 0.19h(-1) for μ(max), 0.7-24.8mg/L for K(s) and 19.6-76.2mg/L for K(i). Androgenic activity using β-galactosidase assay showed that all strains degraded MT to the products with no androgenic potency., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mycolic acids are one of the basic elements of the cell wall structure of bacteria belonging to the suborder Corynebacterineae, constituting from 20% to 40% of dry weight. Additionally, they show high structural diversity within each family and species. Nowadays, profiles of mycolic acids are widely described for the genus Mycobacterium, the causative agent of tuberculosis. However, the suborder Corynebacterineae also includes many representatives of opportunistic human pathogens, e.g. Dietzia, Gordonia, Nocardia and Rhodococcus. Currently, an increased infection risk caused by this group of microorganisms especially in immunocompromised patients has been observed. Better knowledge of mycolic acid profiles for Corynebacterineae may allow identification of mycolic acids as diagnostic markers in the detection of opportunistic bacterial infections. Modern techniques of chemical analysis, including mass spectrometry, may enable the development of new chemotaxonomic methods for the detection and differentiation of bacteria within the suborder Corynebacterineae.

The taxonomic position of two soil actinomycetes, strains A2012(T) and A2019(T), isolated from Turkish soils, was determined using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that the strains belonged to the family Nocardiaceae. Strains A2012(T) and A2019(T) were most closely related to Nocardia caishijiensis DSM 44831(T) (98.9 %) and Nocardia mexicana CIP 108295(T) (98.6 %), respectively; similarity to other type strains of the genus Nocardia ranged from 96.9 to 97.9 %. However, DNA-DNA relatedness and phenotypic data demonstrated that strains A2012(T) and A2019(T) could be clearly distinguished from members of the most closely related Nocardia species. It is evident from the genotypic and phenotypic data that the two isolates represent two novel species of the genus Nocardia. It is proposed, therefore, that strains A2012(T) and A2019(T) be classified in the genus Nocardia as representatives of Nocardia goodfellowii sp. nov. (type strain A2012(T) = DSM 45516(T) = NRRL B-24833(T) = KCTC 19986(T)) and Nocardia thraciensis sp. nov. (type strain A2019(T) = DSM 45517(T) = NRRL B-24834(T) = KCTC 19985(T)), respectively.

A novel application of whole cells of Nocardia corallina B-276 for the deracemisation of ibuprofen is reported. This microorganism successfully hydrolysed ibuprofen nitrile to ibuprofen amide, and ibuprofen amide to ibuprofen, using a suspension of cells in a potassium phosphate buffer solution (0.1 M, pH = 7.0). These results can be explained by the presence of NHase and amidase enzymes, but the reactions are not enantioselective and low ee values were obtained. However, (R)-ibuprofen was isolated with > 99% ee by a deracemisation process catalysed by N. corallina B-276. This is the first report of this kind of catalysis with this microorganism.

Two nonribosomal peptide synthetases (NRPS), NocA and NocB, together comprising five modules, are essential for the biosynthesis of the D,L,D configured tripeptide backbone of the monocyclic β-lactam nocardicin A. We report a double replacement gene strategy in which point mutations were engineered in the two encoding NRPS genes without disruption of the nocABC operon by placing selective markers in adjacent genes. A series of mutants was constructed to inactivate the thiolation (T) domain of each module and to evaluate an HHxxxDR catalytic motif in NocA and an atypical extended histidine motif in NocB. The loss of nocardicin A production in each of the T domain mutants indicates that all five modules are essential for its biosynthesis. Conversely, production of nocardicin A was not affected by mutation of the NocB histidine motif or the R828G mutation in NocA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from L-proline. Nargenicin A(1), a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A(1) in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A(1) was also derived from L-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A(1) is initiated by NgnN4 that catalyzes ATP-dependent activation of L-proline into L-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert L-proline into pyrrole-2-carboxylic acid moiety.

The ability of two recently isolated actinobacteria, that degrade medium and long chain n-alkanes in laboratory water medium, was investigated in soil microcosms using different standard soils that were artificially contaminated with n-alkanes of different length (C(12)- C(20)- C(24)- C(30)). The two strains, identified as Nocardia sp. SoB and Gordonia sp. SoCp, revealed a similar high HC degradation efficiency with an average of 75% alkane degraded after 28 days incubation. A selectivity of bacteria towards n-alkanes of different length was detected as well as a consistent effect of soil texture and other soil physical chemical characteristics on degradation. It was demonstrated the specific aptitude of these selected strains towards specific environmental conditions.

A novel actinobacterium, designated YIM 65623(T), was isolated from a surface-sterilized stem of Artemisia annua L. Strain YIM 65623(T) had morphological, biochemical, physiological and chemotaxonomic properties that were consistent with its classification in the genus Nocardia. Growth occurred with 0-7% (w/v) NaCl (optimum 0-3%), at pH 5.0-9.0 (optimum pH 6.0) and at 10-37 °C (optimum 20-28 °C). Comparative 16S rRNA gene sequence analysis showed that strain YIM 65623(T) constituted a distinct sublineage within the genus Nocardia and displayed 94.1-98.2% sequence similarity to members of established species in the genus Nocardia. However, DNA-DNA relatedness and physiological and biochemical characteristics showed that strain YIM 65623(T) could be differentiated from its closest phylogenetic relatives. The G+C content of the genomic DNA was 69.6 mol%. It is proposed that strain YIM 65623(T) be classified as a representative of a novel species, Nocardia artemisiae sp. nov. The type strain is YIM 65623(T) (=DSM 45379(T) =CCTCC AA 209038(T)).

Bacteria of the genus Nocardia cause opportunistic infections of lung, brain and central nervous system, and cutaneous tissue. They are also producers of antibiotics and industrially important enzymes. As studies describing plasmids in this genus are limited, we have characterized a 4326bp cryptic plasmid pYS1 from Nocardia aobensis IFM 10795. Three open reading frames (ORFs) were predicted. Both sequence analyses and detection of single-stranded intermediates suggested a rolling-circle mechanism as the mode of replication of pYS1. Mutageneses and deletion analyses revealed both the predicted double- and single-stranded origins to be indispensable in replication, suggesting a lack of secondary signals for leading and lagging strand synthesis. The replicon of pYS1 is broad-host-range and compatible to that of pAL5000 of mycobacteria, making it potentially useful in genetic manipulation of various actinomycetes. Insertion analyses showed orf1, despite its sequence similarity to plasmid transfer genes, is involved in plasmid stability rather than conjugation and is lethal in the absence of a functional orf3. This situation is somewhat analogous to the kil/kor system of pIJ101 of Streptomyces, except that orf3 was unrelated to korA and was shown by promoter-probe assays to encode a novel transcriptional repressor negatively regulating orf1 expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aims: Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a cyclic nitramine explosive that is a major component in many high-explosive formulations and has been found as a contaminant of soil and groundwater. The RDX-degrading gene locus xplAB, located on pGKT2 in Gordonia sp. KTR9, is highly conserved among isolates from disparate geographical locations suggesting a horizontal gene transfer (HGT) event. It was our goal to determine whether Gordonia sp. KTR9 is capable of transferring pGKT2 and the associated RDX degradation ability to other bacteria., Methods and Results: We demonstrate the successful conjugal transfer of pGKT2 from Gordonia sp. KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia sp. TW2. Through growth and RDX degradation studies, it was demonstrated that pGKT2 conferred to transconjugants the ability to degrade and utilize RDX as a nitrogen source. The inhibitory effect of exogenous inorganic nitrogen sources on RDX degradation in transconjugant strains was found to be strain specific., Conclusions: Plasmid pGKT2 can be transferred by conjugation, along with the ability to degrade RDX, to related bacteria, providing evidence of at least one mechanism for the dissemination and persistence of xplAB in the environment., Significance and Impact of Study: These results provide evidence of one mechanism for the environmental dissemination of xplAB and provide a framework for future field relevant bioremediation practices., (Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to US Government works.)

Mycobacteria such as M. tuberculosis represent a significant health concern throughout much of the developing world. In mycobacteria and other pathogenic bacteria, an important virulence factor is the ability of the bacterium to obtain iron from its host. One means of obtaining iron is through the use of siderophores. Brasilibactin A is a membrane bound siderophore produced by Nocardia brasiliensis with structural similarity to the mycobactin class of siderophore in mycobacteria. A characterization of the protonation constants and Fe(III) affinity of a water soluble Brasilibactin A analog (Bbtan) has been performed. Using protonation constants and competition with EDTA, the stability constant of the 1 : 1 Fe(III)-Bbtan complex was found to be log β(110) = 26.96. The pFe of Bbtan is 22.73, somewhat low for a proposed siderophore molecule. The redox potential of the Fe-Bbtan complex was found to be -300 mV vs. NHE, very high for an iron-siderophore complex. The combination of relatively low complex stability and ease of iron reduction may play a crucial role in the mechanism of mycobactin siderophore-mediated iron uptake in mycobacteria and related organisms.

Genome shuffling is a recent development in microbiology. The advantage of this technique is that genetic changes can be made in a microorganism without knowing its genetic background. Genome shuffling was applied to the marine derived bacterium Nocardia sp. ALAA 2000 to achieve rapid improvement of ayamycin production. The initial mutant population was generated by treatment with ethyl methane sulfonate (EMS) combined with UV irradiation of the spores, resulting in an improved population (AL/11, AL/136, AL/213 and AL/277) producing tenfold (150 μg/ml) more ayamycin than the original strain. These mutants were used as the starting strains for three rounds of genome shuffling and after each round improved strains were screened and selected based on their ayamycin productivity. The population after three rounds of genome shuffling exhibited an improved ayamycin yield. Strain F3/22 yielded 285 μg/ml of ayamycin, which was 19-fold higher than that of the initial strain and 1.9-fold higher than the mutants used as the starting point for genome shuffling. We evaluated the genetic effect of UV + EMS-mutagenesis and three rounds of genome shuffling on the nucleotide sequence by random amplified polymorphic DNA (RAPD) analysis. Many differences were noticed in mutant and recombinant strains compared to the wild type strain. These differences in RAPD profiles confirmed the presence of genetic variations in the Nocardia genome after mutagenesis and genome shuffling.

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.

Samples of soil, water, and sediments from industrial estates in Lagos were collected and analyzed for heavy metals and physicochemical composition. Bacteria that are resistant to elevated concentrations of metals (Cd(2+), Co(2+), Ni(2+), Cr(6+), and Hg(2+)) were isolated from the samples, and they were further screened for antibiotic sensitivity. The minimum tolerance concentrations (MTCs) of the isolates with dual resistance to the metals were determined. The physicochemistry of all the samples indicated were heavily polluted. Twenty-two of the 270 bacterial strains isolated showed dual resistances to antibiotics and heavy metals. The MTCs of isolates to the metals were 14 mM for Cd(2+), 15 mM for Co(2+) and Ni(2+), 17 mM for Cr(6+), and 10 mM for Hg(2+). Five strains (Pseudomonas aeruginosa, Actinomyces turicensis, Acinetobacter junni, Nocardia sp., and Micrococcus sp.) resisted all the 18 antibiotics tested. Whereas Rhodococcus sp. and Micrococcus sp. resisted 15 mM Ni(2+), P. aeruginosa resisted 10 mM Co(2+). To our knowledge, there has not been any report of bacterial strains resisting such high doses of metals coupled with wide range of antibiotics. Therefore, dual expressions of antibiotics and heavy-metal resistance make the isolates, potential seeds for decommissioning of sites polluted with industrial effluents rich in heavy metals, since the bacteria will be able to withstand in situ antibiosis that may prevail in such ecosystems.

Characterization of the biosynthetic gene cluster of glycosylated antibiotic nocathiacin I (NOC-I) here adds new insights to thiopeptide biosynthesis, showing the NOC-specific tailoring and unusual sugar formation. NOC-I biosynthesis shares the paradigm for forming a common thiopeptide core and the generality for converting to an e series member, as that of the parent compound nosiheptide (NOS). This may permit the production of NOC-I in the genetically amenable, NOS-producing strain by building NOC-specific genes for pathway engineering.

A soil bacterium isolated from oil-polluted sand samples collected in the Saudi Arabian Desert has been determined as Nocardia cyriacigeorgica, which has a high capacity of degrading and utilizing a broad range of hydrocarbons. The metabolic pathways of three classes of hydrocarbons were elucidated by identifying metabolites in cell-free extracts analyzed by GC/MS and HPLC/UV-Vis in comparison with standard compounds. During tetradecane oxidation, tetradecanol; tetradecanoic acid; dodecanoic acid; decanoic acid could be found as metabolites, indicating a monoterminal degradation pathway of n -alkanes. The oxidation of pristane resulted in the presence of pristanoic acid; 2-methylglutaric acid; 4,8-dimethylnonanoic acid; and 2,6-dimethylheptanoic acid, which give rise to a possible mono- and di-terminal oxidation. In case of sec -octylbenzene, eight metabolites were detected including 5-phenylhexanoic acid; 3-phenylbutyric acid; 2-phenylpropionic acid; beta -methylcinnamic acid; acetophenone; beta -hydroxy acetophenone; 2,3-dihydroxy benzoic acid and succinic acid. From these intermediates a new degradation pathway for sec -octylbenzene was investigated. Our results indicate that N. cyriacigeorgica has the ability to degrade aliphatic and branched chain alkanes as well as alkylbenzene effectively and, therefore, N. cyriacigeorgica is probably a suitable bacterium for biodegradation of oil or petroleum products in contaminated soils., (((c) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

The present taxonomic situation of pathogenic actinomycetes including Nocardia was clarified, and the impact of genomic sequence information of Nocardia farcinica IFM 10152 on taxonomic work is introduced. The number of cases of nocardiosis is on the rise along with the increasing number of immunocompromised patients in Japan. From 1999 to 2007, 718 strains of pathogenic actinomycetes were received for identification by Medical Mycology Research Center (MMRC), Chiba University. About 75% of these were classified into Nocardia, major species being N. farcinica, N. nova, and N. brasiliensis. Among the strains classified as Nocardia species, there were some unclassifiable strains and taxonomic studies on these led to the proposal of more than 18 new species, resulting in more than 1/4 of all Nocardia species having been proposed by our group. Recently a new Nocardia species, Nocardia mikamii was proposed by American researchers. A new phylogenetic analysis method using gryB and secA1 genes was proposed for the Nocardia and Gordonia strains.Our whole genome analysis of N. farcinica suggests that the bacterium has unique and characteristic gene profiles, and also suggests that N. farcinica is similar to Mycobacterium tuberculosis. N. farcinica also has siderophore (nocobactin) and mce genes which are similar to mycobactin (siderophore) of M. tuberculosis as virulence factors. Nocardia strains were found to be producers of new secondary metabolites including antifungal, antitumor and immunosuppressive activities.We reported novel antibiotic resistance mechanisms such as ribosylation, glucosylation, phosphorylation and degradation of rifampicin, phosphorylation of aminoglycosides, and glucosylation of macrolide antibiotics. Among rifampicin inactivation mechanisms, ribosylation was found to be through the Arr enzyme that catalyzes ADP-ribosylation of rifampicin in a fast-growing Mycobacterium smegmatis. This unique mechanism has also been reported as an antibiotic resistant mechanism in pathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.Our cooperative work on the elucidation of high-level resistance to the aminoglycoside antibiotic amikacin in N. farcinica with a research group from CDC, USA, revealed the presence of homozygous mutations in the 16R rRNA genes which are responsible for high-level aminoglycoside antibiotic resistance.