Longistylin A from Cajanus cajan (L.) Millsp. disturbs glycerophospholipid metabolism and cytokinin biosynthesis of Nocardia seriolae.

ETHNOPHARMACOLOGICAL RELEVANCE: Nocardiosis is an uncommon infectious disease that bears certain similarities to tuberculosis, with a continuous increase in its incidence and a poor prognosis. In traditional Chinese medicine, the leaves of Cajanus cajan (L.) Millsp. are employed to treat wounds, malaria, coughs, and abdominal pain. AIM OF THE STUDY: In this study, we investigated the effects and mechanisms of longistylin A (LGA), a natural stilbene isolated from C. cajan, as a potential antibiotic against nocardiosis. MATERIALS AND METHODS: LGA was isolated from the leaves of C. cajan and assessed using a minimum bactericidal concentration (MBC) determination against Nocardia seriolae. Multi-omics analysis encompassing genes, proteins, and metabolites was conducted to investigate the impact of LGA treatment on N. seriolae. Additionally, quantitative analysis of 40 cytokinins in N. seriolae mycelium was performed to assess the specific effects of LGA treatment on cytokinin levels. Cryo-scanning electron microscopy was utilized to examine morphological changes induced by LGA treatment, particularly in the presence of exogenous trans-zeatin-O-glucoside (tZOG). The therapeutic effect of LGA was investigated by feeding N. seriolae-infected largemouth bass. RESULTS: LGA exhibited significant efficacy against N. seriolae, with MBC value of 2.56 µg/mL. Multi-omics analysis revealed that LGA disrupted glycerophospholipid metabolism and hormone biosynthesis by notably reducing the expression of glycerol-3-phosphate dehydrogenase and calmodulin-like protein. Treatment with LGA markedly disrupted 12 distinct cytokinins in N. seriolae mycelium. Additionally, the addition of exogenous tZOG counteracted the inhibitory effects of LGA on filamentous growth, resulting in mycelial elongation and branching. Furthermore, LGA treatment improved the survival rate of largemouth bass infected with N. seriolae. CONCLUSIONS: We found for the first time that LGA from C. cajan exhibited significant efficacy against N. seriolae by interfering with glycerophospholipid metabolism and cytokinin biosynthesis.

Genomic insights and anti-phytopathogenic potential of siderophore metabolome of endolithic Nocardia mangyaensis NH1.

Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among actinobacteria, Nocardia is one of the most important agricultural and industrial bacteria. Screening and isolation of Nocardia related bacteria from extreme habitats such as endolithic environments are beneficial for practical applications in agricultural and environmental biotechnology. In this work, bioinformatics analysis revealed that a novel strain Nocardia mangyaensis NH1 has the capacity to produce structurally varied bioactive compounds, which encoded by non-ribosomal peptide synthases (NRPS), polyketide synthase (PKS), and post-translationally modified peptides (RiPPs). Among NRPS, five gene clusters have a sequence homology with clusters encoding for siderophore synthesis. We also show that N. mangyaensis NH1 accumulates both catechol- and hydroxamate-type siderophores simultaneously under iron-deficient conditions. Untargeted LC-MS/MS analysis revealed a variety of metabolites, including siderophores, lipopeptides, cyclic peptides, and indole-3-acetic acid (IAA) in the culture medium of N. mangyaensis NH1 grown under iron deficiency. We demonstrate that four CAS (chrome azurol S)-positive fractions display variable affinity to metals, with a high Fe3+ chelating capability. Additionally, three of these fractions exhibit antioxidant activity. A combination of iron scavenging metabolites produced by N. mangyaensis NH1 showed antifungal activity against several plant pathogenic fungi. We have shown that the pure culture of N. mangyaensis NH1 and its metabolites have no adverse impact on Arabidopsis seedlings. The ability of N. mangyaensis NH1 to produce siderophores with antifungal, metal-chelating, and antioxidant properties, when supplemented with phytohormones, has the potential to improve the release of macro- and micronutrients, increase soil fertility, promote plant growth and development, and enable the production of biofertilizers across diverse soil systems.

Characterization of the genome of a Nocardia strain isolated from soils in the Qinghai-Tibetan Plateau that specifically degrades crude oil and of this biodegradation.

A strain of Nocardia isolated from crude oil-contaminated soils in the Qinghai-Tibetan Plateau degrades nearly all components of crude oil. This strain was identified as Nocardia soli Y48, and its growth conditions were determined. Complete genome sequencing showed that N. soli Y48 has a 7.3 Mb genome and many genes responsible for hydrocarbon degradation, biosurfactant synthesis, emulsification and other hydrocarbon degradation-related metabolisms. Analysis of the clusters of orthologous groups (COGs) and genomic islands (GIs) revealed that Y48 has undergone significant gene transfer events to adapt to changing environmental conditions (crude oil contamination). The structural features of the genome might provide a competitive edge for the survival of N. soli Y48 in oil-polluted environments and reflect the adaptation of coexisting bacteria to distinct nutritional niches.

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.

[Destruction of oil in the presence of Cu2+ and surfactants of Acinetobacter calcoaceticus IMV B-7241, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405].

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.

[SYNTHESIS OF PHYTOHORMONES BY NOCARDIA VACCINII IMV B-7405--PRODUCER OF SURFACTANTS].

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.

[Synthesis of surfactants by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 on industrial waste].

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%).

[Intensification of surfactants synthesis by Nocardia vaccinii K-8 on crude glycerol].

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.

Monitoring bacterial population dynamics using real-time PCR during the bioremediation of crude-oil-contaminated soil.

We evaluated the activity and abundance of the crude oil- degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon (TPH) degradation rate constants (k) of the soils treated with and without H17-1 were 0.103 d-1 and 0.028 d-1, respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA (16S rRNA), alkane monooxygenase (alkB4), and catechol 2,3-dioxygenase (23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil (alpha=0.05, p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.

Uptake of phosphorus by filamentous bacteria and the role of cation on polyphosphates composition.

Many microorganisms have the ability to store phosphorus as polyphosphates in volutin granules. The aim of the research was to characterise the phosphorus sequestered by filamentous microorganisms present in the foam. Also the importance of required cations like potassium and magnesium in the process of phosphorus uptake by filamentous microorganisms was examined. Electron microscopy and energy dispersive X - ray analysis were used to define the composition of polyphosphate granules in filamentous bacteria.

Diesel and kerosene degradation by Pseudomonas desmolyticum NCIM 2112 and Nocardia hydrocarbonoxydans NCIM 2386.

Pseudomonas desmolyticum NCIM 2112 (Pd 2112) and Nocardia hydrocarbonoxydans NCIM 2386 (Nh 2386) demonstrated an ability to degrade diesel and kerosene. Triton X-100 had enhanced the diesel degradation process by reducing the time required for the maximum utilization of total petroleum hydrocarbon. Fourier transform infrared spectroscopy spectrum of degraded diesel indicates the presence of aliphatic and aromatic aldehydes, C=C aromatic nuclei, and substituted benzenes. Surface tension reduction and stable emulsification was increased using consortium when compared to individual strains. Triton X-100 showed increase in microbial attachment to hydrocarbon among the various chemical surfactants tested. For generating a rapid assay to screen microorganisms capable of degrading kerosene, the acetaldehyde produced in the degradation process could be used as an indicator of degradation. These results indicate diesel and kerosene degradation ability of both of the strains.

Hydrocarbon degradation by thermophilic Nocardia otitidiscaviarum strain TSH1: physiological aspects.

Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-contaminated desert soils with ambient temperature of 45-50 degrees C. The first objective of this study was to demonstrate the hydrocarbon-degrading capability of Nocardia otitidiscaviarum TSH1 (DSM 45,036) which grows optimally at 50 degrees C. Analysis of the metabolic profile of the strain TSH1 showed that it could metabolize phenol, intermediate-chain-length n -alkanes and some polycyclic aromatic hydrocarbons (PAHs) ranging in size from two to four fused rings efficiently, but not toluene and xylene. N. otitidiscaviarum TSH1 was able to survive and grow at phenol concentrations up to 875 mg l(-1). For the first time, the physiological response of a thermophilic Nocardia strain to poorly available hydrophobic compounds was also investigated. When grown on a mineral salt medium with hexadecane, N. otitidiscaviarum TSH1 showed very high affinity for the organic phase. Additionally, PAH-grown cells were considerably hydrophobic. The capacity of PAH-utilizing N. otitidiscaviarum TSH1 isolate to produce biosurfactants was also investigated. Fatty acids (C(14)-C(18)) were detected by GC-MS analysis during bacterial growth in PAH supplemented mineral media. High cell surface hydrophobicity and capability of N. otitidiscaviarum TSH1 to degrade different hydrocarbons at 50 degrees C may make it an ideal candidate to treat oil-contaminated desert soils.

Characterization of a moderate thermophilic Nocardia species able to grow on polycyclic aromatic hydrocarbons.

AIMS: Our goal was the characterization of a new moderate thermophilic polycyclic aromatic hydrocarbon (PAH)-utilizing Nocardia strain. METHODS AND RESULTS: A thermophilic bacterium, strain TSH1, was isolated from a contaminated soil. The macroscopic and microscopic features fit well with the description of Nocardia species. The results of 16S rRNA gene analysis showed 100% match to the type strain of N. otitidiscaviarum DSM 43242(T). Strain TSH1 showed the same mycolic acid pattern as the type strain of N. otitidiscaviarum but its fatty acid profile did not permit identification to the species level. The carbon utilization profile of strain TSH1 was different from N. otitidiscaviarum. The results of hydrophobicity measurements showed that PAHs-grown cells were significantly more hydrophobic than LB-grown cells. Furthermore, biosurfactant production was detected during bacterial growth on different culture media. CONCLUSIONS: Strain TSH1 is capable of growing on a range of PAHs. When grown in PAHs-supplemented media, strain TSH1 showed a high affinity for the organic phase, suggesting that it can develop a hydrophobic surface. SIGNIFICANCE AND IMPACT OF THE STUDY: High cell surface hydrophobicity and capability of strain TSH1 to degrade different PAHs at 50 degrees C may make it an ideal candidate to treat PAH-contaminated desert soils.

Engineering away lysosomal junk: medical bioremediation.

Atherosclerosis, macular degeneration, and neurodegenerative diseases such as Alzheimer's disease, are associated with the intracellular accumulation of substances that impair cellular function and viability. Reversing this accumulation may be a valuable therapy, but the accumulating substances resist normal cellular catabolism. On the other hand, these substances are naturally degraded in the soil and water by microorganisms. Thus, we propose the concept of "medical bioremediation," which derives from the successful field of in situ environmental bioremediation of petroleum hydrocarbons. In environmental bioremediation, communities of microorganisms mineralize hydrophobic organics using a series of enzymes. In medical bioremediation, we hope to utilize one or several microbial enzymes to degrade the intracellular accumulators enough that they can be cleared from the affected cells. Here, we present preliminary, but promising results for the bacterial biodegradation of 7-ketocholesterol, the main accumulator of foam cells associated with atherosclerosis. In particular, we report on the isolation of several Nocardia strains able to biodegrade 7-ketocholesterol and as an ester of 7-ketocholoesterol. We also outline key intermediates in the biodegradation pathway, a key step towards identifying the key enzymes that may lead to a therapy.

Effects of anaerobic selector hydraulic retention time on biological foam control and enhanced biological phosphorus removal in a pure-oxygen activated sludge system.

Increased anaerobic selector hydraulic retention times (HRTs) in a high-purity oxygen activated sludge process resulted in an increase in soluble orthophosphate release and biodegradable chemical oxygen demand removal, confirming that enhanced biological phosphorus removal occurs at aeration solids retention times (SRTs) below 1.7 days. Under operating conditions that included biological foam trapping and recycling, an anaerobic selector with HRTs higher than 55 minutes resulted in a decrease in filament counts and effective foam control. Effective norcardioform control is achieved through the combination of metabolic selective pressure and increased soluble organic substrate removal in the anaerobic selector and low aeration SRT.

Metabolism of daidzein by Nocardia species NRRL 5646 and Mortierella isabellina ATCC 38063.

The phytoestrogen daidzein was metabolized by Nocardia species NRRL 5646 to give two metabolites obtained by hydroxylation and methylation. These metabolites were spectrally characterized as 7-methoxy-4'-hydroxyisoflavone (isoformononetin) and 7,8-dimethoxy-4'-hydroxyisoflavone. Mortierella isabellina ATCC 38063 was able to metabolize daidzein to the unusual metabolite daidzein-4'-rhamnopyranoside.

Efficacy of amikacin combinations for nocardiosis.

We isolated five bacterial strains from patients diagnosed as having nocardiosis. Bacterial species were identified based on the similarities in the nucleotide sequences of 16S ribosomal RNAs. Three of the five strains were identified as Nocardia asteroids, but unexpectedly other two were Streptomyces hygroscopicus and Rothia dentocariosa. The latter two species are not members of the family Nocardiaceae. We investigated the susceptibilities of these five strains to the following nine antimicrobial agents: trimethoprim/sulfamethoxazole (TMP/SMX), minocycline (MINO), erythromycin (EM), amikacin (AMK), cefotaxime (CTX), faropenem (FRPM), imipenem (IPM), ciprofloxacin (CPFX), and sparfloxacin (SPFX). The minimum inhibitory concentration (MIC) ranges (mg/ml) were as follows: TMP-SMX, 4- > 32; MINO, 0.125-8; EM, < or = 0.016- > 32; AMK, 1-2; CTX, 0.063- > 32; FRPM, 0.063-16; IPM, 0.125-2; CPFX, 4-32; and SPFX, 0.5-16. Moreover, the synergistic effects of AMK in combination with each of TMP-SMX, MINO, EM, CTX, IPM, and SPFX were investigated by checkerboard synergy testing. No antagonism was recognized for the three N. asteroides strains. Synergistic and additive effects were observed for the combinations of AMK with CTX, IPM, or SPFX.

Oxidation, reduction, and methylation of carnosic acid by Nocardia.

Preparative-scale incubations with Nocardia sp. NRRL 5646 were conducted to produce new derivatives of the abietane diterpene chemoprotectant and antioxidant carnosic acid (1). Reductive biotransformation of the C-20 carboxylic acid functional group followed by biological methylation at the C-11 phenol afforded 4. Oxidative cyclization of 1 to carnosol 5 followed by dihydroxylation at the isopropyl moiety afforded 6. Metabolites 4 and 6 are new carnosic acid derivatives whose structures were confirmed by mass spectrometry and NMR spectroscopic analysis. The radical quenching properties of 4-6 using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging assay showed activities similar to that of mixed tocopherols and carnosic acid.

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213.

In beta-lactam producing microorganisms, the first step in the biosynthesis of the beta-lactam ring is the condensation of three amino acid precursors: alpha-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, alpha-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2.

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.