In vivo 31P and 13C NMR investigations of Rhodococcus rhodochrous metabolism and behaviour during biotransformation processes.

AIMS: The strain Rhodococcus rhodochrous OBT18 was isolated from a water treatment plant used to decontaminate industrial effluents containing benzothiazole derivatives. Aims of the work are to study the central metabolism of this strain and more specifically its behaviour during biodegradation of 2-aminobenzothiazole. METHODS AND RESULTS: In vivo(13)C and (31)P NMR experiments showed that this strain contains storage compounds such as polyphosphates, glycogen and trehalose and produces biosurfactants containing trehalose as sugar unit. Trehalose can be synthesized after reversion of the glycolytic pathway. In vivo(31)P NMR experiments showed that energy metabolism markers such as the intracellular pH and the ATP concentration did not change during biotransformation processes when R. rhodochrous was exposed to potentially toxic compounds including iron complexes and (* )OH radicals. Also R. rhodochrous recovers the normal values of ATP and pH after anoxia/reoxygenation cycle very quickly. CONCLUSIONS: Rhodococcus rhodochrous carbon and energy metabolism is well adapted to different stresses and consequently to live in the environment where conditions are constantly changing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to understand the behaviour of this bacterium in natural environments but also in water treatment plants where iron and UV light are present.

Environmental scenarii for the degradation of oxo-polymers.

The fate of oxo-polymers in nature is strongly dependent on environmental conditions, mainly on the intensity and duration of sunshine, which vary with the season and the climate. In this work, we report the effect of different scenarii on the production and the molecular composition of oligomers released from oxo-biodegradable HDPE films. Under our experimental conditions, the duration of accelerated weathering corresponded to a period of 3 months to 3 years of exposure to outside conditions under temperate climate. In addition, the oligomers were extracted in three different solvents: i) water to mimics the natural environment; ii) acetone and chloroform to identify oligomers trapped in the polymer matrix. The combination of high-resolution mass spectrometry and 1H NMR spectroscopy gives an extensive picture of the relative concentrations and the structural compositions of the extracted oligomers in the different tested conditions. In particular, the masses, the number of oxygen and carbon atoms could be determined for up to 2283 molecules. Globally the concentration and the size of oligomers increased with the duration of extraction, the level of aging of the polymer and the use of non-polar solvents. Surprisingly, the presence of highly oxidized molecules in acetone and chloroform extract, suggested an important swelling of HPDE films in these solvents and a better diffusion of these oligomers in the matrix. In nature, the biodegradability of oligomers could result from processes occurring both at the molecular (oxidation) and the macromolecular (diffusion and release) levels.

Draft Genome Sequence of Pseudomonas syringae PDD-32b-74, a Model Strain for Ice-Nucleation Studies in the Atmosphere.

We report here the whole genome sequence of Pseudomonas syringae PDD-32b-74, a gammaproteobacterium isolated from cloud water. This microorganism is equipped with ice-nucleation protein and biosurfactant genes that could potentially be involved in physicochemical processes in the atmosphere and clouds.

Draft Genome Sequence of Pseudomonas graminis PDD-13b-3, a Model Strain Isolated from Cloud Water.

The whole genome of Pseudomonas graminis PDD-13b-3, a strain of bacteria isolated from cloud water, was sequenced. This showed that this microorganism is equipped with genes that could potentially be involved in its survival in the atmosphere and clouds: those for oxidative stress and carbon starvation responses, DNA repair, and iron uptake.

Draft Genome Sequence of Rhodococcus enclensis 23b-28, a Model Strain Isolated from Cloud Water.

The whole genome of Rhodococcus enclensis 23b-28, a bacterial strain isolated from cloud water, was sequenced. This microorganism is equipped with genes able to degrade aromatic compounds and could thus play a role in complex organic matter decomposition in cloud water.

Characterization of oxidized oligomers from polyethylene films by mass spectrometry and NMR spectroscopy before and after biodegradation by a Rhodococcus rhodochrous strain.

The objective of this work was to develop a new approach to assess the specificity and the efficiency of biodegradation of oxidized oligomers extracted from aged HDPE polyethylene films and to bring insight on the mechanisms occurring during biodegradation. 1H NMR spectroscopy and LC Orbitrap™ mass spectrometry were combined together with data processing using Kendrick mass defect calculation and Van Krevelen Diagram. We showed that the molecular weight of extracted oligomers was lower than 850 Da with maximum chain length of 55 carbon atoms. The oligomers were divided into 11 classes of molecules with different oxidation state ranging from 0 to 10. All classes included series of chemically related compounds including up to 19 molecules. 95% of the soluble oligomers were assimilated by a strain of Rhodococcus rhodocchrous after 240 days of incubation. Large highly oxidized molecules completely disappeared while the other classes of molecules were still represented. Molecules containing 0-1 oxygen atom were less degraded. A strong shift to smaller molecules (<450 Da, 25 carbon atoms) was observed suggesting that longer molecules disappeared more rapidly than the smaller ones. It opens new perspectives on biodegradation processes as not only intracellular ß-oxidation must be considered but also extracellular mechanisms leading to chain cleavages.

Modifications of pH and K+ gradients in Candida albicans blastospores induced by amphotericin B. A 31P NMR and K+ atomic absorption study.

Candida albicans blastospores harvested from 8 h (exponential) or 48 h (stationary) cultures were incubated with increasing doses of amphotericin B (AmB). The time course of H+ influx and K+ efflux was monitored by in vivo 31P NMR and K+ atomic absorption respectively. AmB was shown to be more active on exponential phase cells than on stationary phase cells. For both growth phases, K+ leakage occurred before pH acidification. In light of these results, together with iodoacetate experiments, it seems difficult to assert that K+ leakage is a secondary effect resulting from an increase in the permeability to protons, as formerly proposed. In addition, no H+ over K+ selectivity of pores formed by AmB could be detected. Finally, some unexpected results were afforded by 31P NMR experiments: a broadening of Pi signals was detected on exponential phase cell spectra when the blastospores were incubated with 10(-3) and 10(-4) M AmB reflecting a transient heterogeneity of the intracellular pH within the cell population. For stationary phase blastospores, two subpopulations (IIa and IIb) were detected; population IIb, with a more acidic pHi, was much more sensitive to AmB action.

Intracellular pH of Candida albicans blastospores as measured by laser microspectrofluorimetry and 31P-NMR.

The intracellular pH (pHi) of Candida albicans blastospores harvested from 8 h or 48 h cultures was determined under identical experimental conditions by two different techniques: 31P-NMR and laser microspectrofluorimetry. Time dependence of pHi was monitored by 31P-NMR on the whole cell population. Microspectrofluorimetry, after loading of the cells with SNARF-1, enabled the determination of pHi in isolated cells and its distribution among the cell population. By this method, the vacuolar pH could not be distinguished from the cytoplasmic pH in C. albicans blastospores, but alkalization of pHi was observed at the beginning of germ tubes. The absolute values of pHi determined by 31P-NMR were slightly different from those obtained by laser microspectrofluorimetry. However, the pH distributions in the cell population were converging. For blastospores in exponential phase a gaussian distribution of pHi was observed with both methods, the cells maintained a steady pHi value when the external pH was varied from 5.5 to 8.5. For cells in stationary phase two pools were identified: the combination of the two techniques demonstrated the presence of two different subpopulations. One of these population (with lower pH) was able to commute to the other one with time as shown by 31P-NMR kinetics. This information is reported here for the first time in C. albicans.

Ionophore properties of monensin derivatives studied on human erythrocytes by 23Na NMR and K+ and H+ potentiometry: relationship with antimicrobial and antimalarial activities.

Eight derivatives of monensin with a modified C25-C26 moiety were synthesized. Their ionophore properties were studied on human erythrocytes by measuring Na+ influx with 23Na NMR and concomitant K+ and H+ efflux by potentiometry. Modification of OH-26 led to inversion of selectivity of transport in favor of K+/Na+ in comparison with monensin. This selectivity disappeared by suppression of the C26-OH moiety. Finally the ionophore ability was lost if the head-to-tail chelation of the monensin skeleton was prevented by blocking the terminal OH-25 and -26 functions. All the compounds were inactive on Gram-negative bacteria and fungi. MIC measured on Bacillus cereus showed that derivatives with increased K+/Na+ selectivity were clearly the most active against Bacillus growth. Most of the compounds showed potential antimalarial properties in the nanomolar range when tested in vitro against Plasmodium falciparum. The IC50S measured were correlated with the whole Na+ and K+ transport efficiency rather than with the ionic selectivity. In both cases determination of initial fluxes of transport for both cations (Na+ and K+) was necessary to investigate the relationship between biological and ionophore properties.

Syntheses and biological activities (topoisomerase inhibition and antitumor and antimicrobial properties) of rebeccamycin analogues bearing modified sugar moieties and substituted on the imide nitrogen with a methyl group.

As a part of studies on structure-activity relationships, several potential topoisomerase I inhibitors were prepared. Different analogues of the antitumor antibiotic rebeccamycin substituted on the imide nitrogen with a methyl group were synthesized. These compounds bore either the sugar residue of rebeccamycin, with or without the chlorine atoms on the indole moieties, or modified sugar residues (galactopyranosyl, glucopyranosyl, or fucopyranosyl) linked to the aglycone via a beta- or alpha-N-glycosidic bond. Their inhibitory properties toward protein kinase C, topoisomerase I, and topoisomerase II were examined, and their DNA-binding properties were investigated. Their in vitro antitumor activities against murine B16 melanoma and P388 leukemia cells were determined. Their antimicrobial activities were tested against Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, Gram-negative bacterium Escherichia coli, and yeast Candida albicans. These compounds are inactive toward topoisomerase II but inhibit topoisomerase I. A substitution with a methyl group on the imide nitrogen led to a loss of proteine kinase C inhibition in the maleimide indolocarbazole series but did not prevent topoisomerase I inhibition. Compounds possessing a beta-N-glycosidic bond, which fully intercalated into DNA, were more efficient at inhibiting topoisomerase I than their analogues with an alpha-N-glycosidic bond; however, both were equally toxic toward P388 leukemia cells. Dechlorinated rebeccamycin possessing a methyl group on the imide nitrogen was about 10 times more efficient in terms of cytotoxicity and inhibition of topoisomerase I than the natural metabolite.

Structure-activity relationships in a series of substituted indolocarbazoles: topoisomerase I and protein kinase C inhibition and antitumoral and antimicrobial properties.

A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I and II were tested together with their in vitro antitumor efficiency against murine B16 melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid side effects expected with protein kinase C inhibitors, we introduced substitution on the maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these structures were inefficient on topoisomerase II, and some of them exhibited a strong activity against topoisomerase I. Generally, dechlorinated compounds were found to be more active than chlorinated analogues against both purified topoisomerase I and protein kinase C. On the other hand, opposite results were obtained in the cell antiproliferative assays. These results suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage of carbon-chlorine bonds inside the cell.

Syntheses and biological activities of rebeccamycin analogues. Introduction of a halogenoacetyl substituent.

In the course of structure-activity relationships on rebeccamycin analogues, a series of compounds bearing a halogenoacetyl substituent were synthesized with the expectation of increasing the interaction with DNA, possibly via covalent reaction with the double helix. Two rebeccamycin analogues bearing an acetyl instead of a bromoacetyl substituent were prepared to gain an insight into the role of the halogen atom. The new compounds show very little effect on protein kinase C and no covalent reaction with DNA was detected. However, the drugs behave as typical topoisomerase I poisons, and they are significantly more toxic toward P388 leukemia cells than to P388/CPT5 cells resistant to camptothecin. The introduction of a bromo- or chloro-acetyl substituent does not affect the capacity of the drug to interfere with topoisomerase I either in vitro or in cells. One of the bromoacetyl derivatives, compound 8, is the most cytotoxic rebeccamycin derivative among the hundred of derivatives we have synthesized to date. In addition, we determined the antimicrobial activities against two Gram-positive bacteria, Bacillus cereus and Streptomyces chartreusis, and against the Gram-negative bacterium Escherichia coli. The effect of the drugs on Candida albicans yeast growth and their anti-HIV-1 activities were also measured.

Synthesis, mode of action, and biological activities of rebeccamycin bromo derivatives.

Bromo analogues of the natural metabolite rebeccamycin with and without a methyl substituent on the imide nitrogen were synthesized. The effects of the drugs on protein kinase C, the binding to DNA, and the effect on topoisomerase I were determined. The drugs' uptake and their antiproliferative activities against P388 leukemia cells sensitive and resistant to camptothecin, their antimicrobial activity against a Gram-positive bacterium (B. cereus), and their anti-HIV-1 activity were measured and compared to those of the chlorinated and dechlorinated analogues. Dibrominated imide 5 shows a remarkable activity against topoisomerase I, affecting both the kinase and DNA cleavage activity of the enzyme. The marked cytotoxic potency of this compound depends essentially on its capacity to inhibit topoisomerase I.

Syntheses and biological evaluation of indolocarbazoles, analogues of rebeccamycin, modified at the imide heterocycle.

A series of 10 indolocarbazole derivatives, analogues to the antitumor antibiotic rebeccamycin, bearing modifications at the imide heterocycle were synthesized. They bear an N-methyl imide, N-methyl amide, or anhydride function instead of the original imide. Their inhibitory potencies toward topoisomerase I were examined using a DNA relaxation assay and by analyzing the drug-induced cleavage of 32P-labeled DNA. Protein kinase C (PKC) inhibition and interaction with DNA were also studied together with the in vitro antiproliferative activities against B16 melanoma and P388 leukemia cells. The antimicrobial activities against two Gram-positive bacteria (Bacillus cereus and Streptomyces chartreusis), a Gram-negative bacterium (Escherichia coli), and a yeast (Candida albicans) were tested as well as their antiviral activities toward HIV-1. The efficiency of the anhydride compounds was compared to that of the parent compound rebeccamycin and its dechlorinated analogue. All the compounds studied were inactive against PKC. The structural requirements for PKC and topoisomerase I inhibition are markedly different. In sharp contrast with the structure-PKC inhibition relationships, we found that an anhydride function does not affect topoisomerase I inhibition, whereas a methyl group on the indole nitrogen prevents the poisoning of topoisomerase I. The compounds exhibiting a marked toxicity to P388 leukemia cells had little or no effect on the growth of P388CPT5 cells which are resistant to the topoisomerase I inhibitor camptothecin. This study reinforces the conclusion that the DNA-topoisomerase I cleavable complex is the primary cellular target of the indolocarbazoles and significantly contributes to their cytotoxicity and possibly to their weak but noticeable anti-HIV-1 activities. The structure-activity relationships are also discussed.

Synthesis and biological activities of indolocarbazoles bearing amino acid residues.

Three indolocarbazole compounds bearing a tripeptide or a lysine group attached to one of the indole nitrogens via a propylamino chain and two rebeccamycin derivatives bearing a lysine residue on the sugar moiety were synthesised with the aim of improving the binding to DNA and the antiproliferative activities. Four tumour cell lines, from murine L1210 leukemia, human HT29 colon carcinoma, A549 non-small cell lung carcinoma and K-562 leukemia, were used to evaluate the cytotoxicity of the drugs. Their effects on the cell cycle of L1210 cells and their antimicrobial properties against two Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, a Gram-negative bacterium Escherichia coli and a yeast Candida albicans were also investigated.

Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors.

The antimicrobial activities of twenty-two substances structurally related to staurosporine, aglycone in the indolocarbazole and bis-indole series were examined against Streptomyces chartreusis and Streptomyces griseus, Bacillus cereus, Escherichia coli, Candida albicans and Botrytis cinerea. Inhibition of sporulation was examined also on the two species of Streptomyces. Unlike literature reports for efficient protein kinase inhibitors, staurosporine and K-252a, no evident correlation could be found either between protein kinase inhibitory potencies and inhibition of sporulation of the Streptomyces species, or protein kinase inhibitory potencies and growth of all microorganisms tested. A weak activity against C. albicans was observed for the chloro-indolocarbazole compounds as already reported for structurally related substances from the cyanobacterium Tolypothrix tjipanasensis.

Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors. II. Substitution on maleimide nitrogen with functional groups bearing a labile hydrogen.

New compounds, structurally related to the potent protein kinase C inhibitor staurosporine, and substituted on the imide nitrogen with a functional group bearing a labile hydrogen (hydroxymethyl, amino, hydroxy), were synthesized. Their in vitro inhibitory potencies towards protein kinase C and protein kinase A showed that N-hydroxymethyl and N-hydroxy substitution, unlike alkyl substitution, can provide efficient protein kinase C inhibitors. The antimicrobial activities of these new compounds against Streptomyces chartreusis and Streptomyces griseus, Bacillus cereus, Escherichia coli, Candida albicans and Botrytis cinerea were examined. They proved to be inactive against E. coli and two fungi. The results suggest that there is no link between in vitro inhibition of protein kinase C and inhibition of growth and sporulation of the two Streptomyces tested. Unlike indolocarbazole maleimides, bis-indole maleimides are active against the two Streptomyces species.

Microbial conversion of nigericin in three successive steps, by Sebekia benihana.

A strain of Sebekia benihana NRRL 11111 was found to transform nigericin in three successive steps, giving three compounds which were isolated. Their structure were determined by IR, 1H and 13C NMR, and fast atom bombardment mass spectra. The first compound resulted from the reduction of the terminal hemiketal ring it was then transformed into the two other compounds as a result of the oxidation of methyl (C-33) into a CH2OH and COOH group respectively. All these products had lost the ionophoric and antibiotic properties of nigericin and thus were products of a detoxification process.

Syntheses and antimicrobial activities of five-membered ring heterocycles coupled to indole moieties.

Indole-substituted oxazolidinones, oxazolones, pyrrolidinone, imidazolidinone and imidazolones were synthesized. Their inhibitory potencies towards protein kinase C and protein kinase A were determined and their in vitro activities against Streptomyces chartreusis, Streptomyces griseus, Bacillus cereus, Candida albicans and Escherichia coli were examined. The inhibition of Streptomyces sporulation observed for some of them could not be linked to in vitro protein kinase C inhibition. All proved inactive against C. albicans but three of them exhibited a marked activity towards E. coli. This effect extends to other Gram-negative bacteria.

Degradation products of a phenylurea herbicide, diuron: synthesis, ecotoxicity, and biotransformation.

The degradation products of diuron (photoproducts and metabolites), already described in the literature, were synthesized in order to carry out further investigations. Their ecotoxicity was determined using the standardized Microtox test, and most of the derivatives presented a nontarget toxicity higher than that of diuron. Therefore, the biotransformation of these compounds was tested with four fungal strains and a bacterial strain, which were known to be efficient for diuron transformation. With the exception of the 3,4-dichlorophenylurea, all the degradation products underwent other transformations with most of the strains tested, but no mineralization was observed. For many of them, the biodegradation compound for which the toxicity was important was 3,4-dichlorophenylurea. This study underlines the importance of knowing the nature of the degradation products, which has to be kept in mind while analyzing natural water samples or soil samples.