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.

First evaluation of the effect of microorganisms on steady state hydroxyl radical concentrations in atmospheric waters.

Clouds are complex multiphasic media where efficient chemical reactions take place and where microorganisms have been found to be metabolically active. Hydroxyl radical is the main oxidant in cloud water, and more generally in the atmosphere, during the day and drives the cloud oxidative capacity. However, only one measurement of the steady state hydroxyl radical concentrations in cloud water has been reported so far. Cloud chemistry models are used to estimate the hydroxyl radical concentrations with values ranging from 10-12 to 10-15 M that are surely overestimated due to a lack of knowledge about the speciation of the organic matter acting as a sink for hydroxyl radicals. The aim of this work is to quantify the concentration of hydroxyl radicals at steady state in rain and cloud waters and to measure the impact of native microflora on this concentration. First, the non-toxicity of terephthalic acid as probe is controlled before the analysis in real atmospheric water samples. Higher concentrations of hydroxyl radicals are found in cloud waters than in rain waters, with a mean value "1.6 ±â€¯1.5" × 10-16 M and "7.2 ±â€¯5.0" × 10-16 M for rain and cloud waters respectively and no real impact of microorganisms was observed. This method allows the measurement of steady state hydroxyl radical levels at very low concentrations (down to 10-17 M) and it is biocompatible, fast and easy to handle. It is a useful tool, complementary to other methods, to give a better overview of atmospheric water oxidant capacity.

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.

Detection of semi-volatile compounds in cloud waters by GC×GC-TOF-MS. Evidence of phenols and phthalates as priority pollutants.

Although organic species are transported and efficiently transformed in clouds, more than 60% of this organic matter remains unspeciated. Using GCxGC-HRMS technique we were able to detect and identify over 100 semi-volatile compounds in 3 cloud samples collected at the PUY station (puy de Dôme mountain, France) while they were present at low concentrations in a very small sample volume (<25 mL of cloud water). The vast majority (∼90%) of the detected compounds was oxygenated, while the absence of halogenated organic compounds should be specially mentioned. This could reflect both the oxidation processes in the atmosphere (gas and water phase) but also the need of the compounds to be soluble enough to be transferred and dissolved in the cloud droplets. Furans, esters, ketones, amides and pyridines represent the major classes of compounds demonstrating a large variety of potential pollutants. Beside these compounds, priority pollutants from the US EPA list were identified and quantified. We found phenols (phenol, benzyl alcohol, p-cresole, 4-ethylphenol, 3,4-dimethylphenol, 4-nitrophenol) and dialkylphthalates (dimethylphthalate, diethylphthalate, di-n-butylphthalate, bis-(2-ethylhexyl)-phthalate, butylbenzylphthalate, di-n-octyl phthalate). In general, the concentrations of phthalates (from 0.09 to 52 µg L-1) were much higher than those of phenols (from 0.03 to 0.74 µg L-1). To our knowledge phthalates in clouds are described here for the first time. We investigated the variability of phenols and phthalates concentrations with cloud air mass origins (marine vs continental) and seasons (winter vs summer). Although both factors seem to have an influence, it is difficult to deduce general trends; further work should be conducted on large series of cloud samples collected in different geographic areas and at different seasons.

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.

Study by NMR of the mode of action of monensin on Streptococcus faecalis de-energized and energized cells.

Streptococcus faecalis was used as a bacterial model for studying the mode of action of monensin by NMR investigations. Experiments were carried out in two states, characterized by several complementary methods: (i) the resting (de-energized) cell which was considered as an inert biological membrane, on which cationic transport induced by the ionophore alone can be investigated; (ii) the active (energized) cell where the ionophore-sensitive response of the living organism, particularly the cation pumps and the glycolysis, is probed. Studies of resting cells were performed, with changing external ionic concentrations, in the presence of monensin, which is preferentially a sodium carrier. Internal and external Na+ and H+ were followed by corresponding 23Na and 31P (inorganic phosphate) NMR resonances, K+ fluxes were measured by atomic absorption. It was shown that the induced cationic movements were linked to the existing ionic gradients for K+ and Na+. 31P and 13C NMR spectra for the intermediary metabolites detected in active cells showed that glycolysis is dramatically modified in the presence of monensin.

Conditions modulating the ionic selectivity of transport by monensin examined on Enterococcus hirae (Streptococcus faecalis) by 23Na-NMR and K+ atomic absorption.

Factors likely to modulate the ionic selectivity of monensin were examined on Enterococcus hirae (Streptococcus faecalis) in two states previously characterized: the resting (de-energized) cell and the active (energized) cell. Internal and external Na+ were followed by corresponding 23Na-NMR resonances K+ concentrations were measured by atomic absorption. For a given cellular population of de-energized cells, the apparent transport rates and the final cationic concentrations reached at the steady state were decreasing with the ionophore dose. Monensin was selective for sodium only at low concentrations, in the range 1 mM-10(-4) mM the transport was depending on the effective cationic gradients. Comparison of the activity curves for two cell populations (7.10(9) and 7.10(10) cells/ml) showed the importance of the ratios of monensin/mg phospholipid and also of the ratios of external/internal volumes. On energized cells, except for low monensin concentrations, the main effect was a K(+)-induced efflux and not a Na+ influx. Two factors were modulating the resulting selectivity of this ionophore: the response of the intrinsic bacterial carriers and the generation of the gradients (mainly the external pH) which were favourable to a K+/Na+ transport. Once again the results obtained for two cell populations could be compared, the determining factors were the ratio external/internal volume and the generation of the pH gradient.

Na+ and K+ transport by 4-chlorophenylurethane-monensin in Enterococcus hirae de-energized and energized cells studied by 23Na-NMR and K+ atomic absorption.

Na+ and K+ movements induced by 4-chlorophenylurethane-monensin, which presents an inverted ion selectivity (K+ > Na+) in model systems compared with monensin, were followed on Enterococcus hirae cells by 23Na-NMR and K+ atomic absorption. For de-energized cells, the urethane derivative is much more selective for K+ than monensin, but only at low concentrations (10(-3)-10(-4) mM). For higher concentrations, as previously shown for monensin, the sodium and potassium movements are driven by the ion gradients present. On energized cells, both K+ and Na+ gradients were highly perturbed, and this can be related to the higher toxicity in mice and bacteria for this derivative.

Re-investigation of glucose metabolism in Fibrobacter succinogenes, using NMR spectroscopy and enzymatic assays. Evidence for pentose phosphates phosphoketolase and pyruvate formate lyase activities.

The glucose metabolism of Fibrobacter succinogenes S85 was studied in detail; key intermediates and alternative pathways were evidenced by NMR and/or enzymatic assays. A high phosphoketolase activity was detected in four strains of Fibrobacter under strictly anaerobic conditions, with ribose-5-phosphate as substrate, no activity was evidenced with fructose-6-phosphate. This is the first report of a pentose phosphates phosphoketolase in bacteria unable to use pentoses. In contrast, the Entner-Doudoroff pathway and the oxidative branch of the pentose phosphate pathway could not be evidenced. Incubation of living cells of F. succinogenes with Na2(13)CO3 confirmed the incorporation of 13CO2 in the carboxylic group of succinate. The presence of fumarase was evidenced by in vivo 4C-NMR using 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO): the enzyme showed a high reversibility under physiological conditions. The production of formate from glucose catabolism was evidenced by enzymatic assay and by NMR and a pyruvate formate lyase activity was detected using strictly anaerobic conditions.

Characterization of the potent in vitro and in vivo antimalarial activities of ionophore compounds.

Large-scale in vitro screening of different types of ionophores previously pinpointed nine compounds that were very active and selective in vitro against Plasmodium falciparum; their in vitro and in vivo antimalarial effects were further studied. Addition of the ionophores to synchronized P. falciparum suspensions revealed that all P. falciparum stages were sensitive to the drugs. However, the schizont stages were three- to ninefold more sensitive, and 12 h was required for complete parasite clearance. Pretreatment of healthy erythrocytes with toxic doses of ionophores for 24 to 48 h showed that the activity was not due to an irreversible effect on the host erythrocyte. No preferential ionophore adsorption in infected or uninfected erythrocytes occurred. On the other hand, ionophore molecules strongly bound to serum proteins since increasing the serum concentration from 2 to 50% led to almost a 25-fold parallel increase in the ionophore 50% inhibitory concentration. Mice infected with the malaria parasites Plasmodium vinckei petteri or Plasmodium chabaudi were successfully treated with eight ionophores in a 4-day suppressive test. The 50% effective dose after intraperitoneal administration ranged from 0.4 to 4.1 mg/kg of body weight, and the therapeutic indices were about 5 for all ionophores except monensin A methyl ether, 5-bromo lasalocid A, and gramicidin D, whose therapeutic indices were 12, 18, and 344, respectively. These three compounds were found to be curative, with no recrudescence. Gramicidin D, which presented impressive antimalarial activity, requires parenteral administration, while 5-bromo lasalocid A has the major advantage of being active after oral administration. Overall, the acceptable levels of toxicity and the good in vivo therapeutic indices in the rodent model highlight the interesting potential of these ionophores for the treatment of malaria in higher animals.

[Synthesis of a DNA fragment containing dihydro-5,6 thymine]. / Synthèse d'un fragment de DNA comportant la dihydro-5,6 thymine.

The preparation of a pentadecanucleoside tetradecaphosphate containing the modified base 5,6-dihydrothymine (DHT) is reported herein. The synthesis was performed with a mixture of diastereoisomers (5R and 5S), obtained by catalytic hydrogenation of thymidine. The phosphorylated protected monomer was characterized by proton NMR and FAB mass spectrometry. It was introduced into the DNA fragment (poly dT) by the solid phase phosphotriester approach. After deprotection, the determination of the site of the modified base in the chain was made using different methods. The classical chemical sequencing method of Maxam and Gilbert showed no difference with respect to thymine moieties in the pentadecamer. To characterize the presence and the location of the modified base DHT, the 32P 5'-end labelled pentadecamer was heated in formic acid at 90 degrees C and length separated by polyacrylamide gel electrophoresis. The Electronic Impact mass spectrometry under pyrolytic conditions of the 15-mer showed that the modified base was present in the deprotected DNA fragment.

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.

Differentiation of mobile and immobile pesticides on anionic clays by 1H HR MAS NMR spectroscopy.

Adsorbed vs. intercalated MCPA (4-chloro-2-methylphenoxyacetic acid) in highly hydrated clays taken as a soil model were clearly distinguished by 1H HR MAS NMR; adsorbed herbicide gave sharp signals indicating high mobility while intercalated herbicide gave very wide unresolved spectra due to its strong interaction with the solid matrix.

13C and 1H NMR study of cellulose metabolism by Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.

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.