Cyanobacteria and Algae in Clouds and Rain in the Area of puy de Dôme, Central France.

The atmosphere contains diverse living microbes, of which the heterotrophic community has been the best studied. Microbes with other trophic modes, such as photoautotrophy, have received much less attention. In this study, culture-independent and dependent methods were used to examine the presence and diversity of oxygenic photoautotrophic microbes in clouds and rain collected at or around puy de Dôme Mountain, central France. Cloud water was collected from the summit of puy de Dôme (1,465 m above sea level [a.s.l.]) for cultivation and metagenomic analysis. Cyanobacteria, diatoms, green algae, and other oxygenic photoautotrophs were found to be recurrent members of clouds, while green algae affiliated with the Chlorellaceae were successfully cultured from three different clouds. Additionally, rain samples were collected below the mountain from Opme meteorological station (680 m a.s.l.). The abundance of chlorophyll a-containing cells and the diversity of cyanobacteria and green algae in rain were assessed by flow cytometry and amplicon sequencing. The corresponding downward flux of chlorophyll a-containing organisms to the ground, entering surface ecosystems with rain, varied with time and was estimated to be between ∼1 and >300 cells cm-2 day-1 during the sampling period. Besides abundant pollen from Pinales and Rosales, cyanobacteria of the Chroococcidiopsidales and green algae of the Trebouxiales were dominant in rain samples. Certain members of these taxa are known to be ubiquitous and stress tolerant and could use the atmosphere for dispersal. Overall, our results indicate that the atmosphere carries diverse, viable oxygenic photoautotrophic microbes and acts as a dispersal vector for this microbial guild.IMPORTANCE Information regarding the diversity and abundance of oxygenic photoautotrophs in the atmosphere is limited. More information from diverse locations is needed. These airborne organisms could have important impacts upon atmospheric processes and on the ecosystems they enter after deposition. Oxygenic photoautotrophic microbes are integral to ecosystem functioning, and some have the potential to affect human health. A better understanding of the diversity and the movements of these aeolian dispersed organisms is needed to understand their ecology, as well as how they could affect ecosystems and human health.

Metabolomic study of the response to cold shock in a strain of Pseudomonas syringae isolated from cloud water.

INTRODUCTION: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level. OBJECTIVES: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock. METHODS: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA). RESULTS: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function. CONCLUSIONS: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.

Siderophores in Cloud Waters and Potential Impact on Atmospheric Chemistry: Production by Microorganisms Isolated at the Puy de Dôme Station.

A total of 450 bacteria and yeast strains isolated from cloud waters sampled at the puy de Dôme station in France (1465 m) were screened for their ability to produce siderophores. To achieve this, a high-throughput method in 96-well plates was adapted from the CAS (chrome azurol S) method. Notably, 42% of the isolates were siderophore producers. This production was examined according to the phyla of the tested strains and the type of chelating functional groups (i.e., hydroxamate, catechol, and mixed type). The most active bacteria in the clouds belong to the γ-Proteobacteria class, among which the Pseudomonas genus is the most frequently encountered. γ-Proteobacteria are produced in the majority of mixed function siderophores, such as pyoverdines, which bear a photoactive group. Finally, siderophore production was shown to vary with the origin of the air masses. The organic speciation of iron remains largely unknown in warm clouds. Our results suggest that siderophores could partly chelate Fe(III) in cloud waters and thus potentially impact the chemistry of the atmospheric aqueous phase.

Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds.

Within cloud water, microorganisms are metabolically active and, thus, are expected to contribute to the atmospheric chemistry. This article investigates the interactions between microorganisms and the reactive oxygenated species that are present in cloud water because these chemical compounds drive the oxidant capacity of the cloud system. Real cloud water samples with contrasting features (marine, continental, and urban) were taken from the puy de Dôme mountain (France). The samples exhibited a high microbial biodiversity and complex chemical composition. The media were incubated in the dark and subjected to UV radiation in specifically designed photo-bioreactors. The concentrations of H(2)O(2), organic compounds, and the ATP/ADP ratio were monitored during the incubation period. The microorganisms remained metabolically active in the presence of ()OH radicals that were photo-produced from H(2)O(2). This oxidant and major carbon compounds (formaldehyde and carboxylic acids) were biodegraded by the endogenous microflora. This work suggests that microorganisms could play a double role in atmospheric chemistry; first, they could directly metabolize organic carbon species, and second, they could reduce the available source of radicals through their oxidative metabolism. Consequently, molecules such as H(2)O(2) would no longer be available for photochemical or other chemical reactions, which would decrease the cloud oxidant capacity.

Biotransformation of various saccharides and production of exopolymeric substances by cloud-borne Bacillus sp. 3B6.

The ability of Bacillus sp. 3B6, a bacterial strain isolated from cloudwaters, to biotransform saccharides present in the atmosphere was evaluated using in situ 1D and 2D NMR spectroscopy. Bacillus is one of the genera most frequently described in the air and in atmospheric waters. Sugars present in these environments have a biogenic origin; they include alditols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Bacillus sp. 3B6 was able to efficiently metabolize sugars, which could thus provide sources of energy for this bacterium and allow it to live and to be metabolically active in warm clouds. In addition, a number of these saccharides (L-arabitol, D-fructose, sucrose, D-glucose, cellotetraose, cellulose, and starch) were transformed to EPSs (exopolymeric substances). We have clearly identified the structure of two EPSs as 1,6-α-galactan and partially acetylated polyethylene glycol. 1,6-α-Galactan is a newly described polymer. The production of EPSs might protect this bacterium under hostile cloud environment conditions, including low nutrient availability, cold temperature and freeze-thaw processes, UV and radical exposure, and evaporation-condensation processes and thus desiccation and osmolarity changes. EPSs could also have a potential role in atmospheric processes because they can be considered as secondary organic aerosols and efficient cloud condensation nuclei.

Complete Genome of Sphingomonas aerolata PDD-32b-11, Isolated from Cloud Water at the Summit of Puy de Dôme, France.

The complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy.

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water.

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dôme (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

Contribution of microbial activity to carbon chemistry in clouds.

The biodegradation of the most abundant atmospheric organic C1 to C4 compounds (formate, acetate, lactate, succinate) by five selected representative microbial strains (three Pseudomonas strains, one Sphingomonas strain, and one yeast strain) isolated from cloud water at the puy de Dôme has been studied. Experiments were first conducted under model conditions and consisted of a pure strain incubated in the presence of a single organic compound. Kinetics showed the ability of the isolates to degrade atmospheric compounds at temperatures representative of low-altitude clouds (5 degrees C and 17 degrees C). Then, to provide data that can be extrapolated to real situations, microcosm experiments were developed. A solution that chemically mimicked the composition of cloud water was used as an incubation medium for microbial strains. Under these conditions, we determined that microbial activity would significantly contribute to the degradation of formate, acetate, and succinate in cloud water at 5 degrees C and 17 degrees C, with lifetimes of 0.4 to 69.1 days. Compared with the reactivity involving free radicals, our results suggest that biological activity drives the oxidation of carbonaceous compounds during the night (90 to 99%), while its contribution accounts for 2 to 37% of the reactivity during the day, competing with photochemistry.

Metabolic modulations of Pseudomonas graminis in response to H2O2 in cloud water.

In cloud water, microorganisms are exposed to very strong stresses especially related to the presence of reactive oxygen species including H2O2 and radicals, which are the driving force of cloud chemistry. In order to understand how the bacterium Pseudomonas graminis isolated from cloud water respond to this oxidative stress, it was incubated in microcosms containing a synthetic solution of cloud water in the presence or in the absence of H2O2. P. graminis metabolome was examined by LC-MS and NMR after 50 min and after 24 hours of incubation. After 50 min, the cells were metabolizing H2O2 while this compound was still present in the medium, and it was completely biodegraded after 24 hours. Cells exposed to H2O2 had a distinct metabolome as compared to unexposed cells, revealing modulations of certain metabolic pathways in response to oxidative stress. These data indicated that the regulations observed mainly involved carbohydrate, glutathione, energy, lipid, peptides and amino-acids metabolisms. When cells had detoxified H2O2 from the medium, their metabolome was not distinguishable anymore from unexposed cells, highlighting the capacity of resilience of this bacterium. This work illustrates the interactions existing between the cloud microbial metabolome and cloud chemistry.

Comparison of microbial and photochemical processes and their combination for degradation of 2-aminobenzothiazole.

The transformation of 2-aminobenzothiazole (ABT) was studied under various conditions: (i) a photodegradation process at a lambda of >300 nm in the presence of an Fe(III)-nitrilotriacetic acid complex (FeNTA), (ii) a biodegradation process using Rhodococcus rhodochrous OBT18 cells, and (iii) the combined processes (FeNTA plus Rhodococcus rhodochrous in the presence or absence of light). The transformation of ABT in the combined system, with or without light, was much more efficient (99% degradation after 25 h) than in the separated systems (37% photodegradation and 26% biodegradation after 125 h). No direct photolysis of ABT was observed. The main result seen is the strong positive impact of FeNTA on the photodegradation, as expected, and on the biotransformation efficiency of ABT, which was more surprising. This positive impact of FeNTA on the microbial metabolism was dependent on the FeNTA concentration. The use of UV high-performance liquid chromatography, liquid chromatography-electrospray ionization mass spectrometry, and in situ (1)H nuclear magnetic resonance provided evidence of the intermediary products and thus established transformation pathways of ABT in the different processes. These pathways were identical whether the degradation process was photo- or biotransformation. A new photoproduct was identified (4OH-ABT), corresponding to a hydroxylation reaction on position 4 of the aromatic ring of ABT.

Fate of the nitrilotriacetic acid-Fe(III) complex during photodegradation and biodegradation by Rhodococcus rhodochrous.

Aminopolycarboxylic acids are ubiquitous in natural waters and wastewaters. They have the ability to form very stable water-soluble complexes with many metallic di- or trivalent ions. The iron complex nitrilotriacetic acid-Fe(III) (FeNTA) has been previously shown to increase drastically the rate of photo- and biodegradation of 2-aminobenzothiazole, an organic pollutant, by Rhodococcus rhodochrous. For this paper, the fate of FeNTA was investigated during these degradation processes. First, it was shown, using in situ (1)H nuclear magnetic resonance, that the complex FeNTA was biodegraded by Rhodococcus rhodochrous cells, but the ligand (NTA) alone was not. This result indicates that FeNTA was transported and biotransformed inside the cell. The same products, including iminodiacetic acid, glycine, and formate, were obtained during the photo- and biodegradation processes of FeNTA, likely because they both involve oxidoreduction mechanisms. When the results of the different experiments are compared, the soluble iron, measured by spectrophotometry, was decreasing when microbial cells were present. About 20% of the initial iron was found inside the cells. These results allowed us to propose detailed mechanistic schemes for FeNTA degradation by solar light and by R. rhodochrous.

Synthesis and biological evaluation of diversely substituted indolin-2-ones.

The synthesis of indolin-2-one derivatives substituted in the 3-position by an aminomethylene group bearing either an ornithine or a lysine residue is described. The inhibitory activities of these compounds toward a panel of eight kinases were examined. Furthermore, the antibacterial activities of the prepared compounds were tested against two Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, a Gram-negative bacterium Escherichia coli and a yeast Candida albicans.

Identification of new metabolic pathways in the enantioselective fungicide tebuconazole biodegradation by Bacillus sp. 3B6.

The use of triazole fungicides in various fields ranging from agriculture to therapy, can cause long-term undesirable effects on different organisms from various environmental compartments and lead to resistance phenomena (even in humans) due to their extensive use and persistence. Their occurrence in various water bodies has increased and tebuconazole, in particular, is often detected, sometimes in high concentration. Only a few bacterial and fungal strains have been isolated and found to biotransform this fungicide, described as not easily biodegradable. Nevertheless, the knowledge of efficient degrading-strains and metabolites potentially formed could improve bioremediation process and global overview of risk assessment. Therefore, a broad screening of microorganisms, isolated from various environmental compartments or from commercially-available strain collections, allowed us to find six bacterial strains able to biotransform tebuconazole. The most efficient one was studied further: this environmental strain Bacillus sp. 3B6 biotransforms the fungicide enantioselectively (ee = 18%) into two hydroxylated metabolites, one of them being transformed in its turn to alkene by a biotic dehydration reaction. This original enantioselective pathway shows that racemic pesticides should be treated by the environmental risk assessment authorities as a mixture of two compounds because persistence, biodegradation, bioaccumulation and toxicity often show chiral dependence.

Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures.

This work constitutes the first large report on aerobic cultivable microorganisms present in cloud water. Seven cloud-event samples were collected at the Puy de Dôme summit, and cultivation was performed leading to the isolation of 71 bacterial, 42 fungal and 15 yeast strains. Most of the fungi isolated were of Cladosporium or Trametes affiliation, and yeasts were of Cryptococcus affiliation. Bacteria, identified on the basis of their 16S rRNA gene sequence, were found to belong to Actinobacteria, Firmicutes, Proteobacteria (Alpha, Beta and Gamma subclasses) and Bacteroidetes phyla, and mainly to the genera Pseudomonas, Sphingomonas, Staphylococcus, Streptomyces, and Arthrobacter. These strains appear to be closely related to some bacteria described from cold environments, water (sea and freshwater), soil or vegetation. Comparison of the distribution of Gram-negative vs. Gram-positive bacteria shows that the number of Gram-negative bacteria is greater in summer than in winter. Finally, a very important result of this study concerns the ability of half of the tested strains to grow at low temperatures (5 degrees C): most of these are Gram-negative bacteria, and a few are even shown to be psychrophiles. On the whole, these results give a good picture of the microbial content of cloud water in terms of classification, and suggest that a large proportion of bacteria present in clouds have the capacity to be metabolically active there. This is of special interest with respect to the potential role of these microorganisms in atmospheric chemistry.

Bacterial characterization of the snow cover at Spitzberg, Svalbard.

A sampling campaign was organized during spring 2004 in Spitzberg, Svalbard, in the area around the scientific base of Ny-Alesund, to characterize the snow pack bacterial population. Total bacteria counts were established by 4',6-diamino-2-phenylindole (DAPI) in the seasonal snow pack bordering the sea. On the sea shore, bacterial concentration was about 6 x 10(4) cells mL(-1), without any significant variation according to depth. In the accumulation snow layer of the glacier, concentrations were about 2 x 10(4 )cells mL(-1), except in the 2003 summer layer, where it reached 2 x 10(5) cells mL(-1), as the result of cell multiplication allowed by higher temperature and snow melting. Strains isolated from the seasonal snow pack were identified from their 16S rRNA gene sequences, and lodged in GenBank. They belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, Firmicutes and Actinobacteria. They are closely related to cold environment bacteria, as revealed by phylogenetic tree constructions, and two appear to be of unknown affiliation. Using 1H nuclear magnetic resonance, it was shown that these isolates have the capacity to degrade organic compounds found in Arctic snow (propionate, acetate and formate), and this can allow them to develop when snow melts, and thus to be actively involved in snow chemistry.

Thin bacteria/Layered Double Hydroxide films using a layer-by-layer approach.

This paper reports the design of thin bacteria/Layered Double Hydroxides (LDH) films in which bacterial cells of Pseudomonas sp. strain ADP were assembled alternatively with Mg2Al-NO3 LDH nanosheets by a layer-by-layer deposition method. The UV-Vis spectroscopy was used to monitor the assembly process, showing a progressive increase in immobilized bacteria amount upon deposited cycles. The {ADP/LDH}n film was characterized by X-ray diffraction, infrared spectroscopy, scanning electron microscopy and atomic force microscopy. The metabolic activity of immobilized bacteria was determined using chronoamperometry by measuring the biochemical oxygen demand in presence of glucose using an artificial electron acceptor (Fe(CN)6(3-)) at 0.5V/Ag-AgCl. A steady current of 0.250µAcm(-2) was reached in about 30s after the addition of 5mM glucose.

Bacteria encapsulated in layered double hydroxides: towards an efficient bionanohybrid for pollutant degradation.

A soft chemical process was successfully used to immobilize Pseudomonas sp. strain ADP (ADP), a well-known atrazine (herbicide) degrading bacterium, within a Mg2Al-layered double hydroxide host matrix. This approach is based on a simple, quick and ecofriendly direct coprecipitation of metal salts in the presence of a colloidal suspension of bacteria in water. It must be stressed that by this process the mass ratio between inorganic and biological components was easily tuned ranging from 2 to 40. This ratio strongly influenced the biological activity of the bacteria towards atrazine degradation. The better results were obtained for ratios of 10 or lower, leading to an enhanced atrazine degradation rate and percentage compared to free cells. Moreover the biohybrid material maintained this biodegradative activity after four cycles of reutilization and 3 weeks storage at 4°C. The ADP@MgAl-LDH bionanohybrid materials were completely characterized by X-ray diffraction (XRD), FTIR spectroscopy, thermogravimetric analysis and scanning and transmission electronic microscopy (SEM and TEM) evidencing the successful immobilization of ADP within the inorganic matrix. This synthetic approach could be readily extended to other microbial whole-cell immobilization of interest for new developments in biotechnological systems.

Synthesis and biological evaluation of oxindoles and benzimidazolinones derivatives.

The synthesis of new oxindoles and benzimidazolinones derivatives bearing a sugar residue on the aromatic nitrogen is described. The presence of the glycoside moiety should enhance the solubility of these heterocyclic compounds and/or improve the interaction with the active site of the biological targets. The inhibitory activities of these new compounds toward five kinases were examined: KDR (VEGFR-2), FGFR-1, PDGFR-beta, EGFR and Tie 2. Furthermore, the antibacterial activities of the prepared compounds were tested against two Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, a Gram-negative bacterium Escherichia coli and a yeast Candida albicans.

Isolation, characterization and diuron transformation capacities of a bacterial strain Arthrobacter sp. N2.

A bacterial strain able to transform diuron was isolated from a soil by enrichment procedures. Strain isolation was realized by plating on minimal-agarose medium spread with this herbicide and selecting the colonies surrounded by a clear thin halo. One strain was characterized and identified as an Arthrobacter sp. It metabolized diuron and the final transformation product, 3,4-dichloroaniline, was produced in stoichiometric amounts. The transformation of diuron at different concentrations was more efficient in the presence of alternative sources of carbon and nitrogen. The bacterial activity was also evaluated in soil microcosms with a consequent disappearance of diuron and concomitant appearance of 3,4-dichloroaniline, of which the concentration decreased thereafter. Bacterial cells inoculated in the microcosms survived as viable but eventually nonculturable cells.

Biotransformation of phenylurea herbicides by a soil bacterial strain, Arthrobacter sp. N2: structure, ecotoxicity and fate of diuron metabolite with soil fungi.

In order to assess the influence of the aromatic substitution on the ability of a soil bacterial strain, Arthrobacter sp. N2, to degrade phenylurea herbicides, biotransformation assays were performed in mineral medium with resting cells of this soil bacterial strain on three phenylurea herbicides (diuron, chlorotoluron and isoproturon). Each herbicide considered, led to the formation of only one metabolite detected by HPLC analysis. After isolation, the metabolites were identified by NMR and MS, as the corresponding substituted anilines. According to the Microtox test (realized on the bacterium Vibrio fischeri), these metabolites presented non-target toxicity far more important (up to 600 times higher for 4-isopropylaniline) than the parent molecule. For isoproturon and chlorotoluron, the amount of substituted anilines obtained at the end of the biotransformation was very low, whereas the biotransformation of diuron into 3,4-dichloroaniline was almost quantitative. In this last case, the degradation product accumulated in the medium. In soil, other microorganisms are present that might degrade it. So the biotransformation of 3,4-dichloroaniline was then tested with four fungal strains: Aspergillus niger, Beauveria bassiana, Cunninghamella echinulata var. elegans and Mortierella isabellina. The aniline was further transformed with all the microorganisms tested. Only one metabolite was detected by HPLC analysis and after isolation, it was identified to be 3,4-dichloroacetanilide. This acetylated compound led to biological effects less important on V. fischeri than 3,4-dichloroaniline. These results stress the importance of identifying the degradation products to assess the impact of a polluting agent. Indeed, the pollutant may undergo transformation yielding compounds more toxic than the parent molecule.