Viability and stress state of bacteria associated with primary production or zooplankton-derived suspended particulate matter in summer along a transect in Baffin Bay (Arctic Ocean).

In the framework of the GreenEdge Project (whose the general objective is to understand the dynamic of the phytoplankton spring bloom in Arctic Ocean), lipid composition and viability and stress state of bacteria were monitored in sea ice and suspended particulate matter (SPM) samples collected in 2016 along a transect from sea ice to open water in Baffin Bay (Arctic Ocean). Lipid analyses confirmed the dominance of diatoms in the bottommost layer of ice and suggested (i) the presence of a strong proportion of micro-zooplankton in SPM samples collected at the western ice covered St 403 and St 409 and (ii) a high proportion of macro-zooplankton (copepods) in SPM samples collected at the eastern ice covered St 413 and open water St 418. The use of the propidium monoazide (PMA) method allowed to show a high bacterial mortality in sea ice and in SPM material collected in shallower waters at St 409 and St 418. This mortality was attributed to the release of bactericidal free fatty acids by sympagic diatoms under the effect of light stress. A strong cis-trans isomerization of bacterial MUFAs was observed in the deeper SPM samples collected at the St 403 and St 409. It was attributed to the ingestion of bacteria stressed by salinity in brine channels of ice by sympagic bacterivorous microzooplankton (ciliates) incorporating trans fatty acids of their preys before to be released in the water column during melting. The high trans/cis ratios also observed in SPM samples collected in the shallower waters at St 413 and St 418 suggest the presence of positively or neutrally buoyant extracellular polymeric substances (EPS)-rich particles retained in sea ice and discharged (with bacteria stressed by salinity) in seawater after the initial release of algal biomass. Such EPS particles, which are generally considered as ideal vectors for bacterial horizontal distribution in the Arctic, appeared to contain a high proportion of dead and non-growing bacteria.

Dissolved organic matter released by two marine heterotrophic bacterial strains and its bioavailability for natural prokaryotic communities.

Marine heterotrophic prokaryotes (HP) play a key role in organic matter processing in the ocean; however, the view of HP as dissolved organic matter (DOM) sources remains underexplored. In this study, we quantified and optically characterized the DOM produced by two single marine bacterial strains. We then tested the availability of these DOM sources to in situ Mediterranean Sea HP communities. Two bacterial strains were used: Photobacterium angustum (a copiotrophic gammaproteobacterium) and Sphingopyxis alaskensis (an oligotrophic alphaproteobacterium). When cultivated on glucose as the sole carbon source, the two strains released from 7% to 23% of initial glucose as bacterial derived DOM (B-DOM), the quality of which (as enrichment in humic or protein-like substances) differed between strains. B-DOM induced significant growth and carbon consumption of natural HP communities, suggesting that it was partly labile. However, B-DOM consistently promoted lower prokaryotic growth efficiencies than in situ DOM. In addition, B-DOM changed HP exoenzymatic activities, enhancing aminopeptidase activity when degrading P. angustum DOM, and alkaline phosphatase activity when using S. alaskensis DOM, and promoted differences in HP diversity and composition. DOM produced by HP affects in situ prokaryotic metabolism and diversity, thus changing the pathways for DOM cycling (e.g. respiration over biomass production) in the ocean.

Use of organic exudates from two polar diatoms by bacterial isolates from the Arctic Ocean.

Global warming affects primary producers in the Arctic, with potential consequences for the bacterial community composition through the consumption of microalgae-derived dissolved organic matter (DOM). To determine the degree of specificity in the use of an exudate by bacterial taxa, we used simple microalgae-bacteria model systems. We isolated 92 bacterial strains from the sea ice bottom and the water column in spring-summer in the Baffin Bay (Arctic Ocean). The isolates were grouped into 42 species belonging to Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Forty strains were tested for their capacity to grow on the exudate from two Arctic diatoms. Most of the strains tested (78%) were able to grow on the exudate from the pelagic diatom Chaetoceros neogracilis, and 33% were able to use the exudate from the sea ice diatom Fragilariopsis cylindrus. 17.5% of the strains were not able to grow with any exudate, while 27.5% of the strains were able to use both types of exudates. All strains belonging to Flavobacteriia (n = 10) were able to use the DOM provided by C. neogracilis, and this exudate sustained a growth capacity of up to 100 times higher than diluted Marine Broth medium, of two Pseudomonas sp. strains and one Sulfitobacter strain. The variable bioavailability of exudates to bacterial strains highlights the potential role of microalgae in shaping the bacterial community composition. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

Changes in bacterial community metabolism and composition during the degradation of dissolved organic matter from the jellyfish Aurelia aurita in a Mediterranean coastal lagoon.

Spatial increases and temporal shifts in outbreaks of gelatinous plankton have been observed over the past several decades in many estuarine and coastal ecosystems. The effects of these blooms on marine ecosystem functioning and particularly on the dynamics of the heterotrophic bacteria are still unclear. The response of the bacterial community from a Mediterranean coastal lagoon to the addition of dissolved organic matter (DOM) from the jellyfish Aurelia aurita, corresponding to an enrichment of dissolved organic carbon (DOC) by 1.4, was assessed for 22 days in microcosms (8 l). The high bioavailability of this material led to (i) a rapid mineralization of the DOC and dissolved organic nitrogen from the jellyfish and (ii) the accumulation of high concentrations of ammonium and orthophosphate in the water column. DOM from jellyfish greatly stimulated heterotrophic prokaryotic production and respiration rates during the first 2 days; then, these activities showed a continuous decay until reaching those measured in the control microcosms (lagoon water only) at the end of the experiment. Bacterial growth efficiency remained below 20%, indicating that most of the DOM was respired and a minor part was channeled to biomass production. Changes in bacterial diversity were assessed by tag pyrosequencing of partial bacterial 16S rRNA genes, DNA fingerprints, and a cultivation approach. While bacterial diversity in control microcosms showed little changes during the experiment, the addition of DOM from the jellyfish induced a rapid growth of Pseudoalteromonas and Vibrio species that were isolated. After 9 days, the bacterial community was dominated by Bacteroidetes, which appeared more adapted to metabolize high-molecular-weight DOM. At the end of the experiment, the bacterial community shifted toward a higher proportion of Alphaproteobacteria. Resilience of the bacterial community after the addition of DOM from the jellyfish was higher for metabolic functions than diversity, suggesting that jellyfish blooms can induce durable changes in the bacterial community structure in coastal lagoons.

Genome Sequence of Maribius sp. Strain MOLA 401, a Marine Roseobacter with a Quorum-Sensing Cell-Dependent Physiology.

Maribius sp. strain MOLA401 is an alphaproteobacterium isolated from a coral reef lagoon located in New Caledonia, France. We report the genome sequence and its annotation which, interestingly, reveals the presence of genes involved in quorum sensing. This is the first report of a full genome within the genus Maribius.

Shotgun redox proteomics: identification and quantitation of carbonylated proteins in the UVB-resistant marine bacterium, Photobacterium angustum S14.

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.

A new, sensitive marine microalgal recombinant biosensor using luminescence monitoring for toxicity testing of antifouling biocides.

In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC(50)) after only 2 days (diuron, 5.65 ± 0.44 µg/liter; Irgarol 1015, 0.76 ± 0.10 µg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.

Proteome analysis of the UVB-resistant marine bacterium Photobacterium angustum S14.

The proteome of the marine bacterium Photobacterium angustum S14 was exposed to UVB and analyzed by the implementation of both the post-digest ICPL labeling method and 2D-DIGE technique using exponentially growing cells. A total of 40 and 23 proteins were quantified in all replicates using either the ICPL or 2D-DIGE methods, respectively. By combining both datasets from 8 biological replicates (4 biological replicates for each proteomics technique), 55 proteins were found to respond significantly to UVB radiation in P. angustum. A total of 8 UVB biomarkers of P. angustum were quantified in all replicates using both methods. Among them, the protein found to present the highest increase in abundance (almost a 3-fold change) was RecA, which is known to play a crucial role in the so-called recombinational repair process. We also observed a high number of antioxidants, transport proteins, metabolism-related proteins, transcription/translation regulators, chaperonins and proteases. We also discuss and compare the UVB response and global protein expression profiles obtained for two different marine bacteria with trophic lifestyles: the copiotroph P. angustum and oligotroph Sphingopyxis alaskensis.

Influence of growth temperature and starvation state on survival and DNA damage induction in the marine bacterium Sphingopyxis alaskensis exposed to UV radiation.

Despite the considerable volume of literature describing the individual effects of temperature or UV light on aquatic bacteria, little is known about their combined effects. The current study was conducted to learn about the effects of growth temperature and duration of starvation on the response of a marine bacterium, Sphingopyxis alaskensis to UV-B or simulated solar radiation. Cells grown at 12 degrees C or 24 degrees C, and harvested at early or late stationary phase, were exposed to UV-B or simulated solar radiation (>290 nm). The predominant forms of UV-induced DNA damage, namely cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PP s), were quantified using a HPLC-mass spectrometry. While the commonly accepted view that DNA damage induced by UV-B radiation is temperature-independent, we observed in S. alaskensis that the yield of photoproducts for 12 degrees C was generally lower than for cells grown at 24 degrees C. The relative distribution of DNA photoproducts also varied with growth temperature, with an increased formation of TC 6-4PP for late compared to early stationary phase cells. In contrast, with the exception of cultures grown at 12 degrees C exposed to simulated solar radiation, the duration of stationary phase had no effect on total photoproduct formation. Collectively, these data indicate that growth temperature has more effect than duration of starvation on the formation of photoproducts in S. alaskensis.

Remarkable resistance to UVB of the marine bacterium Photobacterium angustum explained by an unexpected role of photolyase.

DNA damage and cell survival was assessed in the marine bacteria, Photobacterium angustum (GC%=39.6) and Sphingopyxis alaskensis (GC%=65.5) following UVB irradiation and recovery in the presence or absence of visible light. The extent of bipyrimidine photoproduct formation was analyzed by HPLC-MS/MS. S. alaskensis was chosen as a reference species since it was previously shown to be photoresistant. Interestingly, P. angustum exhibited an even higher level of survival to UVB irradiation than S. alaskensis. This higher photoresistance was associated with a decrease in the rate of formation of cyclobutane pyrimidine dimers (CPDs) at high UVB doses. Despite different distributions in UVB-induced lesions, the survival difference between the two marine bacteria could not be accounted for by qualitative differences in either photoreactivation or the rate of nucleotide excision repair of the photoproducts arising from the different bipyrimidine doublets (TT, CT, TC and CC). Dark repair was found to be much more efficient for P. angustum than S. alaskensis but the corresponding rate of photoproduct removal was lower than that observed at high UVB doses. We propose that the increased resistance of P. angustum under high UVB doses results from a UVB-induction of CPD photolyase(s) that may directly repair DNA damage and/or act indirectly by enhancing the rate of nucleotide excision repair.

Sunlight-induced DNA damage in marine micro-organisms collected along a latitudinal gradient from 70 degrees N to 68 degrees S.

We examined ultraviolet radiation (UVR)-induced DNA damage in marine micro-organisms collected from surface seawater along a latitudinal transect in the Central Pacific Ocean from 70 degrees N to 68 degrees S. Samples were collected predawn and incubated under ambient UVR in transparent incubators at in situ temperatures until late afternoon at which time they were filtered into primarily bacterioplankton and eukaryotic fractions. Cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts [(6-4)PDs] were quantified in DNA extracts using radioimmunoassays. UVB was lowest in the polar regions and highest near the equator and correlations between UVB and DNA damage were observed. The eukaryotic fraction showed significant CPDs across the entire transect; (6-4)PDs were detected only in the tropics. The bacterial fraction showed no accumulation of (6-4)PDs at any latitude, although residual (6-4)PDs were observed. Bacterial cell volumes were greatest in the sub-Arctic and northern temperate latitudes and lower in the tropics and southern hemisphere, a unique observation that parallels Bergmann's rule. A strong negative correlation was observed between cell volume and CPDs. The environmental impact of solar UVR on marine micro-organisms in the open ocean is complex and our results suggest that several factors such as DNA repair, cell size, temperature, salinity, nutrients and species composition are important in determining relative sensitivity.

Ultraviolet radiation in the rhône river lenses of low salinity and in marine waters of the northwestern mediterranean sea: attenuation and effects on bacterial activities and net community production.

The high content in nutrients of freshwater outflows induces highly productive and buoyant plumes spreading over marine waters (MW). As a consequence, the growth of organisms developing in these low-salinity waters (LSW) might be potentially affected by UV-R (280-400 nm). This study investigated the penetration of UV-R and its impact on net community production (NCP) and bacterial protein (B(PROT)S) and DNA (B(DNA)S) synthesis in mesotrophic-LSW formed from the Rhône River and in oligotrophic MW of the Northwestern Mediterranean Sea (Gulf of Lions) in May 2006. High concentrations of chlorophyll a (up to 8 microg L(-1)) measured in the LSW (<37.8 psu, 0-10 m) were the main factor influencing the diffuse attenuation coefficients (K(d)) of both UV-R and photosynthetically active radiation (PAR). The mean ratio of the K(d) measured between the LSW and the MW increased with wavelength from 2.4 at 305 nm to 2.9 at 380 nm and 3.1 for PAR indicating more similarity in the UV region. NCP was severely inhibited by UV-R at the surface of the LSW, whereas no effect was measured in the surrounding MW. In contrast, B(PROT)S and B(DNA)S were affected deeper by UV-R in the MW (up to 8 m depth) compared to the LSW where inhibition was only observed at the surface. Differences in response of bacteria in LSW and MW are largely explained by differences in UV-R transparency; however, transplant experiments indicate that bacterial assemblages from the MW were also more sensitive to UV-R than those present in the LSW. We also observed that higher activity of bacteria after nutrient additions increased their sensitivity to UV-R during the day, but favored their recovery during the night incubation period for both LSW and MW. Results suggest that riverine and nutrient inputs may alter the effects of UV-R on microbial activity by attenuating the UV-R penetration and by modifying the physiology of bacteria.

Physiological alteration of the marine bacterium Vibrio angustum S14 exposed to simulated sunlight during growth.

Growth experiments on the marine bacterium Vibrio angustum S14 were conducted under four light conditions using a solar simulator: visible light (V), V + ultraviolet A (UV-A), V + UV-A + UV-B radiation, and dark. Growth was inhibited mainly by UV-B and slightly by UV-A. UV-B radiation induced filaments containing multiple genome copies with low cyclobutane pyrimidine dimers. These cells did not show modifications in cellular fatty acid composition in comparison with dark control cultures and decreased in size by division after subsequent incubation in the dark. A large portion of the bacterial population grown under visible light showed an alteration in cellular DNA fluorescence as measured by flow cytometry after SYBR-Green I staining. This alteration was not aggravated by UV-A and was certainly due to a change in DNA topology rather than DNA deterioration because all the cells remained viable and their growth was not impaired. Ecological consequences of these observations are discussed.

Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts.

Solar UV radiation is a major mutagen that damages DNA through the formation of dimeric photoproducts between adjacent thymine and cytosine bases. A major effect of the GC content of the genome is thus anticipated, in particular in prokaryotes where this parameter significantly varies among species. We quantified the formation of UV-induced photolesions within both isolated and cellular DNA of bacteria of different GC content. First, we could unambiguously show the favored formation of cytosine-containing photoproducts with increasing GC content (from 28 to 72%) in isolated DNA. Thymine-thymine cyclobutane dimer was a minor lesion at high GC content. This trend was confirmed by an accurate and quantitative analysis of the photochemical data based on the exact dinucleotide frequencies of the studied genomes. The observation of the effect of the genome composition on the distribution of photoproducts was then confirmed in living cells, using two marine bacteria exhibiting different GC content. Because cytosine-containing photoproducts are highly mutagenic, it may be predicted that species with genomes exhibiting a high GC content are more susceptible to UV-induced mutagenesis.

[Isolation and characterization of marine oligotrophic bacteria]. / Isolement et caractérisation de bactéries marines oligotrophes.

A significant part of the world ocean is characterized by low absolute nutrients and chlorophyll concentrations. In these oligotrophic environments, bacteria are very abundant and play a vital role in the remineralization of the dissolved organic matter. Bacteria adapted to oligotrophic waters differ from those adapted to richer environments by some genetic and metabolic characteristics. Culture techniques in bacteriology are based on rich media and do not allow the growth of most marine bacteria. New techniques have been developed for the culture of oligotrophic bacteria, which allow to isolate unknown bacteria. Pelagibacter ubique and Sphingopyxis alaskensis belong to these bacteria recently isolated from the marine environment and their study yielded better understanding of how marine bacteria adapt to oligotrophic conditions.

Photodegradation of sulcotrione in various aquatic environments and toxicity of its photoproducts for some marine micro-organisms.

Photochemical behaviour of sulcotrione, a triketone herbicide, was studied in a variety of aqueous solutions including natural waters (sea and river) under laboratory conditions. Photodegradation experiments were carried out under two irradiation systems (UV-B and simulated solar radiation) in order to evaluate kinetics of active ingredient. The degradation kinetics, more rapid under UV-B radiation than solar simulator, followed a first-order reaction (photolysis half-lives ranged between 3 and 50 h) and appeared strongly dependent on water origin, pH value and molecular structure of the herbicide. Dissolved organic matter showed a retarding effect while low concentrations of nitrate ions had no effect on photolysis rate. Identification of photoproducts indicated that hydrolysis, a pH-dependent process (no degradation at pH >6 but at pH=3, k=0.0344 h(-1)), could be photoassisted. These results were compared to those of mesotrione, another triketone herbicide, which appeared more stable under UV-B irradiation. Toxicological studies on two marine heterotrophic bacteria and one cyanobacterium showed absence of effects up to 100 microgL(-1) for both sulcotrione and its photoproducts.

A survey on bacteria inhabiting the sea surface microlayer of coastal ecosystems.

Bacterial populations inhabiting the sea surface microlayer from two contrasted Mediterranean coastal stations (polluted vs. oligotrophic) were examined by culturing and genetic fingerprinting methods and were compared with those of underlying waters (50 cm depth), for a period of two years. More than 30 samples were examined and 487 strains were isolated and screened. Proteobacteria were consistently more abundant in the collection from the pristine environment whereas Gram-positive bacteria (i.e., Actinobacteria and Firmicutes) were more abundant in the polluted site. Cythophaga-Flavobacter-Bacteroides (CFB) ranged from 8% to 16% of total strains. Overall, 22.5% of the strains showed a 16S rRNA gene sequence similarity only at the genus level with previously reported bacterial species and around 10.5% of the strains showed similarities in 16S rRNA sequence below 93% with reported species. The CFB group contained the highest proportion of unknown species, but these also included Alpha- and Gammaproteobacteria. Such low similarity values showed that we were able to culture new marine genera and possibly new families, indicating that the sea-surface layer is a poorly understood microbial environment and may represent a natural source of new microorganisms. Genetic fingerprinting showed, however, no consistent differences between the predominant bacterial assemblages from surface microlayer and underlying waters, suggesting that the presence of a stable and abundant neustonic bacterial community is not a common trait of coastal marine environments.

Resistance of marine bacterioneuston to solar radiation.

A total of 90 bacterial strains were isolated from the sea surface microlayer (i.e., bacterioneuston) and underlying waters (i.e., bacterioplankton) from two sites of the northwestern Mediterranean Sea. The strains were identified by sequence analysis, and growth recovery was investigated after exposure to simulated solar radiation. Bacterioneuston and bacterioplankton isolates were subjected to six different exposure times, ranging from 0.5 to 7 h of simulated noontime solar radiation. Following exposure, the growth of each isolate was monitored, and different classes of resistance were determined according to the growth pattern. Large interspecific differences among the 90 marine isolates were observed. Medium and highly resistant strains accounted for 41% and 22% of the isolates, respectively, and only 16% were sensitive strains. Resistance to solar radiation was equally distributed within the bacterioneuston and bacterioplankton. Relative contributions to the highly resistant class were 43% for gamma-proteobacteria and 14% and 8% for alpha-proteobacteria and the Cytophaga/Flavobacterium/Bacteroides (CFB) group, respectively. Within the gamma-proteobacteria, the Pseudoalteromonas and Alteromonas genera appeared to be highly resistant to solar radiation. The majority of the CFB group (76%) had medium resistance. Our study further provides evidence that pigmented bacteria are not more resistant to solar radiation than nonpigmented bacteria.