Bacterial single-cell approaches to the relationship between diversity and fucntion in the sea

We have measured several variables related to the C and S cycling in a coastal Mediterranean environment, Blanes Bay. The values of the variables considered are chlorophyll, phytoplankton biomass, primary production (particulate and dissolved), bacterial production, bacterial respiration, glucose incorporation, grazing on bacteria and determination of the nutrient limiting bacterial growth. For the S cycle we have focussed in the production, incorporation and turnover of DMS and DMSP, with also the determination of DMSO.

Information in GenBank on 16S rDNA sequences of cultured and uncultured marine bacteria has been synthesized in a geographic database showing the worldwide distribution of 16S rDNA sequences obtained by the scientific community so far. The database is publicly available via http://www.icm.csic.es/bio/projects/basics/ and is a valuable tool for scientists to analyse bacterial diversity and distribution in European and Global marine waters.

The role of the activity of microorganisms on carbon biogeochemistry was investigated during a 1.5 year period in the Bay of Villefranche. DOC concentrations ranged from 56 to 108 µmol per liter. DOC concentration was lowest in winter and increased during the after the spring bloom, which was therefore as expected in the relative oligotrophic and deep bay the major source of organic matter. The ratio of gross primary production (GPP) to respiration was > 1 during the first half year of the study period and < 1 afterwards. This indicates a shift from autotrophy to heterotrophy of the system in the study period with no clear seasonal pattern. One of the conclusions from this study is that the metabolic balance and its effect on carbon biogeochemsirty are difficult to predict. Across the study period, bacterial production represented between 18% and 70% of gross primary production and reached highest values (ca. 40-70%) during the phytoplankton blooms investigated. There were also differences in bacterial production and respiration between two consecutive blooms. Highest respiration was measured in association to forest fires, which introduced black carbon and nutrients (NO3 and PO4) into the water column. A Sahara dust wet depostion events also caused bacterial respiration to increase by 60-90%. The data suggest that the effects of short-term events such as forest fires, local upwelling, rain fall and Sahara dust events on carbon biogeochemistry have not been appreciated sufficiently.

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells have been analysed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analysed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.

We undertook weekly measurements in relation to sulphur and carbon biogeochemistry at Station L4 (50º15'N, 04º13'W) in waters south of Plymouth in the western English Channel between January 2003 and July 2004. The following variables were determined : water temperature, salinity, fluorescence, chlorophyll a, microsporine-like amino acid (MAA) concentrations, FRRF measurements of photophysiology, phytoplankton biomass, flow cytometric enumeration of nano- and pico-phytoplankton and bacterioplankton, particulate DMSP, dissolved DMSP and DMS. We undertook weekly measurements in relation to sulphur and carbon biogeochemistry at Station L4 (50º15'N, 04º13'W) in waters south of Plymouth in the western English Channel between January 2003 and July 2004. The following variables were determined: water temperature, salinity, fluorescence, chlorophyll a, microsporine-like amino acid (MAA) concentrations, FRRF measurements of photophysiology, phytoplankton biomass, flow cytometric enumeration of nano- and pico-phytoplankton and bacterioplankton, particulate DMSP, dissolved DMSP and DMS.

A protocol is presented for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes (FISH) and signal amplification (catalysed reporter deposition, CARD). The FISH probes are labeled with the enzyme horseradish peroxidase. A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multi-color CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups of bacteria. This method serves as a tool for rapid identification of marine bacteria newly developed during the BASICS project. The protocol provides a standardized approach that allows scientists from different laboratories to produce comparable information on the abundances of different bacterial species in marine waters. It thus serves as a methodological base for a comparative monitoring of microbes in different European Seas. It is the central tool for a dissemination of this method for bacterial single cell identification in specific workshops on FISH planned within the framework of the project and thereafter.

A protocol was optimised for the efficient extraction of DNA from seawater. It was found that a number of different steps in the extraction of DNA from seawater needed optimisation. The protocol will be valuable to researchers working with molecular-based methods in seawater.

We have developed and written a series of protocols for using flow cytometry as a tool to investigate the distribution, abundance and biomass of microorganisms in fresh- and marine waters. The protocols are available through the Basics website, and provide the readers with easy-to-use step by step guidelines.

Dimethylsulphonioproprionate (DMSP) is one of the most fascinating molecules in the oceans. It is principally an osmolyte or compatible solute and is synthesised by a variety of microalgae and several higher plants. DMSP can be broken down by enzymatic cleavage to produce the climatically active gas, dimethyl sulphide (DMS) and acrylate. In the surface oceans, bacteria play an important role in determining how much DMSP is converted to DMS and hence, how much DMS has the potential to enter the atmosphere. Bacteria also regulate the flux of DMS from the ocean to the atmosphere by consuming DMS itself. The most sensitive means of quantifying how much DMSP is utilised by bacteria and transformed to DMS, and of quantifying the consumption rates of DMS by bacteria is to use 35S-radiotracer techniques. 35-S DMSP is not available commercially. We have developed a modification of procedures used by Dr. Andrew Hanson, University of Florida, Gainsville and Dr. Ron Kiene, University of South Alabama to synthesise 35S-DMSP and 35S-DMS to a high purity (>95%), allowing their use as radiotracers of bacterial metabolism. The synthesised radiotracer has been used successfully to determine DMSP and DMS turnover rates by bacterioplankton in the waters of Plymouth Sound, and in the waters off the Iberian Peninsula. The synthesis utilises an amino acid oxidase reaction to convert labelled methionine to methiolpropionate (MTP). MTP is then methylated to DMSP. Purification of the products involves ion exchange purification and HPLC seperation procedures. Typical labelled DMSP- yields from the radiolabelled methionine ranges from 5 to 12%.

The diversity of Bacteria and Archaea was investigated during a 1.5 years study in the Bay of Villefranche and sequence information is available for the phylogenetic affiliation of the detected phylotypes. Between 17 and 30 bands (phylotypes) were detected. Using cluster analysis, phylotypes grouped into specific periods such as phytoplankton bloom, Synechococcus bloom, summer and fall/winter. However, this grouping differed for waters above and below the thermocline. Specific phylotypes responded characteristically to short-term changes such as blooms, local upwellings, heavy rainfall, forest fires and Sahara dust depositions. These results indicate that unpredictable short-term events play a significant and previously not sufficiently considered role for sustaining diversity in coastal microbial food webs. Diversity was not linked to ecosystem functions using conventional analysis such as multiple regression analysis, however, when artificial neural networks were used, bacterial production and respiration were linked strongly but in a non-linearizable way to bacterial diversity. This suggests a complex pattern between diversity and ecosystem function, which has to be considered in assessing the effects of global change.

The method uses the technique of X-Ray Fluorescence (XRF)in transmission electron microscopy to detect the elemental composition of individual micro-organisms. Sample preparation involves no incubation step and the method is therefore thought to be free of the complications related to growth, predation and wall effects in most bio-assays. With a thin-window detector, the biologically light element carbon and nitrogen can be included, and the method thus provides C,N,P content and stoichiometry of individual cells, believed to reflect their nutrient stress. A previously unknown effect of growth conditions on the Mg/Na content of bacteria seems to be related to growth limitation by organic-C. The present working theory is that the production of organic osmolytes is too expensive in C-limited situation, and that an increase in Mg-content somehow is related stabilization of macromolecules and/or production of inorganic compatible solutes under C-limitation. Since the ecosystem states with carbon versus mineral nutrient limitation of heterotrophic bacteria is believed to influence the ability of bacteria to consume DOC, the method has potential applications in studies such as that of the oceanic C-cycle and in studies of organic pollution degradation (e.g. oil spill clean-up).

While molecular methods give information on microbial diversity in terms of number and type of co-existing species, they do not per se give an answer to the question of what controls the diversity and the success of particular species. While traditional competition theory to a large extent has focussed on the importance of being small to be an efficient competitor for dissolved nutrients, we have shown that this is not the case if a non-limiting substrate can be used to increase size. In this case an osmotrop0h organism can gain competitive advantage from becoming large. Since increasing size also has been postulated to protect from heavy predation pressure by microzooplankton, the result is a possible strategy that simultaneously optimizes competition and defense. The theory explains the success of osmotrophic microorganisms such as large bacteria under conditions with excess organic-C and diatoms under conditions with excess Si. The theory thus improves our ability to explain observed patterns in microbialø biodiversity.

An important query in marine microbial ecology is at what temporal and spatial scales variations in the structure of natural bacterial communities occur. Samples of different volumes were collected at different spatial (vertical and horizontal) and temporal (from hours to seasons) scales along a transect between a coastal station (26m depth) and an offshore Microbial Observatory (1000 m depth) in the NW Mediterranean Sea. The structure of the bacterial communities was determined by capillary electrophoresis single strand conformation polymorphism (CE SSCP) fingerprinting of polymerase chain reaction (PCR)-amplified 16S rDNA. This technique is a powerful tool to compare natural microbial assemblages at different spatial or temporal scales. Similar bacterial assemblages were found up to 3.7 km from the coastal station, whereas significant changes were found over greater distances (from 9.3 to 33.3 km). Although the bacterial community structure did not change with depth at coastal and shallow stations, vertical changes were found at deeper stations, most likely due to vertical variations in physico-chemical and biogeochemical parameters. Temporal changes were mainly related to environmental variations that occurred at a seasonal scale and during phytoplankton blooms. Finally, we suggest that long-term studies, at least in the NW Mediterranean Sea, should involve a minimum sampling time scale of 2 wk and a shorter time-scale when environmental changes are detected by the real-time monitoring of a few basic parameters (i.e. fluorescence, temperature, salinity). Sampling strategies should also include different depths depending on the vertical structure of the water column, based on the same basic parameters.

We have described bacterial diversity in Blanes Bay by means of: 4 clon libraries, a DGGE with cutting and sequencing of bands, use of CARD-FISH probes, and the isolation of bacteria in culture. The results are available from the authors as a series of images or as a series of 16SrRNA sequences submitted to GenBank.

We have measured the effect of UV radiation using a single-cell level approach on heterotrophic bacteria from a Mediterranean coastal station. We performed short-term experiments exposing samples to natural light conditions and studied the effects of PAR and UV radiation on bacterioplankton after exposure with the use of cutoff filters. We used a combination of physiological probes and flow cytometry to assess the effects on the activity and integrity of the cells. Heterotrophic bacterial activity measured by CTC reduction decreased after sunlight exposure with a concomitant inhibition on total and cell-specific leucine incorporation. In contrast, no apparent effect was detected on cell viability as assessed by the simultaneous staining with propidium iodine and SybrGreen. We applied the MAR-FISH technique in order to test the sensitivity of the different bacterial groups to the sunlight radiation. Bacteria labelled with the Gamma-Proteobacteria probes appeared to be resistant to the radiation, with no detectable change in their activity after the exposure. On the contrary, the alpha-Proteobacteria bacteria exhibited a higher sensitivity to the radiation decreasing significantly their activity as measured by the cell-specific incorporation of labelled aminoacids and ATP. This trend was recurrent over two different seasonal periods. Our results suggest that UV radiation is an important modulator of short-term changes in single-cell activity of bacterioplankton, and naturally dominating phylogenetically distinct groups of bacteria have different sensitivity to radiation.

This protocol allows to set up a flow cytometer protocol for counting viruses in liquid solutions. The protocol has been developed for a Becton Dickinson FACSCalibur and might thus be modified for other flow cytometers. Note that the settings of the flow cytometer might be slightly different between flow cytometers of the same brand. This method allows for a rapid and precise quantification of viruses. Thus, a large scale assessment of the effect of viruses on bacterial production and diversity and their role in the carbon cycle is possible. A protocol can be obtain upon contact (Markus Weinbauer: wein@obs-vlfr.fr).

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