Stéphan Jacquet

Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget.

ABSTRACT The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.

DIEL PATTERNS OF GROWTH AND DIVISION IN MARINE PICOPLANKTON IN CULTURE

The effect of a 12:12‐h light:dark (LD) cycle on the phasing of several cell parameters was explored in a variety of marine picophytoplanktonic strains. These included the photosynthetic prokaryotes Prochlorococcus (strains MED 4, PCC 9511, and SS 120) and Synechococcus (strains ALMO 03, ROS 04, WH 7803, and WH 8103) and five picoeukaryotes (Bathycoccus prasinos Eikrem et Throndsen, Bolidomonas pacifica Guillou et Chrétiennot‐Dinet, Micromonas pusilla Manton et Parke, Pelagomonas calceolata Andersen et Saunders, and Pycnococcus provasolii Guillard et al.). Flow cytometric analysis was used to determine the relationship between cell light scatter, pigment fluorescence, DNA (when possible), and the LD cycle in these organisms. As expected, growth and division were tightly coupled to the LD cycle for all of these strains. For both Prochlorococcus and picoeukaryotes, chl and intracellular carbon increased throughout the light period as estimated by chl fluorescence and light scatter, respectively. In response to cell division, these parameters decreased regularly during the early part of the dark period, a decrease that either continued throughout the dark period or stopped for the second half of the dark period. For Synechococcus, the decrease of chl and scatter occurred earlier (in the middle of the light period), and for some strains these cellular parameters remained constant throughout the dark period. The timing of division was very similar for all picoeukaryotes and occurred just before the subjective dusk, whereas it was more variable between the different Prochlorococcus and Synechococcus strains. The burst of division for Prochlorococcus SS 120 and PCC 9511 was recorded at the subjective dusk, whereas the MED 4 strain divided later at night. Synechococcus ALMO 03, ROS 04, and WH 7803, which have a low phycourobilin to phycoerythrobilin (PUB:PEB) ratio, divided earlier, and their division was restricted to the light period. In contrast, the high PUB:PEB Synechococcus strain WH 8103 divided preferentially at night. There was a weak linear relationship between the FALSmax:FALSmin ratio and growth rate calculated from cell counts (r = 0.83, n = 11, P < 0.05). Because of the significance of picoplanktonic populations in marine systems, these results should help to interpret diel variations in oceanic optical properties in regions where picoplankton dominates.

Cell Cycle Regulation by Light in Prochlorococcus Strains

ABSTRACT The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m−2 s−1, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. ShiftingProchlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.

Effects of ultraviolet radiation on marine virus-phytoplankton interactions

Abstract Ambient ultraviolet radiation (UVR) is harmful to many biological systems and increased UVR, due to a reduced ozone layer, may have many unforeseen consequences. Viruses are the most abundant biological particles in the sea and are thought to play an important role in the structure and functioning of aquatic ecosystems. Although an increasing number of studies have been published during the last 15 years, aquatic viral ecology is still in its infancy and little is known about the effect of environmental factors on virus life cycle and host-virus interactions. Using flow cytometry, we have investigated the effect of UVR (UVB intensity: 0.22 W m(-2) and UVA/UVB ratio approximately 30) on five different cultured marine phytoplankton host-virus systems (CeV-Chrysochromulina ericina, EhV-Emiliania huxleyi, MpV-Micromonas pusilla, PpV-Phaeocystis pouchetii and PoV-Pyramimonas orientalis). Viruses appear to be susceptible to UV, but also they might provide some protection to their hosts. It is shown that (i) some of the investigated microalgae that have been co-cultured with viruses are less sensitive (e.g. P. pouchetii, M. pusilla) to UVB stress compared to susceptible microalgae (i.e. virus-free cultures), (ii) different viruses have different sensitivities to UVB in terms of both their abundance patterns (no effect for most of them except EhV) and infectivity (from no effect for PoV, to complete inactivation for PpV), (iii) UVA has no effect on host-virus interactions. Our results show UVB to be a potentially important factor in the regulation of virus-host interactions in surface waters.

Comparative effects of the quality and quantity of light and temperature on the growth of Planktothrix agardhii and P. rubescens1

The effects of temperature, light intensity, and quality on the growth of the cyanobacteria Planktothrix agardhii (Gomont) Anag. et Komárek and P. rubescens (Gomont) Anag. et Komárek were assessed in batch cultures. The relative competitiveness of the green‐pigmented P. agardhii and the red‐pigmented P. rubescens was evaluated in separate and mixed cultures, under different light intensities and qualities (green, red, and white), and at two different temperatures, chosen as representative of the natural conditions favoring the respective blooms of each species. In monocultures, the P. rubescens strain appeared to be particularly well adapted to low intensities of green light and displayed a strong photoinhibition under high irradiance levels. The P. agardhii strain appeared less specialized with regard to light quality and also less sensitive to photoinhibition at higher irradiances. In competition experiments, temperature (15°C vs. 25°C) was the most important parameter in determining relative fitness of the species and competitive success. At 15°C, P. rubescens appeared to be much more competitive than P. agardhii, while P. agardhii was more competitive at 25°C. Under low irradiance, however, the pigmentation of these strains was of primary importance in determining the outcomes of the competition experiments. On the basis of our experimental results and on field observations, we propose that the successful growth leading to the proliferation of these two differently pigmented strains may largely depend on the combined conditions of light and temperature. The two strains, being genetically close relatives, could therefore be considered as two ecotypes that are adapted to different light and temperature environments. Competition experiments showed that the combination of these parameters largely controls the success of one strain in comparison to the other.

Diel Expression of Cell Cycle-Related Genes in Synchronized Cultures of Prochlorococcus sp. Strain PCC 9511

The cell cycle of the chlorophyll b-possessing marine cyanobacterium Prochlorococcus is highly synchronized under natural conditions. To understand the underlying molecular mechanisms we cloned and sequenced dnaA and ftsZ, two key cell cycle-associated genes, and studied their expression. An axenic culture of Prochlorococcus sp. strain PCC 9511 was grown in a turbidostat with a 12 h-12 h light-dark cycle for 2 weeks. During the light periods, a dynamic light regimen was used in order to simulate the natural conditions found in the upper layers of the worlds oceans. This treatment resulted in strong cell cycle synchronization that was monitored by flow cytometry. The steady-state mRNA levels of dnaA and ftsZ were monitored at 4-h intervals during four consecutive division cycles. Both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase. Western blot experiments indicated that the peak of FtsZ concentration occurred at night, i.e., at the time of cell division. Thus, the transcript accumulation of genes involved in replication and division is coordinated in Prochlorococcus sp. strain PCC 9511 and might be crucial for determining the timing of DNA replication and cell division.

Microbial Community Structure and Dynamics in the Largest Natural French Lake (Lake Bourget)

We investigated the dynamics and diversity of heterotrophic bacteria, autotrophic and heterotrophic flagellates, and ciliates from March to July 2002 in the surface waters (0–50 m) of Lake Bourget. The heterotrophic bacteria consisted mainly of “small” cocci, but filaments (>2 μm), commonly considered to be grazing-resistant forms under increased nanoflagellate grazing, were also detected. These elongated cells mainly belonged to the Cytophaga-Flavobacterium (CF) cluster, and were most abundant during spring and early summer, when mixotrophic or heterotrophic flagellates were the main bacterial predators. The CF group strongly dominated fluorescent in situ hybridization–detected cells from March to June, whereas clear changes were observed in early summer when Beta-proteobacteria and Alpha-proteobacteria increased concomitantly with maximal protist grazing pressures. The analysis of protist community structure revealed that the flagellates consisted mainly of cryptomonad forms. The dynamics of Cryptomonas sp. and Dinobryon sp. suggested the potential importance of mixotrophs as consumers of bacteria. This point was verified by an experimental approach based on fluorescent microbeads to assess the potential grazing impact of all protist taxa in the epilimnion. From the results, three distinct periods in the functioning of the epilimnetic microbial loop were identified. In early spring, mixotrophic and heterotrophic flagellates constituted the main bacterivores, and were regulated by the availability of their resources mainly during April (phase 1). Once the “clear water phase” was established, the predation pressure of metazooplankton represented a strong top-down force on all microbial compartments. During this period only mixotrophic flagellates occasionally exerted a significant bacterivory pressure (phase 2). Finally, the early summer was characterized by the highest protozoan grazing impact and by a rapid shift in the carbon pathway transfer, with a fast change-over of the main predators contribution, i.e., mixotrophic, heterotrophic flagellates and ciliates in bacterial mortality. The high abundance of ciliates during this period was consistent with the high densities of resources (heterotrophic nanoflagellates, algae, bacteria) in deep layers containing the most chlorophyll. Bacteria, as ciliates, responded clearly to increasing phytoplankton abundance, and although bacterial grazing impact could vary largely, bacterial abundance seemed to be primarily bottom-up regulated (phase 3).

Viral abundance, production, decay rates and life strategies (lysogeny versus lysis) in Lake Bourget (France).

We have investigated the ecology of viruses in Lake Bourget (France) from January to August 2008. Data were analysed for viral and bacterial abundance and production, viral decay, frequency of lysogenic cells, the contribution of bacteriophages to prokaryotic mortality and their potential influence on nutrient dynamics. Analyses and experiments were conducted on samples from the epilimnion (2 m) and the hypolimnion (50 m), taken at the reference site of the lake. The abundance of virus-like particles (VLP) varied from 3.4 × 10⁷to 8.2 × 10⁷ VLP ml⁻¹; with the highest numbers and virus-to-bacterium ratio (VBR = 69) recorded in winter. Viral production varied from 3.2 × 10⁴ VLP ml⁻¹  h⁻¹ (July) to 2 × 10⁶ VLP ml⁻¹ h⁻¹ (February and April), and production was lower in the hypolimnion. Viral decay rate reached 0.12-0.15 day⁻¹, and this parameter varied greatly with sampling date and methodology (i.e. KCN versus filtration). Using transmission electron microscopy (TEM) analysis, viral lysis was responsible for 0% (January) to 71% (February) of bacterial mortality, while viral lysis varied between 0% (April) and 53% (January) per day when using a modified dilution approach. Calculated from viral production and burst size, the virus-induced bacterial mortality varied between 0% (January) and 68% (August). A weak relationship was found between the two first methods (TEM versus dilution approach). Interestingly, flow cytometry analysis performed on the dilution experiment samples revealed that the viral impact was mostly on high DNA content bacterial cells whereas grazing, varying between 8.3% (June) and 75.4% (April), was reflected in both HDNA and LDNA cells equally. The lysogenic fraction varied between 0% (spring/summer) and 62% (winter) of total bacterial abundance, and increased slightly with increasing amounts of mitomycin C added. High percentages of lysogenic cells were recorded when bacterial abundance and activity were the lowest. The calculated release of carbon and phosphorus from viral lysis reached up to 56.5 µgC l⁻¹ day⁻¹ (assuming 20 fgC cell⁻¹) and 1.4 µgP l⁻¹ day⁻¹ (assuming 0.5 fgP cell⁻¹), respectively, which may represent a significant fraction of bacterioplankton nutrient demand. This study provides new evidence of the quantitative and functional importance of the virioplankton in the functioning of microbial food webs in peri-alpine lakes. It also highlights methodologically dependent results.

Flow cytometry sorting of freshwater phytoplankton

We used the flow sorting capacities of a benchtop FACSCalibur flow cytometer to analyze the phytoplankton community of four different aquatic ecosystems. We show that despite the high optical, mechanistic, and hydrodynamic stress for the cells while sorted, most of the targeted populations could be isolated and grew in mixed culture media subsequent to sorting. Forty-five phytoplankton taxa were isolated, including green algae (29 species), cyanobacteria (eight), diatoms (seven), and cryptomonads (one). The isolation success average was high since 80% of the total sorted populations grew successfully and 47% constituted monocultures. It is noteworthy, however, that some groups could not be isolated, as for example colonial cyanobacteria, chrysophytes, euglenophytes, desmids, or dinoflagellates, and some species such as Cryptomonas sp. were very sensitive to the sorting process. It is proposed that flow cytometric analysis of freshwater phytoplankton might be a relevant tool for water managers and could be applied in some specific cases, such as early monitoring of blooming taxa or basic bio-monitorings of key species. The higher isolation average obtained from the flow sorting can also be powerful for the physiological or molecular study of some taxa after their cultivation.

Trophic interactions between viruses, bacteria and nanoflagellates under various nutrient conditions and simulated climate change

Population dynamics in the microbial food web are influenced by resource availability and predator/parasitism activities. Climatic changes, such as an increase in temperature and/or UV radiation, can also modify ecological systems in many ways. A series of enclosure experiments was conducted using natural microbial communities from a Mediterranean lagoon to assess the response of microbial communities to top-down control [grazing by heterotrophic nanoflagellates (HNF), viral lysis] and bottom-up control (nutrients) under various simulated climatic conditions (temperature and UV-B radiations). Different biological assemblages were obtained by separating bacteria and viruses from HNF by size fractionation which were then incubated in whirl-Pak bags exposed to an increase of 3°C and 20% UV-B above the control conditions for 96 h. The assemblages were also provided with an inorganic and organic nutrient supply. The data show (i) a clear nutrient limitation of bacterial growth under all simulated climatic conditions in the absence of HNF, (ii) a great impact of HNF grazing on bacteria irrespective of the nutrient conditions and the simulated climatic conditions, (iii) a significant decrease in burst size (BS) (number of intracellular lytic viruses per bacterium) and a significant increase of VBR (virus to bacterium ratio) in the presence of HNF, and (iv) a much larger temperature effect than UV-B radiation effect on the bacterial dynamics. These results show that top-down factors, essentially HNF grazing, control the dynamics of the lagoon bacterioplankton assemblage and that short-term simulated climate changes are only a secondary effect controlling microbial processes.