Distinctive Patterns in the Taxonomical Resolution of Bacterioplankton in the Sediment and Pore Waters of Contrasted Freshwater Lakes.

Bacteria assemblages in lake sediments play a key role in various biogeochemical processes, yet their association with interstitial pore waters has been scarcely investigated. In this study, we utilized Illumina next-generation amplicon sequencing of the 16S rRNA gene to characterize the seasonal bacterial communities in the sediments and pore waters of three contrasted temperate freshwater lakes, namely Pavin, Aydat, and Grangent (French Massif Central). Despite occupying seemingly similar habitats, bacterial communities differed substantially between sediments and pore waters at all seasons with low sharing of operational taxonomic units (OTUs, 6.7 to 20.3%) between them. Sediment-associated bacteria were more rich and diverse than pore water bacteria, indicating a high heterogeneity in the sediment microhabitat. The changes in both sediment and pore water bacterial communities were lake and season specific. The bacterial community showed distinct differences between the lakes, with larger presence of strict anaerobes such as Syntrophus, Syntrophorhabdus, and Sulfuricurvum in the pore water and sediments of Pavin responsible for carbon and sulfur cycling. In both Aydat and Grangent, the hgcI_clade dominated throughout the study period in the pore waters. The higher representation of lesser-known transient members of lake communities such as Methylotenera in the pore waters of Aydat, and Clostridium and Sulfuricurvum in the pore and sediments of Grangent, respectively, were observed during the period of temporary anoxia in summer caused by lake stratification. Our study revealed that in the investigated lakes, the prevailing environmental factors across time and space structured and influenced the adaptation of bacterial communities to specific ecological niches.

Taxonomical Resolution and Distribution of Bacterioplankton Along the Vertical Gradient Reveals Pronounced Spatiotemporal Patterns in Contrasted Temperate Freshwater Lakes.

We examined the relationship between viruses and co-occurring bacterial communities across spatiotemporal scale in two contrasting freshwater lakes, namely meromictic Lake Pavin and dimictic Lake Aydat (Central France). Next-generation sequencing of 16S rRNA genes suggested distinct patterns in bacterioplankton community composition (BCC) between the lakes over depths and seasons. BCC were generally dominated by members of Actinobacteria, Proteobacteria, and Bacteroidetes covering about 95% of all sequences. Oxygen depletion at the bottom waters in Aydat and existence of permanent anoxia in the monimolimnion of Pavin resulted in the occurrence and dominance of lesser known members of lake communities such as Methylotenera, Methylobacter, Gallionella, Sulfurimonas, and Syntrophus in Pavin and Methylotenera and Sulfuritalea in Aydat. Differences in BCC appeared strongly related to dissolved oxygen concentration, temperature, viral infection, and virus-to-bacteria ratio. UniFrac analysis indicated a clear distinction in BCC when the percentage of viral infected bacterial cells and virus-to-bacteria ratio exceeded a threshold level of 10% and 5, respectively, suggesting a link between viruses and their potential bacterial host communities. Our study revealed that in both the lakes, the prevailing environmental factors across time and space structured and influenced the adaptation of bacterial communities to specific ecological niches.

Functional Responses of Bacterioplankton Diversity and Metabolism to Experimental Bottom-Up and Top-Down Forcings.

We conducted an experimental approach using microcosms to simultaneously examine the functional response of natural freshwater bacterial assemblages to the impact of resources (nutrients) and top-down factors (viruses and grazers) on bacterial physiological state and their community structure. Addition of organic and inorganic nutrients led to the proliferation of high nucleic acid content bacterial cells accompanied by high bacterial growth efficiency (considered as proxy of bacterial carbon metabolism) estimates, suggesting that this subgroup represented the most active fraction of bacterial community and had a high capacity to incorporate carbon into its biomass. However, their rapid growth induced the pressure of viral lytic infection which led to their lysis toward the end of the experiment. In microcosms with flagellates plus viruses, and with viruses alone, the selective removal of metabolically active high nucleic acid cells through viral lysis benefitted the less active low nucleic acid content cells, perhaps via the use of lysis products for its growth and survival. Changes in bacterial physiological state in microcosms were reflected in their community structure which was examined using 16S ribosomal RNA (rRNA) gene sequencing by Illumina's Miseq platform. Chao estimator and Shannon diversity index values suggested that bacterial species richness was highest in the presence of both the top-down factors, indicating a tighter control of bacterioplankton dominants within a relatively stable bacterial community. The increase in bacterial metabolism with nutrient addition followed by subsequent lysis of bacterial dominants indicate that both resources and top-down factors work in concert for the sustenance of stable bacterial communities.

Linking host prokaryotic physiology to viral lifestyle dynamics in a temperate freshwater lake (Lake Pavin, France).

In aquatic ecosystems, fluctuations in environmental conditions and prokaryotic host physiological states can strongly affect the dynamics of viral life strategies. The influence of prokaryote physiology and environmental factors on viral replication cycles (lytic and lysogeny) was investigated from April to September 2011 at three different strata (epi, meta, and hypolimnion) in the mixolimnion of deep volcanic temperate freshwater Lake Pavin (France). Overall, the euphotic region (epi and metalimnion) was more dynamic and showed significant variation in microbial standing stocks, prokaryotic physiological state, and viral life strategies compared to the aphotic hypolimnion which was stable within sampled months. The prokaryotic host physiology as inferred from the nucleic acid content of prokaryotic cells (high or low nucleic acid) was strongly regulated by the chlorophyll concentration. The predominance of the high nucleic acid (HNA) prokaryotes (cells) over low nucleic acid (LNA) prokaryotes (cells) in the spring (HNA/LNA = 1.2) and vice versa in the summer period (HNA/LNA = 0.4) suggest that the natural prokaryotic communities underwent major shifts in their physiological states during investigated time period. The increase in the percentage of inducible lysogenic prokaryotes in the summer period was associated with the switch in the dominance of LNA over HNA cells, which coincided with the periods of strong resource (nutrient) limitation. This supports the idea that lysogeny represents a maintenance strategy for viruses in unproductive or harsh nutrient/host conditions. A negative correlation of percentage of lysogenic prokaryotes with HNA cell abundance and chlorophyll suggest that lysogenic cycle is closely related to prokaryotic cells which are stressed or starved due to unavailability of resources for its growth and activity. Our results provide support to previous findings that changes in prokaryote physiology are critical for the promotion and establishment of lysogeny in aquatic ecosystems, which are prone to constant environmental fluctuations.

Resources drive trade-off between viral lifestyles in the plankton: evidence from freshwater microbial microcosms.

Viruses have evolved different life strategies for coping with environmental challenges and this is a key explanation for their omnipresence in aquatic systems. However, factors that determine the balance between lytic versus lysogenic decision within natural virioplankton are poorly documented, primarily in freshwaters. This study was designed to investigate the experimental short-term (24 h incubation) effects of added organic and inorganic nutrients on the two viral lifestyles in nutrient-depleted freshwater microbial (i.e. <0.8 microm fraction) microcosms, using mitomycin C as prophage inductor agent. In the absence of mitomycin, viral lytic production increased as a functional response to the strong stimulation of bacterial growth rates (0.7-0.8 day(-1)) by the added nutrients, primarily the organic nutrients which appeared scarcer than inorganic nutrients and was related to the sampling period and the geomorphological peculiarities of Lake Pavin. In the presence of mitomycin, temperate phage production (frequency of lysogenically infected bacterial cells, FLC=17-19% of total cells) significantly exceeded lytic production (frequency of lytically infected bacterial cells, FIC=9-11%) in natural samples (i.e. without nutrient additions) as a result of enhanced prophage induction, which relatively increased with the decreasing contact probability between viruses and their potential hosts. In contrast, addition of nutrients drastically reduced FLC (<4%) and increased FIC (>22%). Both variables were antagonistically correlated as was the correlation between FLC and bacterial growth rates, supporting the idea that lysogeny may represent a maintenance strategy for viruses in harsh nutrient/host conditions which appeared as major instigators of the trade-off between the two viral lifestyles. Overall, at the community level, we reject the hypothesis that nutrients but mitomicyn C stimulate temperate phage induction, and retained the hypotheses that nutrients rather (i) stimulate lytic viruses via enhanced host growth and (ii) when limiting, promote lysogenic conversion in natural waters.

Differential impact of top-down and bottom-up forces in structuring freshwater bacterial communities.

Limited data exist on the simultaneous impact of bottom-up (nutrients) and top-down (viruses and heterotrophic nanoflagellates) forces in shaping freshwater bacterial communities. In our laboratory microcosms, nutrient additions (organic and inorganic) and viral reduction approach led to the proliferation of high nucleic acid (HNA) bacterial subpopulation without an increase in phage abundance. High viral-mediated bacterial lysis in the presence of nanoflagellates yielded high proportion of low nucleic acid bacterial subpopulation. 16S rRNA gene sequence analysis indicated that members of classes Proteobacteria and Bacteroidetes evoked differential responses to nutrients and mortality forces, thereby resulting in differences (P < 0.001) in bacterial community composition and diversity, as observed from analysis of similarities and UniFrac analysis. Bacterial species richness (Chao) and diversity (Shannon) index was significantly higher (P < 0.001) in the presence of both the top-down factors and viruses alone, whereas lower host diversity was observed under nutrient relaxation of growth-limiting substrates due to the explosive growth of opportunistic HNA bacterial subpopulation. Our results are in agreement with the theoretical model of 'killing the winner', where the availability of growth-limiting substrates can act as a stimulating factor for host community composition while top-down forces can operate in the control of host diversity.

Viral and grazer regulation of prokaryotic growth efficiency in temperate freshwater pelagic environments.

In aquatic systems, limited data exists on the impact of mortality forces such as viral lysis and flagellate grazing when seeking to explain factors regulating prokaryotic metabolism. We explored the relative influence of top-down factors (viral lysis and heterotrophic nanoflagellate grazing) on prokaryotic mortality and their subsequent impact on their community metabolism in the euphotic zone of 21 temperate freshwater lakes located in the French Massif Central. Prokaryotic growth efficiency (PGE, index of prokaryotic community metabolism) determined from prokaryotic production and respiration measurements varied from 5 to 74% across the lakes. Viral and potential grazer-induced mortality of prokaryotes had contrasting impact on PGE. Potential flagellate grazing was found to enhance PGE whereas viral lysis had antagonistic impacts on PGE. The average PGE value in the grazing and viral lysis dominated lake water samples was 35.4% (±15.2%) and 17.2% (±8.1%), respectively. Selective viral lysis or flagellate grazing on prokaryotes together with the nature of contrasted substrates released through mortality processes can perhaps explain for the observed variation and differences in PGE among the studied lakes. The influences of such specific top-down processes on PGE can have strong implications on the carbon and nutrient fluxes in freshwater pelagic environments.

Bacterial growth efficiency in a tropical estuary: seasonal variability subsidized by allochthonous carbon.

Bacterial growth efficiency (BGE) is a key factor in understanding bacterial influence on carbon flow in aquatic ecosystems. We report intra-annual variability in BGE, and bacteria-mediated carbon flow in the tropical Mandovi and Zuari estuaries (southwest India) and the adjoining coastal waters (Arabian Sea). BGE ranged from 3% to 61% and showed clear temporal variability with significantly (ANOVA, p < 0.01) higher values in the estuaries (mean, 28 +/- 14%) than coastal waters (mean, 12 +/- 6%). The greater variability of BGE in the estuaries than coastal waters suggest some systematic response to nutrient composition and the variability of dissolved organic matter pools, as BGE was governed by bacterial secondary production (BP). Monsoonal rains and its accompanied changes brought significant variability in BGE and bacterial productivity/primary productivity (BP/PP) ratio when compared to nonmonsoon seasons in the estuaries and coastal waters. High BP/PP ratio (>1) together with high carbon flux through bacteria (>100% of primary productivity) in the estuarine and coastal waters suggests that bacterioplankton consumed dissolved organic carbon in excess of the amount produced in situ by phytoplankton of this region, which led to the mismatch between primary production of carbon and amount of carbon consumed by bacteria. Despite the two systems being subsidized by allochthonous inputs, the low BGE in the coastal waters may be attributable to the nature and time interval in the supply of allochthonous carbon.

Phage bacteriolysis, protistan bacterivory potential, and bacterial production in a freshwater reservoir: coupling with temperature.

Phage abundance and infection of bacterioplankton were studied from March to November 2003 in the Sep Reservoir (Massif Central, France), together with temperature, chlorophyll, bacteria (abundance and production), and heterotrophic nanoflagellates (abundance and potential bacterivory). Virus abundance (VA) ranged from 0.6 to 13 x 10(10) viruses l(-1), exceeding bacterial abundance (BA) approximately sixfold on average. In terms of carbon, viruses corresponded to up to 25% of bacterial biomass. A multiple regression model indicated that BA was the best predictor for VA (R(2) = 0.75). The frequency of infected bacteria (estimated from the percentage of visibly infected cells) varied from 1% to 32% and was best explained by a combination of temperature (R(2) = 0.20) and bacterial production (R(2) = 0.25). Viruses and flagellates contributed about equally to bacterial mortality. Both factors destroyed 55% of bacterial production, with a shift from phage bacteriolysis in early spring to protistan bacterivory in late summer. The vertical differences in most of the biological variables were not significant, contrasting with the seasonal differences (i.e., spring vs. summer-autumn). All biological variables under study were indeed significantly coupled to temperature. We regarded this to be the consequence of the enhanced discharge of the reservoir in 2003 (compared to previous years). This substantially weakened the stability and the thermal inertia of the water column, thereby establishing temperature as a stronger forcing factor in setting the conditions for optimal metabolic activity of microbial communities.