Diversity and Evolution of Pigment Types in Marine Synechococcus Cyanobacteria.

Synechococcus cyanobacteria are ubiquitous and abundant in the marine environment and contribute to an estimated 16% of the ocean net primary productivity. Their light-harvesting complexes, called phycobilisomes (PBS), are composed of a conserved allophycocyanin core, from which radiates six to eight rods with variable phycobiliprotein and chromophore content. This variability allows Synechococcus cells to optimally exploit the wide variety of spectral niches existing in marine ecosystems. Seven distinct pigment types or subtypes have been identified so far in this taxon based on the phycobiliprotein composition and/or the proportion of the different chromophores in PBS rods. Most genes involved in their biosynthesis and regulation are located in a dedicated genomic region called the PBS rod region. Here, we examine the variability of gene content and organization of this genomic region in a large set of sequenced isolates and natural populations of Synechococcus representative of all known pigment types. All regions start with a tRNA-PheGAA and some possess mobile elements for DNA integration and site-specific recombination, suggesting that their genomic variability relies in part on a "tycheposon"-like mechanism. Comparison of the phylogenies obtained for PBS and core genes revealed that the evolutionary history of PBS rod genes differs from the core genome and is characterized by the co-existence of different alleles and frequent allelic exchange. We propose a scenario for the evolution of the different pigment types and highlight the importance of incomplete lineage sorting in maintaining a wide diversity of pigment types in different Synechococcus lineages despite multiple speciation events.

Resource availability drives bacterial succession during leaf-litter decomposition in a bromeliad ecosystem.

Despite the growing number of investigations on microbial succession during the last decade, most of our knowledge on primary succession of bacteria in natural environments comes from conceptual models and/or studies of chronosequences. Successional patterns of litter-degrading bacteria remain poorly documented, especially in undisturbed environments. Here we conducted an experiment with tank bromeliads as natural freshwater microcosms to assess major trends in bacterial succession on two leaf-litter species incubated with or without animal exclusion. We used amplicon sequencing and a co-occurrence network to assess changes in bacterial community structure according to treatments. Alpha-diversity and community complexity displayed the same trends regardless of the treatments, highlighting that primary succession of detrital-bacteria is subject to resource limitation and biological interactions, much like macro-organisms. Shifts in bacterial assemblages along the succession were characterized by an increase in uncharacterized taxa and potential N-fixing bacteria, the latter being involved in positive co-occurrence between taxa. These findings support the hypothesis of interdependence between taxa as a significant niche-based process shaping bacterial communities during the advanced stage of succession.

Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea.

To test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacterial amoA transcripts. Our results showed a succession of different nitrifiers from river to sea with bacterial amoA transcripts dominating in the freshwater station while archaeal transcripts were predominant in the marine station. The 16S rRNA sequence analysis revealed that Thaumarchaeota marine group I (MGI) were the most abundant overall but other archaeal groups like Methanosaeta were also potentially active in winter (December-March) and Euryarchaeota marine group II (MGII) were dominant in seawater in summer (April-August). Each station also contained different Thaumarchaeota MGI phylogenetic clusters, and the clusters' microdiversity was associated to specific environmental conditions suggesting the presence of ecotypes adapted to distinct ecological niches. The amoA and ureC transcript dynamics further indicated that some of the Thaumarchaeota MGI subclusters were involved in ammonia oxidation through the hydrolysis of urea. Our findings show that ammonia-oxidizing Archaea and Bacteria were adapted to contrasted conditions and that the Thaumarchaeota MGI diversity probably corresponds to distinct metabolisms or life strategies.

Temporal dynamics of active Archaea in oxygen-depleted zones of two deep lakes.

Deep lakes are of specific interest in the study of archaeal assemblages as chemical stratification in the water column allows niche differentiation and distinct community structure. Active archaeal community and potential nitrifiers were investigated monthly over 1 year by pyrosequencing 16S rRNA transcripts and genes, and by quantification of archaeal amoA genes in two deep lakes. Our results showed that the active archaeal community patterns of spatial and temporal distribution were different between these lakes. The meromictic lake characterized by a stable redox gradient but variability in nutrient concentrations exhibited large temporal rearrangements of the dominant euryarchaeal phylotypes, suggesting a variety of ecological niches and dynamic archaeal communities in the hypolimnion of this lake. Conversely, Thaumarchaeota Marine Group I (MGI) largely dominated in the second lake where deeper water layers exhibited only short periods of complete anoxia and constant low ammonia concentrations. Investigations conducted on archaeal amoA transcripts abundance suggested that not all lacustrine Thaumarchaeota conduct the process of nitrification. A high number of 16S rRNA transcripts associated to crenarchaeal group C3 or the Miscellaneous Euryarchaeotic Group indicates the potential for these uncharacterized groups to contribute to nutrient cycling in lakes.

Bacterial communities associated with Microcystis colonies differ from free-living communities living in the same ecosystem.

The search for a better understanding of why cyanobacteria often dominate phytoplankton communities in eutrophic freshwater ecosystems has led to a growing interest in the interactions between cyanobacteria and bacteria. Against this background, we studied the location of bacteria within Microcystis colonies, and compared the structural and phylogenetic diversity of Microcystis-attached and free-living bacterial communities living in the same French lake, the Villerest reservoir. Using transmission electron microscopy, we show that most of the bacteria inside the colonies were located close to detrital materials that probably resulted from lysis of Microcystis cells. The 16S rRNA sequencing approach revealed a clear distinction between the attached and free-living communities at the levels of both their general structure and their operational taxonomic unit (OTU) composition. In particular, Microcystis colonies appeared to be depleted of Actinobacteria, but conversely enriched in Gammaproteobacteria, in particular when the bloom was declining. At the OTU level, a clear distinction was also found between attached and free-living bacteria, and new clades were identified among our sequences. All these findings suggest that Microcystis colonies constitute a distinct habitat for bacteria living in freshwater ecosystems, and that direct and indirect interactions (cell lysis, nutrient recycling, etc.) may occur between them inside these colonies.

Structure of the rare archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters.

Marine Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, but details regarding their community structure and long-term seasonal activity and dynamics remain largely unexplored. In this study, we monitored the interannual archaeal community composition of abundant and rare biospheres in northwestern Mediterranean Sea surface waters by pyrosequencing 16S rDNA and rRNA. A detailed analysis of the rare biosphere structure showed that the rare archaeal community was composed of three distinct fractions. One contained the rare Archaea that became abundant at different times within the same ecosystem; these cells were typically not dormant, and we hypothesize that they represent a local seed bank that is specific and essential for ecosystem functioning through cycling seasonal environmental conditions. The second fraction contained cells that were uncommon in public databases and not active, consisting of aliens to the studied ecosystem and representing a nonlocal seed bank of potential colonizers. The third fraction contained Archaea that were always rare but actively growing; their affiliation and seasonal dynamics were similar to the abundant microbes and could not be considered a seed bank. We also showed that the major archaeal groups, Thaumarchaeota marine group I and Euryarchaeota group II.B in winter and Euryarchaeota group II.A in summer, contained different ecotypes with varying activities. Our findings suggest that archaeal diversity could be associated with distinct metabolisms or life strategies, and that the rare archaeal biosphere is composed of a complex assortment of organisms with distinct histories that affect their potential for growth.

Dynamics of ammonia-oxidizing Archaea and Bacteria in contrasted freshwater ecosystems.

Thaumarchaeota have been recognized as the main drivers of aerobic ammonia oxidation in many ecosystems. However, little is known about the role of ammonia-oxidizing Archaea (AOA) and Bacteria (AOB) in lacustrine ecosystems. In this study, the photic zone of three contrasted freshwater ecosystems located in France was sampled during two periods: winter homothermy (H) and summer thermal stratification (TS), to investigate the distribution of planktonic AOA and AOB. We showed that AOB were predominant in nutrient-rich ecosystems, whereas AOA dominated when ammonia concentrations were the lowest and during winter, which could provide a favorable environment for their growth. Moreover, analyses of archaeal libraries revealed the ubiquity of the thaumarchaeal I.1a clade associated with higher diversity of AOA in the most nutrient-poor lake. More generally, this work assesses the presence of AOA in lakes, but also highlights the existence of clades typically associated with lacustrine and hot spring ecosystems and specific ecological niches occupied by these microorganisms.

Temporal dynamics and phylogenetic diversity of free-living and particle-associated Verrucomicrobia communities in relation to environmental variables in a mesotrophic lake.

In the present study, the abundance and phylogenetic diversity of free-living and particle-associated Verrucomicrobia were investigated in a mesotrophic lake by quantitative PCR and sequencing of the 16S rRNA gene. The relative verrucomicrobial 16S rRNA gene abundance accounted for 0.02% to 1.98% of the particle-associated bacteria and 0.52% to 1.64% of the free-living bacteria. In total, 71 operational taxonomic units (OTUs) (n = 303 clones) were identified for particle-associated bacteria, and 59 OTUs (n = 292 clones) were identified for the free-living fraction. This study determined six new putative freshwater Verrucomicrobia clusters. Of these newly defined clusters, two were exclusively represented by particle-associated bacteria (FukuS27, BourFIV). The freshwater Verrucomicrobia clusters CRE-PA29, FukuN18 and CL120-10 appeared to be dominant, comprising 22.3%, 16.15% and 14.61% of the total retrieved OTUs, respectively. The seasonal dynamics of phytoplankton communities resulted in changes in the distinct bacterial phylotypes for both the particle-associated and free-living verrucomicrobial communities. According to canonical correspondence analysis, the diversity of the particle-associated verrucomicrobial communities appeared to be primarily influenced by phytoplankton richness, rotifer abundance and inorganic nutrients, whereas the free-living fraction was correlated with the biomass dynamics of some phytoplankton classes (Chlorophyceae, Chrysophaceae, Desmidiaceae and Zygnemataceae).

Diversity and dynamics of free-living and particle-associated Betaproteobacteria and Actinobacteria in relation to phytoplankton and zooplankton communities.

The diversity of attached and free-living Actinobacteria and Betaproteobacteria, based on 16S rRNA gene sequences, was investigated in a mesotrophic lake during two periods of contrasting phytoplankton dominance. Comparison analyses showed a phylogenetic difference between attached and free-living communities for the two bacterial groups. For Betaproteobacteria, the betaI clade was detected at all sampling dates in free-living and attached bacterial communities and was the dominant clade contributing to 57.8% of the total retrieved operational taxonomic units (OTUs). For Actinobacteria, the acIV cluster was found to be dominant, followed by acI contributing to 45% and 25% of the total retrieved OTUs, respectively. This study allows the determination of eight new putative clades among the Betaproteobacteria termed lbI-lbVIII and a new putative clade named acLBI belonging to the Actinobacteria. The seasonal dynamics of phytoplankton and zooplankton communities have been reflected as changes in distinct bacterial phylotypes for both attached and free-living communities. For attached communities, relationships were observed between Actinobacteria and Chrysophyceae, and between Betaproteobacteria and Dinophyceae and Chlorophyceae biomass. On the other hand, within free-living communities, few actinobacterial clades were found to be dependent on either nutrients or phytoplankton communities, whereas Betaproteobacteria were mainly associated with biological parameters (i.e. phytoplankton and copepod communities).

Metaproteomic and metagenomic analyses of defined oceanic microbial populations using microwave cell fixation and flow cytometric sorting.

A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems.

Diel rhythmicity in amino acid uptake by Prochlorococcus.

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and (35)S-methionine and (3)H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10(4) times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G(2) cell cycle stage were consistently 2.2 times higher than those of cells at the G(1) stage. Furthermore, S+G(2) cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean.

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.

Microbial control of phosphate in the nutrient-depleted North Atlantic subtropical gyre.

Little is known about the dynamics of dissolved phosphate in oligotrophic areas of the world's oceans, where concentrations are typically in the nanomolar range. Here, we have budgeted phosphate uptake by the dominant microbial groups in order to assess the effect of the microbial control of this depleted nutrient in the North Atlantic gyre. Low concentrations (2.2 +/- 1.2 nM) and rapid microbial uptake (2.1 +/- 2.4 nM day(-1)) of bioavailable phosphate were repeatedly determined in surface waters of the North Atlantic oligotrophic gyre during spring and autumn research cruises, using a radiotracer dilution bioassay technique. Upper estimates of the concentration of bioavailable phosphate were 7-55% of the dissolved mineral phosphate suggesting that a considerable part of the chemically measured nanomolar phosphate was in a form unavailable for direct microbial uptake. A 1:1 relationship (r(2) = 0.96, P < 0.0001) was observed between the bioavailable total phosphate uptake and the phosphate uptake of all the flow sorted bacterioplankton cells, demonstrating that bacterioplankton were the main consumers of phosphate. Within the bacterioplankton a group of heterotrophic bacteria and Prochlorococcus phototrophic cyanobacteria, were the two major competing groups for bioavailable phosphate. These heterotrophic bacteria had low nucleic acid content and 60% of them comprised of SAR11 clade cells based on the results of fluorescence in situ hybridization. Each of the two competing bacterial groups was responsible for an average of 45% of the phosphate uptake, while Synechococcus cyanobacteria (7%) and picoplanktonic algae (0.3%) played minor roles in direct phosphate uptake. We have demonstrated that phosphate uptake in the oligotrophic gyre is rapid and dominated by two bacterial groups rather than by algae.

Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313.

Cyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low- to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.

Two-component systems in Prochlorococcus MED4: genomic analysis and differential expression under stress.

Two-component signal transduction systems, composed of histidine sensory kinases and response regulators, constitute a key element of the mechanism by which bacteria sense and acclimatize to changes in their environment. The availability of whole genome sequences permits a detailed analysis of these genes in cyanobacteria. In the present paper, we focus mainly on Prochlorococcus MED4, a strain adapted to surface oceanic conditions, for which six putative response regulators (rer) and five putative histidine kinases (hik) were identified. These numbers are comparable to those found in the other marine picocyanobacteria but much lower than those found in freshwater cyanobacteria. Moreover, the diversity of these genes is low in Prochlorococcus since most histidine kinases are related to a single group (type I) and most response regulators to a single family (OmpR). Under standard conditions, quantitative reverse transcription polymerase chain reaction revealed that one hik (hik03) and two rer (rer04 and rer05) genes were expressed at relatively high levels compared to the other two-component system genes. In response to high light exposure, a moderate increase (>5-fold) was observed in the expression of some putative rer genes (rer01, rer04, rer05, and rer06), whereas a smaller increase (<3-fold) in hik03 and hik04 mRNA levels was detected. In contrast, both cold and heat shocks decreased rather than increased the expression of most hik and rer genes.