Cecilia Alonso

Distinct flavobacterial communities in contrasting water masses of the North Atlantic Ocean

Members of the class Flavobacteria in the phylum Bacteroidetes are among the most abundant picoplankton in coastal and polar oceans. Their diversity is high in marine waters. However, quantitative information about distribution patterns of flavobacterial clades is scarce. We analyzed the diversity and clade-specific abundances of individual Flavobacteria in different oceanic provinces in the North Atlantic Ocean. Samples were taken along the 30°W meridian between the East Greenland current and the North Atlantic subtropical gyre. Comparative sequence analysis of 16S ribosomal RNA (rRNA) gene libraries revealed high diversity and significant spatial variability within the class Flavobacteria. Published and newly designed oligonucleotide probes were used to enumerate eleven flavobacterial clades by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). We found that different provinces harbor distinct flavobacterial communities. Clade DE2 accounted for a substantial fraction of total Flavobacteria only in the Polar Biome (BPLR), whereas the VISION clades VIS1 and VIS4 significantly increased in the Arctic (ARCT) province. Members of the genus Polaribacter were the most abundant clade in all the water masses analyzed, with highest absolute numbers in BPLR and ARCT. We improved the CARD-FISH protocol to quantify the rare clades VIS2, VIS3, VIS5 and VIS6, which were present in abundances below 0.5%. They all showed pronounced regional distribution patterns. Microscopic analysis proved a specific enrichment of Flavobacteria in the phycosphere of nanophytoplankton of BPLR and ARCT. Our results suggest that different marine flavobacterial clades have distinct niches and different life strategies.

Incorporation of glucose under anoxic conditions by bacterioplankton from coastal North Sea surface waters.

ABSTRACT It has been hypothesized that the potential for anaerobic metabolism might be a common feature of bacteria in coastal marine waters (L. Riemann and F. Azam, Appl. Environ. Microbiol. 68: 5554-5562, 2002). Therefore, we investigated whether different phylogenetic groups of heterotrophic picoplankton from the coastal North Sea were able to take up a simple carbon source under anoxic conditions. Oxic and anoxic incubations (4 h) or enrichments (24 h) of seawater with radiolabeled glucose were performed in July and August 2003. Bacteria with incorporated substrate were identified by using a novel protocol in which we combined fluorescence in situ hybridization and microautoradiography of cells on membrane filters. Incorporation of glucose under oxic and anoxic conditions was found in α-Proteobacteria, γ-Proteobacteria, and the Cytophaga-Flavobacterium cluster of the Bacteroidetes at both times, but not in marine Euryarchaeota. In July, the majority of cells belonging to the α-proteobacterial Roseobacter clade showed tracer incorporation both in oxic incubations and in oxic and anoxic enrichments. In August, only a minority of the Roseobacter cells, but most bacteria affiliated with Vibrio spp., were able to incorporate the tracer under either condition. A preference for glucose uptake under anoxic conditions was observed for bacteria related to Alteromonas and the Pseudoalteromonas-Colwellia group. These genera are commonly considered to be strictly aerobic, but facultatively fermentative strains have been described. Our findings suggest that the ability to incorporate substrates anaerobically is widespread in pelagic marine bacteria belonging to different phylogenetic groups. Such bacteria may be abundant in fully aerated coastal marine surface waters.

Blooms of single bacterial species in a coastal lagoon of the southwestern Atlantic Ocean.

ABSTRACT We investigated seasonal differences in community structure and activity (leucine incorporation) of the planktonic bacterial assemblage in the freshwater and brackish-water zones of a shallow coastal lagoon of the southwestern Atlantic Ocean. Alphaproteobacteria formed the dominant microbial group in both zones throughout the sampling period. After an intrusion of marine water, members of the SAR11 lineage became abundant in the brackish-water zone. These bacteria were apparently distributed over the lagoon during the following months until they constituted almost 30% of all prokaryotic cells at both sampling sites. At the first sampling date (March 2003) a single alphaproteobacterial species unrelated to SAR11, Sphingomonas echinoides, dominated the microbial assemblages in both zones of the lagoon concomitantly with a bloom of filamentous cyanobacteria. Pronounced maxima of leucine incorporation were observed once in each zone of the lagoon. In the freshwater zone, this highly active microbial assemblage was a mix of the typical bacteria lineages expected in aquatic systems. By contrast, a single bacterial genotype with >99% similarity to the facultative pathogen gammaproteobacterial species Stenotrophomonas maltophilia formed >90% of the bacterial assemblage (>107 cell ml−1) in the brackish-water zone at the time point of highest bacterial leucine incorporation. Moreover, these bacteria were equally dominant, albeit less active, in the freshwater zone. Thus, the pelagic zone of the studied lagoon harbored repeated short-term blooms of single bacterial species. This finding may have consequences for environmental protection.

Concentration-Dependent Patterns of Leucine Incorporation by Coastal Picoplankton

ABSTRACT Coastal pelagic environments are believed to feature concentration gradients of dissolved organic carbon at a microscale, and they are characterized by pronounced seasonal differences in substrate availability for the heterotrophic picoplankton. Microbial taxa that coexist in such habitats might thus differ in their ability to incorporate substrates at various concentrations. We investigated the incorporation patterns of leucine in four microbial lineages from the coastal North Sea at concentrations between 0.1 and 100 nM before and during a spring phytoplankton bloom. Community bulk incorporation rates and the fraction of leucine-incorporating cells in the different populations were analyzed. Significantly fewer bacterial cells incorporated the amino acid before (13 to 35%) than during (23 to 47%) the bloom at all but the highest concentration. The incorporation rate per active cell in the prebloom situation was constant above 0.1 nM added leucine, whereas it increased steeply with substrate concentration during the bloom. At both time points, a high proportion of members of the Roseobacter clade incorporated leucine at all concentrations (55 to 80% and 86 to 94%, respectively). In contrast, the fractions of leucine-incorporating cells increased substantially with substrate availability in bacteria from the SAR86 clade (8 to 31%) and from DE cluster 2 of the Flavobacteria-Sphingobacteria (14 to 33%). The incorporation patterns of marine Euryarchaeota were between these extremes (30 to 56% and 48 to 70%, respectively). Our results suggest that the contribution of microbial taxa to the turnover of particular substrates may be concentration dependent. This may help us to understand the specific niches of coexisting populations that appear to compete for the same resources.

Ecophysiological differences of betaproteobacterial populations in two hydrochemically distinct compartments of a subtropical lagoon

We studied the population sizes and substrate incorporation patterns of three phylogenetic groups of Betaproteobacteria in a coastal subtropical lagoon that is characterized by a sharp transition from humic freshwater to turbid brackish water. Various cellular processes were addressed by short-term incubations with four radiolabelled compounds and microautoradiographic assessment of substrate incorporation. Group-specific differences in the abundances and the respective physiological state of the three populations were observed upon transfer from the humic-rich compartment to the main body of the lagoon (estimated at 1-2 days). Members of the clade B of Polynucleobacter (PnecB) experienced only an insignificant change in cell numbers, but displayed a general metabolic downshift, carbon metabolism (glucose incorporation) being most affected. By contrast, bacteria from the closely related Polynucleobacter C clade (PnecC) clearly differed in total abundances and in the numbers of DNA-synthesizing or glucose incorporating cells. At the same time, PnecC bacteria maintained comparable levels of protein synthesis (leucine uptake) in both lagoon compartments, and the proportion of cells incorporating N-acetylglucosamine was even higher in the main body of the lagoon. Members of the R-BT lineage showed little changes in cell numbers, DNA synthesis and carbon metabolism. Altogether, the observed patterns of substrate metabolism suggest that different bacterial populations in the lagoon undergo specific physiological adjustments in response to changing environmental conditions.

Environmental dynamics as a structuring factor for microbial carbon utilization in a subtropical coastal lagoon

Laguna de Rocha belongs to a series of shallow coastal lagoons located along South America. It is periodically connected to the sea through a sand bar, exhibiting a hydrological cycle where physicochemical and biological gradients are rapidly established and destroyed. Its most frequent state is the separation of a Northern zone with low salinity, high turbidity and nutrient load, and extensive macrophyte growth, and a Southern zone with higher salinity and light penetration, and low nutrient content and macrophyte biomass. This zonation is reflected in microbial assemblages with contrasting abundance, activity, and community composition. The physicochemical conditions exerted a strong influence on community composition, and transplanted assemblages rapidly transformed to resembling the community of the recipient environment. Moreover, the major bacterial groups responded differently to their passage between the zones, being either stimulated or inhibited by the environmental changes, and exhibiting contrasting sensitivities to gradients. Addition of allochthonous carbon sources induced pronounced shifts in the bacterial communities, which in turn affected the microbial trophic web by stimulating heterotrophic flagellates and virus production. By contrast, addition of organic and inorganic nutrient sources (P or N) did not have significant effects. Altogether, our results suggest that (i) the planktonic microbial assemblage of this lagoon is predominantly carbon-limited, (ii) different bacterial groups cope differently with this constraint, and (iii) the hydrological cycle of the lagoon plays a key role for the alleviation or aggravation of bacterial carbon limitation. Based on these findings we propose a model of how hydrology affects the composition of bacterioplankton and of carbon processing in Laguna de Rocha. This might serve as a starting hypothesis for further studies about the microbial ecology of this lagoon, and of comparable transitional systems.

HISH-SIMS analysis of bacterial uptake of algal-derived carbon in the Río de la Plata estuary.

One of the main goals of microbial ecologists is to assess the contribution of distinct bacterial groups to biogeochemical processes, e.g. carbon cycling. Until very recently, it was not possible to quantify the uptake of a given compound at single cell level. The advent of nano-scale secondary-ion mass spectrometry (nanoSIMS), and its combination with halogen in situ hybridization (HISH) opened up this possibility. Despite its power, difficulties in cell identification during analysis of environmental samples might render this approach challenging for certain applications. A pilot study, designed to quantify the incorporation of phytoplankton-derived carbon by the main clades of heterotrophic aquatic bacteria (i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes), is used to exemplify and suggest potential solutions to these technical difficulties. The results obtained indicate that the main aquatic bacterial clades quantitatively differ in the incorporation of algae-derived organic matter. From the methodological point of view, they highlight the importance of the concentration of the target cells, which needs to be sufficient to allow for a rapid mapping under the nanoSIMS. Moreover, when working with highly productive waters, organic and inorganic particles pose a serious problem for cell recognition based on HISH-SIMS. In this work several technical suggestions are presented to minimize the above mentioned difficulties, including alternatives to improve the halogen labeling of the cells and proposing the use of a combination of FISH and HISH along with a mapping system. This approach considerably enhances the reliability of cell identification and the speed of the subsequent nanoSIMS analysis in such complex samples.

A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria

Chitin is the second most abundant polymer on Earth, playing a crucial role in the biogeochemical cycles. A core issue for studying its processing in aquatic systems is the identification and enumeration of chitin-containing particles and organisms, ideally in a manner that can be directly linked to bulk chitin quantification. The aim of this study was the development of such a technique. We successfully combined the methodology of bulk chitin determination using wheat germ agglutinin (FITC-WGA) for staining chitin-containing particles and organisms along with CARD-FISH staining of either chitin-containing eukaryotic cells or bacteria associated with them. Environmental chitin staining was successfully applied to natural water samples. Fungal hyphae, diatoms, and dinoflagellates, sestonic aggregates and chitin-containing structures derived from metazoa were observed. Also, hybridized bacteria attached to chitinaceous debris were clearly visualized. Finally, as proof of principle, cultured yeast cells were simultaneously-targeted by FITC-WGA and the EUK516 probe without exhibiting any interference between both stains. The presented approach appears as a powerful tool to evaluate the contribution of different size classes and organisms to chitin production and consumption, opening the possibility for application of single-cell approaches targeting the ecophysiology of chitin transformations in aquatic systems.