Kinetic modulation of bacterial hydrolases by microbial community structure in coastal waters.

In this study, we hypothesized that shifts in the kinetic parameters of extracellular hydrolytic enzymes may occur as a consequence of seasonal environmental disturbances and would reflect the level of adaptation of the bacterial community to the organic matter of the ecosystem. We measured the activities of enzymes that play a key role in the bacterial growth (leucine aminopeptidase, ß- and α-glucosidases) in surface coastal waters of the Eastern Cantabrian Sea and determined their kinetic parameters by computing kinetic models of distinct complexity. Our results revealed the existence of two clearly distinct enzymatic systems operating at different substrate concentrations: a high-affinity system prevailing at low substrate concentrations and a low-affinity system characteristic of high substrate concentrations. These findings could be the result of distinct functional bacterial assemblages growing concurrently under sharp gradients of high-molecular-weight compounds. We constructed an ecological network based on contemporaneous and time-delayed correlations to explore the associations between the kinetic parameters and the environmental variables. The analysis revealed that the recurring phytoplankton blooms registered throughout the seasonal cycle trigger the wax and wane of those members of the bacterial community able to synthesize and secrete specific enzymes.

Prokaryotic Capability to Use Organic Substrates Across the Global Tropical and Subtropical Ocean.

Prokaryotes play a fundamental role in decomposing organic matter in the ocean, but little is known about how microbial metabolic capabilities vary at the global ocean scale and what are the drivers causing this variation. We aimed at obtaining the first global exploration of the functional capabilities of prokaryotes in the ocean, with emphasis on the under-sampled meso- and bathypelagic layers. We explored the potential utilization of 95 carbon sources with Biolog GN2 plates® in 441 prokaryotic communities sampled from surface to bathypelagic waters (down to 4,000 m) at 111 stations distributed across the tropical and subtropical Atlantic, Indian, and Pacific oceans. The resulting metabolic profiles were compared with biological and physico-chemical properties such as fluorescent dissolved organic matter (DOM) or temperature. The relative use of the individual substrates was remarkably consistent across oceanic regions and layers, and only the Equatorial Pacific Ocean showed a different metabolic structure. When grouping substrates by categories, we observed some vertical variations, such as an increased relative utilization of polymers in bathypelagic layers or a higher relative use of P-compounds or amino acids in the surface ocean. The increased relative use of polymers with depth, together with the increases in humic DOM, suggest that deep ocean communities have the capability to process complex DOM. Overall, the main identified driver of the metabolic structure of ocean prokaryotic communities was temperature. Our results represent the first global depiction of the potential use of a variety of carbon sources by prokaryotic communities across the tropical and the subtropical ocean and show that acetic acid clearly emerges as one of the most widely potentially used carbon sources in the ocean.

Recurrent seasonal changes in bacterial growth efficiency, metabolism and community composition in coastal waters.

The microbial response to environmental changes in coastal waters of the eastern Cantabrian Sea was explored for four years by analysing a broad set of environmental variables along with bacterial community metabolism and composition. A recurrent seasonal cycle emerged, consisting of two stable periods, characterized by low bacterial metabolic activity (winter) from October to March, and high bacterial metabolic activity (summer) from May to August. These two contrasting periods were linked by short transition periods in April (TA ) and September (TS ). The phylogenetic groups Alphaproteobacteria and Bacteroidetes were dominant during winter and summer respectively, and their recurrent alternation was mainly driven by the bloom of eukaryotic phytoplankton before TA and the bloom of prokaryotic phytoplankton before TS . Bacterial growth efficiency remained high and stable during the winter and summer periods but dropped during the two short transition periods. Our results suggest that bacterial growth efficiency should be considered a very resilient property that reflects different stages in the adaptation of the bacterial community composition to the environmental changes occurring throughout the seasonal cycle in this coastal ecosystem.

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities.

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature-sensitivity, as represented by the activation energies (Ea ), of extracellular ß-glucosidase (ßG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These Ea were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting Ea in the subtropical and tropical ocean, with ßG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication.

Release and consumption of D-amino acids during growth of marine prokaryotes.

Analysis of the composition of the marine-dissolved organic matter has highlighted the importance of D-amino acids, whose origin is attributed mainly to the remains of bacterial peptidoglycan released as a result of grazing or viral lysis. However, very few studies have focused on the active release of D-amino acids by bacteria. With this purpose, we measured the concentration of dissolved amino acids in both enantiomeric forms with two levels of complexity: axenic cultures of Vibrio furnissii and Vibrio alginolyticus and microcosms created from marine microbial assemblages (Biscay Bay, Cantabrian Sea) with and without heterotrophic nanoflagellates (HNFs). Axenic cultures showed that only D-Ala was significantly released and accumulated in the medium up to a concentration of 120 nM, probably as a consequence of the rearrangement of peptidoglycan. The marine microbial assemblages showed that only two D-amino acids significantly accumulated in the environment, D-Ala and D-aspartic acid (Asp), in both the absence and presence of HNFs. The D/L ratio increased during the incubation and reached maximum values of 3.0 to 4.3 for Ala and 0.4 to 10.6 for Asp and correlated with prokaryotic and HNF abundance as well as the rate of prokaryotic thymidine and leucine incorporation. Prokaryotes preferentially consumed L-amino acids, but the relative uptake rates of D-Ala significantly increased in the growth phase. These results demonstrate that bacteria can release and consume D-amino acids at high rates during growth, even in the absence of viruses and grazers, highlighting the importance of bacteria as producers of dissolved organic matter (DOM) in the sea.

Changes in bacterial metabolism as a response to dissolved organic matter modification during protozoan grazing in coastal Cantabrian and Mediterranean waters.

We explored how marine dissolved organic matter (DOM) altered by bacterial growth and protozoan grazing modify the metabolism of Southeastern Cantabrian Sea (CS) and NW Mediterranean Sea (MS) coastal bacterial communities. Major metabolic features were measured in treatments with half of the natural water replaced by water with different DOM quality, characterized by fluorescent DOM analysis and collected from key times of the predator-prey curve. In both ecosystems, protozoan-altered DOM led to similar increases in bacterial carbon demand (238% and 213%) and decreases in bacterial growth efficiency (BGE: 56% for the CS and 46% for the MS). These low BGEs were caused by similar bacterial production but much higher bacterial respiration rates, which in turn were positively related to aminopeptidase activity. However, in the CS bacterial community dominated by Bacteroidetes (41%), the enhanced hydrolytic activity was produced at a lower metabolic cost than in the MS, dominated by SAR11 (47%), which suggests a better adaptation of Bacteroidetes to the DOM altered during protozoan grazing. These results highlight protozoan grazing as a relevant factor influencing BGE in coastal ecosystems, and relate bacterial community composition to the major metabolic processes that result after a change in the quality of marine DOM.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria.

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the L- and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the L- and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment (AU)

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