Links between bacterial diversity and carbon cycling in the sea

Memoria de tesis doctoral presentada por Laura Alonso Sáez para optar al grado de Doctora en Ciencias de Mar por la Universitat Politécnica de Catalunya (UPC), realizada bajo la dirección del Dr. Josep Maria Gasol Piqué del Institut de Ciències del Mar (ICM-CSIC).-- 247 pages[EN] Although our knowledge about bacterial diversity in marine waters and that about the role of bacteria in the oceanic carbon cycling have greatly advanced in the last decades, very little is known about the linkage between those important characteristics, particularly under in situ conditions. The study of such relationship is hampered by the difficulties in obtaining pure cultures from representative marine bacteria, which would provide information about their metabolic capacities. The recent development of single-cell techniques has allowed studying the activity of marine populations in situ, allowing advances on this topic. We are starting now to know what is the specific role of specific groups of bacteria in carbon cycling, and the relationship between bacterial community assemblage and the global magnitude and patterns of carbon cycling in the ocean. In this study, we assessed the linkage between bacterial diversity and function along two gradients in marine waters: a temporal gradient (a seasonal study in a coastal Mediterranean station), and a spatial gradient covering different trophic states in oceanic waters (from the Cape Blanc upwelling to the N Atlantic subtropical gyre). We first described exhaustively bacterial assemblage structure at both sites, with the simultaneous use of clone libraries, fluorescence in situ hybridization with specific probes and fingerprinting of genes encoding the 16S rRNA. Since both studies were focused on surface bacterial assemblages, Archaea were minor components and we focused on Bacteria. We also exhaustively described the use of carbon by bacteria at both sites, taking special care in identifying and constraining the limitations in the methodologies used for that purpose. Afterwards, we performed comparative studies in which changes in bacterial assemblage structure were systematically compared to changes in a range of in situ measurements of bacterial carbon processing, including bacterial respiration and production, since this type of comparisons in marine field studies are absent from the literature. Several similarities were found between both systems studied, in terms of bacterial community composition, such as the increasing abundances of the Roseobacter group in high-nutrients waters, and the predominance of SAR11 bacteria in less productive waters. However, and with the use of multivariate statistics and Mantel tests, a tight linkage between bacterial diversity and function could not be proved in neither of the gradients. This lack of pattern could be due to several reasons, including the fact that the bulk measurements that describe carbon function (bacterial biomass production or respiration) are so general and important for ecosystem maintenance that they are redundantly performed by different bacterial groups. However, a more specific study using a single-cell level approach (microautoradiography combined to fluorescent in situ hybridization) showed that different bacterial populations had significantly distinct patterns of carbon use along the seasonal study in Blanes Bay, which suggests that different bacterial groups are not equal in terms of carbon utilization. Finally, we studied the effect of a variable environmental factor, such as UV and PAR radiation, as it inhibited or stimulated the different bacterial groups. Environmental conditions were shown to affect differently the activity of specific bacterial groups and, thus, their role in the processing of carbon in the marine planktonic environment[ES] A pesar del gran avance en nuestro conocimiento sobre la diversidad bacteriana marina y el papel de las bacterias en el ciclo de carbono oceánico, aún se sabe muy poco sobre la relación entre estas dos importantes características en condiciones naturales. El estudio de esta relación se ha visto limitado por la dificultad para obtener cultivos puros de bacterias representativas del medio marino, lo que nos permitiría disponer de información sobre sus capacidades metabólicas. El reciente desarrollo de técnicas de resolución individual ha permitido el estudio de la actividad de poblaciones bacterianas in situ, y recientemente, hemos comenzado a conocer cual es el papel de grupos específicos de bacterias marinas en el ciclo de carbono. En esta tesis, se estudia la relación entre diversidad bacteriana y función a lo largo de dos gradientes en aguas marinas: un gradiente temporal (estudio estacional en una estación costera del Mediterráneo), y un gradiente espacial que cubre diferentes estados tróficos en aguas oceánicas (desde el afloramiento de Cabo Blanco hasta el giro subtropical del Atlántico Norte). Primeramente, se ha descrito exhaustivamente la estructura de la comunidad bacteriana en ambos lugares de muestreo, utilizando simultáneamente diversas técnicas genéticas como bibliotecas génicas, hibridación in situ con sondas fluorescentes y “fingerprint” de genes que codifican el RNA ribosómico 16S. Dado que los estudios se concentran en aguas superficiales, las Archaeas son poco representativas y nos concentramos en las bacterias. También se ha estudiado exhaustivamente el uso bacteriano de carbono a lo largo de ambos gradientes, con especial énfasis en la identificación de las limitaciones en las metodologías utilizadas. Tras estos estudios descriptivos, se llevaron a cabo estudios comparativos en los que los cambios en la estructura de la comunidad bacteriana fueron sistemáticamente comparados a los cambios en las medidas de procesamiento de carbono, incluyendo producción y respiración bacteriana. Este tipo estudios no se habían realizado anteriormente en muestras naturales marinas. Encontramos diversas similitudes entre los dos sistemas estudiados en cuanto a la estructura de la comunidad bacteriana, como el aumento en la proporción de bacterias del grupo Roseobacter en aguas ricas en nutrientes, y la predominancia del grupo SAR11 en aguas menos productivas. Sin embargo, mediante el uso de técnicas estadísticas multivariantes y el test de Mantel, no se pudo probar una fuerte relación entre diversidad y función en ninguno de los gradientes. Esta ausencia de relación podría deberse a varias razones, incluyendo el hecho de que las medidas utilizadas para describir la función en el ciclo de carbono (producción de biomasa bacteriana y respiración) son tan generales y cruciales para el mantenimiento del ecosistema que son llevadas a cabo de forma redundante por los distintos grupos bacterianos. Sin embargo, en un segundo estudio mucho mas específico, se empleó una técnica de resolución individual (microautoradiografía combinada con hibridación in situ con sondas fluorescentes) y se mostró que distintas poblaciones bacterianas llevan a cabo distintos patrones de uso de carbono a lo largo del estudio estacional en la Bahía de Blanes, lo que sugiere que los grupos bacterianos no son equivalentes en la utilización de carbono. Finalmente, estudiamos el efecto de una variable ambiental, la luz ultravioleta y la radiación PAR, y encontramos que inhibía o estimulaba diferencialmente la actividad especifica de varios grupos bacterianos, y por tanto su papel en el procesamiento de carbono en el medio planctónico marino[CAT] El nostre coneixement sobre la diversitat bacteriana al mar i el del paper que els bacteris tenen en el cicle del carboni han avançat molt en les darreres dècades, tot i això, es coneix ben poca cosa de la relació entre aquests dues conceptes. L’estudi de la relació entre diversitat i funcionament de l’ecosistema es veu limitat per les dificultats d’obtenir cultius purs de bacteris marins representatius, que podrien oferir informació sobre les seves capacitats metabòliques. El desenvolupament recent de tècniques d’anàlisi a nivell individual ha permès l’estudi de l’activitat in situ de les poblacions marines, permetent avenços en aquesta relació diversitatfunció. Comencem a saber quin és el paper específic de grups concrets de bacteris en la circulació de carboni a l’oceà. En aquest treball, hem analitzat la relació entre diversitat bacteriana i funcionament de l’ecosistema al llarg de dos gradients en medi marí: un gradient temporal (estudi estacional en una estació costanera mediterrània), i un gradient espacial que comprèn diferents estats tròfics en aigües oceàniques (prop de l’aflorament de Cap Blanc al gir subtropical de l’Atlàntic Nord). Primer descrivim exhaustivament l’estructura de la comunitat de bacteris als dos llocs, mitjançant l’elaboració simultània de biblioteques de clons, hibridació in situ amb sondes fluorescents i emprentes genètiques dels gens que codifiquen per l’rRNA 16S. Com que tots dos estudis es centren en comunitats bacterianes d’aigües superficials, els Arqueus són poc representatius i ens hem centrat en els bacteris. També hem descrit exhaustivament la utilització de carboni per part dels bacteris als dos indrets, prenent especial cura amb identificar i restringir les limitacions en la metodologia utilitzada per aquestes mesures. Després hem fet estudis comparatius en els quals els canvis en l’estructura de la comunitat bacteriana s’han comparat sistemàticament amb els canvis en un seguit de mesures del processament de carboni per part dels microorganismes, incloent-hi la respiració i la producció bacteriana, car aquesta mena de comparacions són infreqüents a la literatura. Els dos sistemes estudiants comparteixen algunes característiques, com ara les abundàncies creixents de bacteris del grup de Roseobacter en aigües plenes de nutrients, i la predominança de bacteris del grup SAR11 a les aigües menys productives. Tanmateix, i amb la utilització de tècniques estadístiques multivariants i testos de Mantel, no hem pogut provar cap relació estreta entre diversitat bacteriana i funcionament a cap dels dos gradients. Aquesta mancança de relació pot ser deguda a diverses raons, entre elles el fet que les mesures a l’engròs per descriure la funció dels bacteris en el cicle del carboni (producció de biomassa o respiració bacterianes) són tan generals i tan importants per al manteniment de l’ecosistema que són realitzades de forma redundant per diferents grups bacterians. De tota manera, un estudi més concret, utilitzant una aproximació a nivell de cèl·lules individuals (microautoradiografia combinada amb hibridació in situ amb sondes fluorescents) ha mostrat que diferents poblacions bacterianes tenen diferents patrons d’utilització de carboni al llarg de l’estudi estacional de la Badia de Blanes, la qual cosa suggereix que els diferents grups bacterians no utilitzen de la mateixa manera el carboni. Finalment, hem estudiat l’efecte inhibidor o estimulador d’una variable ambiental, la radiació PAR i la UV, sobre els diferents grups bacterians. Hem pogut mostrar com aquestes condicions ambientals afectaven de forma diferent l’activitat dels diferents grups bacterians i, per tant, el seu paper en el processament de carboni en el medi planctònic maríPeer Reviewe

Significant year-round effect of small mixotrophic flagellates on bacterioplankton in an oligotrophic coastal system

14 pages, 7 figures, 1 tableThe seasonal variation in the grazing effect of mixotrophic flagellates on bacterioplankton was assessed during an annual cycle in an oligotrophic coastal station in the northwest Mediterranean Sea. Ingestion rates of fluorescently labeled bacteria were estimated for different size categories of phytoflagellates (PF) and heterotrophic flagellates (HF) in short-term experiments and compared with long-term grazing estimates and published empirical models. The mixotrophic flagellates included haptophyte-like cells, cryptophytes, and dinoflagellates. The group-specific grazing rates (SGR) averaged 1.1 (3–5 µm PF), 1.3 (5–20 µm PF), 4.0 (<5 µm HF), and 15.4 bacteria individual-1 h-1 (5–20 µm HF). Lower SGR but higher abundances of PF resulted in an average mixotroph contribution of 50% to the total flagellate grazing. Remarkably, the effect was relatively high all through the year (35–65%). Regardless of the presence of chloroplasts, flagellates <5 mm in size accounted, on average, for about 80% of total flagellate bacterivory and ingested a large percentage of their cell carbon per day from bacteria. Soluble reactive phosphorus concentration was negatively correlated with the ingestion rate of both groups of PF, suggesting that mixotrophic flagellates would be using their phagotrophic capability to obtain phosphorus when this nutrient is limiting. HF grazing activity showed a marked seasonality, with grazing being higher during the warmer seasons, and clearance rates were positively correlated with water temperature. Total bacterivory accounted for most of the bacterial production. Short-term and long-term bacterivory measurements were highly correlated, confirming that the smallest flagellates were the main causative agent of bacterial loss. The bacterivory values were also well correlated to a published empirical model that considers HF as the only bacterivorous. However, this model underestimated (up to 50%) total flagellate grazing during periods of high effect of mixotrophic flagellatesThis study was supported mainly by a EU project (EVK3-CT-2002-00078) and a Spanish post-doctoral fellowship (SB2001-0166) and also partially funded by three projects supported by the Spanish government (REN2001-2120/MAR; CTM2004-12631/MAR; CTM2004-02586/MAR)Peer reviewe

High bicarbonate assimilation in the dark by Arctic bacteria

10 páginas, 4 figuras, 1 tabla.Although both autotrophic and heterotrophic microorganisms incorporate CO2 in the dark through different metabolic pathways, this process has usually been disregarded in oxic marine environments. We studied the significance and mediators of dark bicarbonate assimilation in dilution cultures inoculated with winter Arctic seawater. At stationary phase, bicarbonate incorporation rates were high (0.5–2.5 μg C L−1 d−1) and correlated with rates of bacterial heterotrophic production, suggesting that most of the incorporation was due to heterotrophs. Accordingly, very few typically chemoautotrophic bacteria were detected by 16S rRNA gene cloning. The genetic analysis of the biotin carboxylase gene accC putatively involved in archaeal CO2 fixation did not yield any archaeal sequence, but amplified a variety of bacterial carboxylases involved in fatty acids biosynthesis, anaplerotic pathways and leucine catabolism. Gammaproteobacteria dominated the seawater cultures (40–70% of cell counts), followed by Betaproteobacteria and Flavobacteria as shown by catalyzed reporter deposition fluorescence in situ hybridization (CARDFISH). Both Beta- and Gammaproteobacteria were active in leucine and bicarbonate uptake, while Flavobacteria did not take up bicarbonate, as measured by microautoradiography combined with CARDFISH. Within Gammaproteobacteria, Pseudoalteromonas-Colwellia and Oleispira were very active in bicarbonate uptake (ca. 30 and 70% of active cells, respectively), while the group Arctic96B-16 did not take up bicarbonate. Our results suggest that, potentially, the incorporation of CO2 can be relevant for the metabolism of specific Arctic heterotrophic phylotypes, promoting the maintenance of their cell activity and/or longer survival under resource depleted conditions.This work is a contribution to the International Polar Year – Circumpolar Flaw Lead system study (IPY-CFL 2007/2008) lead by D. Barber (University of Manitoba) supported through grants from the Canadian IPY Federal Program Office, the National Sciences and Engineering Research Council, grant BOREAL (CLG2007-28872-E/ANT) from the Spanish Ministry of Science and Innovation to C.P.-A., and grants from the Swedish Research Council to S.B and L.A.S. L.A.S. was supported by a Marie Curie Intraeuropean Fellowship (CHEMOARC PIEF-GA-2008- 221121), E.O.C by the Spanish grant CGL2009-13318- BOS, and P. E. G by a Marie Curie grant (CRENARC MEIF-CT-2007-040247).Peer reviewe

Testicular Germ Cell Tumours and Proprotein Convertases

Testicular Germ Cell Tumours (TGCT) are widely considered a “curable cancer” due to their exceptionally high survival rate, even if it is reduced by many years after the diagnosis due to metastases and relapses. The most common therapeutic approach to TGCTs has not changed in the last 50 years despite its multiple long-term side effects, and because it is the most common malignancy in young Caucasian men, much research is needed to better the quality of life of the many survivors. Proprotein Convertases (PC) are nine serine proteases responsible for the maturation of inactive proproteins with many diverse functions. Alterations in their expression have been associated with various diseases, including cancer and inflammation. Many of their substrates are adhesion molecules, metalloproteases and proinflammatory molecules, all of which are involved in tumour development. Inhibition of certain convertases has also been shown to slow tumour formation, demonstrating their involvement in this process. Considering the very established link between PCs and inflammation-related malignancies and the recent studies carried out into the immune microenvironment of TGCTs, the study of the involvement of PCs in testicular cancer may open up avenues for being both a biomarker for diagnosis and a therapeutic target.This research was funded by the University of the Basque Country UPV/EHU grant number GIU 20/050

Temperature enhances the functional diversity of dissolved organic matter utilization by coastal marine bacteria

Although bulk bacterial metabolism in response to temperature has been determined for different oceanic regions, the impact of temperature on the functional diversity of dissolved organic matter (DOM) utilization has been largely unexplored. Here, we hypothesized that besides modifying the rates of carbon utilization, temperature can also alter the diversity of substrates utilized. The patterns of utilization of 31 model DOM compounds (as represented in Biolog EcoPlates™) by bacterioplankton were assessed using inocula from surface waters of the southern Bay of Biscay continental shelf over 1 year. Bacteria utilized more polymers and carbohydrates in late spring and summer than in winter, likely reflecting changes in substrate availability linked to the release and accumulation of DOM in phytoplankton post-bloom conditions. Seawater temperature correlated positively with the number of substrates utilized (i.e. functional richness) and this relationship was maintained in monthly experimental incubations spanning 3°C below and above in situ values. The enhancement of functional richness with experimental warming displayed a unimodal response to ambient temperature, peaking at 16°C. This temperature acted as a threshold separating nutrient-sufficient from nutrient-deficient conditions at the study site, suggesting that trophic conditions will be critical in the response of microbial DOM utilization to future warming

Chitosan Films Incorporated with Exopolysaccharides from Deep Seawater Alteromonas sp.

Two Alteromonas sp. strains isolated from deep seawater were grown to promote the production of exopolysaccharides (EPS, E611 and E805), which were incorporated into chitosan solutions to develop films. The combination of the major marine polysaccharides (chitosan and the isolated bacterial EPS) resulted in the formation of homogenous, transparent, colorless films, suggesting good compatibility between the two components of the film-forming formulation. With regards to optical properties, the films showed low values of gloss, in the range of 5–10 GU, indicating the formation of non-glossy and rough surfaces. In addition to the film surface, both showed hydrophobic character, with water contact angles higher than 100 º, regardless of EPS addition. Among the two EPS under analysis, chitosan films with E805 showed better mechanical performance, leading to resistant, flexible, easy to handle films.This research was funded by the Economic Development and Infrastructures Department of the Basque Government (grant number KK-2019/00076) and the Quality and Food Industry Department of the Basque Government (grant number 22-2018-00078)

Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH

We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4′,6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (>20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversit

Novel interactions between phytoplankton and bacteria shape microbial seasonal dynamics in coastal ocean waters

Trophic interactions between marine phytoplankton and heterotrophic bacteria are at the base of the biogeochemical carbon cycling in the ocean. However, the specific interactions taking place between phytoplankton and bacterial taxa remain largely unexplored, particularly out of phytoplankton blooming events. Here, we applied network analysis to a 3.5-year time-series dataset to assess the specific associations between different phytoplankton and bacterial taxa along the seasonal scale, distinguishing between free-living and particle-attached bacteria. Using a newly developed network post-analysis technique we removed bacteria-phytoplankton correlations that were primarily driven by environmental parameters, to detect potential biotic interactions. Our results indicate that phytoplankton dynamics may be a strong driver of the inter-annual variability in bacterial community composition. We found the highest abundance of specific bacteria-phytoplankton associations in the particle-attached fraction, indicating a tighter bacteria-phytoplankton association than in the free-living fraction. In the particle-associated fraction we unveiled novel potential associations such as the one between Planctomycetes taxa and the diatom Leptocylindrus spp. Consistent correlations were also found between free-living bacterial taxa and different diatoms, including novel associations such as those between SAR11 with Naviculales diatom order, and between Actinobacteria and Cylindrotheca spp. We also confirmed previously known associations between Rhodobacteraceae and Thalassiosira spp. Our results expand our view on bacteria-phytoplankton associations, suggesting that taxa-specific interactions may largely impact the seasonal dynamics of heterotrophic bacterial communities

Sample dilution and bacterial community composition influence empirical leucine-to-carbon conversion factors in surface waters of the world's oceans

Research articleThe transformation of leucine incorporation into prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world’s oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e. removal of grazers; F treatment) or filtration combined with dilution (i.e. relieving also resource competition; FD treatment). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu-1 and were significantly lower in the filtered and diluted (FD) than in the filtered (F) treatments. Also, changes in bacterial community composition during the incubations, as assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA), were stronger in the FD than in the F treatments, as compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCFs determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus) and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCFs variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts although resulting into relatively low carbon production rates in the oligotrophic ocean.En prensa3,829