This research studied the seasonal and spatial dynamics for abundance of picophytoplanktons (including Prochlorococcus spp., Synechococcus spp. and picoeukaryotes) in the South China Sea (SCS) and the Kuroshio. Waters were collected during five cruises between August 2009 and December 2010. Growth rates were determined in two size fractioned waters, <2 um and <10 um, after incubation. The differences of growth rates between the two size fractions were defined as the grazing rates. Before the incubation, waters were enriched with FeCl3, EDTA, or NH4Cl to examine the possible shortage of Fe or nitrogen. Abundances of picophytoplanktons and nanoflagellates were examined using a flow-cytometry and a microscope, respectively. Prochlorococcus was more abundant in the warm than the cold seasons and in the Kuroshio and the basin of the SCS than in the shelf and slope of the SCS. In the high abundance seasons/regions, low irradiance enhanced the growth rates of Prochlorococcus. Although both of the growth rates and grazing rates were high during then, the growth rates were found higher than the grazing rates. Addition of EDTA enhanced the growth rates that was likely attributed to its chelating with toxic trace metals (such as Cd2+, Cd2+) and/or with growth necessity trace metals (such as Co2+). The seasonal/spatial distributions for Synechococcus were in contrast to that of Prochlorococcus. High growth rates of Synechococcus were related to high nitrate concentrations and the low irradiance. The growth rates were higher than the grazing rates in the high nitrogen seasons/regions when/where irradiance was also relatively low. EDTA also enhanced the growth of Synechococcus, and was likely due to its chelating to remove Cd2+ and/or to retain Co2+. Distributions of picoeukaryotes were similar to that of Synechococcus. Factors affected its dynamics were not clear because of its complicated compositions.

Würzburg, Universiẗat, Diss., 2004. / Erscheinungsjahr an der Haupttitelstelle: 2004.

Ribonuklease P (RNase P) ist eine essentielle Endonuklease, welche die 5'-Flanke von pre-tRNAs entfernt. Die RNase P RNA des Cyanobakteriums Prochlorococcus marinus ist in vitro katalytisch aktiv und bevorzugt in heterologen Prozessierungssystemen Substrate mit vollständigem 3’-CCA-Ende. Diese Substratspezifität widerspricht den Erwartungen, da tRNAs in P. marinus nicht mit dem CCA-Ende codiert sind und die RNase P RNA auch nicht das GGU-Bindungsmotiv für diese CCA-Enden aufweist. Um die Substratspezifität und Aufbau des Ribozym-Substrat-Komplex von P. marinus RNase P RNA im homologen System untersuchen zu können, wurden Transkriptionsklone für P. marinus pre- und mat-tRNAArgCCU konstruiert, mit denen nach entsprechender Restriktionshydrolyse Transkripte mit stufenweise verkürzten 3’-CCA-Ende synthetisiert werden können. Durch enzymkinetische Untersuchungen der Prozessierung durch P. marinus RNase P RNA wurde unter steady-state-Bedingungen für pre-tRNACCA eine Michaelis-Menten Konstante von 6,92 µM ermittelt. Die Entfernung von A76 und C75 des 3’-CCA-Endes führt zu einer Erhöhung der KM (7,13 µM bzw. 19,68µM). Diese Substrate werden folglich weniger stark gebunden, was sich auch in der freien Bindungsenthalpie von 0,02 und 0,65 kcal/mol ausdrückt. Die Entfernung des vollständigen 3’-CCA-Endes führt zu einer erheblichen Erniedrigung der KM (0,83µM) und zu einer energetisch begünstigten, stärkeren Substratbindung (–1,31 kcal/mol). P. marinus RNase RNase P RNA zeigt folglich bei der in vitro Prozessierung im homologen System unter steady-state-Bedingungen eine Substratspezifität für das Substrat mit deletiertem 3’-CCA-Ende. Durch die Methode des Crosslinking, die in dieser Arbeit etabliert und optimiert wurde, können RNA-Protein und RNA-RNA Interaktionen nachgewiesen werden. Mit ihr wurde die Bindung von Substrat und Produkt im Komplex mit der RNase P RNA untersucht. Durch interne Modifizierung der P. marinus RNase P RNA-Komponente mit dem photosensiblen Nukleotidanalogon s4U wurden Kontaktstellen in 5’-Flanke, Acceptor-Stamm, D-Stamm, D-Schleife, Anticodon-Schleife und in der variablen Schleife der P. marinus pre-tRNAArg identifiziert. Diese lokalisierten Kontaktstellen stehen denen in der 5’-Flanke, dem Acceptor-Stamm und der 3’-Flanke, wie sie für den Ribozym-Substrat-Komplex mit E. coli RNase P RNA identifiziert wurden, gegenüber. In P. marinus RNase P RNA werden folglich alternative Kontaktstellen zur Substratbindung benutzt. Mit Hilfe der hier überexprimierten E. coli Nukleotidyltransferase, konnte pre- und mat-tRNAArg durch eine neue Synthesestrategie am 3’-CCA-Ende mit dem Crosslink-Reagenz Azidophenacyl (APA) modifiziert werden. Durch die Positionierung von APA am 5’-Terminus von pre- und mat-tRNAArg wurden weitere modifizierte tRNAs synthetisiert. Durch Crosslink-Experimente im homologen P. marinus System mit diesen modifizierten pre- und mat-tRNAArg-Varianten wurden die selben Regionen des katalytischen Zentrums (J18/2, Region P15/P16, J5/15) der RNase P RNA identifiziert, wie sie von E. coli und B. subtilis RNase P RNA bekannt sind. Dies bedeutet, dass die 5’-Flanke, die Prozessierungsstelle und das 3’-CCA-Ende der tRNAs auf einer vergleichbaren Oberfläche positioniert werden wie in anderen Ribozymen. Durch die fehlende Fixierung des 3’-CCA-Endes über Basenpaarungen mit dem GGU-Bindungsmotiv werden die tRNAs in P. marinus RNase P RNA weniger starr an das Ribozym gebunden und das 3’-CCA-Ende besitzt eine flexiblere Positionierung im Komplex mit dem Ribozym. Die Existenz unterschiedlicher Crosslink-Muster in P6, P18, J5/15 und J3/4 zeigt, dass pre-tRNAs und reife tRNAs durch verschiedene Modi an das P. marinus Ribozym gebunden werden. Die Identifizierung von vernetzten Nukleotiden in P15, J15/16, P16 und J16/15, die mit vergleichbaren modifizierten tRNAs in E. coli RNase P RNA nicht gefunden wurden, belegen, dass in P. marinus RNase P RNA ein anderer Produkt-Bindungs-Modus existiert als in E. coli. Erstmals konnten in dieser Arbeit auch zu erwartende Interaktionen mit dem katalytischen Zentrum identifiziert werden, die in bisherigen Crosslink-Experimenten in E. coli und B. subtilis RNase P RNA nicht oder nur geringfügig auftraten. Um die erhaltenen Ergebnisse besser veranschaulichen zu können, wurde mit dem Programm ERNA 3D ein Raumstrukturmodell für P. marinus RNase P RNA und tRNAArg erstellt. Die RNase P RNA der Cyanellen von Cyanophora paradoxa, ist in vitro katalytisch inaktiv. Um zu klären, ob die fehlende Ribozym-Aktivität dieser RNase P RNA auf eine fehlerhafte Substratbindung zurückzuführen ist, sollten Crosslink-Experimente mit den modifizierten P. marinus tRNAArg durchgeführt werden. Es konnte gezeigt werden, dass 5’- und 3’-modifizierte pre-tRNAs in C. paradoxa in einem anderen Modus gebunden werden, als durch die katalytisch aktive P. marinus RNase P RNA. / Ribonuclease P (RNase P) is the essential endonuclease responsible for the removal of the 5’-flank of precursor tRNAs. The RNase P RNA from the cyanobacterium Prochlorococcus marinus shows in vitro catalytic activity and specificity for heterologous substrates containing the complete 3’-CCA end. This preference is in contrast to the fact that the P. marinus RNase P RNA does not possess the binding motif for the CCA terminus, which is not encoded in tRNA genes in this organism. To analyse the substrate specificity and architecture of the ribozyme-substrate-complex of P. marinus RNase P RNA in a homologous system, transcription clones for P. marinus pre- and mat-tRNAArg were generated to obtain different transcripts with stepwise shortened 3’-CCA ends. In the kinetic analysis of P. marinus RNase P RNA, the Michaelis constant (KM) for pre-tRNACCA was 6,92 µM, as determined under steady-state conditions. The subsequent deletion of A76 and C75 from the 3’-CCA end results in an increase of KM (7,13 µM and 19,69 µM, respectively). These substrates are bound less strongly, which is expressed in loss of binding energy (0,02 and 0,65 kcal/mol, respectively).The removal of the complete CCA end results in an considerable decrease of KM (0,83 µM) and an energetically favoured and stronger binding of substrate (–1,31 kcal/mol). In conclusion, in the homologous in vitro system, P. marinus RNase P RNA has a preference for substrate lacking the 3’-CCA end. The method of Crosslinking, which was established and optimised in this work, is generally used to determine RNA-protein and RNA-RNA interactions. This method was used to examine the binding of substrate and product in the complex composed with RNase P RNA. P. marinus RNase P RNA was internally modified with the photoinducible nucleotide analogue s4U. With this modified RNA, interactions of the 5’-flank, acceptor stem, D-stem and loop, anticodon loop and variable loop of pre-tRNAArg with RNase P RNA were detected. These contacts are in contrast to signals in the 5’-flank, acceptor stem and 3’-flank which have been identified in the ribozyme-substrate-complexes of E. coli RNase P RNA. Thus, in P. marinus RNase P RNA, alternative interactions are used for substrate binding. Using purified recombinant E. coli Nucleotidyltransferase, pre- and mat-tRNAArg were modified at the 3’-CCA end by a new strategy using the crosslink-reagent azidophenacyl (APA). Additional modified tRNAs were obtained by positioning the APA-reagent at the 5’-end. In the homologous P. marinus system, crosslinking experiments with the modified tRNAs identified the same regions of the catalytic centre (J18/2, region P15/P16, J5/15) which have been established in E. coli and B. subtilis RNase P RNA. This observation indicates that 5’-flank, cleavage site and 3’-CCA end are positioned on a similar surface, as in the other ribozymes. Due to the missing interaction between the GGU motif and the CCA end, tRNAs are bound less rigid to the ribozyme in P. marinus and the 3’-CCA end is more flexible in the complex. Different crosslink patterns in P6, P18, J5/15 and J3/4 indicate that pre-tRNAs and mat-tRNAs are bound in a different mode by P. marinus RNase P RNA. The identification of crosslinked nucleotides in P15, J15/16, P16 and J16/15 which are not observed with analogous modified tRNAs in E. coli RNase P RNA, show that a different mode of product binding exists in P. marinus RNase P RNA. For the first time, interactions within the catalytic centre could be identified which had been anticipated, but were only weakly detectable in the E. coli and B. subtilis RNase P RNAs. The crosslinks in P4, J3/4 and P3 are a distinctive feature, which is supported by mutational studies, phosphorothioate interference and NAIM analysis. To obtain a good visualization of the crosslinking results, a 3D-model of P. marinus RNase P RNA and tRNAArg was created with the program ERNA-3D. RNase P RNA from the cyanelles of Cyanophora paradoxa does not show catalytic activity in vitro. To establish whether the lack of substrate binding ability is the reason for the missing ribozyme activity, crosslinking experiments with the modified P. marinus tRNAArg were done. 5’- and 3’- modified pre-tRNAArg are bound by cyanelle RNase P RNA in a different mode than by the catalytically active P. marinus RNase P RNA.

Aquatic primary productivity mainly depends on pelagic phytoplankton. The globally abundant marine picocyanobacteria Prochlorococcus comprises a significant fraction of the photosynthetic biomass in most tropical, oligotrophic oceans. The Red Sea is an enclosed narrow body of water characterized by continuous solar irradiance, and negligible annual rainfall, in addition to elevated temperatures and salinity levels, which mimics a global warming scenario. Analysis of 16S rRNA sequences of bacterioplankton communities indicated the predominance of a high-light adapted ecotype (HL II) of Prochlorococcus at the surface of the Northern and Central Red Sea. To this end, we analyzed the distribution of Prochlorococcus at multiple depths within and below the euphotic zone in different regions of the Red Sea, using clone libraries of the 16S–23S rRNA internal transcribed spacer (ITS) region. Results indicated a high diversity of Prochlorococcus ecotypes at the 100 m depth in the water column and an unusual dominance of HL II-related sequences in deeper waters of the Red Sea. To further investigate the microdiversity of Prochlorococcus over a wider biogeographical scope, we used a 454-pyrosequencing approach to analyze rpoC1 gene pyrotags. Samples were collected from the surface of the water column to up to 500 m at 45 stations that span the Red Sea’s main basin from 4 north to south. Phylogenetic analysis of abundant rpoC1 OTUs revealed genotypes of recently discovered strains that belong to the high-light and lowlight clades. In addition, we used a rapid community-profiling tool (GraftM) and quantitatively analyzed rpoC1 gene abundance from 45 metagenomes to assess the Prochlorococcus community structure across vertical and horizontal physicochemical gradients. Results revealed the clustering of samples according to their depth and a strong influence on ecotypic distribution by temperature and oxygen levels. In efforts to better understand how the cells survive the unusual features of the Red Sea, a Prochlorococcus strain of the HL II adapted clade from the euphotic zone was cultured, enabling morphological analyses and growth rates measurements for the strain. In addition, we successfully sequenced and annotated the genome of the strain, which was then used for genomic comparison with other ecotypes. Interestingly, the set of unique genes identified in the draft genome included genes encoding proteins involved in salt tolerance mechanisms. The expression level and pattern of these genes in the Red Sea water column was explored through metatranscriptomic mapping and revealed their occurrence throughout, independent of the diel cycle. This led to the hypothesis that Prochlorococcus populations in the highly saline Red Sea are able to biosynthesize additional compatible solutes via several pathways to counterbalance the effects of salt stress. The results presented in this dissertation provide the first glimpse on how the environmental parameters of the Red Sea can affect the evolution, diversity and distribution patterns of Prochlorococcus ecotypes.

<p>While much is known about the abundance and genetic diversity of environmental microbial communities, little is known about their taxon-specific activity. In this thesis I address this gap using a model marine microbe, the cyanobacterium <italic>Prochlorococcus spp.</italic>, which numerically dominates tropical and subtropical open oceans and encompasses a group of genetically defined clades that are ecologically distinct. Ribosomal RNA is a promising indicator of in situ activity because of its essential role in protein synthesis as well as its phylogenetic information, which could be used to distinguish clades among mixed populations. Here I show that, in a laboratory system the specific growth rate of representative <italic>Prochlorococcus</italic> strains could be quantitative predicted from cellular rRNA content (assessed by RT-qPCR), cell size (assessed by flow cytometry) and temperature. Applying this approach in the field, I show unique clade-specific activity patterns for <italic>Prochlorococcus</italic>. For example, vertically within the euphotic zone, eHL-II activity is strongly impacted by light and is consistent with patterns of photosynthesis and on a horizontal transect from Hawaii to San Diego, eHL-I and eHL-II activities exhibit significant transitions and appear to be regulated by temperature, nutrient and vertical mixing gradients. Using ribosomal tag pyrosequencing of DNA and RNA, I have extended our observation to the Eubacterial community and described the biomass distribution (rDNA) and activity (rRNA) patterns from two representative depths (25 and 100 m) at a well-studied oligotrophic ocean station. These results show that for some populations the abundances and activities are significantly uncoupled, which suggests substantial top-down controls or physical transport processes. Further exploring the taxon-specific activity patterns along with abundances and environmental variables across time and space is essential to better understanding the dynamics of a complex microbial system as well as predicting the consequences of environmental change.</p> / Dissertation

Population dynamics of picophytoplanktons, including Prochlorococcus, Synechococcus, and picoeukaryotes, were investigated in the upstream Kuroshio. Data were collected during eight cruises between July 2007 and May 2009. Sampling stations were located along 21¢X55¡¦N and between 121¢X00¡¦E and 122¢X10¡¦E in the Kuroshio off the Southeast Taiwan. Monitoring experiments including light shadding experiment, nutrient enrichment, temperature control, and grazing experiments were conducted to better understand the mechanisms that affect the growths of the picophytoplanktons. The abundances of the picophytoplanktons were measured using a flow cytometry.Water column integrated (0~200 m) abundance of Prochlorococcus was higher (26.63 ¡Ó 3.87 ¡Ñ 1012 cells m-2) in spring than either summer (19.07 ¡Ó 4.08 ¡Ñ 1012 cells m-2), autumn (16.05 ¡Ó 2.80 ¡Ñ 1012 cells m-2), or winter (17.89 ¡Ó 5.41 ¡Ñ 1012 cells m-2). During winter, the abundance was significantly (p<0.05) higher at the offshore station (17.89 ¡Ó 5.41 ¡Ñ 1012 cells m-2) than the inshore station (3.19 ¡Ó 2.07 ¡Ñ 1012 cells m-2). The abundance of Prochlorococcus was positively related to water temperature, nitracline depth (Dni), and euphotic depth (Deu), and negatively to surface concentration of N+N or SRP. Prochlorococcus was abundant (>100 ¡Ñ 103 cells ml-1) in the upper 100-m water column. Its maximum (200~300 ¡Ñ 103 cells ml-1) often occurred at the depth shallower than 75 m. The cell density sustained at >25 ¡Ñ 103 cells ml-1 between 100~150 m and was almost nil at the depth deeper than 150 m. There was no significant seasonal differences for either the abundances of Synechococcus (0.32~1.07 ¡Ñ 1012 cells m-2) or picoeukaryotes (0.16~0.24 ¡Ñ 1012 cells m-2). During winter, the abundances of Synechococcus was significantly (p<0.05) higher in the offshore Kuroshio water (2.94 ¡Ó 0.32 ¡Ñ 1012 cells m-2) than that of the inshore Kuroshio water. Similar trend of offshore (0.52 ¡Ó 0.05 ¡Ñ 1012 cells m-2) higher than the inshore was observed for picoeukaryotes in winter. The dynamics of Synechococcus abundance was positively related to surface SRP concentration and negatively to Dni. The picoeukaryotes abundance was positively related to surface N+N concentration, and SRP and negatively to Temp, Dni, and Deu. Vertical distribution of Synechococcus showed that the maximum abundance often occurred above 75 m, but was almost nil below 100 m. By contrast, the maximum abundance for picoeukaryotes often occurred between 50~125 m. The abundance of Synechococcus was positively related to the abundance of picoeukaryotes. And their abundance were negatively related to that of Prochlorococcus. Many environmental factors fluctualed parallelly. Dynamics of surface Temp, Dni and Deu were positively correlated to each other and either of them was negatively correlated to the dynamics of surface concentration of N+N or SRP. Surface N+N was positively correlated with surface SRP. The result of light shadding experiment showed that Prochlorococcus and picoeukaryotes, compared to Synechococcus, were much sensitive to high intensity of light. This suggest that Synechococcus was more tolerant to high light intensity or required more light energy than Prochlorococcus or picoeukaryotes. The results of nutrient enrichment experiments showed that addition of EDTA significantly enhanced the growth of three groups of picophytoplanktons. However, there was no significant difference after addition of either nitrate, Fe, or Cu. Prochlorococcus grew better at 27 ¢XC than 30 ¢XC in the temperature experiment. But there was no difference in the growth rate between 27 ¢XC and 30 ¢XC for Synechococcus or picoeukaryotes The result of grazing experiment showed that there was no difference between the growth rate with and without grazers in the incubation for any of the three groups of picophytoplanktons.

This research investigated the seasonal and spatial distributions of picophytoplankton, including Prochlorococcus, Synechococcus and picoeukaryotes, in the northern South China Sea. Monitoring experiments including light intensity control and nutrient enrichment were conducted concurrently with on board sampling to examine factors affecting their cell densities dynamics. Quantification of cell numbers was carried out by flow cytometry. Averaged Synechococcus abundance in the South China Sea was 1¡Ñ104 cells ml-1, high in winter (1.37¡Ó0.30¡Ñ104 cells ml-1) and low in summer or fall (0.51¡Ó0.13¡Ñ104 cells ml-1 and 0.53¡Ó0.22¡Ñ104 cells ml-1, respectively). During a same season of the year, there was more Synechococcus in the shelf-slope region than in the basin. The cell density in summer, but not in winter, was significantly positively related to surface water nutrient concentration. Nutrient enrichment experiment carried out in winter also indicated that the growth of Synechococcus did not respond to addition of nitrate. On the other hand, Synechococcus seemed to prefer high illumination. In the light intensity experiment, Synechococcus collected from surface water grew better at 100% surface illumination than <100% light intensities. Synechococcus collected from deep water grew best at 30% and 18% of surface illuminations. Vertically, Synechococcus concentrated mostly in surface water with maximum cell number occurring at the surface or a few meters deep. Nutrient enrichment experiment in winter also showed that Synechococcus responded significantly to iron addition. Average cell density of picoeukaryotes was always less than 0.5¡Ñ104 cells ml-1, being high in winter (0.46¡Ó0.10¡Ñ104 cells ml-1) and low in summer or fall (0.15¡Ó0.02¡Ñ104 cells ml-1 and 0.19¡Ó0.03¡Ñ104 cells ml-1, respectively). Picoeukaryotes was always more concentrated in the shelf-slope region than in the basin, especially in winter when cell density in the shelf-slope region was 0.70¡Ó0.11¡Ñ104 cells ml-1. Although in winter picoeukaryotes was significantly positively related to surface water nutrient concentration, enrichments of nitrate or iron did not enhance their growth. Prochlorococcus had a cell density > 5.5¡Ñ104 cells ml-1 in the euphotic zone, and distributed as deep as 200 m. Light intensity monitoring experiment showed that Prochlorococcus from surface water grew better under high illumination than those from deep water and vice versa. Under 9% of surface illumination, deepwater Prochlorococcus population showed a positive growth, corresponding well with its deep distribution. Nutrient enrichment experiment conducted in winter showed that Prochlorococcus did not respond to enrichment of nitrate or iron.

Picoplankton, cells between 0.2 - 2 μm, play a vital role in the carbon flow and nutrient cycling in marine food webs. Auto- and heterotrophic picoplankton dominate the biomass of oligotrophic tropical and subtropical oceans. However, little is known about their vertical distribution, changes in space and time and their relationships with environmental variables in the central Red Sea. The goal of this Ph.D. dissertation is to obtain baseline knowledge about their abundance, cellular characteristics (cell size, relative pigment and nucleic acid content) and biomass at seasonal and high-frequency temporal resolution (every 2 hours). This dissertation also aims at assessing picoplankton responses to separate and joint effects of nutrients additions (inorganic, organic and mixed) and temperature in order to be able to predict the relative contribution of eutrophication and warming in the future standing stocks of picoplankton in the Red Sea. I conducted a total of 63 vertical profiles (15 at around noon plus 48 more from the high-frequency diel samplings) from the surface down to the bottom (ca. 700 m) at a station situated 6 km off the coast of King Abdullah Economic City (KAEC) in the central Red Sea and performed 4 nutrient and temperature experiments lasting each 6 days with surface waters from the harbor of King Abdullah University of Science and Technology (KAUST). Flow cytometry allowed me to consistently identify five groups of autotrophs (Prochlorococcus, two populations of Synechococcus separated by their relative phycoerythrin fluorescence, and two differently-sized groups of picoeukaryotes) and two groups of heterotrophic prokaryotes characterized by their different relative nucleic acid content. One of the most surprising findings is the relatively lower abundances and to a lesser extent also growth rates of picoplankton compared with other tropical and subtropical oceans. Seasonality in environmental conditions emerged as an important factor in the response of picoplankton to nutrient additions and temperature. Picoplankton mostly responded to inorganic and mixed nutrient additions rather than warming. Overall, the information provided in this dissertation fills the gap of a critical component of Red Sea pelagic ecosystems and expands the information available on picoplankton communities in tropical waters.

No description available.

No description available.