ALBERT

Beschreibung: The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH4 and CO2 assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO2 reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ13C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of 14C from radiolabeled CH4 or CO2 revealed one hot spot for methanotrophy and CO2 fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with 14CH4 or 14CO2 revealed that there was interconversion of CH4 and CO2. The level of CO2 reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.

Beschreibung: The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro 13CH4 labeling study (δ13CH4, ∼5,400‰) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the 13C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Δ11]; difference between the δ13C at the start and the end of the experiment [Δδ13Cstart-end], ∼160‰). In contrast, bacterial glycerol diethers exhibited only slight changes in δ13C (Δδ13Cstart-end, ∼10‰). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Δδ13Cstart-end, ∼25‰), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the δ13C (Δδ13Cstart-end, ∼2‰). Moreover, an increase in the uptake of 13C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in 13C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater 13C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.

Beschreibung: A nodule-shaped microbial mat was found subsurface in sediments of a gas seep in the anoxic Black Sea. This mat was dominated by ANME-1 archaea and consumed methane and sulfate simultaneously. We propose that such subsurface mats represent the initial stage of previously investigated microbial reefs.

Beschreibung: In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for 〉90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.

Beschreibung: We studied the effect of the quorum-sensing (QS) blockers 5-hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2(5H)-one (FUR1), (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl]furan-2(5H)-one (FUR2) and triclosan (TRI) on the formation of bacterial biofilms, and the effect of these biofilms on the larval attachment of the polychaete Hydroides elegans and the bryozoan Bugula neritina. 14-day-old subtidal biofilms were harvested from artificial substrata and were allowed to develop in the laboratory with and without QS blockers. QS blockers inhibited the production of violacein by the QS reporter strain Chromobacterium violaceum CV026 and did not affect the metabolic activity of bacteria in multispecies biofilms. At a concentration of 10(-3) M all three tested compounds inhibited the establishment of microbial communities, but at one of 10(-4) M only FUR2 inhibited establishment. The tested QS blockers caused changes in bacterial density and bacterial community structure, as revealed by terminal restriction fragment length polymorphism and FISH. The groups most affected by QS blockers were Alphaproteobacteria, Gammaproteobacteria and the Cytophagales. Larvae of H. elegans and B. neritina avoided settling on biofilms that had developed in the presence of QS blockers. Our results suggest that QS blockers directly control the formation of multi-species biofilms, and indirectly - by means of biofilm properties - affect larval attachment on these modified biofilms.

Beschreibung: The diversity of Cyanobacteria in water and sediment samples from four representative sites of the Salar de Huasco was examined using denaturing gradient gel electrophoresis and analysis of clone libraries of 16S rRNA gene PCR products. Salar de Huasco is a high altitude (3800 m altitude) saline wetland located in the Chilean Altiplano. We analyzed samples from a tributary stream (H0) and three shallow lagoons (H1, H4, H6) that contrasted in their physicochemical conditions and associated biota. Seventy-eight phylotypes were identified in a total of 268 clonal sequences deriving from seven clone libraries of water and sediment samples. Oscillatoriales were frequently found in water samples from sites H0, H1 and H4 and in sediment samples from sites H1 and H4. Pleurocapsales were found only at site H0, while Chroococcales were recovered from sediment samples of sites H0 and H1, and from water samples of site H1. Nostocales were found in sediment samples from sites H1 and H4, and water samples from site H1 and were largely represented by sequences highly similar to Nodularia spumigena. We suggest that cyanobacterial communities from Salar de Huasco are unique - they include sequences related to others previously described from the Antarctic, along with others from diverse, but less extreme environments.

Beschreibung: As ecosystem engineers, seagrasses are angiosperms of paramount ecological importance in shallow shoreline habitats around the globe. Furthermore, the ancestors of independent seagrass lineages have secondarily returned into the sea in separate, independent evolutionary events. Thus, understanding the molecular adaptation of this clade not only makes significant contributions to the field of ecology, but also to principles of parallel evolution as well. With the use of Dr. Zompo, the first interactive seagrass sequence database presented here, new insights into the molecular adaptation of marine environments can be inferred. The database is based on a total of 14 597 ESTs obtained from two seagrass species, Zostera marina and Posidonia oceanica, which have been processed, assembled and comprehensively annotated. Dr. Zompo provides experimentalists with a broad foundation to build experiments and consider challenges associated with the investigation of this class of non-domesticated monocotyledon systems. Our database, based on the Ruby on Rails framework, is rich in features including the retrieval of experimentally determined heat-responsive transcripts, mining for molecular markers (SSRs and SNPs), and weighted key word searches that allow access to annotation gathered on several levels including Pfam domains, GeneOntology and KEGG pathways. Well established plant genome sites such as The Arabidopsis Information Resource (TAIR) and the Rice Genome Annotation Project are interfaced by Dr. Zompo. With this project, we have initialized a valuable resource for plant biologists in general and the seagrass community in particular. The database is expected to grow together with more data to come in the near future, particularly with the recent initiation of the Zostera genome sequencing project. The Dr. Zompo database is available at http://drzompo.uni-muenster.de/

Beschreibung: Understanding the factors that influence the distribution and abundance of marine diazotrophs is important in order to assess their role in the oceanic nitrogen cycle. Environmental DNA samples from four cruises to the North Atlantic Ocean, covering a sampling area of 0°N to 42°N and 67°W to 13°W, were analyzed for the presence and amount of seven nifH phylotypes using real-time quantitative PCR and TaqMan probes. The cyanobacterial phylotypes dominated in abundance (94% of all nifH copies detected) and were the most widely distributed. The filamentous cyanobacterial type, which included both Trichodesmium and Katagnymene, was the most abundant (51%), followed by group A, an uncultured unicellular cyanobacterium (33%), and gamma A, an uncultured gammaproteobacterium (6%). Group B, unicellular cyanobacterium Crocosphaera, and group C Cyanothece-like phylotypes were not often detected (6.9% and 2.3%, respectively), but where present, could reach high concentrations. Gamma P, another uncultured gammaproteobacterium, was seldom detected (0.5%). Water temperature appeared to influence the distribution of many nifH phylotypes. Very high (up to 1 × 106 copies liter−1) nifH concentrations of group A were detected in the eastern basin (25 to 17°N, 27 to 30°W), where the temperature ranged from 20 to 23°C. The highest concentrations of filamentous phylotypes were measured between 25 and 30°C. The uncultured cluster III phylotype was uncommon (0.4%) and was associated with mean water temperatures of 18°C. Diazotroph abundance was highest in regions where modeled average dust deposition was between 1 and 2 g/m2/year.

Beschreibung: Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.

Beschreibung: Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission

Beschreibung: Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed “IF clusters,” that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.

Beschreibung: Numerous marine sponges harbor enormous amounts of as-yet-uncultivated bacteria in their tissues. There is increasing evidence that these symbionts play an important role in the synthesis of protective metabolites, many of which are of great pharmacological interest. In this study, genes for the biosynthesis of polyketides, one of the most important classes of bioactive natural products, were systematically investigated in 20 demosponge species from different oceans. Unexpectedly, the sponge metagenomes were dominated by a ubiquitously present, evolutionarily distinct, and highly sponge-specific group of polyketide synthases (PKSs). Open reading frames resembling animal fatty acid genes were found on three corresponding DNA regions isolated from the metagenomes of Theonella swinhoei and Aplysina aerophoba. Their architecture suggests that methyl-branched fatty acids are the metabolic product. According to a phylogenetic analysis of housekeeping genes, at least one of the PKSs belongs to a bacterium of the Deinococcus-Thermus phylum. The results provide new insights into the chemistry of sponge symbionts and allow inference of a detailed phylogeny of the diverse functional PKS types present in sponge metagenomes. Based on these qualitative and quantitative data, we propose a significantly simplified strategy for the targeted isolation of biomedically relevant PKS genes from complex sponge-symbiont associations.

Beschreibung: The cold-water coral Lophelia pertusa (Scleractinia, Caryophylliidae) is a key species in the formation of cold-water reefs, which are among the most diverse deep-sea ecosystems. It occurs in two color varieties: white and red. Bacterial communities associated with Lophelia have been investigated in recent years, but the role of the associated bacteria remains largely obscure. This study uses catalyzed reporter deposition fluorescence in situ hybridization to detect the in situ location of specific bacterial groups on coral specimens from the Trondheimsfjord (Norway). Two tissue-associated groups were identified: (i) bacteria on the host's tentacle ectoderm, “Candidatus Mycoplasma corallicola,” are flasklike, pointed cells and (ii) endoderm-associated bona fide TM7 bacteria form long filaments in the gastral cavity. These tissue-bound bacteria were found in all coral specimens from the Trondheimsfjord, indicating a closer relationship with the coral compared to bacterial assemblages present in coral mucus and gastric fluid.

Beschreibung: To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.

Beschreibung: Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

Beschreibung: Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --〉 malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.

Beschreibung: Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications.

Beschreibung: Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo 13C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO2 fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO2 acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the α-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.

Beschreibung: The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

Beschreibung: The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Schöhsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Schöhsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Schöhsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environments

Beschreibung: The taxonomy of two sympatric surf clams Donax marincovichi Coan, 1983 and Donax obesulus Reeve, 1854, inhabiting the coastal Humboldt Current Upwelling System is revisited. Because both species are exploited by artisanal fisheries, it is essential to verify that they are indeed distinct species that have to be managed separately. In this study, both taxa were sampled across their shared distributional area and specimens were indentified according to their respective morphological characteristics. Although width/height and height/length ratios revealed significant differences within sampling areas, the two morphotypes were frequently incongruent for taxonomically important morphometric parameters. In addition, they showed no significant mitochondrial genetic differentiation within or among populations and exhibited indistinguishable sperm ultrastructure. We conclude that the two morphotypes do not represent distinct species and should be included together under D. obesulus.

Beschreibung: Sulfur-oxidizing epsilonproteobacteria are common in a variety of sulfidogenic environments. These autotrophic and mixotrophic sulfur-oxidizing bacteria are believed to contribute substantially to the oxidative portion of the global sulfur cycle. In order to better understand the ecology and roles of sulfur-oxidizing epsilonproteobacteria, in particular those of the widespread genus Sulfurimonas, in biogeochemical cycles, the genome of Sulfurimonas denitrificans DSM1251 was sequenced. This genome has many features, including a larger size (2.2 Mbp), that suggest a greater degree of metabolic versatility or responsiveness to the environment than seen for most of the other sequenced epsilonproteobacteria. A branched electron transport chain is apparent, with genes encoding complexes for the oxidation of hydrogen, reduced sulfur compounds, and formate and the reduction of nitrate and oxygen. Genes are present for a complete, autotrophic reductive citric acid cycle. Many genes are present that could facilitate growth in the spatially and temporally heterogeneous sediment habitat from where Sulfurimonas denitrificans was originally isolated. Many resistance-nodulation-development family transporter genes (10 total) are present; of these, several are predicted to encode heavy metal efflux transporters. An elaborate arsenal of sensory and regulatory protein-encoding genes is in place, as are genes necessary to prevent and respond to oxidative stress.

Beschreibung: Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.

Beschreibung: The pseudocolonial coral Lophelia pertusa (Scleractinia, Caryophylliidae) is a eurybathic, stenothermal cosmopolitan cold-water species. It occurs in two color varieties, white and red. L. pertusa builds vast cold-water coral reefs along the continental margins, which are among the most diverse deep-sea habitats. Microbiology of L. pertusa has been in scientific focus for only a few years, but the question of whether the coral holds a host-specific bacterial community has not been finally answered. Bacteria on coral samples from the Trondheimsfjord (Norway) were characterized by the culture-independent 16S rRNA gene-based techniques terminal restriction fragment length polymorphism and sequence analysis. L. pertusa revealed a high microbial richness. Clone sequences were dominated by members of the Alpha- and Gammaproteobacteria. Other abundant taxa were Bacteroidetes, Actinobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes. The bacterial community of L. pertusa not only differed conspicuously from that of the environment but also varied with both the location and color variety of its host. Therefore, the microbial colonization cannot be termed “specific” sensu stricto. However, similarities to other coral-bacterium associations suggest the existence of “cold-water coral-specific” bacterial groups sensu lato. L. pertusa-associated bacteria appear to play a significant role in the nutrition of their host by degradation of sulfur compounds, cellulose, chitin, and end products of the coral's anaerobic metabolism. Some coral-associated microbes were regarded as opportunistic pathogens. Dominance of mixotrophic members of the Rhodobacteraceae in white L. pertusa could explain the wider dispersal of this phenotype by supplementary nutrition.

Beschreibung: Microbial isolate Z143-1 found to be associated with an unidentified tunicate was characterized due to its significant antimicrobial activity. Z143-1 is similar to Pseudovibrio ascidiaceicola and Pseudovibrio denitrificans in morphological, physiological and biochemical characteristics, except for its ability to ferment glucose and produce a characteristic red pigment. Fatty acid methyl ester analysis revealed a predominance of the fatty acid 18:1 ω7c at 80.55%, at levels slightly lower than the Pseudovibrio denitrificans type strain DN34T (87.7%). The mol% G+C of Z143-1 is 54.02, relatively higher than the Pseudovibrio denitrificans type strain DN34T and Pseudovibrio ascidiaceicola with mol% G+C of 51.7 and 51.4, respectively. However, phylogenetic analysis of the 16S rRNA gene sequence of Z143-1 showed 100% similarity with the Pseudovibrio denitrificans type strain DN34T. In this study, the bacterium Z143-1 is reported as a new strain of Pseudovibrio denitrificans. While there is no report of a secondary metabolite for Pseudovibrio denitrificans, Z143-1 produces the red pigment heptylprodigiosin, also known as 16-methyl-15-heptyl-prodiginine, which shows anti-Staphylococcus aureus activity.

Beschreibung: Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC50], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahydroxyflavone (IC50s, 0.5 μg/ml) and catechol (IC50, 0.8 μg/ml). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC50s less than 5.0 μg/ml. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC50s of 0.6, 0.7, 0.8, and 1.0 μg/ml, respectively. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compounds lacked cytotoxicity in vitro and in vivo. By screening a large number of flavonoids and analogues, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quantitative structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivatives as potent and effective antiprotozoal agents.

Beschreibung: The basal position of the Mamiellales (Prasinophyceae) within the green lineage makes these unicellular organisms key to elucidating early stages in the evolution of chlorophyll a/b-binding light-harvesting complexes (LHCs). Here, we unveil the complete and unexpected diversity of Lhc proteins in Ostreococcus tauri, a member of the Mamiellales order, based on results from complete genome sequencing. Like Mantoniella squamata, O. tauri possesses a number of genes encoding an unusual prasinophyte-specific Lhc protein type herein designated "Lhcp". Biochemical characterization of the complexes revealed that these polypeptides, which bind chlorophylls a, b, and a chlorophyll c-like pigment (Mg-2,4-divinyl-phaeoporphyrin a 5 monomethyl ester) as well as a number of unusual carotenoids, are likely predominant. They are retrieved to some extent in both reaction center I (RCI)- and RCII-enriched fractions, suggesting a possible association to both photosystems. However, in sharp contrast to previous reports on LHCs of M. squamata, O. tauri also possesses other LHC subpopulations, including LHCI proteins (encoded by five distinct Lhca genes) and the minor LHCII polypeptides, CP26 and CP29. Using an antibody against plant Lhca2, we unambiguously show that LHCI proteins are present not only in O. tauri, in which they are likely associated to RCI, but also in other Mamiellales, including M. squamata. With the exception of Lhcp genes, all the identified Lhc genes are present in single copy only. Overall, the discovery of LHCI proteins in these prasinophytes, combined with the lack of the major LHCII polypeptides found in higher plants or other green algae, supports the hypothesis that the latter proteins appeared subsequent to LHCI proteins. The major LHC of prasinophytes might have arisen prior to the LHCII of other chlorophyll a/b-containing organisms, possibly by divergence of a LHCI gene precursor. However, the discovery in O. tauri of CP26-like proteins, phylogenetically placed at the base of the major LHCII protein clades, yields new insight to the origin of these antenna proteins, which have evolved separately in higher plants and green algae. Its diverse but numerically limited suite of Lhc genes renders O. tauri an exceptional model system for future research on the evolution and function of LHC components. © The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

Beschreibung: Background During the Ordovician the global diversity increased dramatically at family, genus and species levels. Partially the diversification is explained by an increased nutrient, and phytoplankton availability in the open water. Cephalopods are among the top predators of todays open oceans. Their Ordovician occurrences, diversity evolution and abundance pattern potentially provides information on the evolution of the pelagic food chain. Methodology/Principal Findings We reconstructed the cephalopod departure from originally exclusively neritic habitats into the pelagic zone by the compilation of occurrence data in offshore paleoenvironments from the Paleobiology Database, and from own data, by evidence of the functional morphology, and the taphonomy of selected cephalopod faunas. The occurrence data show, that cephalopod associations in offshore depositional settings and black shales are characterized by a specific composition, often dominated by orthocerids and lituitids. The siphuncle and conch form of these cephalopods indicate a dominant lifestyle as pelagic, vertical migrants. The frequency distribution of conch sizes and the pattern of epibionts indicate an autochthonous origin of the majority of orthocerid and lituitid shells. The consistent concentration of these cephalopods in deep subtidal sediments, starting from the middle Tremadocian indicates the occupation of the pelagic zone early in the Early Ordovician and a subsequent diversification which peaked during the Darriwilian. Conclusions/Significance The exploitation of the pelagic realm started synchronously in several independent invertebrate clades during the latest Cambrian to Middle Ordovician. The initial rise and diversification of pelagic cephalopods during the Early and Middle Ordovician indicates the establishment of a pelagic food chain sustainable enough for the development of a diverse fauna of large predators. The earliest pelagic cephalopods were slowly swimming vertical migrants. The appearance and early diversification of pelagic cephalopods is interpreted as a consequence of the increased food availability in the open water since the latest Cambrian.

Beschreibung: Mechanisms that affect thermal tolerance of ectothermic organisms have recently received much interest, mainly due to global warming and climate-change debates in both the public and in the scientific community. In physiological terms, thermal tolerance of several marine ectothermic taxa can be linked to oxygen availability, with capacity limitations in ventilatory and circulatory systems contributing to oxygen limitation at extreme temperatures. The present review briefly summarizes the processes that define thermal tolerance in a model cephalopod organism, the cuttlefish Sepia officinalis, with a focus on the contribution of the cephalopod oxygen-carrying blood pigment, hemocyanin. When acutely exposed to either extremely high or low temperatures, cuttlefish display a gradual transition to an anaerobic mode of energy production in key muscle tissues once critical temperatures (Tcrit) are reached. At high temperatures, stagnating metabolic rates and a developing hypoxemia can be correlated with a progressive failure of the circulatory system, well before Tcrit is reached. However, at low temperatures, declining metabolic rates cannot be related to ventilatory or circulatory failure. Rather, we propose a role for hemocyanin functional characteristics as a major limiting factor preventing proper tissue oxygenation. Using information on the oxygen binding characteristics of cephalopod hemocyanins, we argue that high oxygen affinities (= low P50 values), as found at low temperatures, allow efficient oxygen shuttling only at very low venous oxygen partial pressures. Low venous PO2s limit rates of oxygen diffusion into cells, thus eventually causing the observed transition to anaerobic metabolism. On the basis of existing blood physiological, molecular, and crystallographical data, the potential to resolve the role of hemocyanin isoforms in thermal adaptation by an integrated molecular physiological approach is discussed.

Beschreibung: Recent molecular studies investigating higher-level phylogenetics of coleoid cephalopods (octopuses, squids and cuttlefishes) have produced conflicting results. A wide range of sequence alignment and analysis methods are used in cephalopod phylogenetic studies. The present study investigated the effect of commonly used alignment and analysis methods on higher-level cephalopod phylogenetics. Two sequence homology methods: (1) eye alignment, (2) implied alignment, and three analysis methods: (1) parsimony, (2) maximum likelihood, (3) Bayesian methodologies, were employed on the longest sequence dataset available for the coleoid cephalopods, comprising three mitochondrial and six nuclear loci. The data were also tested for base composition heterogeneity, which was detected in three genes and resolved using RY coding. The Octopoda, Argonautoidea, Oegopsida and Ommastrephidae are monophyletic in the phylogenies resulting from each of the alignment and analysis combinations. Furthermore, the Bathyteuthidae are the sister taxon of the Oegopsida in each case. However many relationships within the Coleoidea differed depending upon the alignment and analysis method used. This study demonstrates how differences in alignment and analysis methods commonly used in cephalopod phylogenetics can lead to different, but often highly supported, relationships.

Beschreibung: Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study.

Beschreibung: To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of 〈26°C, and were sometimes recovered from waters with detectable nitrate concentrations.

Beschreibung: A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.

Beschreibung: The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, α-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly β-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.