Distribución y degradación de las cianotoxinas microcistina y cilidrospermopsina en embalses = distribution and degradaton of the cyanobacterial

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 15-09-200

Novel Cardiolipins from Uncultured Methane-Metabolizing Archaea

Novel cardiolipins from Archaea were detected by screening the intact polar lipid (IPL) composition of microbial communities associated with methane seepage in deep-sea sediments from the Pakistan margin by high-performance liquid chromatography electrospray ionization mass spectrometry. A series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol, BPG) represented 0.5% to 5% of total archaeal IPLs. These molecules are similar to the recently described cardiolipin analogues with four phytanyl chains from extreme halophilic archaea. It is worth noting that cardiolipin analogues from the seep archaeal communities are composed of four isoprenoidal chains, which may contain differences in chain length (20 and 25 carbon atoms) and degrees of unsaturation and the presence of a hydroxyl group. Two novel diether lipids, structurally related to the BPGs, are described and interpreted as degradation products of archaeal cardiolipin analogues. Since archaeal communities in seep sediments are dominated by anaerobic methanotrophs, our observations have implications for characterizing structural components of archaeal membranes, in which BPGs are presumed to contribute to modulation of cell permeability properties. Whether BPGs facilitate interspecies interaction in syntrophic methanotrophic consortia remains to be tested

Overwintering populations of Anabaena, Aphanizomenon and Microcystis as potential inocula for summer blooms

Overwintering cyanobacterial populations of Nostocales and Microcystis were investigated in six freshwater reservoirs in Northwestern Spain during two consecutive winters. Surface sediments hosted 103–105 akinetes mL21 and 102–104 Microcystis colonies mL21. Sediments from deeper areas close to dam accumulated 2-fold (Microcystis) and 11-fold (akinetes) greater concentrations than those at the shallower upstream areas. Anabaena spp. and Microcystis aeruginosa dominated the sediment pool, with minor amounts of akinetes of Aphanizomenon (Aph. flos-aquae, Aph. gracile) and benthic Nostocales (Cylindrospermum, Nostoc and Trichormus). Our study confirms the dual benthic-pelagic overwintering of Anabaena, Aphanizomenon and Microcystis, found in the pelagial at 7.5–9.88C. This study also provides an insight into the little known annual cycle of potential cyanotoxin-producers Aph. gracile and Anabaena circinalis. Our estimates show that: (i) only a small fraction (,1%) of the sediment pool of akinetes and Microcystis was resupended in the bottom water during winter which, however, may be sufficient inocula to build up the summer maxima under realistic in situ growth rates; and (ii) the time required for the development of summer populations is mainly driven by growth rates, and therefore by the environmental conditions faced by the inoculum, with a lower influence (although greater for Microcystis than for Nostocales) of the inoculum sizeSamuel Cirés and Lars Wörmer were supported by a FPU grant from Ministerio de Ciencia e Innovación (MICINN, Spain). This work was partially funded by the Confederacion Hidrográfica del Cantábrico and Confederación Hidrográfica del Miño-Sil (MMAMRM, Spain) projects Exp. 01.812.008/0411 and Exp. 01.812-008/0481

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing Nostocales cyanobacteria from Mediterranean Europe (Spain)

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg-1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporumThis study was partially funded by grants from the German Ministry of Education, Science and Research (BMBF; 0330792) and the Kompetenzzentrum Wasser Berlin GmbH with financial support from Veolia Water and the Berliner Wasserbetriebe. We thank the United Research Services España S.L. company for partially funding this study. We are grateful to the Spanish public entities Canal de Isabel II, C. H. del Norte (Ministerio de Medio Ambiente), and CEDEX (Ministerio de Fomento) and to the United Research Services España S.L. company for providing water samples. We are grateful to Elena Galán and Celia Ratón (Universidad Autónoma de Madrid, Madrid, Spain) for their valuable help with strain isolation and toxin analysis. Finally, we thank two anonymous reviewers for their constructive comments on earlier versions of the manuscrip

Global Diversity of Microbial Communities in Marine Sediment

Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species–area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 103 to 6.10 × 105 and 3.28 × 104 to 2.46 × 106 amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth’s global biosphere

Ecotoxicity assessment of microcystins from freshwater samples using a bioluminescent cyanobacterial bioassay

The hepatotoxic cyanotoxins microcystins (MCs) are emerging contaminants naturally produced by cyanobacteria. Yet their ecological role remains unsolved, previous research suggests that MCs have allelopathic effects on competing photosynthetic microorganisms, even eliciting toxic effects on other freshwater cyanobacteria. In this context, the bioluminescent recombinant cyanobacterium Anabaena sp. PCC7120 CPB4337 (hereinafter Anabaena) was exposed to extracts of MCs. These were obtained from eight natural samples from freshwater reservoirs that contained MCs with a concentration range of 0.04–11.9 μg MCs L−1. MCs extracts included the three most common MCs variants (MC-LR, MC-RR, MC-YR) in different proportions (MC-LR: 100–0%; MC-RR: 100–0%; MC-YR: 14.2–0%). The Anabaena bioassay based on bioluminescence inhibition has been successfully used to test the toxicity of many emerging contaminants (e.g., pharmaceuticals) but never for cyanotoxins prior to this study. Exposure of Anabaena to MCs extracts induced a decrease in its bioluminescence with effective concentration decreasing bioluminescence by 50% ranging from 0.4 to 50.5 μg MC L−1 in the different samples. Bioluminescence responses suggested an interaction between MCs variants which was analyzed via the Additive Index method (AI), indicating an antagonistic effect (AI < 0) of MC-LR and MC-RR present in the samples. Additionally, MC extracts exposure triggered an increase of intracellular free Ca2+ in Anabaena. In short, this study supports the use of the Anabaena bioassay as a sensitive tool to assess the presence of MCs at environmentally relevant concentrations and opens interesting avenues regarding the interactions between MCs variants and the possible implication of Ca2+ in the mode of action of MCs towards cyanobacteri

Sedimentation Patterns of Toxin-Producing Microcystis Morphospecies in Freshwater Reservoirs

toxin

The ABC Transporter Components HgdB and HgdC are Important for Glycolipid Layer Composition and Function of Heterocysts in Anabaena sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium able to fix atmospheric nitrogen in semi-regularly spaced heterocysts. For correct heterocyst function, a special cell envelope consisting of a glycolipid layer and a polysaccharide layer is essential. We investigated the role of the genes hgdB and hgdC, encoding domains of a putative ABC transporter, in heterocyst maturation. We investigated the subcellular localization of the fusion protein HgdC-GFP and followed the differential expression of the hgdB and hgdC genes during heterocyst maturation. Using a single recombination approach, we created a mutant in hgdB gene and studied its phenotype by microscopy and analytical chromatography. Although heterocysts are formed in the mutant, the structure of the glycolipid layer is aberrant and also contains an atypical ratio of the two major glycolipids. As shown by a pull-down assay, HgdB interacts with the outer membrane protein TolC, which indicates a function as a type 1 secretion system. We show that the hgdB-hgdC genes are essential for the creation of micro-oxic conditions by influencing the correct composition of the glycolipid layer for heterocyst function. Our observations confirm the significance of the hgdB-hgdC gene cluster and shed light on a novel mode of regulation of heterocyst envelope formation