RESUMEN

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RESUMEN

There is growing evidence for bacteria playing a role in the pathogenesis and formation of pigmented gallstones from humans. These studies mainly involved cultivation of gallstone-associated bacteria and 16S rRNA profiling, providing an indirect link between processes involved in gallstone formation by the bacteria in-situ. Here, we provide functional metagenomic evidence of a range of genes involved in bile stress response, biofilm formation, and anaerobic energy metabolism by Gram-negative Klebsiella in pigmented gallstones from a 76-year-old male patient. Klebsiella was also present in one cholesterol-type stone in a 30-year-old female patient who had additional cholesterol gallstones characterised by Gram-positive bacteria. Pigmented stones further revealed a predominance of genes involved in carbohydrate metabolism, whilst cholesterol stones indicated a profile dominanted by protein metabolism possibly reflecting known chemical differences between Gram-negative and Gram-positive biofilm matrices. Archaeal genes were not detected. Complementary carbon and hydrogen isotopic analyses of cholesterol within the patients' stones revealed homogeneity, suggesting a common diet or cholesterol biosynthesis pathway that has little influence on microbial composition. This pilot study provides a framework to study microbial processes that play a potential role in gallstone formation across markedly different types of stones and patient backgrounds.

Asunto(s)

Bacterias/genética , Colesterol/genética , Cálculos Biliares/microbiología , Metagenómica , Adulto , Anciano , Bacterias/clasificación , Bacterias/aislamiento & purificación , Biopelículas/crecimiento & desarrollo , Femenino , Cálculos Biliares/genética , Cálculos Biliares/patología , Humanos , Klebsiella/genética , Klebsiella/patogenicidad , Masculino , Pigmentación/genética , ARN Ribosómico 16S/genética

RESUMEN

Recent studies have shown that ancient plankton DNA can be recovered from Holocene lacustrine and marine sediments, including from species that do not leave diagnostic microscopic fossils in the sediment record. Therefore, the analysis of this so-called fossil plankton DNA is a promising approach for refining paleoecological and paleoenvironmental information. However, further studies are needed to reveal whether DNA of past plankton is preserved beyond the Holocene. Here, we identified past eukaryotic plankton members based on 18S rRNA gene profiling in eastern Mediterranean Holocene and Pleistocene sapropels S1 (~9 ka), S3 (~80 ka), S4 (~105 ka), and S5 (~125 ka). The majority of preserved ~400- to 500-bp-long 18S rDNA fragments of microalgae that were studied in detail (i.e. from haptophyte algae and dinoflagellates) were found in the youngest sapropel S1, whereas their specific lipid biomarkers (long-chain alkenones and dinosterol) were also abundant in sediments deposited between 80 and 124 ka BP. The late-Pleistocene sediments mainly contained eukaryotic DNA of marine fungi and from terrestrial plants, which could have been introduced via the river Nile at the time of deposition and preserved in pollen grains. A parallel analysis of Branched and Isoprenoid Tetraethers (i.e. BIT index) showed that most of the organic matter in the eastern Mediterranean sediment record was of marine (e.g. pelagic) origin. Therefore, the predominance of terrestrial plant DNA over plankton DNA in older sapropels suggests a preferential degradation of marine plankton DNA.

Asunto(s)

Dinoflagelados/genética , Sedimentos Geológicos/química , Haptophyta/genética , Paleontología/métodos , Reacción en Cadena de la Polimerasa/métodos , Benzopiranos/análisis , Biomarcadores/análisis , Biomasa , Colestenos/análisis , Electroforesis en Gel de Gradiente Desnaturalizante/métodos , Dinoflagelados/química , Dinoflagelados/clasificación , Éteres/análisis , Eucariontes/química , Eucariontes/clasificación , Eucariontes/genética , Fósiles , Haptophyta/química , Haptophyta/clasificación , Sustancias Húmicas/análisis , Cetonas/análisis , Mar Mediterráneo , Filogenia , Fitoplancton/química , Fitoplancton/clasificación , Fitoplancton/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificación

RESUMEN

Recent work has shown that paleoenvironmental genomics, i.e. the application of genomic tools to analyze preserved DNA in sedimentary records, is a promising approach to reconstruct the diversity of past planktonic communities. This provides information about past ecological and environmental changes. A major advantage of this approach is that individual species, including those that did not leave other characteristic markers, can be identified. In this study, we determined which dinoflagellate marker (i.e. 18S rDNA, dinosterol or dinocysts) provided the most detailed information about the late-Holocene succession of dinoflagellates in an Antarctic Fjord (Ellis Fjord, Vestfold Hills). The preserved rDNA revealed two intervals in the 2750-year-old sediment record. The dinoflagellate diversity was the highest until approximately 1850 cal yr bp and included phylotypes related to known dinosterol producers. A lower concentration of dinosterol in sediments <1850 cal yr bp coincided with a community shift towards a predominance of the autotrophic sea-ice dinoflagellate Polarella glacialis, which is not a source of dinosterol. Remarkably, cultures of P. glacialis are known to produce other diagnostic sterols, but these were not recovered here. In addition, conspicuous resting cysts of P. glacialis were not preserved in the analyzed sediments. Overall, dinocysts were rare and the paleoenvironmental genomics approach revealed the highest diversity of dinoflagellates in Ellis Fjord, and was the only approach that recorded a shift in dinoflagellate composition at approximately 1850 cal yr bp indicative of a colder climate with more extensive ice cover - this timing coincides with a period of changing climate reported for this region.

Asunto(s)

ADN Protozoario/aislamiento & purificación , Dinoflagelados/genética , Sedimentos Geológicos/parasitología , Lípidos/aislamiento & purificación , Animales , Regiones Antárticas , Biodiversidad , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Datos de Secuencia Molecular , Filogenia , ARN Protozoario/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN

RESUMEN

The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of approximately 3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unusually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

Asunto(s)

Ecosistema , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Agua de Mar/química , Agua de Mar/microbiología , Recuento de Colonia Microbiana , Medios de Cultivo , Dermatoglifia del ADN/métodos , ADN Ribosómico/análisis , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/crecimiento & desarrollo , Mar Mediterráneo , ARN Ribosómico 16S/genética

RESUMEN

Hydrolytic exoenzymes as indicators of metabolically active bacteria were investigated in four consecutive sapropel layers collected from bathyal sediments of the eastern Mediterranean Sea. For comparison, the organic carbon-poor layers between the sapropels, sediment from the anoxic Urania basin, and sediments of intertidal mud flats of the German Wadden Sea were also analyzed. The sapropel layers contained up to 1.5. 10(8) bacterial cells cm(-3), whereas cell numbers in the intermediate layers were lower by a factor of 10. In sapropels, the determination of exoenzyme activity with fluorescently labeled substrate analogues was impaired by the strong adsorption of up to 97% of the enzymatically liberated fluorophores (4-methylumbelliferone [MUF] and 7-amino-4-methylcoumarin [MCA]) to the sediment particles. Because all established methods for the extraction of adsorbed fluorophores proved to be inadequate for sapropel sediments, we introduce a correction method which is based on the measurement of equilibrium adsorption isotherms for both compounds. Using this new approach, high activities of aminopeptidase and alkaline phosphatase were detected even in a 124,000-year-old sapropel layer, whereas the activity of beta-glucosidase was low in all layers. So far, it had been assumed that the organic matter which constitutes the sapropels is highly refractory. The high potential activities of bacterial exoenzymes indicate that bacteria in Mediterranean sapropels are metabolically active and utilize part of the subfossil kerogen. Since a high adsorption capacity was determined not only for the low-molecular-weight compounds MUF and MCA but also for DNA, the extraordinarily strong adsorption of structurally different substrates to the sapropel matrix appears to be the major reason for the long-term preservation of biodegradable carbon in this environment.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Aminopeptidasas/metabolismo , Bacterias/enzimología , Sedimentos Geológicos/microbiología , beta-Glucosidasa/metabolismo , Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Recuento de Colonia Microbiana , Cumarinas/metabolismo , Colorantes Fluorescentes/metabolismo , Fósiles , Himecromona/metabolismo , Cinética , Mar Mediterráneo , Agua de Mar/microbiología , Microbiología del Agua

RESUMEN

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and beta-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, beta-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species.

Asunto(s)

Bacterias/clasificación , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , ADN Ribosómico/genética , Ecosistema , ARN Ribosómico 16S/genética , Microbiología del Agua , Bacterias/genética , Bacterias/aislamiento & purificación , Cartilla de ADN , Desulfovibrio/genética , Desulfovibrio/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Agua Dulce , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Agua de Mar , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico

RESUMEN

Molecular remains of purple sulfur bacteria (Chromatiaceae) were detected in Holocene sediment layers of a meromictic salt lake (Mahoney Lake, British Columbia, Canada). The carotenoid okenone and bacteriophaeophytin a were present in sediments up to 11,000 years old. Okenone is specific for only a few species of Chromatiaceae, including Amoebobacter purpureus, which presently predominates in the chemocline bacterial community of the lake. With a primer set specific for Chromatiaceae in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of four different Chromatiaceae species were retrieved from different depths of the sediment. One of the sequences, which originated from a 9, 100-year-old sample, was 99.2% identical to the 16S rRNA gene sequence of A. purpureus ML1 isolated from the chemocline. Employing primers specific for A. purpureus ML1 and dot blot hybridization of the PCR products, the detection limit for A. purpureus ML1 DNA could be lowered to 0.004% of the total community DNA. With this approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that A. purpureus ML1 constituted at least a part of the ancient purple sulfur bacterial community. The concentrations of A. purpureus DNA and okenone in the sediment were not correlated, and the ratio of DNA to okenone was much lower in the subfossil sediment layers (2.7 . 10(-6)) than in intact cells (1.4). This indicates that degradation rates are significantly higher for genomic DNA than for hydrocarbon cell constituents, even under anoxic conditions and at the very high sulfide concentrations present in Mahoney Lake.

Asunto(s)

Chromatiaceae/clasificación , Chromatiaceae/genética , Fósiles , Filogenia , ARN Ribosómico 16S/genética , Secuencia de Bases , Chromatiaceae/aislamiento & purificación , Cartilla de ADN , ADN Ribosómico/genética , Agua Dulce/microbiología , Alemania , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/genética , Microbiología del Suelo , Especificidad de la Especie