Bulk sediment characteristics and geomicrobiology of sediment core 5022-1J from Laguna Potrok Aike, southern Patagonia

Living microorganisms inhabit every environment of the biosphere but only in the last decades their importance governing biochemical cycles in deep sediments has been widely recognized. Most investigations have been accomplished in the marine realm whereas there is a clear paucity of comparable studies in lacustrine sediments. One of the main challenges is to define geomicrobiological proxies that can be used to identify different microbial signals in the sediments. Laguna Potrok Aike, a maar lake located in Southeastern Patagonia, has an annually not stratifying cold water column with temperatures ranging between 4 and 10 °C, and most probably an anoxic water/sediment interface. These unusual features make it a peculiar and interesting site for geomicrobiological studies. Living microbial activity within the sediments was inspected by the first time in a sedimentary core retrieved during an ICDP-sponsored drilling operation. The main goals to study this cold subsaline environment were to characterize the living microbial consortium; to detect early diagenetic signals triggered by active microbes; and to investigate plausible links between climate and microbial populations. Results from a meter long gravity core suggest that microbial activity in lacustrine sediments can be sustained deeper than previously thought due to their adaptation to both changing temperature and oxygen availability. A multi-proxy study of the same core allowed defining past water column conditions and further microbial reworking of the organic fraction within the sediments. Methane content shows a gradual increase with depth as a result of the fermentation of methylated substrates, first methanogenic pathway to take place in the shallow subsurface of freshwater and subsaline environments. Statistical analyses of DGGE microbial diversity profiles indicate four clusters for Bacteria reflecting layered communities linked to the oxidant type whereas three clusters characterize Archaea communities that can be linked to both denitrifiers and methanogens. Independent sedimentary and biological proxies suggest that organic matter production and/or preservation have been lower during the Medieval Climate Anomaly (MCA) coinciding with a low microbial colonization of the sediments. Conversely, a reversed trend with higher organic matter content and substantial microbial activity characterizes the sediments deposited during the Little Ice Age (LIA). Thus, the initial sediments deposited during distinctive time intervals under contrasting environmental conditions have to be taken into account to understand their impact on the development of microbial communities throughout the sediments and their further imprint on early diagenetic signals.

Caractérisation moléculaire de la biodiversité des Fusarium associés à la fusariose de l'asperge (Asparagus officinalis L.) au Québec

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Caractérisation moléculaire de la biodiversité des Fusarium associés à la fusariose de l'asperge (Asparagus officinalis L.) au Québec

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Geomicrobiology, bulk sediment characteristics and pore water chemistry of sediment cores from Laguna Potrok Aike, southern Patagonia

Authigenic minerals can form in the water column and sediments of lakes, either abiotically or mediated by biological activity. Such minerals have been used as paleosalinity and paleoproductivity indicators and reflect trophic state and early diagenetic conditions. They are also considered potential indicators of past and perhaps ongoing microbial activity within sediments. Authigenic concretions, including vivianite, were described in late glacial sediments of Laguna Potrok Aike, a maar lake in southernmost Argentina. Occurrence of iron phosphate implies specific phosphorus sorption behavior and a reducing environment, with methane present. Because organic matter content in these sediments was generally low during glacial times, there must have been alternative sources of phosphorus and biogenic methane. Identifying these sources can help define past trophic state of the lake and diagenetic processes in the sediments. We used scanning electron microscopy, phosphorus speciation in bulk sediment, pore water analyses, in situ ATP measurements, microbial cell counts, and measurements of methane content and its carbon isotope composition (d13C CH4) to identify components of and processes in the sediment. The multiple approaches indicated that volcanic materials in the catchment are important suppliers of iron, sulfur and phosphorus. These elements influence primary productivity and play a role in microbial metabolism during early diagenesis. Authigenic processes led to the formation of pyrite framboids and revealed sulfate reduction. Anaerobic oxidation of methane and shifts in pore water ion concentration indicated microbial influence with depth. This study documents the presence of active microbes within the sediments and their relationship to changing environmental conditions. It also illustrates the substantial role played by microbes in the formation of Laguna Potrok Aike concretions. Thus, authigenic minerals can be used as biosignatures in these late Pleistocene maar sediments.

Microbial community of black band disease on infection, healthy and dead part of scleractinian Montipora sp. colony at Seribu islands, Indonesia

It is crucial to understand the microbial community associated with the host when attempting to discern the pathogen responsible for disease outbreaks in scleractinian corals. This study determines changes in the bacterial community associated with Montipora sp. in response to black band disease in Indonesian waters. Healthy, diseased, and dead Montipora sp. (n = 3 for each sample type per location) were collected from three different locations (Pari Island, Pramuka Island, and Peteloran Island). DGGE (Denaturing Gradient Gel Electrophoresis) was carried out to identify the bacterial community associated with each sample type and histological analysis was conducted to identify pathogens associated with specific tissues. Various Desulfovibrio species were found as novelty to be associated with infection samples, including Desulfovibrio desulfuricans, Desulfovibrio magneticus, and Desulfovibrio gigas, Bacillus benzoevorans, Bacillus farraginis in genus which previously associated with pathogenicity in corals. Various bacterial species associated with uninfected corals were lost in diseased and dead samples. Unlike healthy samples, coral tissues such as the epidermis, endodermis, zooxanthellae were not present on dead samples under histological observation. Liberated zooxanthellae and cyanobacteria were found in black band diseased Montipora sp. samples.

Exploring hydrocarbonoclastic bacterial communities in the estuarine surface microlayer

Bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) in the estuarine surface microlayer (SML) of the Ria de Aveiro, Portugal—which is chronically polluted with oil hydrocarbons (OH)—were isolated and characterized; Pseudomonas was dominant among the PAH-degrading bacteria. Screening for PAH dioxygenase genes detected almost identical nahAc genes (encoding the alpha subunits of naphthalene dioxygenase) in 2 phylogenetically distinct isolates: Pseudomonas sp. and an unknown species of the family Enterobacteriaceae; this suggested that horizontal transfer of nah genes might be involved in PAH degradation in the SML. We also investigated the effect of PAH contamination on the spatial variability of the bacterioneuston along a gradient of pollution in the estuarine system of the Ria de Aveiro. Culture-independent techniques—fluorescence in situ hy - bridization (FISH) and denaturing-gradient gel electrophoresis (DGGE)—revealed a similar structure among the bacterioneuston communities along the estuary. In contrast, we detected differences in the relative abundance and diversity of organisms of the Gammaproteobacteria, including those of the genus Pseudomonas (which belongs to the Gammaproteobacteria). This is the first insight into the hydrocarbonoclastic bacterial communities in the SML of an estuarine area polluted with hydrocarbons. Our findings highlight the importance of SML-adapted hydrocarbonoclastic bacterioneuston as a potential source of new PAH-degrading bacteria (including new pseudomonads) with potential use in the bioremediation of hydrocarbon-polluted ecosystems.

Degradation of Biofumigant Isothiocyanates and Allyl Glucosinolate in Soil and Their Effects on the Microbial Community Composition

Brassicales species rich in glucosinolates are used for biofumigation, a process based on releasing enzymatically toxic isothiocyanates into the soil. These hydrolysis products are volatile and often reactive compounds. Moreover, glucosinolates can be degraded also without the presence of the hydrolytic enzyme myrosinase which might contribute to bioactive effects. Thus, in the present study the stability of Brassicaceae plant-derived and pure glucosinolates hydrolysis products was studied using three different soils ( model biofumigation). In addition, the degradation of pure 2-propenyl glucosinolate was investigated with special regard to the formation of volatile breakdown products. Finally, the influence of pure glucosinolate degradation on the bacterial community composition was evaluated using denaturing gradient gel electrophoresis of 16S rRNA gene amplified from total community DNA. The model biofumigation study revealed that the structure of the hydrolysis products had a significant impact on their stability in the soil but not the soil type. Following the degradation of pure 2-propenyl glucosinolate in the soils, the nitrile as well as the isothiocyanate can be the main degradation products, depending on the soil type. Furthermore, the degradation was shown to be both chemically as well as biologically mediated as autoclaving reduced degradation. The nitrile was the major product of the chemical degradation and its formation increased with iron content of the soil. Additionally, the bacterial community composition was significantly affected by adding pure 2-propenyl glucosinolate, the effect being more pronounced than in treatments with myrosinase added to the glucosinolate. Therefore, glucosinolates can have a greater effect on soil bacterial community composition than their hydrolysis products.

Anaerobic oxidation of methane at a marine methane seep in a forearc sediment basin off Sumatra, Indian Ocean

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by C-13-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO2 was confirmed in a C-13-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 x 10(9) cells cm(-3) sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (delta-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

Intraspecific Relationships in Paracalanus quasimodo [Calinoideae] and Temora turbinata [Calinoideae] along the Southeastern Coast of the United States

Paracalanus quasimodo and Temora turbinata are two calanoid copepods prominent in the planktonic communities of the southeastern United States. Despite their prominence, the species and population level structure of these copepods is yet unexplored. The phylogeographic, temporal and phylogenetic structure of P. quasimodo and T. turbinata are examined in my study. Samples were collected from ten sites along the Gulf of Mexico and Florida peninsular coasts. Three sites were sampled quarterly for two years. Individuals were screened for unique ITS-1 sequences with denaturing gradient gel electrophoresis. Unique variants were sequenced at the nuclear ITS-1 and mitochondrial COI loci. Sampling sites were analyzed for pairwise community differences and for variances between geographic and temporal groupings. Genetic variants were analyzed for phylogenetic and coalescent topology. Paracalanus quasimodo is highly structured geographically with populations divided between the Gulf of Mexico, temperate Atlantic and subtropical Atlantic, in addition to isolation by distance. No significant differences were detected between the T. turbinata samples. Both P. quasimodo and T. turbinata are stable within sites over time and between sites within a sampling period, with two exceptions. The first was a pilot sample from Miami taken two years prior to the general sampling whose community showed significant differences from most of the other Miami samples. Paracalanus quasimodo had a positive correlation of Fst with time. The second was high temporal variability detected in the samples from Fort Pierce. Phylogenetically, both P. quasimodo and T. turbinata were in well supported, congeneric clades. Paracalanus quasimodo was not monophyletic, divided into two well-supported clades. Temora turbinata variants were in one clade with insignificant support for topology within the clade and very little intraspecific variation. Paracalanus quasimodo and T. turbinata populations show opposite trends. Paracalanus quasimodo occurs near shore and shows population structure mediated by hydrological features and distance, both geographic and temporal. The phylogeny shows two deeply divergent clades suggestive of cryptic speciation. In contrast, T. turbinata populations range further offshore and show little geographic or temporal structure. However, the low genetic variation detected in this region suggests a recent bottleneck event.

Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialiswere collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

Sugarcane trash levels in soil affects the fungi but not bacteria in a short-term field experiment.

The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments.

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

Comparative analysis of two-dimensional electrophoresis maps (2-DE) of Helicobacter pylori from Brazilian patients with chronic gastritis and duodenal ulcer: a preliminary report

Helicobacter pylori is a bacterium recognized as the major cause of peptic ulcer and chronic gastritis. Recently, a proteome-based approach was developed to investigate pathogenic factors related to H. pylori. In this preliminary study, H. pylori strains were isolated from gastric biopsies of patients with chronic gastritis and duodenal ulcers. A partial proteomic analysis of H. pylori strains was performed by bacterial lyses and proteins were separated by two-dimensional gel electrophoresis (2-DE). A comparative analysis was performed to verify a differential protein expression between these two 2-DE maps. These data should be useful to clarify the role of different proteins related to bacterial pathogenesis. This study will be completed using a larger number of samples and protein identification of H. pylori by MALDI-TOF mass spectrometry.

Purification and characterization of (Na+ + K+)-ATPase from toad kidney

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.

Simulations of electrophoretic collisions of DNA knots with gel obstacles

Gel electrophoresis can be used to separate nicked circular DNA molecules of equal length but forming different knot types. At low electric fields, complex knots drift faster than simpler knots. However, at high electric field the opposite is the case and simpler knots migrate faster than more complex knots. Using Monte Carlo simulations we investigate the reasons of this reversal of relative order of electrophoretic mobility of DNA molecules forming different knot types. We observe that at high electric fields the simulated knotted molecules tend to hang over the gel fibres and require passing over a substantial energy barrier to slip over the impeding gel fibre. At low electric field the interactions of drifting molecules with the gel fibres are weak and there are no significant energy barriers that oppose the detachment of knotted molecules from transverse gel fibres.