Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.

Spatial and Temporal Variation of Archaeal, Bacterial and Fungal Communities in Agricultural Soils

Background: Soil microbial communities are in constant change at many different temporal and spatial scales. However, the importance of these changes to the turnover of the soil microbial communities has been rarely studied simultaneously in space and time. Methodology/Principal Findings: In this study, we explored the temporal and spatial responses of soil bacterial, archaeal and fungal beta-diversities to abiotic parameters. Taking into account data from a 3-year sampling period, we analyzed the abundances and community structures of Archaea, Bacteria and Fungi along with key soil chemical parameters. We questioned how these abiotic variables influence the turnover of bacterial, archaeal and fungal communities and how they impact the long-term patterns of changes of the aforementioned soil communities. Interestingly, we found that the bacterial and fungal b-diversities are quite stable over time, whereas archaeal diversity showed significantly higher fluctuations. These fluctuations were reflected in temporal turnover caused by soil management through addition of N-fertilizers. Conclusions: Our study showed that management practices applied to agricultural soils might not significantly affect the bacterial and fungal communities, but cause slow and long-term changes in the abundance and structure of the archaeal community. Moreover, the results suggest that, to different extents, abiotic and biotic factors determine the community assembly of archaeal, bacterial and fungal communities.

Characterization of Immobilized Biomass by Amplified rDNA Restriction Analysis (ARDRA) in an Anaerobic Sequencing-Batch Biofilm Reactor (ASBBR) for the Treatment of Industrial Wastewater

The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR-laboratory scale- 14L) containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2.L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2.L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.

Brazilian research on extremophiles in the context of astrobiology

Extremophiles are organisms adapted to grow at extreme ranges of environmental variables, such as high or low temperatures, acid or alkaline medium, high salt concentration, high pressures and so forth. Most extremophiles are micro-organisms that belong to the Archaea and Bacteria domains, and are widely spread across the world, which include the polar regions, volcanoes, deserts, deep oceanic sediments, hydrothermal vents, hypersaline lakes, acid and alkaline water bodies, and other extreme environments considered hostile to human life. Despite the tropical climate, Brazil has a wide range of ecosystems which include some permanent or seasonally extreme environments. For example, the Cerrado is a biome with very low soil pH with high Al+3 concentration, the mangroves in the Brazilian coast are anaerobic and saline, Pantanal has thousands of alkaline-saline lakes, the Caatinga arid and hot soils and the deep sea sediments in the Brazilian ocean shelf. These environments harbour extremophilic organisms that, coupled with the high natural biodiversity in Brazil, could be explored for different purposes. However, only a few projects in Brazil intended to study the extremophiles. In the frame of astrobiology, for example, these organisms could provide important models for defining the limits of life and hypothesize about life outside Earth. Brazilian microbiologists have, however, studied the extremophilic micro-organisms inhabiting non-Brazilian environments, such as the Antarctic continent. The experience and previous results obtained from the Brazilian Antarctic Program (PROANTAR) provide important results that are directly related to astrobiology. This article is a brief synopsis of the Brazilian experience in researching extremophiles, indicating the most important results related to astrobiology and some future perspectives in this area. Received 29 February 2012, accepted 25 May 2012, first published online 11 July 2012

Microbial characterization and removal of anionic surfactant in an expanded granular sludge bed reactor

This study evaluated linear alkylbenzene sulfonate removal in an expanded granular sludge bed reactor with hydraulic retention times of 26 h and 32 h. Sludge bed and separator phase biomass were phylogenetically characterized (sequencing 16S rRNA) and quantified (most probable number) to determine the total anaerobic bacteria and methanogenic Archaea. The reactor was fed with a mineral medium supplemented with 14 mg l(-1) LAS, ethanol and methanol. The stage I-32 h consisted of biomass adaptation (without LAS influent) until reactor stability was achieved (COD removal >97%). In stage II-32 h, LAS removal was 74% due to factors such as dilution, degradation and adsorption. Higher HRT values increased the LAS removal (stage III: 26 h - 48% and stage IV: 32 h - 64%), probably due to increased contact time between the biomass and LAS. The clone libraries were different between samples from the sludge bed (Synergitetes and Proteobacteria) and the separator phase (Firmicutes and Proteobacteria) biomass. (C) 2011 Elsevier Ltd. All rights reserved.

Shifts in phylogenetic diversity of archaeal communities in mangrove sediments at different sites and depths in southeastern Brazil

This study focused on the structure and composition of archaeal communities in sediments of tropical mangroves in order to obtain sufficient insight into two Brazilian sites from different locations (one pristine and another located in an urban area) and at different depth levels from the surface. Terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene fragments was used to scan the archaeal community structure, and 16S rRNA gene clone libraries were used to determine the community composition. Redundancy analysis of T-RFLP patterns revealed differences in archaeal community structure according to location, depth and soil attributes. Parameters such as pH, organic matter, potassium and magnesium presented significant correlation with general community structure. Furthermore, phylogenetic analysis revealed a community composition distributed differently according to depth where, in shallow samples, 74.3% of sequences were affiliated with Euryarchaeota and 25.7% were shared between Crenarchaeota and Thaumarchaeota, while for the deeper samples, 24.3% of the sequences were affiliated with Euryarchaeota and 75.7% with Crenarchaeota and Thaumarchaeota. Archaeal diversity measurements based on 16S rRNA gene clone libraries decreased with increasing depth and there was a greater difference between depths (<18% of sequences shared) than sites (>25% of sequences shared). Taken together, our findings indicate that mangrove ecosystems support a diverse archaeal community; it might possibly be involved in nutrient cycles and are affected by sediment properties, depth and distinct locations. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes

Abstract Background The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Results Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. Conclusions Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.

Characterization of immobilized biomass by amplified rDNA restriction analysis (ARDRA) in an anaerobic sequencing-batch biofilm reactor (ASBBR) for the treatment of industrial wastewater

The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR- laboratory scale- 14L )containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2·L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2·L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.

Theoretical studies of Membrane Proteins : Properties, Prediction Methods and Genome-wide analysis

Membrane proteins are a large and important class of proteins. They are responsible for several of the key functions in a living cell, e.g. transport of nutrients and ions, cell-cell signaling, and cell-cell adhesion. Despite their importance it has not been possible to study their structure and organization in much detail because of the difficulty to obtain 3D structures. In this thesis theoretical studies of membrane protein sequences and structures have been carried out by analyzing existing experimental data. The data comes from several sources including sequence databases, genome sequencing projects, and 3D structures. Prediction of the membrane spanning regions by hydrophobicity analysis is a key technique used in several of the studies. A novel method for this is also presented and compared to other methods. The primary questions addressed in the thesis are: What properties are common to all membrane proteins? What is the overall architecture of a membrane protein? What properties govern the integration into the membrane? How many membrane proteins are there and how are they distributed in different organisms? Several of the findings have now been backed up by experiments. An analysis of the large family of G-protein coupled receptors pinpoints differences in length and amino acid composition of loops between proteins with and without a signal peptide and also differences between extra- and intracellular loops. Known 3D structures of membrane proteins have been studied in terms of hydrophobicity, distribution of secondary structure and amino acid types, position specific residue variability, and differences between loops and membrane spanning regions. An analysis of several fully and partially sequenced genomes from eukaryotes, prokaryotes, and archaea has been carried out. Several differences in the membrane protein content between organisms were found, the most important being the total number of membrane proteins and the distribution of membrane proteins with a given number of transmembrane segments. Of the properties that were found to be similar in all organisms, the most obvious is the bias in the distribution of positive charges between the extra- and intracellular loops. Finally, an analysis of homologues to membrane proteins with known topology uncovered two related, multi-spanning proteins with opposite predicted orientations. The predicted topologies were verified experimentally, providing a first example of "divergent topology evolution".

Ecology of an uncultured heterotrophic flagellate lineage: MAST-4

Programa de oceanografía

Biometanazione della frazione organica di rifiuti solidi urbani da raccolta indifferenziata mediante codigestione con refluo zootecnico o addizione di solfuro di sodio

Il presente elaborato è stato finalizzato allo sviluppo di un processo di digestione anaerobica della frazione organica dei rifiuti solidi urbani (FORSU oppure, in lingua inglese OFMSW, Organic Fraction of Municipal Solid Waste) provenienti da raccolta indifferenziata e conseguente produzione di biogas da impiegarsi per il recupero energetico. Questo lavoro rientra nell’ambito di un progetto, cofinanziato dalla Regione Emilia Romagna attraverso il Programma Regionale per la Ricerca Industriale, l’Innovazione e il Trasferimento Tecnologico (PRRIITT), sviluppato dal Dipartimento di Chimica Applicata e Scienza dei Materiali (DICASM) dell’Università di Bologna in collaborazione con la Facoltà di Ingegneria dell’Università di Ferrara e con la società Recupera s.r.l. che applicherà il processo nell’impianto pilota realizzato presso il proprio sito di biostabilizzazione e compostaggio ad Ostellato (FE). L’obiettivo è stato la verifica della possibilità di impiegare la frazione organica dei rifiuti indifferenziati per la produzione di biogas, e in particolare di metano, attraverso un processo di digestione anaerobica previo trattamento chimico oppure in codigestione con altri substrati organici facilmente fermentabili. E’ stata inoltre studiata la possibilità di impiego di reattori con biomassa adesa per migliorare la produzione specifica di metano e diminuire la lag phase. Dalla sperimentazione si può concludere che è possibile giungere allo sviluppo di metano dalla purea codigerendola assieme a refluo zootecnico. Per ottenere però produzioni significative la quantità di solidi volatili apportati dal rifiuto non deve superare il 50% dei solidi volatili complessivi. Viceversa, l’addizione di solfuri alla sola purea si è dimostrata ininfluente nel tentativo di sottrarre gli agenti inibitori della metanogenesi. Inoltre, l’impiego di supporti di riempimento lavorando attraverso processi batch sequenziali permette di eliminare, nei cicli successivi al primo, la lag phase dei batteri metanogeni ed incrementare la produzione specifica di metano.

Starvation's role in plankton metabolism

[EN]Starvation at all scales of plankton from archaea to medusae is the prevailing condition in marine ecosystems. Such nutrient-limitation will shift the physiological state in these organisms with accompanying changes in their physiology and biochemistry. Here, we review our laboratory’s progress in documenting these changes associated with starvation in a range of marine organisms. Specifically, we focused on respiration, ammonium excretion, CO2 production, RQ, respiratory ETS activity, isocitrate dehydrogenase and glutamate dehydrogenase activity in the mysid, Leptomysis lingvura, a dinoflagellate, Oxyrrhis marina and two bacteria, Vibrio natriegens, and Pseudomonas nautica

Studio della subunità epsilon della DNA polimerasi III di Escherichia coli: stabilità e interazione con la subunità polimerasica

Faithful replication of DNA from one generation to the next is crucial for long-term species survival. Genomic integrity in prokaryotes, archaea and eukaryotes is dependent on efficient and accurate catalysis by multiple DNA polymerases. Escherichia coli possesses five known DNA polymerases (Pol). DNA polymerase III holoenzyme is the major replicative polymerase of the Escherichia coli chromosome (Kornberg, 1982). This enzyme contains two Pol III cores that are held together by a t dimer (Studwell-Vaughan and O’Donnell, 1991). The core is composed of three different proteins named α-, ε- and θ-subunit. The α-subunit, encoded by dnaE, contains the catalytic site for DNA polymerisation (Maki and Kornberg, 1985), the ε-subunit, encoded by dnaQ, contains the 3′→5′ proofreading exonuclease (Scheuermann, et al., 1983) and the θ-subunit, encoded by hole, that has no catalytic activity (Studwell-Vaughan, and O'Donnell, 1983). The three-subunit α–ε–θ DNA pol III complex is the minimal active polymerase form purified from the DNA pol III holoenzyme complex; these three polypeptides are tightly associated in the core (McHenry and Crow, 1979) Despite a wealth of data concerning the properties of DNA polymerase III in vitro, little information is available on the assembly in vivo of this complex enzyme. In this study it is shown that the C-terminal region of the proofreading subunit is labile and that the ClpP protease and the molecular chaperones GroL and DnaK control the overall concentration in vivo of ε. Two α-helices (comprising the residues E311-M335 and G339-D353, respectively) of the N-terminal region of the polymerase subunit were shown to be essential for the binding to ε. These informations could be utilized to produce a conditional mutator strain in which proofreading activity would be titrated by a a variant that can only bind e and that is polymerase-deficient. In this way the replication of DNA made by DNA Pol-III holoenzyme would accordingly become error-prone.

Symbiotische Mikroorganismen aus dem Termitendarm

Symbiotische Mikroorganismen aus dem Termitendarm Es ist eine bekannte Tatsache, dass die Hauptaufgabe der Darmflora der niederen Termite im Abbau von Holz liegt. Im Laufe der Millionen Jahre alten Entwicklung der Termiten hat sich in ihrem Darm ein Ökosystem aufgebaut, das aus Protozoen, Archaeen, Bakterien und Hefen besteht. Ziel der vorliegenden Arbeit war die teilweise Erforschung der symbiotischen Zusammenhänge in diesem ökologischen System. Es wurden dabei zwei Gebiete genauer untersucht: Cellulolytische Bakterien im Darm von niederen Termiten Die bisher vorherrschende Meinung sah in den Protozoen die Hauptkomponenten des Celluloseabbaus in Termiten. In dieser Arbeit gelang es 164 cellulolytische Bakterienstämme aus sieben niederen Termitenarten zu isolieren und zu identifizieren. Diese Vielzahl cellulolytischer Bakterienarten könnte ein Indiz dafür sein, dass Bakterien beim Holzabbau von Termiten eine effizientere Rolle spielen als angenommen. Oberflächenbakterien von Mixotricha paradoxa, einem Flagellaten aus dem Darm der niederen Termite Mastotermes darwiniensisMixotricha paradoxa ist ein Beispiel der seltenen Form einer Bewegungssymbiose zwischen Protozoen und Bakterien. Der Flagellat wird von Spirochäten, die auf seiner Oberfläche befestigt sind, vorangetrieben. Zusätzlich leben noch stäbchenförmige Bakterien auf der Hülle. Drei Spirochätenarten und das stäbchenförmige Bakterium konnten identifiziert und lokalisiert werden. Es wird angenommen, dass alle drei Spirochätenarten Mitglieder der Bewegungssymbiose sind.

Charakterisierung von S-Layer-Proteinen bei Prokaryoten

S-Layer sind kristalline Proteinschichten, die als Komponenten von Zellwänden in allen Zweigen der Bakterien und Archaebakterien vorkommen. Aus der Domäne der Archaea wurden die S-Layer-Proteine mesophiler, thermophiler und hyperthermophiler Methanococcales verglichen. Die Zellwand dieser Organismen besteht nur aus einer S-Layerschicht, die die Zellen vor äußeren Einflüssen schützt. Analog zu den Methanococcales wurden S-Layer-Proteine mesophiler und thermophiler Vertreter aus der Familie der Bacillaceae verglichen.Ziel dieser Arbeit war es, die S-Layer-Gensequenzen von Methanotorris igneus, Methanothermococcus thermolithotrophicus, Methanococcus vannielii, Geobacillus stearothermophilus, Bacillus sphaericus und Bacillus fusiformis zu ermitteln. Durch Vergleiche der Primärsequenzen mesophiler und (hyper)thermophiler S-Layer-Proteine sollten Hinweise auf thermostabilisierende Faktoren abgeleitet werden. Durch Verwendung geeigneter bio-chemischer und gentechnischer Arbeitsmethoden wurden die Gen- und Proteinsequenzen der S-Layer-Proteine ermittelt. Die unbekannten Genbereiche wurden durch die Entwicklung einer modifizierten Zwei- und Drei-Schritt-PCR ermittelt.Die Sequenzanalyse der archaebakteriellen und bakteriellen S-Layer-Proteine erbrachte nur für erstere Hinweise auf thermostabilisierende Faktoren. Die (hyper)thermophilen S-Layer-Hüllproteine aus der Ordnung Methanococcales zeigten gegenüber den mesophilen folgende Unterschiede:1. Zunahme von geladenen Resten;2. Abnahme von Alanin und unpolaren Resten3. Vorhandensein von Cystein4. Erhöhte Anzahl an N-glykosidischen BindungsstellenDas hyperthermophile S-Layer-Protein von Mcc. jannaschii wurde in Escherichia coli erfolgreich expremiert. Für das native Hüllprotein wurde, als ein möglicherweise weiterer thermostabilisierender Faktor eine Glykosilierung detektiert. Zudem wies das native S-Layer-Protein eine Konformationsänderung im Verlauf einer Temperaturerhöhung, bei verschiedenen pH-Werten und in Anwesenheit zweiwertigen Mangans auf. Auch das Expressionsprotein zeigte im Verlauf der Temperaturerhöhung und bei verschiedenen pH-Werten eine Konformationsänderung. Mn2+ hatte dagegen keinen Effekt und eine Glykosi-lierung war nicht nachweisbar.Die transkriptionellen und translationellen Erkennungsregionen der S-Layer-Gene aus der Ordnung Methanococcales wurden bestimmt. Basierend auf Sequenzähnlichkeiten und Gemeinsamkeiten der archaebakteriellen S-Layer-Proteine wurden diese in vier Gruppen eingeteilt.Aus dem bakteriellen Zweig wurde die Gen- und Primärsequenzen der S-Layer-Proteine von Gb. stearothermophilus DSM 2358, B. sphaericus DSM 396, B. fusiformis B3 und DSM 2898T ermittelt. Die Proteine wiesen z. T. eine geringere molekulare Masse auf als die be-kannten S-Layer-Hüllproteine aus der Familie der Bacillaceae. Das Protein von B. sphaericus DSM 396 zeigte eine Diskrepanz zwischen der theoretischen (85 kDA) und experimentell (120 kDa) ermittelten Molmasse. Erstmals wurde für die S-Layer-Proteine von B. fusiformis B3 und DSM 2898T eine vermutliche Dimerbildung festgestellt. Basierend auf den N-terminalen Sequenzähnlichkeiten wurden die S-Layer-Proteine aus der Familie der Bacillaceae in vier Gruppen eingeteilt.