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Methods for extracting 'omes from microbialites

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... Unfortunately, it should be noted that the use of only metagenomic analysis may not reveal the full diversity of microorganisms in the study area. Several factors influence the DNA extraction processes (for example, the high amounts of EPS produced by Nostocales and Chroococcales [40] and a high concentration of Mg in nature samples [41,42]. A bias in relative abundances can be expected if only molecular methods are considered [43]. ...
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The role of cyanobacterial communities in the formation of carbonate sediments (ancient and modern) is not completely clear. We studied the cyanobacterial communities connected with carbonate sediments of the freshwater bodies feeding the historical Peterhof fountains (Saint-Petersburg, Russia). Cyanobacterial communities were studied by metagenome analysis and optical microscopy. Carbonates associated with cyanobacterial communities (both in situ and in vitro) were studied by powder X-ray diffraction analysis, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Raman spectroscopy. The interconnection between the mineral composition of carbonate sediments and inhabiting microorganism species was established. The leading role of cyanobacteria in carbonate biomineralization in fresh water of Peterhof fountains water supply system was shown. Cyanobacteria of 24 genera were revealed in sediments composed of calcite and aragonite. The crystallization of carbonates on the surface of 13 species of cyanobacteria was found. Using model experiments, a significant contribution of cyanobacterial species of the Oscillatoriaceae family (Phormidium spp., Lyngbya sp., Oscillatoriaformosa) to carbonate biomineralization is demonstrated.
... By fine-tuning three critical parameters, including the grinding duration and vibrational frequency, as well as lysis temperature and duration, the sizes of genomic DNA fragments ranging from 79 to 145 kb can be obtained (Penouilh-Suzette et al., 2020). More detailed information of the methods for genome DNA extraction can refer to a recent review (Gomez-Acata et al., 2019). However, no method can be universally applicable to all microorganisms. ...
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Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.
... The genomic DNA of fecal samples was extracted using the cetyltrimethylammonium bromide (CATB) method (Gomez-Acata et al., 2019), and then the purity and concentration of the DNA were detected using agarose gel electrophoresis. The diluted genomic DNA was used as a template for PCR amplification. ...
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An unhealthy diet has been linked to increased incidence of chronic diseases. To investigate the relationship between diet and intestinal inflammation, mice in two experimental groups were fed on a high-fat diet or high-fructose diet, respectively. The result showed that the defecation volume of the experimental groups was significantly reduced compared with that of the control group, and the levels of pro-inflammatory cytokines (interleukin (IL)-1β and IL-6) and IgG in serum were increased significantly. In addition, inflammatory cell infiltration was observed in intestinal tissue, indicating that a high-fructose or high-fat diet can lead to constipation and inflammation. Further analysis showed that the microbial composition of the experimental groups changed significantly, including a decrease of the Bacteroidetes/Firmicutes ratio and increased levels of Bacteroides, Akkermansia, Lactobacillus, and Ruminococcus, which might be associated with inflammation. The results of pro-inflammatory metabolites analysis showed that the levels of arachidonic acid, stearic acid, and indoxylsulfuric acid were significantly increased in the experimental groups, which were related significantly to Bacteroides, Enterococcus, and Akkermansia. Meanwhile, the content of 5-hydroxytryptamine (5-HT) was significantly decreased, which might cause constipation by reducing intestinal peristalsis. Moreover, transplantation of fecal bacteria from inflammatory mice caused constipation and inflammation in normal mice, which could be relieved by feeding a normal diet. The results of the present study indicated that changes in intestinal microbiota and microbial metabolites may underlie chronic intestinal inflammation and constipation caused by high-fructose and high-fat diets.
... The high amounts of EPS produced by endolithic taxa such as Nostocales and Chroococcales can hinder DNA extraction (Wu & Xi, 2009). Also, a high concentration of Mg contained in microbialites can interfere with amplification reactions (Wade & Garcia-Pichel, 2003;Gómez-Acata et al., 2019). Therefore, a bias in relative abundances can be expected if only molecular methods are considered. ...
Article
We analysed and characterized the cyanobacteria in microbialites of Alchichica crater lake over a depth gradient, from 3 to 30 m. A polyphasic approach was followed using morphometry, optical and epifluorescence microscopy, and molecular methods. Regions V3-V4 of the 16S rRNA conserved for cyanobacteria were amplified and used for clone library construction. Independently, a NGS library was constructed using the V4 region of the 16S rRNA. To complement the analysis, eight monocultures of cyanobacteria were isolated from microbialites, which were also characterized by microscopy and 16S rRNA sequencing. In total, we described 18 genera of cyanobacteria isolated from microbialites of Alchichica crater lake. The distribution pattern suggests that on the surface there were mostly Nostocales and filamentous Synechococcales, while at depth we found single-celled Synechococcales. Pleurocapsales and Chroococcales were found at all depths, increasing in abundance at 20 and 30 m.
... The study of their microbial diversity started with morphological descriptions using different microscopic techniques (i.e., light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser confocal microscopy). Molecular biology and Next Generation Sequencing techniques (NGS) have allowed the characterization of microbial communities through gene amplification from environmental DNA Coman et al. 2015;Lindsay et al. 2017;Valdespino-Castillo et al. 2018;Gómez-Acata et al. 2019). ...
Chapter
Extant stromatolites have been considered ecological similes to their ancient counterparts. We now know that these microbial assemblages are composed of a great diversity of microbes, which couple and intertwine their metabolic capabilities to create self-sustained microbial ecosystems. Presently, stromatolites thrive in a vast diversity of aquatic environments including freshwater, hypersaline, coastal lagoons, alkaline lakes, oligotrophic pools, abandoned pits, few marine systems, and brackish waters. In this chapter, we will summarize the research that has been done in two stromatolite-harboring sites in Mexico: the alkaline crater-lake Alchichica and the oligotrophic karstic coastal lagoon of Bacalar. Alchichica is located in the Transvolcanic belt in Central Mexico. It is a maar-alkaline crater lake (salinity 8.5 gl−1, pH 9.5) with water chemistry determined by high contents of carbonates, sodium, a high Mg/Ca ratio, and particularly low Ca2+ concentrations (~0.3 mM). Two main stromatolite-types, as defined by mineralogy, texture, and microbial composition, develop along its periphery from surface to over 30 m in depth. Alchichica is a modern environment that resembles Precambrian oceanic conditions. Stromatolites from Alchichica have been dated radiometrically in ~1.1–2.8 ka BP. Bacalar is a coastal lagoon located in the Yucatan Peninsula, which is a carbonate platform that emerged above sea level during the Oligocene. The Bacalar lagoon has high carbonate concentrations as a result of the influx of groundwater (salinity 1.2 gl−1, pH 7.6–8.3). It holds the largest freshwater stromatolite structures known, which have been radiometrically dated in ~6.8–9.2 ka BP. Their mineralogy, shape, and texture are similar along the lagoon’s coast, but microbial composition changes, possibly due to anthropogenic impact.
... The semi-closed nature of stromatolites coupled with their high levels of productivity and rapid cycling of major elements renders stromatolites amenable to meta-omics and systems biology approaches to identify potential organic molecular biosignatures. Stromatolites have been subject of numerous multiomic analyses to more fully characterize the genomes, transcriptomes, and metabolomes of stromatolite-forming taxa in a diverse range of habitats (Desnues et al. 2008;Breitbart et al. 2009;Khodadad and Foster 2012;Mobberley et al. 2012Mobberley et al. , 2015Peimbert et al. 2012;Edgcomb et al. 2013Edgcomb et al. , 2014Saghaï et al. 2015;White et al. 2015White et al. , 2016White et al. , 2018Cerqueda-Garcia and Falcon 2016;Ruvindy et al. 2016;Warden et al. 2016;Casaburi et al. 2016;Louyakis et al. 2017Louyakis et al. , 2018Babilonia et al. 2018;Gomez-Acata et al. 2019). For example, a comparative metagenomic analysis of the stromatolites of Hamelin Pool revealed distinctive genetic enrichments within the stromatolite-forming mats that strongly correlated with water depth within the pool (Babilonia et al. 2018). ...
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For millennia, humanity has looked to stars and wondered, “Are we alone in the universe?” Although this question was initially the purview of philosophers, now, with leaps in scientific and technological advances, we have changed the nature of this question from existential to empirical. Today, the question “Are we alone?” serves as a crux to the field of astrobiology. To search for life elsewhere in the universe, we must first understand how life originates and evolves on Earth but also how biology leaves behind residual signatures of its existence. To address these questions, many astrobiology researchers have targeted stromatolite-forming communities as model ecosystems to explore how microbe–mineral interactions, under a range of environmental conditions, can lead to the formation of biosignatures. Stromatolites are depositional structures formed by the activities and interactions of microbes and have a fossil record dating back billions of years. Due to their long evolutionary history and abundance on the modern Earth, research on the biological, chemical and geological processes of stromatolite formation have provided important insights into the field of astrobiology, including the diversity and preservation of biosignatures. In this chapter, we examine the range of biosignatures found in stromatolites and how these markers improve our understanding of the past, present, and future of life in the context of astrobiology. We also discuss whether stromatolite research can play a role in the future exploration of habitable worlds in our own solar system and beyond.
... The study of their microbial diversity started with morphological descriptions using different microscopic techniques (i.e., light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser confocal microscopy). Molecular biology and Next Generation Sequencing techniques (NGS) have allowed the characterization of microbial communities through gene amplification from environmental DNA Coman et al. 2015;Lindsay et al. 2017;Valdespino-Castillo et al. 2018;Gómez-Acata et al. 2019). ...
Chapter
Deep hydrothermal systems result from the magmatic and tectonic activity of the ocean floor. This deep extreme biosphere represents a unique oasis of life driven by sulfur-based chemosynthesis instead of photosynthesis. The organisms inhabiting these systems are adapted to cope with harsh environmental conditions such as the absence of sunlight, high temperatures and hydrostatic pressures, and elevated concentrations of hydrogen sulfide, as well as high concentrations of heavy metals. Therefore, this biome is different from any other environment on modern Earth. As expected from such conditions, chemoautotrophic prokaryotes are the leading primary producers at the base of the food web considered as an analog to the oldest signs of life on Earth. Herein, we discuss prokaryotic diversity and community structure from the newly discovered hydrothermal systems in the Alarcón Rise (AR), the Pescadero Basin (PB), and the Pescadero Transform Fault (PTF) at the mouth of the Gulf of California, Mexico, using 16S rRNA gene amplicon Illumina sequencing. Despite the spatial proximity of the studied vent systems (<100 km), they differ considerably in their physical, chemical, geological settings, and biotic characteristics. Our results indicated that beta prokaryotic diversity is associated to the sampling source, suggesting a strong effect of environmental conditions in shaping microbial distribution. The most abundant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, and Epsilonbacteraeota. Also, we found evidence on the oxidation of methane as a prevalent process in PB and PTF, since methylotrophic bacteria and Atribacteria were abundant, in contrast to AR basalt-hosted system. Bacteria associated with the sulfur cycle, in particular sulfur compounds reducing and sulfur compounds oxidizing bacteria predominated in all samples, confirming the importance of sulfur supporting vent communities. It is possible that vent systems played a significant role in the origins of life on Earth. Hence, they represent useful models when searching for life elsewhere in the universe.
... The study of their microbial diversity started with morphological descriptions using different microscopic techniques (i.e., light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and laser confocal microscopy). Molecular biology and Next Generation Sequencing techniques (NGS) have allowed the characterization of microbial communities through gene amplification from environmental DNA Coman et al. 2015;Lindsay et al. 2017;Valdespino-Castillo et al. 2018;Gómez-Acata et al. 2019). ...
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A small community of scientists in Mexico has been contributing to the study of planetary bodies in our Solar System and around other stars, including their potential for habitability. Here, we present particular aspects of this research told as a journey: from the first attempts to reproduce cells and the laboratories where the first Mexican astrobiologists were educated to the sites in Mexico where scientists are studying the extremes of life and likely environments of other planets. We jump to space rocks that narrate the history of the Solar System. Then, we move to Mars and the debate of organics and the Viking experiment to continue with the hidden water oceans of the icy satellites and Titan, an exotic orange satellite with methane lakes, hydrocarbon dunes, and water ice rocks. Our journey continues toward other stars where we search for planets beyond our Solar System, known as exoplanets, that have shown a surprising diversity more familiar to science fiction with hot Jupiters, lava worlds, mini-Neptunes, super-Earths, and potentially habitable worlds.
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The white rocks delineating the littoral of Lake Alchichica have called the attention of locals, foreigners, explorers and curious scientists over the years. More recently, science has revealed that these mineral constructions are hotspots of previously hidden microbial diversity. The metabolism of diverse microbial assemblages mediates the formation of massive biomineral structures of hydromagnesite, calcite and aragonite, reaching more than 40 m in depth. The high taxonomical diversity resembles a high metabolic diversity, reported here by the potential of microbialite prokaryotic communities for biogeochemical cycling. Today, hundreds of the microbialite prokaryotes have no identified matches in the present microbial taxonomic databases. This fact has called the attention of scientists worldwide, and Lake Alchichica has become a model system for microbial ecology, evolution, biogeochemistry and astrobiology studies. Microbialites of Lake Alchichica provide a unique environment for the development of many other endemic species. The conservation of these microbial communities is challenging in the panorama of global change and will require coordinated efforts at all levels of the society.
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Bacterioplankton is an often-underestimated component in aquatic environments, which has major implications for ecosystem functioning. Recent studies highlight its activity and diversity in lake systems. Lake Alchichica is no exception in this matter, and although most microbial surveys have addressed the activity and abundance of phytoplankton, the latest surveys in this maar lake have shed light on its taxonomically and metabolic complex composition. These microbial communities include heterotrophic picoplankton (microorganisms of <2 μm size) with high bacterial and archaeal diversity. The main approaches used to survey the water column of Lake Alchichica include epifluorescence methods, such as fluorescence in situ hybridization (FISH) and microscopic techniques, together with next-generation sequencing (NGS) methods using the 16S rRNA gene marker, and functional gene quantifications. Overall, these studies show how the hydrodynamics, depth, and physicochemical characteristics of the lake relate to the bacterioplankton taxonomic and functional diversity displaying spatial and temporal variations.
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The role of archaea in microbial mats is poorly understood. Delineating the spatial distribution of archaea with mat depth will enable resolution of putative niches in these systems. In the present study, high throughput amplicon sequencing was undertaken in conjunction with analysis of key biogeochemical properties of two mats (smooth and pustular) from Shark Bay, Australia. One-way analysis of similarity tests indicated the archaeal community structures of smooth and pustular mats were significantly different (global R = 1, p = 0.1%). Smooth mats possessed higher archaeal diversity, dominated by Parvarchaeota. The methanogenic community in smooth mats was dominated by hydrogenotrophic Methanomicrobiales, as well as methylotrophic Methanosarcinales, Methanococcales, Methanobacteriales and Methanomassiliicoccaceae. Pustular mats were enriched with Halobacteria and Parvarchaeota. Key metabolisms (bacterial and archaeal) were measured, and the rates of oxygen production/consumption and sulfate reduction were up to four times higher in smooth than in pustular mats. Methane production peaked in the oxic layers and was up to seven-fold higher in smooth than pustular mats. The finding of an abundance of anaerobic methanogens enriched at the surface where oxygen levels were highest, coupled with peak methane production in the oxic zone, suggests putative surface anoxic niches in these microbial mats.
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Interpreting the environmental conditions of ancient microbialites rely on comparisons with modern analogues. Yet, we lack a detailed reference framework relating the chemical and mineralogical composition of modern lacustrine microbialites with the physical and chemical parameters prevailing in the lakes where they form. Here we performed geochemical analyses of water solutions and mineralogical analyses of microbialites in 12 Mexican crater lakes. We found a large diversity of microbialites in terms of mineralogical composition, with occurrence of diverse carbonate phases such as magnesian calcite, monohydrocalcite, aragonite, hydromagnesite, and dolomite as well as authigenic magnesium silicate phases. In parallel, the chemical compositions of the lakes differed particularly by their alkalinity, their concentration of ortho-silicic acid (H4SiO4) and their Mg/Ca ratio. From this study, we infer a minimum alkalinity value for the formation of lacustrine microbialites, as well as several constraints given by the presence of mineral phases on the chemical composition of the lakes in which microbialites formed. Finally, we observe a general correlation between the alkalinity and the sodium content of the lakes. This may relate to variations in evaporation intensity and provide a historical model for lacustrine microbialite formation: microbialite start forming only when the lake is sufficiently old/evaporated.
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Microbialites are an example of complex and diverse microbial assemblages where several metabolic pathways are interconnected for biomass formation coupled to mineral precipitation. Lake Alchichica (Mexico) is an oligotrophic environment where nitrogen (N) and phosphorus alternately limit productivity, and massive microbialite growths are found along the lake's perimeter. Previous studies have described the importance of N2 fixation in these microbialites, although other pathways associated with the N cycle, including denitrification, nitrification and anaerobic ammonium oxidation (anammox), had not been evaluated. This study identified the genetic diversity associated with N cycling in both metagenomic DNA and RNA expression by targeting key genes for nitrogenase (nifH), ammonia monooxygenase (amoA), nitrite oxidoreductase (nxrA, nxrB), hydrazine oxidoreductase (hzo) and nitrite (nirS and nirK) and nitrous oxide (nosZ) reductases. While the genetic potential for N2 fixation, ammonia oxidation, anammox and denitrification was present in the microbialites of Lake Alchichica, the most transcribed pathway was N2 fixation.
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Modern microbial mats are potential analogues of some of Earth's earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.The ISME Journal advance online publication, 29 May 2015; doi:10.1038/ismej.2015.87.
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Modern mineral deposits play an important role in evolutionary studies by providing clues to the formation of ancient lithified microbial communities. Here we report the presence of microbialite-forming microbial mats in different microenvironments at 32ºC, 49ºC and 65ºC around the geothermal spring from an abandoned oil drill in Ciocaia, Romania. The mineralogy and the macro- and microstructure of the microbialites were investigated, together with their microbial diversity based on a 16S rRNA gene amplicon sequencing approach. The calcium carbonate is deposited mainly in the form of calcite. At 32ºC and 49ºC, the microbialites show a laminated structure with visible microbial mat-carbonate crystal interactions. At 65ºC, the mineral deposit is clotted, without obvious organic residues. Partial 16S rRNA gene amplicon sequencing showed that the relative abundance of the phylum Archaea was low at 32ºC (<0.5%) but increased significantly at 65ºC (36%). The bacterial diversity was either similar to other microbialites described in literature (the 32ºC sample) or displayed a specific combination of phyla and classes (the 49ºC and 65ºC samples). Bacterial taxa were distributed among 39 phyla, out of which 14 had inferred abundances >1%. The dominant bacterial groups at 32ºC were Cyanobacteria, Gammaproteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Thermi, Actinobacteria, Planctomycetes and Defferibacteres. At 49ºC, there was a striking dominance of the Gammaproteobacteria, followed by Firmicutes, Bacteroidetes, and Armantimonadetes. The 65ºC sample was dominated by Betaproteobacteria, Firmicutes, [OP1], Defferibacteres, Thermi, Thermotogae, [EM3] and Nitrospirae. Several groups from Proteobacteria and Firmicutes, together with Halobacteria and Melainabacteria were described for the first time in calcium carbonate deposits. Overall, the spring from Ciocaia emerges as a valuable site to probe microbes-minerals interrelationships along thermal and geochemical gradients.
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The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool. EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of blood's DNase. As a result, the endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activities. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma. In conclusion, exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from blood's DNases preanalytical impact in EDTA-plasma. Copyright © 2015. Published by Elsevier Inc.
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Viruses are the most abundant biological entities throughout marine and terrestrial ecosystems, but little is known about virus-mineral interactions or the potential for virus preservation in the geological record. Here we use contextual metagenomic data and microscopic analyses to show that viruses occur in high diversity within a modern lacustrine microbial mat, and vastly outnumber prokaryotes and other components of the microbial mat. Experimental data reveal that mineral precipitation takes place directly on free viruses and, as a result of viral infections, on cell debris resulting from cell lysis. Viruses are initially permineralized by amorphous magnesium silicates, which then alter to magnesium carbonate nanospheres of ~80-200 nm in diameter during diagenesis. Our findings open up the possibility to investigate the evolution and geological history of viruses and their role in organomineralization, as well as providing an alternative explanation for enigmatic carbonate nanospheres previously observed in the geological record.
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Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the lab and the storage of water and/or filtered samples in the lab. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. The present study demonstrates the successful preservation of eDNA at room temperature (20°Celsius) in two lysis buffers, CTAB and Longmire's, over a two-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a Phenol-Chloroform-Isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.This article is protected by copyright. All rights reserved.
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Microbialites are organosedimentary deposits formed from interaction between benthic microbial communities (BMCs) and detrital or chemical sediments. Processes involved in the formation of calcareous microbialites include trapping and binding of detrital sediment (forming microbial boundstones), inorganic calcification (forming microbial tufa), and biologically influenced calcification (forming microbial framestones). Microbialites contrast with other biological sediments in that they are generally not composed of skeletal remains. -from Authors
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Microbialites are organosedimentary structures that are formed through the interaction of benthic microbial communities and sediments and include mineral precipitation. These lithifying microbial mat structures include stromatolites and thrombolites. Exuma Sound in the Bahamas, and Hamelin Pool in Shark Bay, Western Australia, are two locations where significant stands of modern microbialites exist. Although prokaryotic diversity in these structures is reasonably well documented, little is known about the eukaryotic component of these communities and their potential to influence sedimentary fabrics through grazing, binding and burrowing activities. Accordingly, comparisons of eukaryotic communities in modern stromatolitic and thrombolitic mats can potentially provide insight into the coexistence of both laminated and clotted mat structures in close proximity to one another. Here we examine this possibility by comparing eukaryotic diversity based on Sanger and high-throughput pyrosequencing of small subunit ribosomal RNA (18S rRNA) genes. Analyses were based on total RNA extracts as template to minimize input from inactive or deceased organisms. Results identified diverse eukaryotic communities particularly stramenopiles, Alveolata, Metazoa, Amoebozoa and Rhizaria within different mat types at both locations, as well as abundant and diverse signatures of eukaryotes with <80% sequence similarity to sequences in GenBank. This suggests the presence of significant novel eukaryotic diversity, particularly in hypersaline Hamelin Pool. There was evidence of vertical structuring of protist populations and foraminiferal diversity was highest in bioturbated/clotted thrombolite mats of Highborne Cay.
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We evaluated and optimized three rapid methods for extraction of high-quality DNA from carbonate-encrusted microbial communities using modern calcifying oncolites built by cyanobacteria and diatoms in a high-calcium freshwater river. Pulverization, acid (HCl) dissolution, and chelator-mediated (EDTA) dissolution of the carbonate matrix were used and optimized to liberate microbial cells from their mineral encasing. This was followed by cell lysis and DNA extraction and isolation. HCl dissolution yielded no measurable DNA, probably due to hydrolysis, whereas pulverization and EDTA dissolution yielded averages of 3.5 and 7.8 μg per gram of sample, respectively, of high molecular weight DNA. These DNA isolates could be used successfully for PCR-amplification of 16S rRNA gene segments (alleles) and subsequent fingerprinting of the cyanobacterial (including diatoms) and total bacterial communities through denaturing gradient gel electrophoresis (DGGE) separation. Fingerprints showed no differences in microbial community composition between the pulverization and EDTA dissolution methods. While the pulverization method was faster, we demonstrate here that EDTA carbonate dissolution is superior because it preserves vertical stratification of the microbial communities lost using the pulverization method, thus allowing for spatially resolved community analyses.
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Detailed stratigraphic, petrographic, and zircon U–Pb geochronological data are provided for surface outcrops and newly obtained, unweathered drillcore intersections of Earth's oldest fossiliferous sedimentary rocks, the lowermost chert–barite unit of the ca. 3.5Ga Dresser Formation, Warrawoona Group, Pilbara Craton.Results show that the ∼8m thick unit at the drilling locality consists of thinly bedded, originally micritic carbonates deposited under quiet water conditions that are interbedded with volcaniclastic conglomerates and coarse polymict conglomerates (diamictites) deposited during periods of tectonic instability. The presence of rapid vertical and lateral facies changes, tilted bedding in some members, and internal erosional unconformities, combined with analysis of fault offsets, indicate that tectonically unstable periods were caused by growth faulting. Intense hydrothermal fluid flow accompanied episodes of growth faulting and resulted in pulsed, repeated precipitation of silica±barite±sphalerite that alternated with precipitation of pyrite. Clasts of hydrothermal minerals in sandstone and coarse, polymict conglomerate beds at several levels within the unit highlight the repeated nature of hydrothermal fluid circulation during sediment accumulation. Hydrothermal fluid circulation caused widespread acid–sulfate alteration of the footwall, extensive replacement of the newly deposited carbonate sediments by hydrothermal precipitates, and crystallization of hydrothermal chert–barite–pyrite in veins perpendicular to bedding in the footwall and parallel to bedding in the sedimentary unit. The combined evidence points to deposition of the chert–barite unit within an active volcanic caldera. A 10cm thick bed of felsic volcaniclastic tuff within finely bedded carbonates near the top of the unit has yielded a maximum age of deposition of 3481.0±3.6Ma (2σ uncertainty), confirming earlier Pb–Pb age data for the antiquity of these rocks.Putative signs of life are present as stratiform, columnar, domical, and coniform stromatolitic laminates at various levels throughout the unit. Petrographic observations show that red- and black-weathering stromatolitic laminates on the surface consist of pyrite in unweathered drillcore material. Observation of local relics of carbonate between pyrite crystals in these laminates indicates a carbonate protolith prior to replacement by hydrothermal pyrite, which provides support for a biological origin of stromatolitic laminates. Further support is provided by clasts of laminated carbonaceous material in thinly bedded, primary micritic carbonates.Textural analysis of jaspilitic “cherts” near the top of the unit reveal haematite as tiny crystals within recrystallized siderite/dolomite rhombs in carbonate beds affected by hydrothermal silica alteration. The presence of unaltered diagenetic pyrite crystals in the haematite-altered siderite indicates that alteration did not result from oxidizing fluids. Rather, haematite alteration is interpreted as the result of an increase in pH during diagenetic alteration by mildly reducing, silica-rich fluids associated with eruption of overlying basalts, possibly with the influence of microbial activity. This has important implications for the origin of jaspilitic cherts throughout the early Archean record and for atmospheric conditions of early Earth.
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On the Kiritimati atoll, several lakes exhibit microbial mat-formation under different hydrochemical conditions. Some of these lakes trigger microbialite formation such as Lake 21, which is an evaporitic, hypersaline lake (salinity of approximately 170‰). Lake 21 is completely covered with a thick multilayered microbial mat. This mat is associated with the formation of decimeter-thick highly porous microbialites, which are composed of aragonite and gypsum crystals. We assessed the bacterial and archaeal community composition and its alteration along the vertical stratification by large-scale analysis of 16S rRNA gene sequences of the nine different mat layers. The surface layers are dominated by aerobic, phototrophic, and halotolerant microbes. The bacterial community of these layers harbored Cyanobacteria (Halothece cluster), which were accompanied with known phototrophic members of the Bacteroidetes and Alphaproteobacteria. In deeper anaerobic layers more diverse communities than in the upper layers were present. The deeper layers were dominated by Spirochaetes, sulfate-reducing bacteria (Deltaproteobacteria), Chloroflexi (Anaerolineae and Caldilineae), purple non-sulfur bacteria (Alphaproteobacteria), purple sulfur bacteria (Chromatiales), anaerobic Bacteroidetes (Marinilabiacae), Nitrospirae (OPB95), Planctomycetes and several candidate divisions. The archaeal community, including numerous uncultured taxonomic lineages, generally changed from Euryarchaeota (mainly Halobacteria and Thermoplasmata) to uncultured members of the Thaumarchaeota (mainly Marine Benthic Group B) with increasing depth.
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Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.
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De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.
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A railroad causeway across Great Salt Lake, Utah (GSL), has restricted water flow since its construction in 1959, resulting in a more saline North Arm (NA; 24%-31% salinity) and a less saline South Arm (SA; 11%-14% salinity). Here, we characterized microbial carbonates collected from the SA and the NA to evaluate the effect of increased salinity on community composition and abundance and to determine whether the communities present in the NA are still actively precipitating carbonate or if they are remnant features from prior to causeway construction. SSU rRNA gene abundances associated with the NA microbialite were three orders of magnitude lower than those associated with the SA microbialite, indicating that the latter community is more productive. SSU rRNA gene sequencing and functional gene microarray analyses indicated that SA and NA microbialite communities are distinct. In particular, abundant sequences affiliated with photoautotrophic taxa including cyanobacteria and diatoms that may drive carbonate precipitation and thus still actively form microbialites were identified in the SA microbialite; sequences affiliated with photoautotrophic taxa were in low abundance in the NA microbialite. SA and NA microbialites comprise smooth prismatic aragonite crystals. However, the SA microbialite also contained micritic aragonite, which can be formed as a result of biological activity. Collectively, these observations suggest that NA microbialites are likely to be remnant features from prior to causeway construction and indicate a strong decrease in the ability of NA microbialite communities to actively precipitate carbonate minerals. Moreover, the results suggest a role for cyanobacteria and diatoms in carbonate precipitation and microbialite formation in the SA of GSL.
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Microbialites found in the low-light-intensity, hypersaline waters of Storr's Lake (SL), San Salvador Island, the Bahamas, were investigated with respect to their morphology, mineralogy, and microbial diversity. Previously described microbialite morphologies, as well as a newly identified "multi-cuspate" morphology, were observed at various depths. Electron microscopy analysis revealed the presence of angular, blocky, and needle-shaped crystals with mineralized cyanobacterial filaments and remains of exopolymeric substances. X-ray diffraction studies confirmed the presence of both Mg-calcite and aragonite in the plateau-mushroom and pinnacle mound microbialites, whereas only Mg-calcite was identified in the other microbialite morphotypes. A comprehensive molecular analysis using barcoded pyrosequencing of five different microbial mat communities identified at least 12 dominant bacterial phyla. Cyanobacteria were generally low in abundance and ranged from ∼0.01% in the deeper pinnacle mounds to ∼3.2% in the shallow calcareous knobs. Other photosynthetic members included green nonsulfur bacteria of the phylum Chloroflexi and purple sulfur bacteria of the class Gammaproteobacteria. All mat types contained significant amounts of sulfate-reducing and dehalogenating bacteria. The low light intensity reaching the deeper microbialites, the lack of dominant cyanobacteria, and the abundance of sulfate reducers and Chloroflexi collectively suggest that sulfate reduction and anoxygenic photosynthetic processes influence the carbonate biomineralization process in these systems. Key Words: Microbial mats-Microbial diversity-Biomineralization. Astrobiology 16, 282-300.
Article
Lithified microbial structures (microbialites) have been present on Earth for billions of years. Lithification may impose unique constraints on microbes. For instance, when CaCO3 forms, phosphate may be captured via coprecipitation and/or adsorption and potentially rendered unavailable for biological uptake. Therefore, the growth of microbes associated with CaCO3 may be phosphorus-limited. In this study, we compared the effects of resource addition on biogeochemical functions of microbial communities associated with microbialites and photoautotrophic microbial communities not associated with CaCO3 deposition in Río Mesquites, Cuatro Ciénegas, México. We also manipulated rates of CaCO3 deposition in microbialites to determine whether lithification reduces the bioavailability of phosphorus (P). We found that P additions significantly increased rates of gross primary production (F2,13 = 103.9, P < 0.001), net primary production (F2,13 = 129.6, P < 0.0001) and ecosystem respiration (F2,13 = 6.44, P < 0.05) in the microbialites, while P addition had no effect on photoautotrophic production in the non-CaCO3 -associated microbial communities. Growth of the non-CaCO3 -associated phototrophs was only marginally stimulated when nitrogen and P were added simultaneously (F1,36 = 3.98, P = 0.053). In the microbialites, resource additions led to some shifts in the abundance of Proteobacteria, Bacteroidetes and Cyanobacteria but mostly had little effect on bacterial community composition. Ca(2+) uptake rates increased significantly with organic carbon additions (F1,13 = 8.02, P < 0.05). Lowering of CaCO3 deposition by decreasing calcium concentrations in the water led to increased microbial biomass accumulation rates in terms of both organic carbon (F4,48 = 5.23, P < 0.01) and P (F6,48 = 13.91, P < 0.001). These results provide strong evidence in support of a role of lithification in controlling P limitation of microbialite communities.
Article
Cyanobacteria are often the key organisms comprising microbial mats. They form dense micrometer-scale communities in which the full plethora of microbial metabolism can be present. Such mats are therefore excellent model systems and because of their analogy with Precambrian stromatolites they are also attractive subjects for evolutionary studies. Growth and metabolism of the oxygenic phototrophic cyanobacteria enrich the sediment with organic matter. However, in mature mats net growth of cyanobacteria appears to be of less importance. Most of the organic matter produced from photosynthetic CO2 fixation is liberated in the sediment by one of the following: fermentation, photorespiration, pouring out of solutes or secretion of mucus although grazing may also be important. This organic matter is degraded by chemotrophic microorganisms, among which sulphate-reducing bacteria are particularly prominent. The combined activities of the cyanobacteria and sulphate-reducing bacteria result in steep and fluctuating gradients of sulphide and oxygen. Cyanobacteria therefore have to cope with high concentrations of sulphide, oxygen supersaturated - and anoxic conditions. These physicochemical gradients force different functional groups of microorganisms to particular vertical stratified positions in the mat. This, and the fact that accretion of sediment fluctuates, gives rise to one of the most conspicuous properties of microbial mats namely their laminated structure. Modern microbial mats have this laminated structure in common with Precambrian stromatolites. Most modern mats do not lithify but this may also have been the case for Archean microbial mats. Only a few examples of modern calcifying stromatolithic microbial mats are known. A hypothesis has been developed which conceives a role for extracellular polysaccharides in calcification. Extracellular polysaccharides in cyanobacterial mats are often produced as the result of unbalanced growth caused by nitrogen deficiency. The mat organisms are embedded in the extensive polysaccharide matrix that inhibits calcification. All cyanobacterial mats can fix atmospheric dinitrogen, which covers part of their nitrogen demand, but the fluctuating physicochemical gradients limits the efficiency of this process. © 2012 Springer Science+Business Media B.V. All rights reserved.
Article
Modern stromatolites represent ideal ecosystems to understand the biological processes required for the precipitation of carbonate, due to their long evolutionary history and occurrence in a wide range of habitats. However, most of the prior molecular work on stromatolites has focused on understanding the taxonomic complexity and not fully elucidating the functional capabilities of these systems. Here, we begin to characterize the microbiome associated with stromatolites of Little Darby Island, The Bahamas using predictive metagenomics of the 16S rRNA gene coupled with direct whole shotgun sequencing. The metagenomic analysis of the Little Darby stromatolites revealed many shared taxa and core pathways associated with biologically induced carbonate precipitation, suggesting functional convergence within Bahamian stromatolites. A comparison of the Little Darby stromatolites with other lithifying microbial ecosystems also revealed that although factors, such as geographic location and salinity, do drive some differences within the population, there are extensive similarities within the microbial populations. These results suggest that for stromatolite formation, "who" is in the community is not as critical as metabolic activities and environmental interactions. Together, these analyses help improve our understanding of the similarities amongst lithifying ecosystems and provide an important first step in characterizing the shared microbiome of modern stromatolites.
Article
Within the subarctic climate of Clinton Creek, Yukon, Canada, lies an abandoned and flooded open-pit asbestos mine that harbors rapidly growing microbialites. To understand their formation we completed a metagenomic community profile of the microbialites and their surrounding sediments. Assembled metagenomic data revealed that bacteria within the phylum Proteobacteria numerically dominated this system, although the relative abundances of taxa within the phylum varied among environments. Bacteria belonging to Alphaproteobacteria and Gammaproteobacteria were dominant in the microbialites and sediments, respectively. The microbialites were also home to many other groups associated with microbialite formation including filamentous cyanobacteria and dissimilatory sulfate-reducing Deltaproteobacteria, consistent with the idea of a shared global microbialite microbiome. Other members were present that are typically not associated with microbialites including Gemmatimonadetes and iron-oxidizing Betaproteobacteria, which participate in carbon metabolism and iron cycling. Compared to the sediments, the microbialite microbiome has significantly more genes associated with photosynthetic processes (e.g., photosystem II reaction centers, carotenoid, and chlorophyll biosynthesis) and carbon fixation (e.g., CO dehydrogenase). The Clinton Creek microbialite communities had strikingly similar functional potentials to non-lithifying microbial mats from the Canadian High Arctic and Antarctica, but are functionally distinct, from non-lithifying mats or biofilms from Yellowstone. Clinton Creek microbialites also share metabolic genes (R (2) < 0.750) with freshwater microbial mats from Cuatro Ciénegas, Mexico, but are more similar to polar Arctic mats (R (2) > 0.900). These metagenomic profiles from an anthropogenic microbialite-forming ecosystem provide context to microbialite formation on a human-relevant timescale.
Article
Dissolved organic phosphorus utilization by different members of natural communities has been closely linked to microbial alkaline phosphatases whose affiliation and diversity is largely unknown. Here we assessed genetic diversity of bacterial alkaline phosphatases phoX and phoD, using highly diverse microbial consortia (microbialites and bacterioplankton) as study models. These microbial consortia are found in an oligo-mesotrophic soda lake with a particular geochemistry, exhibiting a low calcium concentration and a high Mg : Ca ratio relative to seawater. In spite of the relative low calcium concentration in the studied system, our results highlight the diversity of calcium-based metallophosphatases phoX and phoD-like in heterotrophic bacteria of microbialites and bacterioplankton, where phoX was the most abundant alkaline phosphatase found. phoX and phoD-like phylotypes were more numerous in microbialites than in bacterioplankton. A larger potential community for DOP utilization in microbialites was consistent with the TN : TP ratio, suggesting P limitation within these assemblages. A cross-system comparison indicated that diversity of phoX in Lake Alchichica was similar to that of other aquatic systems with a naturally contrasting ionic composition and trophic state, although no phylotypes were shared among systems. Bacterial assemblages of microbialites and bacterioplankton in the tropical soda lake Alchichica exhibit a vast potential for organic phosphorus utilization by calcium-dependent alkaline phosphatases. Bacterial assemblages of microbialites and bacterioplankton in the tropical soda lake Alchichica exhibit a vast potential for organic phosphorus utilization by calcium-dependent alkaline phosphatases.
Article
A culture-independent multidomain survey of biodiversity in microbialite structures within the cold alkaline Pavilion Lake (British Columbia, Canada) revealed a largely homogenous community at depths from 10 to 30 m. Real-time quantitative PCR was used to demonstrate that bacteria comprised approximately 80%–95% of recoverable phylotypes. Archaeal phylotypes accounted for N = 491) revealed that alphaproteobacterial phylotypes were most abundant. Cyanobacterial phylotypes were highly diverse but resolved into 4 dominant genera: Acaryochloris, Leptolyngbya, Microcoleus, and Pseudanabaena. Interestingly, microbialite cyanobacteria generally affiliated phylogenetically with aquatic and coral cyanobacterial groups rather than those from stromatolites. Other commonly encountered bacterial phylotypes were from members of the Acidobacteria, with relatively low abundance of the Betaproteobacteria, Chloroflexi, Nitrospirae, and Planctomycetes. Archaeal diversity (N = 53) was largely accounted for by Euryarchaeota, with most phylotypes affiliated with freshwater methanogenic taxa.
Article
Pavilion Lake in British Columbia, Canada, is home to modern-day microbialites that are actively growing at multiple depths within the lake. While microbialite morphology changes with depth and previous isotopic investigations suggested a biological role in the formation of these carbonate structures, little is known about their microbial communities. Microbialite samples acquired through the Pavilion Lake Research Project (PLRP) were first investigated for phototrophic populations using Cyanobacteria-specific primers and 16S rRNA gene cloning. These data were expounded on by high-throughput tagged sequencing analyses of the general bacteria population. These molecular analyses show that the microbial communities of Pavilion Lake microbialites are diverse compared to non-lithifying microbial mats also found in the lake. Phototrophs and heterotrophs were detected, including species from the recently described Chloroacidobacteria genus, a photoheterotroph that has not been previously observed in microbialite systems. Phototrophs were shown as the most influential contributors to community differences above and below 25 meters, and corresponding shifts in heterotrophic populations were observed at this interface as well. The isotopic composition of carbonate also mirrored this shift in community states. Comparisons to previous studies indicated this population shift may be a consequence of changes in lake chemistry at this depth. Microbial community composition did not correlate with changing microbialite morphology with depth, suggesting something other than community changes may be a key to observed variations in microbialite structure.
Article
Bacteria adhere to natural and engineered surfaces and develop into mature biofilms encased in self-produced extracellular polymeric substances (EPSs). EPS consists of polysaccharides, proteins, metabolites and extracellular DNA (eDNA). Extracellular DNA release by bacteria is mediated by both quorum-sensing (QS)-dependent and -independent mechanisms. Quorum-sensing-independent mechanisms are responsible for basal levels of eDNA release, whereas QS-dependent mechanisms control the production of prophages, phenazines and proteins involved in cell lysis and subsequent release of elevated amounts of eDNA. Extracellular DNA binds with other biopolymers such as polysaccharides, proteins or metabolites like phenazines, thereby providing structural integrity to EPS. Extracellular DNA promotes attractive acid-base interactions between bacterial cells and between bacteria and surfaces. It therefore plays an essential structural role in stabilising biofilms and protecting bacterial cells from physical and chemical challenges. Accordingly, with current knowledge, it becomes clear that targeting and destroying eDNA in bacterial EPS is a promising strategy for treatment of bacterial-associated infections in a medical context and biofilm control on surfaces to prevent biocorrison in an engineering context. In contrast, the addition of DNA can be applied to engineering of biofilms for beneficial purposes such as remediation of environmental pollutants and electricity or fuel production in bioelectrochemical systems or bioreactors.
Article
Extensive study of modern Bahamian stromatolites has resulted in a comprehensive model for their formation. Modern Bahamian thrombolites—microbial deposits with a mottled, clotted fabric— have not, however, received the same degree of study. Current models link the Bahamian thrombolites with the presence of a mixed- bacterial-and-metaphyte benthic ecosystem, whereas stromatolite for- mation is linked with an almost exclusively bacterial benthic ecosys- tem. By focusing on the preserved fabrics of several specimens, in- cluding an entire column 1.5 meters tall, we have developed a new model for the genesis of the clotted, thrombolitic fabric. Our findings demonstrate that variations in the amount and style of penecontem- poraneous diagenesis, rather than differences in surficial benthic eco- systems, are the predominate cause of the disparate carbonate fabrics present in the Bahamian microbialites examined. More specifically, the irregular, clotted fabric that characterizes the thrombolites is the result of remodeling a precursor fabric. This remodeling is caused by physical and metazoan disruption, penecontemporaneous micri- tization, secondary cementation, and localized carbonate dissolution. This new model of syndepositional remodeling of a laminated fabric to a well-cemented, clotted one may be applicable to some ancient thrombolites.
Article
Revealing the geological history of microbial life is very challenging. Microbes rarely are preserved with morphological fidelity, and even when they are, morphology is a poor guide to phylogeny and metabolism. Biological studies of environments considered analogous to those of paleobiological interest on the ancient Earth can inform interpretations and suggest new approaches. This paper reviews recent advances in our understanding of the biological diversity of two environments relevant to Archean paleobiology: those of extreme acidity and temperature (the Mt. Hood and White Island volcanoes), and high salinity (living stromatolites in Shark Bay). The combination of traditional microbial isolation with the use of modern molecular techniques has revealed that the microbial communities in these environments are much more diverse than originally thought. Through the extraction of whole microbial community DNA, enzymatic amplification of evolutionarily conserved genes, and cloning and sequencing of these genes, more specific and informed inferences concerning functional complexity in these extreme environments have now been made. Studies of the modern stromatolites have demonstrated that they have a very diverse range of micoorganisms, and contrary to previous interpretations, cyanobacteria are not the most abundant microbes present. In addition, many of the microorganisms are unique with no known close relatives, and these microorganisms may also possess novel physiologies vital to the integrity and persistence of stromatolites through space and time. Microbes in the volcanoes studied are present ubiquitously and include geochemically significant sulfur- and iron-cycling taxa. The findings from the studies reviewed here suggest that the Archean biota may have been functionally diverse and much more complex than has yet been revealed. The importance of studying modern analogues is stressed in that the biogeochemical processes occurring in these communities leave morphological, mineralogical, lipid and isotopic signals that could be sought in the rock record.