Spatial Heterogeneity of Crenarchaeal Assemblages Within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P \u3c 0.0001] and least-significant-difference analysis [α = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b

Measuring the Microbial Limit of Detection for a Modified DNA Extraction Protocol

Typical kit-based DNA extraction techniques only isolate ~17% of the total starting DNA from a sample. When analyzing environments with few microbial cells, such as Wind Cave National Park, South Dakota, this approach is ineffective. In this study, we developed a modified DNA extraction protocol in order to increase the limit of detection. We conducted a dilution series using known amounts of Escherichia coli over a range of one million cells (1 x 10^6) down to ten cells. DNA was isolated from each of the aliquots using the modified protocol. We used polymerase chain reactions (PCRs) followed by ethidium bromide staining of agarose gels to test the presence of template DNA. The limit of detection by this method was found to be 1 x 10^5 cells. In future experiments to improve upon this limit of detection, we will modify the centrifugation steps to include faster and longer spins

Evaluation of Pre-Lysis Rinses To Improve DNA Yield and Purity

The widespread sampling of environmental DNA from soils has led to a fundamental shift in our understanding of Earth’s microbiome by identifying previously unknown microbes that have never been grown in the laboratory. Many of the published protocols for the extraction of environmental DNA differ based on the starting material. These differences include changes to buffer composition, detergents, and enzymatic digest. To remove this bias, we sought a standardized protocol for samples from Wind Cave National Park, which includes both above ground prairie soil and paleofill from the deepest depths of the cave system. Pre-rinsing samples with either of two solutions, 100 mM sodium phosphate pH 7.2 (Na3PO4 ) and 100mM Tris pH 8.0, 5mM EDTA, 200 mM sodium chloride (TEN), was compared and the resulting DNA was visualized using agarose gel electrophoresis. Incorporating rinses resulted in darker bands of the expected size (greater than 10 kilobases) and less DNA degradation, meaning the rinses helped increase yield while isolating more intact DNA. In the future, including a pre-lysis rinse will improve the limit of detection in cave samples that contain low microbial abundance, allowing us to better understand microbial species composition in isolated environments

Emerging market financial services development: the case of leasing in Poland and China

The aim of this paper is to present Polish and Chinese developments in this area in the period of 1999–2009 for the former and 2005–2008 the latter and then to compare both markets for 2007 and 2008 using different metrics. The time horizon is limited due to the lack of Chinese data. The results show that both economies used leasing to finance ongoing business and both markets have many common features. The results show also that although the Chinese leasing market is less developed, it catches up with the more developed Polish counterpart

Exploring Microbes at Wind Cave as an Analog for Exobiological Environments off Earth

Subterranean environments on Earth serve as an analog for the study of microbes on other planets. This has become an active area of research with the discovery of exoplanets. To learn about the microbial species living in Wind Cave, we are comparing methods to sample environmental DNA because most microbes cannot be cultivated using standard laboratory methods. We are then probing the environmental DNA with broad primers that are designed to amplify most life and narrower primers such as those specific to the domain Archaea. Of the methods compared, the Qiagen DNeasy Powerbiofilm kit produced the purest template as measured by its ability to be PCR amplified. The next steps are to optimize the DNA testing reactions to limit mispriming. In the future, these methods will be used to determine the identity, quantity, and spatial distribution of microbes in Wind Cave

The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5

In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1E232Q, an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression ofSKD1E232Q in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.Facultad de Ciencias Agrarias y Forestale

Archaeal Assemblages Inhabiting Temperate Mixed Forest Soil Fluctuate in Taxon Composition and Spatial Distribution over Time

This study explored the persistence and spatial distribution of a diverse Archaeal assemblage inhabiting a temperate mixed forest ecosystem. Persistence under native conditions was measured from 2001 to 2010, 2011, and 2012 by comparison of 16S rRNA gene clone libraries. The Archaeal assemblages at each of these time points were found to be significantly different (AMOVA, P<0.01), and the nature of this difference was dependent on taxonomic rank. For example, the cosmopolitan genus g_Ca. Nitrososphaera (I.1b) was detected at all time points, but within this taxon the abundance of s_SCA1145, s_SCA1170, and s_Ca. N. gargensis fluctuated over time. In addition, spatial heterogeneity (patchiness) was measured at these time points using 1D TRFLP-SSCP fingerprinting to screen soil samples covering multiple spatial scales. This included soil collected from small volumes of 3 cubic centimeters to larger scales—over a surface area of 50 m2, plots located 1.3 km apart, and a separate locality 23 km away. The spatial distribution of Archaea in these samples changed over time, and while g_Ca. Nitrososphaera (I.1b) was dominant over larger scales, patches were found at smaller scales that were dominated by other taxa. This study measured the degree of change for Archaeal taxon composition and patchiness over time in temperate mixed forest soil