Natural Communities of Novel Archaea and Bacteria Growing in Cold Sulfurous Springs with a String-of-Pearls-Like Morphology

We report the identification of novel archaea living in close association with bacteria in the cold (approximately 10°C) sulfurous marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. These microorganisms form a characteristic, macroscopically visible structure, morphologically comparable to a string of pearls. Tiny, whitish globules (the pearls; diameter, about 0.5 to 3.0 mm) are connected to each other by thin, white-colored threads. Fluorescent in situ hybridization (FISH) studies have revealed that the outer part of the pearls is mainly composed of bacteria, with a filamentous bacterium predominating. Internally, archaeal cocci are the predominant microorganisms, with up to 10⁷ cells estimated to be present in a single pearl. The archaea appear to be embedded in a polymer of unknown chemical composition. According to FISH and 16S rRNA gene sequence analysis, the archaea are affiliated with the euryarchaeal kingdom. The new euryarchaeal sequence represents a deep phylogenetic branch within the 16S rRNA tree and does not show extensive similarity to any cultivated archaea or to 16S rRNA gene sequences from environmental samples.     

Screening oxime libraries by means of mass spectrometry (MS) binding assays: Identification of new highly potent inhibitors to optimized inhibitors γ-aminobutyric acid transporter 1

Generation and screening of oxime libraries by competitive MS Binding Assays represents a powerful tool for the identification of new compounds, with affinity to mGAT1, the most abundant plasma membrane bound GABA transporter in the CNS. By screening a guvacine derived oxime library, new potent inhibitors of mGAT1 had been revealed. In the present study, oxime libraries generated by reaction of a large excess of a rac-nipecotic acid derivative displaying a hydroxylamine functionality in which various aldehydes under suitable conditions, were examined for new potent inhibitors of mGAT1. The pK_i values obtained of the best hits were compared with those of related compounds displaying a guvacine instead of a nipecotic acid subunit as hydrophilic moiety. Amongst the new compounds one of the most affine ligands of mGAT1 known so far (pK_i?=?8.55?±?0.04) was found.     

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a cis-alkene spacer as GABA uptake inhibitors

To discover new, potent, and selective inhibitors for the murine gamma-aminobutyric acid transporter 4 (mGAT4), the structure-activity relationship (SAR) study of a new cis-alkene analog family based on DDPM-1457 [(S)-2], which previously showed promising inhibitory potency at and subtype selectivity for mGAT4, was conducted. To uncover the importance of the differences between the trans- and the cis-alkene moiety in the spacer, the present publication describes the synthesis of the new compounds via catalytic hydrogenation with Lindlar’s catalyst. The biological results collected by the SAR study revealed that analog rac-7j characterized by a four-instead of a three-carbon atom spacer with a cis double bond applying to the majority of the studied compounds displays a surprisingly high potency at mGAT1 (pIC₅₀?=?6.00?±?0.04) and at the same time a reasonable potency at mGAT4 (pIC₅₀?=?4.82).     

Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy

An atypical peg-like terminal constriction (“peg”) on metaphase chromosomes of the plant genus Oziroë could be identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH). Using high-resolution 3D analytical SEM, the architecture and DNA distribution of the peg-like NOR were characterized as typical for chromosomes, albeit with significantly smaller chromomeres. ISH procedure was improved for SEM concerning signal localization, labeling efficiency, and structural preservation, allowing 3D SEM analysis of the peg-like NOR structure and rDNA distribution for the first time. It could be shown that implementation of FluoroNanogold markers is an attractive tool that allows efficient immunodection in both LM and SEM. A model is proposed for the peg structure and its mode of condensation.     

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.     

Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts

Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces.     

Ontogenetic patterns in prey use by pallid sturgeon in the Missouri River, South Dakota and Nebraska

The pallid sturgeon ( Scaphirhynchus albus ) is an endangered species native to the Missouri and Mississippi rivers. To date, recovery efforts have focused on stocking juvenile fish, but little is known about ontogenetic changes in diet composition. Although diet composition for pallid sturgeon is believed to change from macroinvertebrates to fish, it is unclear at what size and/or age these ontogenetic diet shifts occur. To evaluate diet composition, 29 hatchery-stocked pallid sturgeon (range 356–720 mm fork length [FL]; mean = 549; SE = 23) were collected from the Missouri River downstream of Fort Randall Dam, South Dakota and Nebraska during summer 2006. The majority of pallid sturgeon (72%) were captured within a large delta region formed by the Niobrara River in the headwaters of Lewis and Clark Lake. Predominant prey of pallid sturgeon based on percent occurrence was Ceratopogonidae (81%), Isonychiidae (67%), Chironomidae (52%), and fishes (24%). Percent composition by wet weight showed that diets were composed of fishes (68%), Ephemeroptera (23%), Decapoda (6%), and Diptera (3%). Graphical analysis of combined data showed that mayflies, particularly Isonychiidae, were an important component of pallid sturgeon diets. Nonetheless, the percent composition of fishes in the diet increased with pallid sturgeon body size; for fish > 600 mm FL (5–7 years of age) diets were composed primarily of fish prey (66%, mostly johnny darters Etheostoma nigrum ). These findings highlight the importance of ontogenetic changes in diet composition for pallid sturgeon. Moreover, the unique habitat formed in the delta region is characterized by higher fish and invertebrate densities that may enhance foraging opportunities and thus improve recovery efforts for stocked pallid sturgeon.     

The Uracil DNA Glycosylase UdgB of Mycobacterium smegmatis Protects the Organism from the Mutagenic Effects of Cytosine and Adenine Deamination

Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.Spontaneous hydrolytic deamination of cytosine (C), adenine (A), and guanine (G) gives rise to uracil (U), hypoxanthine (Hx), and xanthine, respectively (3). Because the first two reactions occur at appreciable rates (31) and because their products are mutagenic, living organisms have evolved sophisticated defense mechanisms to deal with this threat to their genomic integrity. Deamination of C to U occurs with the highest frequency. Because U base pairs with A during replication, a failure to remove U from DNA prior to the next round of DNA synthesis gives rise to C→T (or, on the opposite strand, G→A) transition mutations (1). The A-to-Hx reaction is only about 10 times slower than the reaction affecting C and has biological significance due to Hx pairing with cytosine during replication.Similar to other modified DNA bases, U and Hx are removed from DNA by base excision repair. This process is initiated by one of several DNA glycosylases, which removes the aberrant base, giving rise to an abasic (apyrimidinic or apurinic) site (AP site). In the subsequent step, the AP site is cleaved by an AP endonuclease on its 5′ side, giving rise to a single-strand break. The baseless sugar phosphate is then removed by the 5′→3′ exonuclease activity of polymerase I in bacteria or by the N-terminal lyase activity of polymerase β in eukaryotes. The same polymerases then fill in the single nucleotide gap, and the remaining nick is sealed by a DNA ligase (20). Base excision repair can also proceed by mechanisms that differ somewhat from the canonical pathway described above, but these mechanisms do not alter the outcome of the studies described in this work and are not described in detail here.Uracil removal is catalyzed by uracil DNA glycosylases (UDGs). These enzymes possess two highly conserved motifs; motif A activates a catalytic water molecule for nucleophilic attack on the glycosidic bond, and motif B stabilizes the protein-DNA complex (25). To date, five UDG families with somewhat different substrate specificities have been characterized (25, 30). Enzymes belonging to family 1 are the best-characterized enzymes. They can remove uracil from U·A pairs or U·G mispairs in double-stranded DNA (dsDNA) and even more efficiently from single-stranded DNA (ssDNA) substrates (32). Family 2 enzymes excise uracil from U·G mismatches in dsDNA. Because these enzymes have most likely evolved to deal with the deamination of 5-methylcytosine to thymine, they are specific for U·G and T·G mispairs and excise neither uracil nor thymine from ssDNA (21). The active site of these enzymes is altered compared to the active site of family 1 UDGs. Family 3 consists of the single-strand-selective monofunctional UDGs, which appear to be hybrids between members of families 1 and 2 (5). As their name implies, family 3 enzymes are preferentially active on uracil-containing ssDNA substrates. Family 4 enzymes have been identified in hyperthermophiles such as Thermotoga maritima (28), Archaeoglobus fulgidus (29), and Pyrobaculum aerophilum (6). These enzymes act on U opposite A or G and exhibit maximal activity at high temperatures. Family 5 uracil glycosylases (UdgB) have been identified in a limited number of organisms, such as hyperthermophilic archaea and Mycobacterium tuberculosis (7, 30, 37). Unexpectedly, motif A of UdgB enzymes contains no polar amino acid, but motif B resembles motif B of family 1 proteins (30). In vitro assays showed that UdgB removes uracil from both ssDNA and dsDNA (30). Interestingly, this enzyme could also excise Hx from oligonucleotide substrates (30, 36) and is thus the only glycosylase that removes both aberrant pyrimidines and purines from DNA in vitro. One of the goals of the present study was to learn whether this enzyme also processes U and Hx in vivo.As discussed above, uracil is generated in DNA by spontaneous hydrolytic deamination of cytosine. However, it can also be directly incorporated into DNA during replication in the form of dUMP. This process is not mutagenic, because dUMP is incorporated opposite adenine. However, it can be cytotoxic, particularly in cells with an imbalanced dUTP-dTTP pool, such as mutants lacking dUTPase, or in cells in which thymidylate synthase is inhibited by, e.g., 5-fluorodeoxyuridine (5FdUrd) treatment. Deoxyuridine incorporated during DNA synthesis is processed primarily by family 1 UDGs, which have been shown to associate with replicating polymerase complexes. Under normal circumstances, this repair process affects only the newly synthesized strand. However, at high dUTP/dTTP ratios, uracil repair might become saturated, so that some uracil remains in the DNA until the following round of DNA replication. In this scenario, processing of uracil in both template and nascent strands would give rise to double-strand breaks and thus to genomic instability (14, 15). This hypothesis is supported by the finding that organisms lacking an efficient repair system can tolerate considerable replacement of thymine with uracil in their DNA (40). The second goal of the present study was to test whether UdgB, like family 1 UDGs, contributed to the cytotoxicity associated with a dUTP-dTTP pool imbalance.We wanted to study whether the substrates processed by UdgB in vitro are also addressed in vivo by this enzyme. As we were unable to do this with P. aerophilum, we set out to find an organism encoding UdgB that would be more amenable to genetic manipulation. M. tuberculosis encodes two potential UDGs, one belonging to family 1 (ung, M. tuberculosis Rv2976c) and one belonging to family 5 (udgB, M. tuberculosis Rv1259) (30). However, to avoid complications linked to the pathogenicity of this organism, we decided to investigate the role of Ung and UdgB in the nonpathogenic organism Mycobacterium smegmatis, which is, unlike P. aerophilum, amenable to genetic manipulation. Our results provide novel insights into the coordinated action of family 1 and family 5 glycosylases.     

The timely deposition of callose is essential for cytokinesis in Arabidopsis

The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE / AtGSL8 , which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.