Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples

AIMS: Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. METHODS AND RESULTS: The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. CONCLUSIONS: Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. SIGNIFICANCE AND IMPACT OF THE STUDY: The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.     

Direct interaction between uracil-DNA glycosylase and a proliferating cell nuclear antigen homolog in the crenarchaeon Pyrobaculum aerophilum

Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp on duplex DNA. Its homologs, present in Eukarya and Archaea, are part of protein complexes that are indispensable for DNA replication and DNA repair. In Eukarya, PCNA is known to interact with more than a dozen different proteins, including a human major nuclear uracil-DNA glycosylase (hUNG2) involved in immediate postreplicative repair. In Archaea, only three classes of PCNA-binding proteins have been reported previously: replication factor C (the PCNA clamp loader), family B DNA polymerase, and flap endonuclease. In this study, we report a direct interaction between a uracil-DNA glycosylase (Pa-UDGa) and a PCNA homolog (Pa-PCNA1), both from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (T(opt) = 100 degrees C). We demonstrate that the Pa-UDGa-Pa-PCNA1 complex is thermostable, and two hydrophobic amino acid residues on Pa-UDGa (Phe(191) and Leu(192)) are shown to be crucial for this interaction. It is interesting to note that although Pa-UDGa has homologs throughout the Archaea and bacteria, it does not share significant sequence similarity with hUNG2. Nevertheless, our results raise the possibility that Pa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.     

Geobiological analysis using whole genome-based tree building applied to the Bacteria, Archaea, and Eukarya

We constructed genomic trees based on the presence and absence of families of protein‐encoding genes observed in 55 prokaryotic and five eukaryotic genomes. There are features of the genomic trees that are not congruent with typical rRNA phylogenetic trees. In the bacteria, for example, Deinococcus radiodurans associates with the Gram‐positive bacteria, a result that is also seen in some other phylogenetic studies using whole genome data. In the Archaea, the methanogens plus Archaeoglobus form a united clade and the Euryarchaeota are divided with the two Thermoplasma genomes and Halobacterium sp. falling below the Crenarchaeota. While the former appears to be an accurate representation of methanogen‐relatedness, the misplacement of Halobacterium may be an artefact of parsimony. These results imply the last common ancestor of the Archaea was not a methanogen, leaving sulphur reduction as the most geochemically plausible metabolism for the base of the archaeal crown group. It also suggests that methanogens were not a component of the Earth's earliest biosphere and that their origin occurred sometime during the Archean. In the Eukarya, the parsimony analysis of five Eukaryotes using the Crenarchaeota as an outgroup seems to counter the Ecdysozoa hypothesis, placing Caenorhabditis elegans (Nematoda) below the common ancestor of Drosophila melanogaster (Arthropoda) and Homo sapiens (Chordata) even when efforts are made to counter the possible effects of a faster rate of sequence evolution for the C. elegans genome. Further analysis, however, suggests that the gene loss of ‘animal’ genes is highest in C. elegans and is obscuring the relationships of these organisms.     

Terminal addition, the Cambrian radiation and the Phanerozoic evolution of bilaterian form

We examine terminal addition, the process of addition of serial elements in a posterior subterminal growth zone during animal development, across modern taxa and fossil material. We argue that terminal addition was the basal condition in Bilateria, and that modification of terminal addition was an important component of the rapid Cambrian evolution of novel bilaterian morphology. We categorize the often-convergent modifications of terminal addition from the presumed ancestral condition. Our focus on terminal addition and its modification highlights trends in the history of animal evolution evident in the fossil record. These trends appear to be the product of departure from the initial terminal addition state, as is evident in evolutionary patterns within-fossil groups such as trilobites, but is also more generally related to shifts in types of morphologic change through the early Phanerozoic. Our argument is contingent on dates of metazoan divergence that are roughly convergent with the first appearance of metazoan fossils in the latest Proterozoic and Cambrian, as well as on an inference of homology of terminal addition across bilaterian Metazoa.     

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100°C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80–160 mM NaCl and 70°C, PaNth displays DNA glycosylase/β-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.