Ultrastructural localization of cell wall teichoic acids in Streptococcus faecalis by means of concanavalin A

Some teichoic acids are known to be partially substituted by α-D-glucopyranosyl residues such as the teichoic acids of Streptococcus faecalis NCIB 8191. They will, therefore, bind specifically the phytohemagglutinin concanavalin A. Concanavalin A labelled with mercury or colloidal gold coated with concanavalin A has been used to mark isolated cell walls in order to localize the teichoic acids at the ultrastructural level. Besides these two direct marking techniques, the indirect concanavalin A-peroxidase technique (localization of peroxidase by the diaminobenzidine method followed by postosmication) has been applied to thin sections of premarked cells. All three methods gave almost identical results, namely, a dense and homogeneous distribution of the cell wall teichoic acids. In control experiments total inhibition was achieved in the presence of methyl-α-D-mannopyranoside. After trichloroacetic acid or alkali extraction of the teichoic acids from isolated walls no marking could be detected.     

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

The Evolution of the Linnaean Hierarchy

The Linnaean system of classification is a threefold system of theoretical assumptions, sorting rules, and rules of nomenclature. Over time, that system has lost its theoretical assumptions as well as its sorting rules. Cladistic revisions have left it less and less Linnaean. And what remains of the system is flawed on pragmatic grounds. Taking all of this into account, it is time to consider alternative systems of classification.     

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.     

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary,multi‐scale approach

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear‐cutting. Retention of habitat trees and deadwood has since become common practice also in continuous‐cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade‐offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi‐functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio‐economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.     

Manipulation of CD45 antigen in transplantation tolerance

CD45 is known to have tyrosine phosphatase activity for signal transduction of T cells. Immunomodulation of CD45 has been tried to prevent T cell-mediated graft rejection in organ transplantation. In vitro study showed that blockade of CD45RB, an alternative splicing isoform of CD45, inhibited proliferative response of T cells after allogeneic stimulation. Treatment with a monoclonal antibody (mAb) against CD45RB induced long-term allograft acceptance in some mouse organ transplantation models. In a rat heart allograft model, a single injection of anti-rat CD45 (RT7) mAb which bound to allomorphic region of RT7 also induced allograft acceptance. CD45/RT7 is also a useful tool of targeting hematopoietic cells, because of the selective expression on all hematopoietic cells. There are two allomorphic forms of CD45 (RT7a and RT7b) in the rat. Using RT7 system, a rat heart allograft model from RT7a donors to RT7b recipients was designed to test functional relevance of graft-associated hematopoietic cells (microchimerism) to allograft acceptance. Then donor-derived hematopoietic cells were selectively depleted using anti-RT7a mAb in vivo. Depletion on day 0 prevented allograft acceptance and was associated with severe acute or chronic graft rejection, while depletion on day 18 after transplantation showed no effect. This experimental study showed a crucial role of microchimerism in induction phase of allograft acceptance. In conclusion, the CD45/RT7 system is not only a target molecule for tolerance induction, but also an useful tool for experimental models in transplantation immunology. In this review, we introduce basic properties of CD45 and recent results with manipulation of CD45.     

Taxonomic Identity of the Invasive Fruit Fly Pest,Bactrocera invadens: Concordance in Morphometry and DNA Barcoding

In 2003, a new fruit fly pest species was recorded for the first time in Kenya and has subsequently been found in 28 countries across tropical Africa. The insect was described as Bactrocera invadens, due to its rapid invasion of the African continent. In this study, the morphometry and DNA Barcoding of different populations of B. invadens distributed across the species range of tropical Africa and a sample from the pest''s putative aboriginal home of Sri Lanka was investigated. Morphometry using wing veins and tibia length was used to separate B. invadens populations from other closely related Bactrocera species. The Principal component analysis yielded 15 components which correspond to the 15 morphometric measurements. The first two principal axes contributed to 90.7% of the total variance and showed partial separation of these populations. Canonical discriminant analysis indicated that only the first five canonical variates were statistically significant. The first two canonical variates contributed a total of 80.9% of the total variance clustering B. invadens with other members of the B. dorsalis complex while distinctly separating B. correcta, B. cucurbitae, B. oleae and B. zonata. The largest Mahalanobis squared distance (D² = 122.9) was found to be between B. cucurbitae and B. zonata, while the lowest was observed between B. invadens populations against B. kandiensis (8.1) and against B. dorsalis s.s (11.4). Evolutionary history inferred by the Neighbor-Joining method clustered the Bactrocera species populations into four clusters. First cluster consisted of the B. dorsalis complex (B. invadens, B. kandiensis and B. dorsalis s. s.), branching from the same node while the second group was paraphyletic clades of B. correcta and B. zonata. The last two are monophyletic clades, consisting of B. cucurbitae and B. oleae, respectively. Principal component analysis using the genetic distances confirmed the clustering inferred by the NJ tree.