Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues

Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.     

Ecological fits, mis-fits and lotteries involving insect herbivores on the invasive plant, Bunias orientalis

Exotic plants bring with them traits that evolved elsewhere into their new ranges. These traits may make them unattractive or even toxic to native herbivores, or vice versa. Here, interactions between two species of specialist (Pieris rapae and P. brassicae) and two species of generalist (Spodoptera exigua and Mamestra brassicae) insect herbivores were examined on two native crucifer species in the Netherlands, Brassica nigra and Sinapis arvensis, and an exotic, Bunias orientalis. Bu. orientalis originates in eastern Europe and western Asia but is now an invasive pest in many countries in central Europe. P. rapae, P. brassicae and S. exigua performed very poorly on Bu. orientalis, with close to 100% of larvae failing to pupate, whereas survival was much higher on the native plants. In choice experiments, the pierid butterflies preferred to oviposit on the native plants. Alternatively, M. brassicae developed very poorly on the native plants but thrived on Bu. orientalis. Further assays with a German Bu. orientalis population also showed that several specialist and generalist herbivores performed very poorly on this plant, with the exception of Spodoptera littoralis and M. brassicae. Bu. orientalis produced higher levels of secondary plant compounds (glucosinolates) than B. nigra but not S. arvensis but these do not appear to be important factors for herbivore development. Our results suggest that Bu. orientalis is a potential demographic ‘trap’ for some herbivores, such as pierid butterflies. However, through the effects of an evolutionary ‘lottery’, M. brassicae has found its way through the plant’s chemical ‘minefield’.     

Detection of denitrification genes by in situ rolling circle amplification‐fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells

A target‐primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single‐copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′‐3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template for in situ RCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescence in situ hybridization (FISH). The optimized protocol showed high specificity and signal‐to‐noise ratio but low detection frequency (up to 15% for single‐copy genes and up to 43% for the multi‐copy 16S rRNA gene). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA‐FISH was demonstrated on activated sludge by the differential detection of two types of nirS‐defined denitrifiers; one of them was identified as Candidatus Accumulibacter phosphatis by combining in situ RCA‐FISH with 16S rRNA‐targeted FISH. While not suitable for quantification because of its low detection frequency, in situ RCA‐FISH will allow to link metabolic potential with 16S rRNA (gene)‐based identification of single microbial cells.     

Vaccinia-virus-induced cellular contractility facilitates the subcellular localization of the viral replication sites

Poxviruses, such as vaccinia virus (VV), replicate their DNA in endoplasmic-reticulum-enclosed cytoplasmic sites. Here, we compare the dynamics of the VV replication sites with those of the attenuated strain, modified VV Ankara (MVA). By live-cell imaging, small, early replication sites of both viruses undergo motility typical of microtubule (MT)-motor-mediated movement. Over time, growing replication sites of VV collect around the nucleus in a MT-dependent fashion, whereas those of MVA remain mostly scattered in the cytoplasm. Surprisingly, blocking the dynein function does not impair the perinuclear accumulation of large VV replication sites. Live-cell imaging demonstrates that in contrast to small replication sites, large sites do not display MT-motor-mediated motility. Instead, VV infection induces cellular contractility that facilitates the collection of growing replication sites around the nucleus. In a subset of cells (30-40%), this VV-induced contractility is alternated by phases of directed cell migration, suggesting that the two processes may be linked. The MVA-infected cells do not display contractility or cell migration, supporting the idea that these cellular activities facilitate the efficient accumulation of the VV replication sites around the nucleus. We propose that the recently described cytoskeletal rearrangements induced by VV are a prerequisite for the observed cell contractility and migration activities that apparently contribute to the organization of the complex cytoplasmic life cycle of VV.     

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

Background  

The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.     

Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum.

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226-3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K(i)* value of 0.6 nm. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nm. The K(m)/K(i)* value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K(m) for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5'-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.     

Microbial community diversity and composition varies with habitat characteristics and biofilm function in macrophyte‐rich streams

Biofilms in streams play an integral role in ecosystem processes and function yet few studies have investigated the broad diversity of these complex prokaryotic and eukaryotic microbial communities. Physical habitat characteristics can affect the composition and abundance of microorganisms in these biofilms by creating microhabitats. Here we describe the prokaryotic and eukaryotic microbial diversity of biofilms in sand and macrophyte habitats (i.e. epipsammon and epiphyton, respectively) in five macrophyte‐rich streams in Jutland, Denmark. The macrophyte species varied in growth morphology, C:N stoichiometry, and preferred stream habitat, providing a range in environmental conditions for the epiphyton. Among all habitats and streams, the prokaryotic communities were dominated by common phyla, including Alphaproteobacteria, Bacteriodetes, and Gammaproteobacteria, while the eukaryotic communities were dominated by Stramenopiles (i.e. diatoms). For both the prokaryotes and eukaryotes, the epipsammon were consistently the most diverse communities and the epiphytic communities were generally similar among the four macrophyte species. However, the communities on the least complex macrophyte, Sparganium emersum, had the lowest richness and evenness and fewest unique OTUs, whereas the macrophyte with the most morphological complexity, Callitriche spp., had the highest number of unique OTUs. In general, the microbial taxa were ubiquitously distributed across the relatively homogeneous Danish landscape as determined by measuring the similarity among communities (i.e. Sørensen similarity index). Furthermore, we found significant correlations between microbial diversity (i.e. Chao1 rarefied richness and Pielou's evenness) and biofilm structure and function (i.e. C:N ratio and ammonium uptake efficiency, respectively); communities with higher richness and evenness had higher C:N ratios and lower uptake efficiency. In addition to describing the prokaryotic and eukaryotic community composition in stream biofilms, our study indicates that 1) physical habitat characteristics influence microbial diversity and 2) the variation in microbial diversity may dictate the structural and functional characteristics of stream biofilm communities.     

Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes.

Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system. By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions. Nitrification was restricted to a narrow zone of 50 microns on the very top of the film. In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy. Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters. Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm.     

Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.