Specific and sensitive detection of Nosema bombi (Microsporidia : Nosematidae) in bumble bees (Bombus spp.; Hymenoptera : Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 107 down to 10 spores diluted in 100 mu l of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of similar to 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to b more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy. (c) 2005 Elsevier Inc. All rights reserved.

Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences

Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.

Extent and variation of phage-borne bacterial 16S rRNA gene sequences in wastewater environments

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.

rbcL sequences indicate a single evolutionary origin of multinucleate cells in the red algal tribe Callithamnieae.

In the Ceramiaceae, one of the largest families of the red algae, there are from 1 to 4000 nuclei in each vegetative cell, but each tribe is homogeneous with respect to the uninucleate/multinucleate character state, except for the Callithamnieae. The goals of this study were to analyze rbcL gene sequences to clarify the evolution of taxa within the tribe Callithamnieae and to evaluate the potential evolutionary significance of the development of multinucleate cells in certain taxa. The genus Aglaothamnion, segregated from Callithamnion because it is uninucleate, was paraphyletic in all analyses. Callithamnion (including Aristothamnion) was monophyletic although not robustly so, apparently due to variations between taxa in rate of sequence evolution. Morphological synapomorphies were identified at different depths in the tree, supporting the molecular phylogenetic analysis. The uninucleate character state is ancestral in this tribe. The evolution of multinucleate cells has occurred once in the Callithamnieae. Multiple nuclei in each cell may combine the benefits of small C values (rapid cell cycle) with large cells (permitting morphological elaboration) while maintaining a constant ratio of nuclear volume: cytoplasmic volume.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIa in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIa is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIa functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Towards Organic Electronic Materials for Electrically Stimulated Gene Delivery

The cell-specific delivery of polynucleic acids (e.g., DNA, RNA), gene therapy, has the potential to treat various diseases. In this chapter we discuss the use of organic electronic materials as non-viral gene delivery vectors and the great potential for electrochemically triggered gene delivery. We highlight some examples in this chapter based on fullerenes (bucky balls and carbon nanotubes), graphenes and electroactive polymers, particularly those that include experiments in vivo.

CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II

Analysis of the draft genome sequence of the opportunistic pathogen Propionibacterium acnes type strain NCTC 737 (=ATCC 6919) revealed five genes with sequence identity to the co-haemolytic Christie-Atkins-Munch-Peterson (CAMP) factor of Streptococcus agalactiae. The predicted molecular masses for the expressed proteins ranged from 28 to 30 kDa. The genes were present in each of the three recently identified recA-based phylogenetic groupings of P. acnes (IA, IB and 11), as assessed by PCR amplification. Conserved differences in CAMP factor gene sequences between these three groups were also consistent with their previous phylogenetic designations. All type IA, IB and 11 isolates were positive for the co-haemolytic; reaction on sheep blood agar. Immunoblotting and silver staining of SIDS-PAGE gels, however, revealed differential protein expression of CAMP factors amongst the different groups. Type IB and 11 isolates produced an abundance of CAMP factor 1, detectable by specific antibody labelling and silver staining of SDS-PAGE gels. In contrast, abundant CAMP factor production was lacking in type A isolates, although larger amounts of CAMP factor 2 were detectable by immunoblotting compared with type 11 isolates. While the potential role of the abundant CAMP factor 1 in host colonization or virulence remains to be determined, it should be noted that the type strain of P. acnes used in much of the published literature is a type A isolate and is, therefore, lacking in this attribute.

Microbial analysis of soil and groundwater from a gasworks site and comparison to a sequenced biological reactive barrier remediation process (SEREBAR)

Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica.

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martin, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N = 9 isolates gave N. apis RFLPs and sequences, N = 20 isolates gave N. ceranae RFLPs and sequences; 100%, correct classification). We then employed the method to analyze N = 115 isolates from across the world. Our data, combined with N = 36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping. (c) 2007 Elsevier Inc. All rights reserved.

The invasive genus Asparagopsis (Bonnemaisoniaceae, Rhodophyta): Molecular systematics, morphology, and ecophysiology of Falkenbergia isolates

The genus Asparagopsis was studied using 25 Falkenbergia tetrasporophyte strains collected worldwide. Plastid (cp) DNA RFLP revealed three groups of isolates, which differed in their small subunit rRNA gene sequences, temperature responses, and tetrasporophytic morphology (cell sizes). Strains from Australia, Chile, San Diego, and Atlantic and Mediterranean Europe were identifiable as A. armata Harvey, the gametophyte of which has distinctive barbed spines. This species is believed to be endemic to cold-temperate waters of Australia and New Zealand and was introduced into Europe in the 1920s. All isolates showed identical cpDNA RFLPs, consistent with a recent introduction from Australia. Asparagopsis taxiformis (Delile) Trevisan, the type and only other recognized species, which lacks spines, is cosmopolitan in warm-temperate to tropical waters. Two clades differed morphologically and ecophysiologically and in the future could be recognized as sibling species or subspecies. A Pacific/Italian clade had 4-8degrees C lower survival minima and included a genetically distinct apomictic isolate from Western Australia that corresponded to the form of A. taxiformis originally described as A. sanfordiana Harvey. The second clade, from the Caribbean and the Canaries, is stenothermal (subtropical to tropical) with some ecotypic variation. The genus Asparagopsis consists of two or possibly three species, but a definitive taxonomic treatment of the two A. taxiformis clades requires study of field-collected gametophytes.

Phocine distemper virus in seals, East Coast, United States, 2006

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

Hydrobiogeochemical Interactions along “Flow Tubes” Controls: Watershed Scale Contaminant Flow and Transformation at the Oak Ridge IFRC

Geochemical,spectrographic, microbiological and hydrogeologic studies at the ORIFRC site indicate that groundwater transport in structured media may behave as a system of parallel flow tubes. These tubes are preferred flowpaths that enable contaminant transport parallel to bedding planes (strike) over distances of 1000s of meters. A significant flux of groundwater is focused within an interval defined by the interface between the competent bedrock and overlying highly-weathered saprolite, commonly referred to as the"transition zone." Characteristics of this transition zone are dense fractures and the relative absence of weathering products (e.g. clays)results in a significantly higher permeability compared to both the overlying clay-saprolite and underlying bedrock. Several stratabound low seismic velocity zones located below the transition zone were identified during geophysics studies and were also determined to be fractured high permeability preferred contaminant transport pathways during subsequent drilling activities. XANES analysis of precipitates collected from these deeper flow zones indicate 95% or more of the U deposited is U(VI). Linear combination fitting of the EXAFS data shows that precipitates are ~51±5% U(VI)-carbonate-like phase (e.g., liebigite) and ~49±5% U(VI) associated with an iron oxide phase; inclusion of a third component in the fit suggests that up to 15% of the U(VI) may be associated with a phosphate phase or OH- phase (e.g.,schoepite). Although precipitates with similar U(VI)-carbonate and/or phosphate associations were identified in the transition zone pathways,there were also U(VI) complexes adsorbed to mineral surfaces that would tend to be more readily mobilized. Groundwater in the different flow tubes has been determined to consist of different water quality types that vary with the solid phase encountered (e.g., clays, carbonates, clastics) as contaminants migrate along the flow paths. This lateral and vertical variability in geochemistry, particularly pH, has a significant impact on microbiological community composition and activity. Ribosomal RNA gene analyses coupled with physiological and genomic analyses suggest that bacteria from the genus Rhodanobacter(a diverse population of denitrifiers that are moderately acid tolerant) have a high relative abundance in the acidic source zone at the ORIFRC site.Watershed-scale analysis across different flow paths/tubes revealed strong negative correlation between pH and the absolute and relative abundance of Rhodanobacter. Recent studies also confirmed that the ORIFRC site hosts a diverse fungal community, with significant differences observed between acidic (pH <5) and circumneutral (>5) wells. The lack of nitrous oxide reduction capability in fungi, and the detection of denitrification potential in slurry microcosms suggest that fungi may have aheretofore under appreciated role in biogeochemical transformations, with implications forsite remediation and greenhouse gas emissions. Further research is needed to determine if these organisms can influence U(VI) mobility either directly through immobilization or indirectly through the depletion of nitrate.In conclusion, additional studies are required to quantify the processes (e.g., solid phase reactions, recharge, diffusion, microbial interactions) that are occurring along the groundwater flow tubes identified at the ORIFRC so predictive models can be parameterized and used to assess long-term contaminant fate and transport and remedial options.