454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases

Separation of VUV/UV photons and reactive particles in the effluent of a He/O2 atmospheric pressure plasma jet

Cold atmospheric pressure plasmas can be used for treatment of living tissues or for inactivation of bacteria or biological macromolecules. The treatment is usually characterized by a combined effect of UV and VUV radiation, reactive species, and ions. This combination is usually beneficial for the effectiveness of the treatment but it makes the study of fundamental interaction mechanisms very difficult. Here we report on an effective separation of VUV/UV photons and heavy reactive species in the effluent of a micro scale atmospheric pressure plasma jet ($\mu$-APPJ). The separation is realized by an additional flow of helium gas under well-defined flow conditions, which deflects heavy particles in the effluent without affecting the VUV and UV photons. Both components of the effluent, the photons and the reactive species, can be used separately or in combination for sample treatment. The results of treatment of a model plasma polymer film and vegetative Bacillus subtilis and Escherichia coli cells are shown and discussed. A simple model of the He gas flow and reaction kinetics of oxygen atoms in the gas phase and at the surface is used to provide a better understanding of the processes in the plasma effluent. The new jet modification, called X-Jet for its appearance, will simplify the investigation of interaction mechanisms of atmospheric pressure plasmas with biological samples.Comment: 10 pages, 7 figures, submitted to Journal of Physics D: Applied Physic

Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis

Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10¿ccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10¿ccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis

Purification and Reconstitution of the Glutamate Carrier GltT of the Thermophilic Bacterium Bacillus stearothermophilus

An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5α grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost, suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

Fur seal microbiota are shaped by the social and physical environment, show mother‐offspring similarities and are associated with host genetic quality

Despite an increasing appreciation of the importance of host‐microbe interactions in ecological and evolutionary processes, the factors shaping microbial communities in wild populations remain poorly understood. We therefore exploited a natural experiment provided by two adjacent Antarctic fur seal (Arctocephalus gazella) colonies of high and low social density and combined 16S rRNA metabarcoding with microsatellite profiling of mother‐offspring pairs to investigate environmental and genetic influences on skin microbial communities. Seal‐associated bacterial communities differed profoundly between the two colonies, despite the host populations themselves being genetically undifferentiated. Consistent with the hypothesis that social stress depresses bacterial diversity, we found that microbial alpha diversity was significantly lower in the high‐density colony. Seals from one of the colonies that contained a stream also carried a subset of freshwater‐associated bacteria, indicative of an influence of the physical environment. Furthermore, mothers and their offspring shared similar microbial communities, in support of the notion that microbes may facilitate mother‐offspring recognition. Finally, a significant negative association was found between bacterial diversity and heterozygosity, a measure of host genetic quality. Our study thus uncovers a complex interplay between environmental and host genetic effects, while also providing empirical support for the leash model of host control, which posits that bacterial communities are driven not only by bottom‐up species interactions, but also by top‐down host regulation. Taken together, our findings have broad implications for understanding host‐microbe interactions as well as prokaryotic diversity in general

Reliable microsatellite genotyping of the Eurasian badger (Meles meles) using faecal DNA

The potential link between badgers and bovine tuberculosis has made it vital to develop accurate techniques to census badgers. Here we investigate the potential of using genetic profiles obtained from faecal DNA as a basis for population size estimation. After trialling several methods we obtained a high amplification success rate (89%) by storing faeces in 70% ethanol and using the guanidine thiocyanate/silica method for extraction. Using 70% ethanol as a storage agent had the advantage of it being an antiseptic. In order to obtain reliable genotypes with fewer amplification reactions than the standard multiple-tubes approach, we devised a comparative approach in which genetic profiles were compared and replication directed at similar, but not identical, genotypes. This modified method achieved a reduction in polymerase chain reactions comparable with the maximumlikelihood model when just using reliability criteria, and was slightly better when using reliability criteria with the additional proviso that alleles must be observed twice to be considered reliable. Our comparative approach would be best suited for studies that include multiple faeces from each individual. We utilized our approach in a well-studied population of badgers from which individuals had been sampled and reliable genotypes obtained. In a study of 53 faeces sampled from three social groups over 10 days, we found that direct enumeration could not be used to estimate population size, but that the application of mark–recapture models has the potential to provide more accurate results

The Classic: Integration of Deoxyribonucleic Acid Specific for Rous Sarcoma Virus after Infection of Permissive and Nonpermissive Hosts: (RNA tumor viruses/reassociation kinetics/duck cells)

A relatively simple but stringent technique was developed to detect the integration of virus-specific DNA into the genomes of higher organisms. In both permissive (duck) and nonpermissive (mammalian) cells which normally contain no nucleotide sequences specific for Rous sarcoma virus, transformation by the virus results in the appearance of DNA specific for Rous sarcoma virus covalently integrated into strands of host-cell DNA containing reiterated sequences. Early after infection of mouse or duck cells by Rous sarcoma virus, unintegrated DNA specific for the virus can be demonstrated

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins