Genome Sequence of Cupriavidus campinensis Strain G5, a Member of a Bacterial Consortium Capable of Polyethylene Degradation.

Nine different bacterial isolates were recovered from landfills. Each isolate was obtained in pure culture. As a consortium, the bacteria degrade polyethylene. The complete genome sequence of strain G5 was determined by PacBio sequencing. Using the TYGS for taxonomic classification, strain G5 was assigned to the species Cupriavidus campinensis.

Genome Sequence of Pseudomonas veronii Strain G2, a Member of a Bacterial Consortium Capable of Polyethylene Degradation.

Nine different bacterial isolates were recovered from landfills. Each isolate was obtained in pure culture. As a consortium, the bacteria degrade polyethylene. The complete genome sequence of strain G2 was determined by PacBio sequencing. Using the TYGS for taxonomic classification, strain G2 was assigned to the species Pseudomonas veronii.

Genome Sequence of Micromonospora aurantiaca Strain G9, a Member of a Bacterial Consortium Capable of Polyethylene Degradation.

Nine different bacterial isolates were recovered from landfills. Each isolate was obtained in pure culture. As a consortium, the bacteria degrade polyethylene. The complete genome sequence of strain G9 was determined by PacBio sequencing. Using the TYGS server for taxonomic classification, strain G9 was assigned to the species Micromonospora aurantiaca.

Microbial Hotspots in Lithic Microhabitats Inferred from DNA Fractionation and Metagenomics in the Atacama Desert.

The existence of microbial activity hotspots in temperate regions of Earth is driven by soil heterogeneities, especially the temporal and spatial availability of nutrients. Here we investigate whether microbial activity hotspots also exist in lithic microhabitats in one of the most arid regions of the world, the Atacama Desert in Chile. While previous studies evaluated the total DNA fraction to elucidate the microbial communities, we here for the first time use a DNA separation approach on lithic microhabitats, together with metagenomics and other analysis methods (i.e., ATP, PLFA, and metabolite analysis) to specifically gain insights on the living and potentially active microbial community. Our results show that hypolith colonized rocks are microbial hotspots in the desert environment. In contrast, our data do not support such a conclusion for gypsum crust and salt rock environments, because only limited microbial activity could be observed. The hypolith community is dominated by phototrophs, mostly Cyanobacteria and Chloroflexi, at both study sites. The gypsum crusts are dominated by methylotrophs and heterotrophic phototrophs, mostly Chloroflexi, and the salt rocks (halite nodules) by phototrophic and halotolerant endoliths, mostly Cyanobacteria and Archaea. The major environmental constraints in the organic-poor arid and hyperarid Atacama Desert are water availability and UV irradiation, allowing phototrophs and other extremophiles to play a key role in desert ecology.

A chemical and microbial characterization of selected mud volcanoes in Trinidad reveals pathogens introduced by surface water and rain water.

Terrestrial mud volcanoes are unique structures driven by tectonic pressure and fluids from the deep subsurface. These structures are mainly found in active tectonic zones, such as the area near the Los Bajos Fault in Trinidad. Here we report a chemical and microbiological characterization of three mud volcanoes, which included analyses of multiple liquid and solid samples from the mud volcanoes. Our study confirms previous suggestions that at least some of the mud volcano fluids are a mixture of deeper salt-rich water and surficial/precipitation water. No apparent water quality differences were found between sampling sites north and south of a major geological fault line. Microbiological analyses revealed diverse communities, both aerobic and anaerobic, including sulfate reducers, methanogens, carbon dioxide fixing and denitrifying bacteria. Several identified species were halophilic and likely derived from the deeper salt-rich subsurface water, while we also cultivated pathogenic species from the Vibrionaceae, Enterobacteriaceae, Shewanellaceae, and Clostridiaceae. These microorganisms were likely introduced into the mud volcano fluids both from surface water or shallow ground-water, and perhaps to a more minor degree by rain water. The identified pathogens are a major health concern that needs to be addressed.

Transitory microbial habitat in the hyperarid Atacama Desert.

Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity.

Plant growth and arbuscular mycorrhizae development in oil sands processing by-products.

Soil pollutants such as hydrocarbons can induce toxic effects in plants and associated arbuscular mycorrhizal fungi (AMF). This study was conducted to evaluate if the legume Lotus corniculatus and the grass Elymus trachycaulus and arbuscular mycorrhizal fungi could grow in two oil sands processing by-products after bitumen extraction from the oil sands in northern Alberta, Canada. Substrate treatments were coarse tailings sand (CTS), a mix of dry mature fine tailings (MFT) with CTS (1:1) and Pleistocene sandy soil (hydrocarbon free); microbial treatments were without AMF, with AMF and AMF plus soil bacteria isolated from oil sands reclamation sites. Plant biomass, root morphology, leaf water content, shoot tissue phosphorus content and mycorrhizal colonization were evaluated. Both plant species had reduced growth in CTS and tailings mix relative to sandy soil. AMF frequency and intensity in roots of E. trachycaulus was not influenced by soil hydrocarbons; however, it decreased significantly over time in roots of L. corniculatus without bacteria in CTS. Mycorrhizal inoculation alone did not significantly improve plant growth in CTS and tailings mix; however, inoculation with mycorrhizae plus bacteria led to a significantly positive response of both plant species in CTS. Thus, combined inoculation with selected mycorrhizae and bacteria led to synergistic effects. Such combinations may be used in future to improve plant growth in reclamation of CTS and tailings mix.

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

BACKGROUND: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts. METHODS: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed. RESULTS: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects. CONCLUSIONS: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Tracing biogeochemical and microbial variability over a complete oil sand mining and recultivation process.

Recultivation of disturbed oil sand mining areas is an issue of increasing importance. Nevertheless only little is known about the fate of organic matter, cell abundances and microbial community structures during oil sand processing, tailings management and initial soil development on reclamation sites. Thus the focus of this work is on biogeochemical changes of mined oil sands through the entire process chain until its use as substratum for newly developing soils on reclamation sites. Therefore, oil sand, mature fine tailings (MFTs) from tailings ponds and drying cells and tailings sand covered with peat-mineral mix (PMM) as part of land reclamation were analyzed. The sample set was selected to address the question whether changes in the above-mentioned biogeochemical parameters can be related to oil sand processing or biological processes and how these changes influence microbial activities and soil development. GC-MS analyses of oil-derived biomarkers reveal that these compounds remain unaffected by oil sand processing and biological activity. In contrast, changes in polycyclic aromatic hydrocarbon (PAH) abundance and pattern can be observed along the process chain. Especially naphthalenes, phenanthrenes and chrysenes are altered or absent on reclamation sites. Furthermore, root-bearing horizons on reclamation sites exhibit cell abundances at least ten times higher (10(8) to 10(9) cells g(-1)) than in oil sand and MFT samples (10(7) cells g(-1)) and show a higher diversity in their microbial community structure. Nitrate in the pore water and roots derived from the PMM seem to be the most important stimulants for microbial growth. The combined data show that the observed compositional changes are mostly related to biological activity and the addition of exogenous organic components (PMM), whereas oil extraction, tailings dewatering and compaction do not have significant influences on the evaluated compounds. Microbial community composition remains relatively stable through the entire process chain.

A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample.

Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Previous studies suggested that eDNA plays an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Previous methods for eDNA extraction were either not suitable for oligotrophic sediments or only allowed quantification but no genetic analyses. Our procedure is based on cell detachment and eDNA liberation from sediment particles by sequential washing with an alkaline sodium phosphate buffer followed by a separation of cells and eDNA. The separated eDNA is then bound onto silica particles and purified, whereas the intracellular DNA from the separated cells is extracted using a commercial kit. The method provides extra- and intracellular DNA of high purity that is suitable for downstream applications like PCR. Extracellular DNA was extracted from organic-rich shallow sediment of the Baltic Sea, glacially influenced sediment of the Barents Sea and from the oligotrophic South Pacific Gyre. The eDNA concentration in these samples varied from 23 to 626ngg(-1) wet weight sediment. A number of experiments were performed to verify each processing step. Although extraction efficiency is higher than other published methods, it is not fully quantitative.

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity.

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

Simultaneous persistence of multiple genome variants of human parvovirus B19.

The species human parvovirus B19 (B19V) can be divided into three genotypes. In this study, we addressed the question as to whether infection of an individual is restricted to one genotype. As viral DNA is detectable in tissue for long times after acute infection, we examined 87 liver specimens from adults for the presence of B19V DNA. Fifty-nine samples were found to be positive, 32 of them for genotype 1, 27 for genotype 2 and four for genotype 3. In four samples, DNA of two genotypes was detected; samples from three individuals were positive for genotypes 1 and 2 and a sample from one individual was positive for genotypes 1 and 3. Surprisingly, significant sequence heterogeneity was observed at approximately 1 % of the nucleotides of the genotype 1 genomes from individuals with double genotype 1 and 2 infection. Controls using different enzymes for genome amplification and dilutions of the template verified that nucleotide heterogeneity was due to the presence of three or more genome variants of genotype 1. In summary, the evidence shows that individuals can be infected with two different genotypes, and B19V DNA can persist as a population of different genomes. The results may have implications for the understanding of the antiviral immune response and the development of vaccines against B19V.

Persistence of novel human parvovirus PARV4 in liver tissue of adults.

Human parvovirus 4 (PARV4) is a recently identified virus whose biology, epidemiology and pathogenic potential have yet to be determined. Recently, it was reported that PARV4 DNA persists in tissues of some HIV-infected individuals, whilst PARV4 DNA was not detected in tissues of subjects not infected with HIV. In the present study, liver tissue from 87 individuals, none of who were infected with HIV, with the exception of a single subject, was analyzed for the presence of PARV4 DNA. Overall, PARV4 DNA was detected in 13 specimens (15%). In other tissues examined, PARV4 genotype 2 (also termed PARV5) DNA was detected in one of four paired bone marrow specimens. Tissue viral loads did not exceed 100 copies per microg of genomic DNA. In addition, serum samples from 40 of these individuals all tested negative for PARV4 DNA. In the subjects analyzed in this study, PARV4 genotype 2 appeared to be genetically more heterogeneous than PARV4 genotype 1. The results show that PARV4 DNA can be detected in liver, and that infection with PARV4 is not restricted to HIV-infected individuals. Previous studies showing the presence of PARV4 in plasma, suggest that during infection with PARV4, a viraemic stage occurs, allowing systemic spread to a variety of tissues.

Bioportfolio: lifelong persistence of variant and prototypic erythrovirus DNA genomes in human tissue.

Human erythrovirus is a minute, single-stranded DNA virus causing many diseases, including erythema infectiosum, arthropathy, and fetal death. After primary infection, the viral genomes persist in solid tissues. Besides the prototype, virus type 1, two major variants (virus types 2 and 3) have been identified recently, the clinical significance and epidemiology of which are mostly unknown. We examined 523 samples of skin, synovium, tonsil, or liver (birth year range, 1913-2000), and 1,640 sera, by qualitative and quantitative molecular assays for the DNA of human erythroviruses. Virus types 1 and 2 were found in 132 (25%) and 58 (11%) tissues, respectively. DNA of virus type 1 was found in all age groups, whereas that of type 2 was strictly confined to those subjects born before 1973 (P < 0.001). Correspondingly, the sera from the past two decades contained DNA of type 1 but not type 2 or 3. Our data suggest strongly that the newly identified human erythrovirus type 2 as well as the prototype 1 circulated in Northern and Central Europe in equal frequency, more than half a century ago, whereafter type 2 disappeared from circulation. Type 3 never attained wide occurrence in this area during the past > or =70 years. The erythrovirus DNA persistence in human tissues is lifelong and represents a source of information about our past, the Bioportfolio, which, at the individual level, provides a registry of one's infectious encounters, and at the population level, a database for epidemiological and phylogenetic analyses.

Contamination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2.

Human parvovirus B19 (B19) DNA has frequently been detected in plasma-derived coagulation factor concentrates. Furthermore, transmission of B19 infection was observed, indicating presence of the infectious virus despite routine viral inactivation/removal procedures during the manufacturing process. Recently, human parvovirus DNA isolates, variant from B19, have been identified resulting in classification of B19 virus into three distinct genotypes, with all viruses previously classified as B19 belonging to genotype 1. So far, there is no information available on contamination of clotting factor concentrates with genotype 2. Therefore, we analysed 202 different factor concentrate lots for genotype 1 and 2 DNA by PCR. Analysis of one hundred eighty-one lots representing 13 different products, administered over the last three years, was compared to 21 lots (8 products) used until the early 1980s which had not been treated by viral inactivation procedures. Genotype 1 DNA was detected in 77/181 (42.5%) currently administered lots, and 17/21 (81%) previously used lots. The level of genotype 1 DNA contamination was similar in currently and previously administered concentrates. Genotype 2 DNA was found in 5/202 (2.5%) lots, all of which were co-contaminated with genotype 1 DNA. DNA sequence analysis showed that the PCR-double positive concentrates contained typical genotype 1 and genotype 2 DNA. Because genotype 2 appears to cause a similar spectrum of diseases as genotype 1, simultaneous detection of genotype 2 by nucleic acid amplification testing (NAT), now widely applied to plasma pools for genotype 1, would give an added level of safety to blood products.