Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.     

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.     

Metabolically active eukaryotic communities in extremely acidic mine drainage

Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50 degrees C), metal-rich (up to 269 mM Fe(2+), 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name "Acidomyces richmondensis" for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.     

Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community

Microbial community succession was examined over a two-year period using spatially and temporally coordinated water chemistry measurements, metagenomic sequencing, phylogenetic binning and de novo metagenomic assembly in the extreme hypersaline habitat of Lake Tyrrell, Victoria, Australia. Relative abundances of Haloquadratum-related sequences were positively correlated with co-varying concentrations of potassium, magnesium and sulfate, but not sodium, chloride or calcium ions, while relative abundances of Halorubrum, Haloarcula, Halonotius, Halobaculum and Salinibacter-related sequences correlated negatively with Haloquadratum and these same ionic factors. Nanohaloarchaea and Halorhabdus-related sequence abundances were inversely correlated with each other, but not other taxonomic groups. These data, along with predicted gene functions from nearly-complete assembled population metagenomes, suggest different ecological phenotypes for Nanohaloarchaea and Halorhabdus-related strains versus other community members. Nucleotide percent G+C compositions were consistently lower in community metagenomic reads from summer versus winter samples. The same seasonal G+C trends were observed within taxonomically binned read subsets from each of seven different genus-level archaeal groups. Relative seasonal abundances were also linked to percent G+C for assembled population genomes. Together, these data suggest that extreme ionic conditions may exert selective pressure on archaeal populations at the level of genomic nucleotide composition, thus contributing to seasonal successional processes. Despite the unavailability of cultured representatives for most of the organisms identified in this study, effective coordination of physical and biological measurements has enabled discovery and quantification of unexpected taxon-specific, environmentally mediated factors influencing microbial community structure.     

Microbial populations stimulated for hexavalent uranium reduction in uranium mine sediment

Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO(2)) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp. would be a major contributor to U(VI) and sulfate reduction and Clostridium spp. to U(VI) reduction.     

Localization of ERK/MAP kinase is regulated by the alphaherpesvirus tegument protein Us2

Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.     

Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.     

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.     

Effect of Microorganisms and Microbial Metabolites on Apatite Dissolution

The dissolution rate of apatite was determined in batch reactors in organic acid solutions and in microbial cultures. Inoculum for the cultures was from biotite plus apatite crystals from a granite weathering profile in South Eastern Australia. In both the biotic and the abiotic experiments, etching of the apatite surface leads to the formation of elongated spires parallel to the c axis. Apatite dissolution rates in the inorganic, acetate, and oxalate solutions increase as pH decreases from approximately 10 -11 mol/m -2 · s -1 at initial pH 5.5 to 10 -7 mol/m -2 · s -1 at initial pH 2. Under mildly acidic to near neutral pH conditions, both oxalate and acetate increased apatite dissolution by up to an order of magnitude compared to the inorganic conditions. Acetate catalyzed the reaction by forming complexes with Ca, either in solution or at the mineral surfaces. Oxalate forms complexes with Ca as well, and can also affect reaction rates and stoichiometry by forming Ca-oxalate precipitates, thus affecting solution saturation states. In all abiotic experiments, net phosphate release to solution approaches zero even when solutions are apparently undersaturated by several orders of magnitude with respect to the solubility of an ideal fluoroapatite mineral. In the microbial experiments, two enrichment cultures increased both apatite and biotite dissolution by producing organic acids, primarily pyruvate, fermentation products, and oxalate, and by lowering bulk solution pH to between 3 and 5. However, the microorganisms were also able to increase phosphate release from apatite (by two orders of magnitude) without lowering bulk solution pH by producing pyruvate and other compounds.     

Biostimulation induces syntrophic interactions that impact C,S and N cycling in a sediment microbial community

Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO₂ during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO₂ production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO₂ fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.