The location of acid invertase activity and sucrose in the vacuoles of storage roots of beetroot (Beta vulgaris).

Vacuoles were isolated from freshly cut slices of the storage roots of beetroot (Beta vulgaris), and from slices that had been washed in aerated water for 1-3 days. The unique vacuolar location of betanin permitted the use of a correlative method to determine whether sucrose and acid invertase were located in the vacuoles. The specific content (the activity of the enzyme or amount of substrate per mg of protein) and the percentage recoveries for betanin, sucrose and acid invertase were determined for the different fractions obtained during the isolation of the vacuoles. For each fraction the specific content of betanin was plotted against those of sucrose and acid invertase. Similar correlative plots were drawn for the percentage recoveries. For both specific contents and percentage recoveries for correlation coefficients for sucrose and for acid invertase versus betanin were close to unity, and the lines passed near the origins. It is concluded that, in beetroot, most of the sucrose and much of the acid invertase are in the vacuoles. Measurements of vacuolar sucrose and acid invertase in beetroot slices washed for 1-3 days demonstrated an inverse relationship between sucrose content and acid invertase activity.     

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L_2,3 absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)–Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.     

De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities

This study describes reconstruction of two highly unusual archaeal genomes by de novo metagenomic assembly of multiple, deeply sequenced libraries from surface waters of Lake Tyrrell (LT), a hypersaline lake in NW Victoria, Australia. Lineage-specific probes were designed using the assembled genomes to visualize these novel archaea, which were highly abundant in the 0.1–0.8 μm size fraction of lake water samples. Gene content and inferred metabolic capabilities were highly dissimilar to all previously identified hypersaline microbial species. Distinctive characteristics included unique amino acid composition, absence of Gvp gas vesicle proteins, atypical archaeal metabolic pathways and unusually small cell size (approximately 0.6 μm diameter). Multi-locus phylogenetic analyses demonstrated that these organisms belong to a new major euryarchaeal lineage, distantly related to halophilic archaea of class Halobacteria. Consistent with these findings, we propose creation of a new archaeal class, provisionally named ‘Nanohaloarchaea''. In addition to their high abundance in LT surface waters, we report the prevalence of Nanohaloarchaea in other hypersaline environments worldwide. The simultaneous discovery and genome sequencing of a novel yet ubiquitous lineage of uncultivated microorganisms demonstrates that even historically well-characterized environments can reveal unexpected diversity when analyzed by metagenomics, and advances our understanding of the ecology of hypersaline environments and the evolutionary history of the archaea.     

Herpes simplex virus particles are unable to traverse the secretory pathway in the mouse L-cell mutant gro29.

The mouse L-cell mutant gro29 was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1) and is defective in the propagation of HSV-1 and vesicular stomatitis virus (F. Tufaro, M. D. Snider, and S. L. McKnight, J. Cell Biol. 105:647-657, 1987). In this report, we show that gro29 cells harbor a lesion that inhibits the egress of HSV-1 virions during infection. We also found that HSV-1 glycoprotein D was slow to traverse the secretory pathway en route to the plasma membrane of infected gro29 cells. The movement of glycoproteins was not blocked entirely, however, and immunofluorescence experiments revealed that infected gro29 cells contained roughly 10% of the expected amount of glycoprotein D on their cell surface at 12 h postinfection. Furthermore, nucleocapsids and virions assembled inside the cells during infection, suggesting that the lesion in gro29 cells impinged on a late step in virion maturation. Electron micrographs of infected cells revealed that many of the intracellular virions were contained in irregular cytoplasmic vacuoles, similar to those that accumulate in HSV-1-infected cells treated with the ionophore monensin. We conclude from these results that gro29 harbors a defect that blocks the egress of HSV-1 virions from the infected cell without seriously impeding the flux of individual glycoproteins to the cell surface. We infer that HSV-1 maturation and egress require a host cell component that is either reduced or absent in gro29 cells and that this lesion, although not lethal to the host cell, cannot be tolerated by HSV-1 during its life cycle.     

Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation.

The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.     

Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome‐resolved metagenomics

Gold ore processing uses cyanide (CN^?), which often results in large volumes of thiocyanate‐ (SCN^?) contaminated wastewater requiring treatment. Microbial communities can degrade SCN^? and CN^?, but little is known about their membership and metabolic potential. Microbial‐based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN^? and CN^? into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory‐scale bioreactors used to study SCN^? and CN^? degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN^? degradation. Genome‐based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN^? degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.     

Functional metagenomic selection of ribulose 1, 5‐bisphosphate carboxylase/oxygenase from uncultivated bacteria

Ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO₂‐dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO‐encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO₂/O₂ specificity typical of form II enzymes. X‐ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO₂‐fixing enzymes not previously characterized.     

Geomicrobiology of Pyrite (FeS2) Dissolution: Case Study at Iron Mountain,California

Geomicrobiology of pyrite weathering at Iron Mountain, CA, was investigated by molecular biological, surface chemical, surface structural, and solution chemical methods in both laboratory and field-based studies. Research focused at sites both within and peripheral to the ore-body. The acid-generating areas we have examined thus far at Iron Mountain (solution pH <1.0, temperature> 35 C) were populated by species other than Thiobacillus ferrooxidans . 16S rDNA bacterial sequence analysis and domain- and specieslevel oligonucleotide probe-based investigations confirmed the presence of planktonic Leptospirillum ferrooxidans and indicated the existence of other species apparently related to other newly described acidophilic chemolithotrophs. T. ferrooxidans was confined to relatively moderate environments (pH 2-3, 20-30 C) that were peripheral to the orebody. Dissolution rate measurements indicated that, per cell, attached and planktonic species contributed comparably in acid release. Surface colonization experiments in the laboratory and field indicated that attachment was specific to sulfides instead of to silicates, occurred in crystallographically preferred orientations, and, after cell division, resulted in a monolayer of cells at a maximum density of 8 X 106 cells cm2. In situ geochemical characterization throughout the year revealed that the microbial community that controlled acid generation varied and could be correlated with seasonal and spatial fluctuations in geochemical conditions.     

Identification of biofilm matrix-associated proteins from an acid mine drainage microbial community

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ～2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.