Microbial diversity of boron-rich volcanic hot springs of St. Lucia, Lesser Antilles

The volcanic Sulphur Springs, St. Lucia, present an extreme environment due to high temperatures, low pH values, and high concentrations of sulfate and boron. St. Lucia offers some unique geochemical characteristics that may shape the microbial communities within the Sulphur Springs area. We chose six pools representing a range of geochemical characteristics for detailed microbial community analyses. Chemical concentrations varied greatly between sites. Microbial diversity was analyzed using 16S rRNA gene clone library analyses. With the exception of one pool with relatively low concentrations of dissolved ions, microbial diversity was very low, with Aquificales sequences dominating bacterial communities at most pools. The archaeal component of all pools was almost exclusively Acidianus spp. and did not vary between sites with different chemical characteristics. In the pool with the highest boron and sulfate concentrations, only archaeal sequences were detected. Compared with other sulfur springs such as those at Yellowstone, the microbial diversity at St. Lucia is very different, but it is similar to that at the nearby Lesser Antilles island of Montserrat. While high elemental concentrations seem to be related to differences in bacterial diversity here, similarities with other Lesser Antilles sites suggest that there may be a biogeographical component as well.     

Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones

During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.     

Variants of human immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan

By selecting the R5 human immunodeficiency virus type 1 (HIV-1) strain JR-CSF for efficient use of a CCR5 coreceptor with a badly damaged amino terminus [i.e., CCR5(Y14N)], we previously isolated variants that weakly utilize CCR5(Delta18), a low-affinity mutant lacking the normal tyrosine sulfate-containing amino-terminal region of the coreceptor. These previously isolated HIV-1(JR-CSF) variants contained adaptive mutations situated exclusively in the V3 loop of their gp120 envelope glycoproteins. We now have weaned the virus from all dependency on the CCR5 amino terminus by performing additional selections with HeLa-CD4 cells that express only a low concentration of CCR5(Delta18). The adapted variants had additional mutations in their V3 loops, as well as one in the V2 stem (S193N) and four alternative mutations in the V4 loop that eliminated the same N-linked oligosaccharide from position N403. Assays using pseudotyped viruses suggested that these new gp120 mutations all made strong contributions to use of CCR5(Delta18) by accelerating a rate-limiting CCR5-dependent conformational change in gp41 rather than by increasing viral affinity for this damaged coreceptor. Consistent with this interpretation, loss of the V4 N-glycan at position N403 also enhanced HIV-1 use of a different low-affinity CCR5 coreceptor with a mutation in extracellular loop 2 (ECL2) [i.e., CCR5(G163R)], whereas the double mutant CCR5(Delta18,G163R) was inactive. We conclude that loss of the N-glycan at position N403 helps to convert the HIV-1 envelope into a hair-trigger form that no longer requires strong interactions with both the CCR5 amino terminus and ECL2 but efficiently uses either site alone. These results demonstrate a novel functional role for a gp120 N-linked oligosaccharide and a high degree of adaptability in coreceptor usage by HIV-1.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis

The isolation and analysis of glycoproteins by coupling lectin affinity chromatography with MS has emerged as a powerful strategy to study the glycoproteome of mammalian cells. However, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation is a common post-translational modification for plant proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling, and defense against pathogens. Here, we describe a strategy to enrich for and identify secreted plant proteins based on affinity chromatography using the lectin Concanavalin A and two-dimensional liquid chromatography, together with matrix-assisted laser desorption/ionization MS analysis. The value of this approach is illustrated through the characterization of glycoproteins that are expressed in ripe tomato (Solanum lycopersicum) fruit, a developmental stage that is fundamentally linked with significant changes in cell wall structure and composition. This glycoprotein trap strategy allowed the isolation of a sub-proteome with an extremely high proportion of proteins that are predicted to be resident in the cell wall or secretory pathway, and the identification of new putative cell wall proteins.     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

Listening to Puns Elicits the Co-Activation of Alternative Homophone Meanings during Language Production

Recent evidence suggests that lexical-semantic activation spread during language production can be dynamically shaped by contextual factors. In this study we investigated whether semantic processing modes can also affect lexical-semantic activation during word production. Specifically, we tested whether the processing of linguistic ambiguities, presented in the form of puns, has an influence on the co-activation of unrelated meanings of homophones in a subsequent language production task. In a picture-word interference paradigm with word distractors that were semantically related or unrelated to the non-depicted meanings of homophones we found facilitation induced by related words only when participants listened to puns before object naming, but not when they heard jokes with unambiguous linguistic stimuli. This finding suggests that a semantic processing mode of ambiguity perception can induce the co-activation of alternative homophone meanings during speech planning.