The impact of a pathogenic bacterium on a social carnivore population

1. The long-term ecological impact of pathogens on group-living, large mammal populations is largely unknown. We evaluated the impact of a pathogenic bacterium, Streptococcus equi ruminatorum, and other key ecological factors on the dynamics of the spotted hyena Crocuta crocuta population in the Ngorongoro Crater, Tanzania. 2. We compared key demographic parameters during two years when external signs of bacterial infection were prevalent ('outbreak') and periods of five years before and after the outbreak when such signs were absent or rare. We also tested for density dependence and calculated the basic reproductive rate R(0) of the bacterium. 3. During the five pre-outbreak years, the mean annual hyena mortality rate was 0.088, and annual population growth was relatively high (13.6%). During the outbreak, mortality increased by 78% to a rate of 0.156, resulting in an annual population decline of 4.3%. After the outbreak, population size increased moderately (5.1%) during the first three post-outbreak years before resuming a growth similar to pre-outbreak levels (13.9%). We found no evidence that these demographic changes were driven by density dependence or other ecological factors. 4. Most hyenas showed signs of infection when prey abundance in their territory was low. During the outbreak, mortality increased among adult males and yearlings, but not among adult females - the socially dominant group members. These results suggest that infection and mortality were modulated by factors linked to low social status and poor nutrition. During the outbreak, we estimated R(0) for the bacterium to be 2.7, indicating relatively fast transmission. 5. Our results suggest that the short-term 'top-down' impact of S. equi ruminatorum during the outbreak was driven by 'bottom-up' effects on nutritionally disadvantaged age-sex classes, whereas the longer-term post-outbreak reduction in population growth was caused by poor survival of juveniles during the outbreak and subsequent poor recruitment of breeding females. These results suggest synergistic effects of 'bottom-up' and 'top-down' processes on host population dynamics.     

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Disentangling the Complexity of Mitogen-Activated Protein Kinases and Reactive Oxygen Species Signaling

We report here on the identification of the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis (Arabidopsis thaliana) AIR12 (for auxin induced in root cultures). Soybean AIR12, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol modification signal and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON (for dopamine β-monooxygenase N terminus) domain superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, fully reduced by ascorbate, and fully oxidized by monodehydroascorbate radical. AIR12 is a high-potential cytochrome b showing a wide bimodal dependence from the redox potential between +80 mV and +300 mV. Optical absorption and electron paramagnetic resonance analysis indicate that AIR12 binds a single, highly axial low-spin heme, likely coordinated by methionine-91 and histidine-76, which are strongly conserved in AIR12 sequences. Phylogenetic analyses reveal that the auxin-responsive genes AIR12 represent a new family of PM b-type cytochromes specific to flowering plants. Circumstantial evidence suggests that AIR12 may interact with other redox partners within the PM to constitute a redox link between cytoplasm and apoplast.Complex interactions between plant cells and the environment are mediated by the apoplast. The apoplastic liquid phase permeating the cell wall contains relatively low concentrations of solutes (Dietz, 1997). Its composition, although largely determined by the protoplast, is easily perturbed by environmental challenges that can thus be perceived by the apoplast and translated into signals that trigger cell responses (Pignocchi and Foyer, 2003; Foyer and Noctor, 2005). Environmental challenges affecting the apoplast commonly result in an oxidative load, caused, for instance, by pollutants (e.g. ozone; Sandermann, 2008) or by endogenously generated reactive oxygen species (ROS). Several enzymatic and nonenzymatic systems are able to generate ROS in the apoplast (Fry, 1998; Apel and Hirt, 2004), an event that is not restricted to biotic or abiotic stresses (Torres and Dangl, 2005), but also involved in diverse physiological conditions, including stomata closure and cell growth (Foreman et al., 2003; Mori and Schroeder, 2004; Gapper and Dolan, 2006; Schopfer and Liszkay, 2006).Apoplastic reductants not only act as an antioxidant barrier, but they could also modulate oxidative signals, thus actively contributing to plant adaptation to the environment. Ascorbate occurs at 10⁻⁴ to 10⁻³ m concentrations in the apoplast, where it represents the major pool of low-molecular-mass antioxidants (Dietz, 1997; Pignocchi and Foyer, 2003; Padu et al., 2005). Maintenance of the apoplastic ascorbate pool depends on transport systems of the plasma membrane (PM; Horemans et al., 2000). The redox state of the ascorbate in the apoplast is relatively flexible and typically more oxidized than in the symplast (Cordoba-Pedregosa et al., 2003, 2005; de Pinto and De Gara, 2004; Padu et al., 2005; Pignocchi et al., 2006). Ascorbate oxidation can be effected enzymatically by ascorbate oxidase or ascorbate peroxidase, and nonenzymatically by direct interaction with ROS (including ozone; Sandermann, 2008), transition metals (e.g. iron, copper; Fry, 1998), or phenolic radicals (Takahama, 1993). Oxidation of ascorbate gives rise to the monodehydroascorbate (MDA) radical, which can disproportionate into ascorbate and fully oxidized dehydroascorbate. In addition, the apoplastic MDA radical can be reduced back to ascorbate by a trans-PM redox system that uses cytosolic ascorbate as a reductant and involves a high-potential cytochrome b (Horemans et al., 1994). The latter has escaped molecular identification thus far (Trost et al., 2000; Bérczi et al., 2003; Griesen et al., 2004; Preger et al., 2005).It was suggested (Asard et al., 2001) that the trans-PM electron transfer from cytosolic ascorbate to apoplastic MDA may be effected by a cytochrome b561, in analogy to the electron transfer of animal chromaffin vesicles (Kelley and Njus, 1986). Cytochromes b561 are high-potential, transmembrane redox proteins of about 25 kD made of six membrane-spanning α-helices, which bind two hemes b. One heme is predicted to be close to an ascorbate binding site facing the cytosol, whereas the second heme faces the opposite side of the membrane and can be oxidized by either MDA or ferrichelates (Tsubaki et al., 1997; McKie et al., 2001; Bérczi et al., 2005; Kamensky et al., 2007). Plants contain several orthologous genes to animal cytochrome b561 (Asard et al., 2000; Bashtovyy et al., 2003). Arabidopsis (Arabidopsis thaliana) contains four genes belonging to this family (Tsubaki et al., 2005) and one of these (At4g25570, CYBASC1), expressed in recombinant form, showed similar biochemical properties to animal cytochrome b561 (Bérczi et al., 2007). However, the localization in vivo of plant cytochrome b561 is controversial. Arabidopsis CYBASC1 was found to be associated with the tonoplast membrane (Griesen et al., 2004) and annotated in proteomic studies as either a tonoplast protein (Carter et al., 2004; Shimaoka et al., 2004) or a chloroplast protein (Zybailov et al., 2008). Tonoplast localization was also reported for bean (Phaseolus vulgaris) CYBASC1 in etiolated hypocotyls (Preger et al., 2005), whereas a GFP construct of CYBASC1 from wild watermelon (Citrullus lanatus) was shown to be targeted to the PM in transformed onion (Allium cepa) epidermal cells (Nanasato et al., 2005). No data are available for any other isoform of cytochrome b561 in plants.An ascorbate-reducible cytochrome b from enriched PM preparations was purified as a glycosylated protein of 55 to 63 kD (bean hypocotyls; Trost et al., 2000) or 120 kD (Arabidopsis; Bérczi et al., 2003) in SDS-PAGE. The association to the PM of the bean hypocotyl cytochrome was confirmed by analytical Suc gradient centrifugation (Preger et al., 2005). Based on potentiometric redox titrations, both bean and Arabidopsis cytochrome b preparations were suggested to bind two hemes with distant redox potentials (E_m7 +135 and +180/+200 mV). However, the nature of this high-potential cytochrome b of plant PM remained elusive, although clearly different from tonoplast cytochrome b561 (Preger et al., 2005).In this article, we report on the purification, molecular identification, cloning, and biochemical characterization of the major ascorbate-reducible cytochrome b associated with the PM of soybean (Glycine max) etiolated hypocotyls. The coding gene, known as AIR12 (for auxin induced in root cultures), is early expressed during auxin-induced lateral root formation in Arabidopsis (Laskowski et al., 2006). We demonstrate that AIR12 is a member of a new family of ascorbate-reducible cytochromes b specific to flowering plant species. The protein is glycosylated and hydrophilic and predicted to be associated in vivo with the external face of the PM by means of a glycosylphosphatidylinositol (GPI) anchor (Borner et al., 2003). AIR12 has been found to be associated with lipid rafts together with other redox proteins (Lefebvre et al., 2007), which may act as its partners in a possible electron link between apoplast and symplast.     

Land‐use and isolation interact to affect wetland plant assemblages

Different management regimes imposed on similar habitat types provide opportunities to investigate mechanisms driving community assembly and changes in species composition. We investigated the effect of pasture management on vegetation composition in wetlands with varying spatial isolation on a Florida cattle ranch. We hypothesized that increased pasture management intensity would dampen the expected negative effect of wetland isolation on native species richness due to a change from dispersal‐driven community assembly to niche‐driven assembly by accentuated environmental tolerance. We used native plant richness, exotic plant richness and mean coefficient of conservatism (CC) to assess wetland plant assemblage composition. Sixty wetlands were sampled, stratified by three levels of isolation across two pasture management intensities; semi‐native (less intensely managed; mostly native grasses, never fertilized) and agronomically improved (intensely managed, planted with exotic grasses, and fertilized). Improved pasture wetlands had lower native richness and CC scores, and greater total soil phosphorus and exotic species coverage compared to semi‐native pasture wetlands. Increased wetland isolation was significantly associated with decreases in native species richness in semi‐native pasture wetlands but not in improved pasture wetlands. Additionally, the species–area relationship was stronger in wetlands in improved pastures than semi‐native pastures. Our results indicate that a) native species switch from dispersal‐based community assembly in semi‐native pastures to a species‐sorting process in improved pastures, and b) recently‐introduced exotic species already sorted for more intensive management conditions are primarily undergoing dispersal‐based community assembly. That land‐use may alter the relative importance of assembly processes and that different processes drive native and exotic richness has implications for both ecosystem management and restoration planning.     

Proline Is Not Uniquely Capable of Providing the Pivot Point for Domain Swapping in 2G12, a Broadly Neutralizing Antibody against HIV-1

The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the “silent” face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.