Dividing oceans into pools: strategies for the global analysis of bacterial genes

In bacterial pathogenesis, it is often easy to accept the results of large-scale screens without independent verification of the results. How can one critically read this literature? Here we review issues inherent in genome-wide screens in bacteria, focusing on experiments that attempt to comprehensively identify genes required for bacterial growth under specific conditions. Our analysis suggests that the methodologies employed undoubtedly shape the results. It is clear, however, that the question is not which method is better but which provides the data most suited to a given question.     

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.     

COMPARATIVE GENOMIC AND POPULATION GENETIC ANALYSES INDICATE HIGHLY POROUS GENOMES AND HIGH LEVELS OF GENE FLOW BETWEEN DIVERGENT HELIANTHUS SPECIES

While speciation can be found in the presence of gene flow, it is not clear what impact this gene flow has on genome- and range-wide patterns of differentiation. Here we examine gene flow across the entire range of the common sunflower, H. annuus , its historically allopatric sister species H. argophyllus and a more distantly related, sympatric relative H. petiolaris . Analysis of genotypes at 26 microsatellite loci in 1015 individuals from across the range of the three species showed substantial introgression between geographically proximal populations of H. annuus and H. petiolaris , limited introgression between H. annuus and H. argophyllus , and essentially no gene flow between the allopatric pair, H. argophyllus and H. petiolaris. Analysis of sequence divergence levels among the three species in 1420 orthologs identified from EST databases identified a subset of loci showing extremely low divergence between H. annuus and H. petiolaris and extremely high divergence between the sister species H. annuus and H. argophyllus , consistent with introgression between H. annuus and H. petiolaris at these loci. Thus, at many loci, the allopatric sister species are more genetically divergent than the more distantly related sympatric species, which have exchanged genes across much of the genome while remaining morphologically and ecologically distinct.     

TESTING THE LINK BETWEEN POPULATION GENETIC DIFFERENTIATION AND CLADE DIVERSIFICATION IN COSTA RICAN ORCHIDS

Species population genetics could be an important factor explaining variation in clade species richness. Here, we use newly generated amplified fragment length polymorphism (AFLP) data to test whether five pairs of sister clades of Costa Rican orchids that differ greatly in species richness also differ in average neutral genetic differentiation within species, expecting that if the strength of processes promoting differentiation within species is phylogenetically heritable, then clades with greater genetic differentiation should diversify more. Contrary to expectation, neutral genetic differentiation does not correlate directly with total diversification in the clades studied. Neutral genetic differentiation varies greatly among species and shows no heritability within clades. Half of the variation in neutral genetic differentiation among populations can be explained by ecological variables, and species‐level traits explain the most variation. Unexpectedly, we find no isolation by distance in any species, but genetic differentiation is greater between populations occupying different niches. This pattern corresponds with those observed for microscopic eukaryotes and could reflect effective widespread dispersal of tiny and numerous orchid seeds. Although not providing a definitive answer to whether population genetics processes affect clade diversification, this work highlights the potential for addressing new macroevolutionary questions using a comparative population genetic approach.     

Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.     

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.     

Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.Human cytomegalovirus (HCMV) infects many different cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, smooth muscle cells, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (reviewed in references 5, 30, 38, and 45). In the laboratory, HCMV is normally propagated in primary human fibroblasts because most other cell types yield low titers of virus. Commonly studied laboratory strains, such as AD169, were propagated extensively in fibroblasts, and this was accompanied by deletions or mutations in a cluster of 22 genes known as ULb′ (6). These mutations were correlated with the inability to infect other cell types, including endothelial and epithelial cells and monocyte-macrophages. Targeted mutagenesis of three of the ULb′ genes, UL128, UL130, and UL131, abolished infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (13, 15). Restoration of the UL128-131 genes in laboratory strains of HCMV strains restored the capacity to infect endothelial and epithelial cells and other cells (15, 52).The UL128, UL130, and UL131 proteins assemble onto the extracellular domain of HCMV gH/gL (1, 42, 53). For all herpesviruses, gH/gL complexes mediate entry into cells (12, 33, 39), suggesting that gH/gL/UL128-131 might participate in the entry mechanism. Indeed, we demonstrated that gH/gL/UL128-131 mediates entry into epithelial and endothelial cells by using the fusogenic agent polyethylene glycol to force entry of HCMV UL128-131 mutants into these cell types (41). This was consistent with reports that UL128-, UL130-, and UL131-specific antibodies blocked the capacity of HCMV to infect epithelial and endothelial cells but not fibroblasts (1, 53). Furthermore, expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV infection, consistent with saturable gH/gL/UL128-131 receptors (40). Expression of all five proteins was necessary so that the gH/gL/UL128-131 complexes were exported from the endoplasmic reticulum (ER) and could function (40-42, 53). Together, these data suggested that gH/gL/UL128-131 mediates entry into epithelial/endothelial cells but is not required for entry into fibroblasts. By extension, it was reasonable to propose that other forms of gH/gL might facilitate the entry into fibroblasts.The laboratory HCMV strain AD169 is known to express a second gH/gL complex containing glycoprotein O (gO) (21-23, 53). In cells infected with a recombinant AD169 in which the UL131 mutation was repaired, gH/gL/gO complexes were separate from gH/gL/UL128-131 complexes, i.e., gO was not detected following immunoprecipitation (IP) with UL128- and UL130-specifc antibodies, and gO-specific antibodies did not precipitate UL128 and UL130 (53). AD169 and Towne gO⁻ mutants produce small plaques on fibroblast monolayers and low titers of virus, supporting an important, although not essential, role for gH/gL/gO in virus replication in fibroblasts (11, 19). AD169 does not infect endothelial and epithelial cells, so AD169 gO⁻ mutants were not tested on these cells. Jiang et al. described a gO-null mutant derived from an endotheliotropic HCMV strain, TB40/E (27). The TB40/E gO-null mutant spread normally on endothelial cells, suggesting that gO or gH/gL/gO is less important for infection and spread in these cells. Given that the role of gH/gL in entry is highly conserved among the herpesviruses, it seemed likely that gH/gL/gO might be involved in entry into fibroblasts. Consistent with this notion, Paterson et al. showed that anti-gO antibodies decreased fusion from without caused by infection of cells with HCMV AD169 (37). These observations supported our working model in which gH/gL/UL128-131 mediates entry into epithelial and endothelial cells, while gH/gL/gO mediates entry into fibroblasts. There is also the possibility that gH/gL (lacking gO and UL128-131) might be incorporated into the virion envelope, although there is presently no direct evidence for this. Any gH/gL detected biochemically might result from dissociation of gO or UL128-131 during sample preparation and analysis. gH/gL expressed without other HCMV proteins was retained in the ER (42), arguing against incorporation into the virion.Other herpesviruses, e.g., Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-7, use different forms of gH/gL to enter different cell types via different pathways (25, 34, 43). Similarly, HCMV entry into fibroblasts occurs by fusion at the plasma membrane at a neutral pH and does not require gH/gL/UL128-131 (7), whereas entry into epithelial and endothelial cells involves endocytosis and low pH-dependent fusion and requires gH/gL/UL128-131 (41).All of the biochemical analyses of gO in terms of binding to gH/gL and intracellular transport have involved fibroblast-adapted strain AD169 (21-23, 31, 53). These studies indicated that gO is a 110- to 125-kDa glycoprotein encoded by the UL74 gene (22). Glycosidase digestion experiments demonstrated that the gO polypeptide chain is ∼62 to 65 kDa (21-23, 53). Pulse-chase studies showed that gH/gL assembles in the ER as a disulfide-linked heterodimer (28) that subsequently binds to, and establishes disulfides with, gO (22, 23). The 220-kDa immature gH/gL/gO trimer is initially sensitive to endoglycosidase H (endo H), which removes immature N-linked oligosaccharides from glycoproteins present in the ER (22, 23). Transport of gH/gL/gO to the Golgi apparatus is associated with processing of N-linked oligosaccharides to mature forms that resist endo H. Also associated with transport to the Golgi apparatus is the addition of O-linked oligosaccharides and phosphorylation, increasing the molecular weight of gO (after reduction) to 125 to 130 kDa and that of the gH/gL/gO complex to 240 to 260 kDa (22, 23, 29). It is the mature glycoprotein complex, previously known as gCIII, that is trafficked to HCMV assembly compartments for incorporation into the virion envelope (22, 23, 29).In addressing the function of gO, it is important to recognize that AD169 has adapted to replication in fibroblasts, losing expression of UL131 and failing to assemble gH/gL/UL128-131 complexes (6) (15). There seems to be strong pressure to mutate UL128-131, because clinical strain Merlin acquired a UL128 mutation within 5 passages on fibroblasts (2). It is also reasonable to suggest that fibroblast adaptation includes changes in gO. The gO genes (UL74) of several laboratory and clinical strains and clinical isolates are highly variable (up to 25% of amino acids) (10, 35, 37, 47). However, it is important to note that AD169-derived UL131-repair virus can infect epithelial and endothelial cells (52). Thus, if AD169 gO is important for infection of these cells, then gO must be functionally normal in this regard. These differences in laboratory versus clinical HCMV prompted us to characterize the gO molecule expressed by the HCMV strain TR. HCMV TR is a clinical isolate that was stabilized in the form of a bacterial artificial chromosome (BAC) after very limited passage in fibroblasts (35, 41). HCMV TR expresses gH/gL/UL128-131 (42) and infects epithelial and endothelial cells (41) and monocyte-macrophages well (D. Streblow and J. Nelson, unpublished results).Here, we report our biochemical and cell trafficking analyses of the TR gO protein. We were surprised to find that TR gO was not present in extracellular virus particles. In contrast, gO was detected in extracellular AD169 particles, consistent with previous findings (22). TR gO expressed either in HCMV-infected cells or by using nonreplicating Ad vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of TR gO with gH/gL promoted transport of gH/gL beyond the ER, and gO was slowly lost from gH/gL complexes but not secreted from cells and not observed in extracellular virus particles. Thus, TR gO acts as a chaperone. Consistent with this, in the accompanying paper by Wille et al. (54), a TR gO-null mutant was described that secreted extracellular particles containing markedly reduced quantities of gH and gL. The gO⁻ mutant failed to enter fibroblasts and also epithelial and endothelial cells. Together, these results suggest that it is gH/gL, not gH/gL/gO, which is incorporated into HCMV TR virions. It appears that gH/gL is required for entry into fibroblasts, and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

Orthoptera, a new order of pollinator

Background and Aims

Pollinator-mediated selection and evolution of floral traits have long fascinated evolutionary ecologists. No other plant family shows as wide a range of pollinator-linked floral forms as Orchidaceae. In spite of the large size of this model family and a long history of orchid pollination biology, the identity and specificity of most orchid pollinators remains inadequately studied, especially in the tropics where the family has undergone extensive diversification. Angraecum (Vandeae, Epidendroideae), a large genus of tropical Old World orchids renowned for their floral morphology specialized for hawkmoth pollination, has been a model system since the time of Darwin.

Methods

The pollination biology of A. cadetii, an endemic species of the islands of Mauritius and Reunion (Mascarene Islands, Indian Ocean) displaying atypical flowers for the genus (white and medium-size, but short-spurred) was investigated. Natural pollinators were observed by means of hard-disk camcorders. Pollinator-linked floral traits, namely spur length, nectar volume and concentration and scent production were also investigated. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified in natural field conditions weekly during the 2003, 2004 and 2005 flowering seasons (January to March).

Key Results

Angraecum cadetii is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator species was observed, an undescribed species of raspy cricket (Gryllacrididae, Orthoptera). These crickets, which are nocturnal foragers, reached flowers by climbing up leaves of the orchid or jumping across from neighbouring plants and probed the most ‘fresh-looking’ flowers on each plant. Visits to flowers were relatively long (if compared with the behaviour of birds or hawkmoths), averaging 16·5 s with a maximum of 41·0 s. At the study site of La Plaine des Palmistes (Pandanus forest), 46·5 % of flowers had pollen removed and 27·5 % had pollinia deposited on stigmas. The proportion of flowers that set fruit ranged from 11·9 % to 43·4 %, depending of the sites sampled across the island.

Conclusions

Although orthopterans are well known for herbivory, this represents the first clearly supported case of orthopteran-mediated pollination in flowering plants.