Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Fine root distribution of trees and understory in mature stands of maritime pine (Pinus pinaster) on dry and humid sites

Maritime pine (Pinus pinaster) is the main tree cropping species in the Landes of Gascogne forest range in south western France. Soils are nutrient poor, sandy podzosols and site fertility is determined essentially by organic matter content and depth of water table, which is known to limit root growth. We hypothesised, with an ultimate goal of constructing a nutrient uptake model applicable to this region, that the organic top horizons together with the depth of the water table should be the most important parameters related to fine root distribution and presence of associated mycorrhiza. To test this hypothesis, we compared two adult Pinus pinaster stands, contrasting in depth of water table and soil fertility and evaluated fine roots (diameter ≤2 mm) of understory species and fine roots and ectomycorrhizal morphotypes of Pinus pinaster down to 1.2 m, using a soil corer approach. Total fine root biomass of Pinus pinaster was not significantly different between both sites (3.6 and 4.5 t ha⁻¹ for the humid, respectively, dry site), but root distribution was significantly shallower and root diameter increased more with depth at the humid site, presumably due to more adverse soil conditions as related to the presence of a hardpan, higher amount of aluminium oxides and / or anoxia. Fine roots of Pinus pinaster represented only about 30% of total fine root biomass and 15% of total fine root length, suggesting that the understory species cannot be ignored with regards to competition for mineral nutrients and water. A comparison of the ectomycorrhizal morphotypes showed that the humid site could be characterised by a very large proportion of contact exploration types, thought to be more relevant in accessing organic nutrient sources, whereas the dry site had a significantly higher proportion of both long-distance and short-distance exploration types, the latter of which was thought to be more resistant to short-term drought periods. These results partly confirm our hypothesis on root distribution as related to the presence of soil mineral nutrients (i.e. in organic matter), point out the potential role of understory plant species and ectomycorrhizal symbiosis and are a valuable step in building a site-specific nutrient uptake model.     

Dynamic resource allocation between pre- and postcopulatory episodes of sexual selection determines competitive fertilization success

In polyandrous mating systems, male reproductive success depends on both mate-acquisition traits (precopulatory) and sperm competitive abilities (postcopulatory). Empirical data on the interaction between these traits are inconsistent; revealing positive, negative or no relationships. It is generally expected that the investment in pre- and postcopulatory traits is mediated by environmental conditions. To test how dietary resource availability affects sexual ornamentation, sperm quality and their interrelationship in three-spined sticklebacks (Gasterosteus aculeatus), full-sibling groups were raised under three conditions differing in food quantity and/or quality (i.e. carotenoid content): (i) high-quantity/high-quality, (ii) high-quantity/low-quality or (iii) low-quantity/low-quality. After 1 year of feeding, food-restricted males developed a more intense breeding coloration and faster sperm compared with their well-fed brothers, indicating that they allocated relatively more in pre- and postcopulatory traits. Moreover, they outcompeted their well-fed, carotenoid-supplemented brothers in sperm competition trials with equal numbers of competing sperm, suggesting that food-restricted males maximize their present reproductive success. This may result in reduced future reproductive opportunities as food-restricted males suffered from a higher mortality, had an overall reduced body size, and sperm number available for fertilization. In accordance with theory, a trade-off between the investment in pre- and postcopulatory traits was observed in food-restricted males, whereas well-fed males were able to allocate to both traits resulting in a significantly positive relationship.     

Intra- and interspecific host discrimination in Asobara (Hymenoptera) larval endo-parasitoids of Drosophilidae: Comparison between closely related and less closely related species

Host discrimination studies were conducted with different species of Asobara, which are larval endo-parasitoids of Drosophilidae. Results indicated variable host discrimination which depended on the relatedness of the species. The closely related sibling species Asobara tabida (Nees) and A. rufescens (Foerster) were not only capable of intraspecific discrimination, but also avoided multiparasitism by discriminating between unparasitized host larvae and larvae previously parasitized by females of the other species. This ability to discriminate interspecifically does not seem functional as each species occupies its own microhabitat. As it was shown to be absent in less closely related Asobara species we concluded that interspecific discrimination by A. tabida and A. rufescens was due to their close relationship.     

A Kdp-like,high-affinity,K+-translocating ATPase is expressed during growth of Rhodobacter sphaeroides in low potassium media

Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K⁺ uptake system when grown in media with low K⁺ concentrations. Antibodies againts the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli crossreact with a 70.0 kDa R. sphaeroides protein that was expressed only in cells grown in media with low K⁺ concentrations. In membranes derived from R. sphaeroides cells grown with low K⁺ concentrations (induced cells), a high ATPase activity could be detected when assayed in Tris-HCl pH 8.0 containing 1 mM MgSO₄. This ATPase activity increased upon addition of 1 mM KCl from 166 to 289 mol ATP hydrolysed x min^-1 x g protein^-1 (1.7-fold stimulation). The K⁺-stimulated ATPase activity was inhibited approximately 93% by 0.5 mM vanadate but hardly by N,N-dicyclohexylcarbo-diimide (DCCD). These results indicate that the inducible K⁺-ATPase in R. sphaeroides resembles the Kdp K⁺-translocating ATPase of Escherichia coli. This Kdp-like transport system is also expressed in R. capsulatus and Rhodospirillum rubrum during growth in media with low K⁺ concentrations suggesting a wide distribution of this transport system among phototrophic bacteria.Abbreviations electrical potential difference across the cytoplasmic membrane - pH pH difference across the cytoplasmic membrane - BSA bovine serum albumine - PAGE polyacrylamide gel electrophoresis - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - PMSF phenyl-methyl-sulfonyl fluoride - DCCD N,N-dicyclohexylcarbodiimide - AIB 2--aminoisobutyric acid - TMG methyl--d-thiogalactopyranoside     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

The function of host discrimination and superparasitization in parasitoids

Summary Host discrimination, i.e. the ability to distinguish unparasitized hosts from parasitized ones, and to reject the latter for egg laying is present in many parasitic wasp species. This property is classically considered as an example of contest competition, and is supposed to have a number of functions. However, different species do not react to each other's marks and lay eggs in hosts parasitized by the other species. Apparently the marks used for recognition are specific.Multiparasitization is the best strategy when hosts are scarce and the egg supplies of the parasitoids are not limited. Interspecific host discrimination is not an ESS.Superparasitization within one species would have selective advantage if the number of unparasitized hosts is small and the wasp has a reasonable chance to lay her egg in a host that is not parasitized by herself, and if the chance for her offspring to survive the competitive battle with the first parasitoid larva is not too small. This is shown to be the case.However, marks are not individual and wasps cannot distinguish hosts parasitized by themselves from those parasitized by others. The hypothesis is tested that the egg laying strategy (i.e. the decision to superparasitize) of wasps is dependent on the number of conspecifics that is searching simultaneously for hosts, since this determines the chance that a parasitized host encountered by a wasp is parasitized by herself.It is shown that host discrimination cannot be regarded as a case of contest competition. Other aspects of superparasitization, related to interference and population regulation, sex allocation and encapsulation are briefly discussed.