Recombinant and wild-type Pseudomonas aureofaciens strains in soil: survival, respiratory activity and effects on nodulation of whitebean Phaseolus vulgaris L. by Rhizobiutn species

Survival and respiratory activity of a genetically engineered Pseudomonas aureofaciens Ps3732RNL11 were compared to the parental wild-type P. aureofaciens Ps3732RN in loam and sandy loam soils over 17- and 28-day periods. Survival and respiratory activity of P. aureofaciens Ps3732RNL11 was not statistically significantly different from that of P. aureofaciens Ps3732RN. Soil texture had an effect on respiratory activity; carbon dioxide evolution was significantly higher in the sandy loam soil. This effect was observed on days 2, 10 and 18 but not on day 24. The presence of P. aureofaciens Ps3732RNL11 and Ps3732RN did not significantly affect growth of whitebean ( Phaseolus vulgaris L.) in vermiculite, loam, or sandy loam soils. There was no significant difference (95% level) in numbers of nodules produced in the presence of P. aureofaciens Ps3732RNL11 and Ps3732RN as a result of the symbiotic relationship between Rhizobium phaseoli and the whitebean roots in vermiculite. Enumeration of nodules on whitebean roots in loam and sandy loam soils was not conducted due to difficulties in removing intact roots from the soils.     

Distribution and complementarity of hydropathy in multisubunit proteins

A survey of 40 multisubunit proteins and 2 protein-protein complexes was performed to assay quantitatively the distribution of hydropathy among the exterior surface, interior, contact surface, and noncontact exterior surface of the isolated subunits. We suggest a useful way to present this distribution by using a "hydropathy level diagram." Additionally, we have devised a function called "hydropathy complementarity" to quantitate the degree to which interacting surfaces have matching hydropathy distributions. Our survey revealed the following patterns: (1) The difference in hydropathy between the interior and exterior of subunits is a fairly invariant quantity. (2) On average, the hydropathy of the contact surface is higher than that of the exterior surface, but is not greater than that of the protein as a whole. There was variation, however, among the proteins. In some instances, the contact surface was more hydrophilic than the noncontact exterior, and in a few cases the contact surface was as hydrophobic as the protein interior. (3) The average interface manifests significant hydropathy complementarity, signifying that proteins interact by placing hydrophobic centers of one surface against hydrophobic centers of the other surface, and by similarly matching hydrophilic centers. As a measure of recognition and specificity, hydropathy complementarity could be a useful tool for predicting correct docking of interacting proteins. We suggest that high hydropathy complementarity is associated with static inflexible interactions. (4) We have found that some subunits that bind predominantly through hydrophilic forces, such as hydrogen bonds, ionic pairs, and water and metal bridges, are involved in dynamic quaternary organization and allostery.     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Plant-attracted ants affect arthropod community structure but not necessarily herbivory

Abstract.  1. The effectiveness of ants as plant defenders is equivocal for plants that attract ants via extrafloral nectaries (EFNs).
2. This study focused on the myrmecophilic savannah tree Pseudocedrela kotschyi that attracts ants to EFNs and on the arthropod fauna associated with P. kotschyi . Herbivory and arthropod community composition were compared between trees that were dominated by one of three congeneric ant species, Camponotus acvapimensis , C. rufoglaucus , and C. sericeus , and between trees where ants were experimentally excluded and untreated control trees.
3. Short-term ant-exclusion experiments failed to demonstrate a consistent effect of ants on herbivory.
4. Plants dominated by different ant species differed significantly in leaf damage caused by herbivorous insects. The relative ranking of herbivory levels of the trees dominated by different ant species was persistent in three consecutive years.
5. Ants significantly reduced the abundance of different arthropod groups (Araneae, Blattodea, Coleoptera, Homoptera, non-ant Hymenoptera). Other groups, including important herbivores, seemed not to be affected (Lepidoptera, Orthoptera, Thysanoptera, Heteroptera).
6. The study suggests that the presence of ants only benefits plants when specific ant species are attracted, and protection by these ants is not counterbalanced by their negative effect on other beneficial arthropods.     

Searching behaviour of an omnivorous predator for novel and native host plants of its herbivores: a study on arthropod colonization of eucalyptus in Brazil

Adaptation to novel host plants is a much‐studied process in arthropod herbivores, but not in their predators. This is surprising, considering the attention that has been given to the role of predators in host range expansion in herbivores; the enemy‐free space hypothesis suggests that plants may be included in the host range of herbivores because of lower predation and parasitism rates on the novel host plants. This effect can only be important if natural enemies do not follow their prey to the novel host plant, at least not immediately, thus allowing the herbivores to adapt to the novel host plant. Hence, depending on the speed with which natural enemies follow their prey to a new host plant, enemy‐free space on novel host plants may only exist for a limited period. This situation may presently be occurring in a system consisting of the herbivorous moth Thyrinteina arnobia Stoll (Lepidoptera: Geometridae) that attacks various species of Myrtaceae, such as guava (Psidium guajava L.) and jaboticaba (Myrciaria spp.), in Brazil. Since the introduction of eucalyptus (Myrtaceae) species into this country some 100 years ago, the moth has included this plant species in its host range and frequently causes outbreaks, a phenomenon that does not occur on the native host plant species. This suggests that the natural enemies that attack the herbivore on native species are not very effective on the novel host. We tested this hypothesis by studying the searching behaviour of one of the natural enemies, the omnivorous predatory bug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae). When offered a choice between plants of the two species, the predators (originally collected in eucalyptus plantations) preferred guava to eucalyptus when both plant species were clean, infested with herbivores, or damaged by herbivores but with herbivores removed prior to the experiments. The bugs preferred herbivore‐damaged to clean guava, and showed a slight preference for damaged to clean eucalyptus. These results may explain the lack of impact of predatory arthropods on herbivore populations on eucalyptus and suggests that eucalyptus may offer an enemy‐free space for herbivores.     

All-atom folding of the three-helix HIV accessory protein with an adaptive parallel tempering method

All-atom protein structure prediction from the amino acid sequence alone remains an important goal of biophysical chemistry. Recent progress in force field development and validation suggests that the PFF01 free-energy force field correctly predicts the native conformation of various helical proteins as the global optimum of its free-energy surface. Reproducible protein structure prediction requires the availability of efficient optimization methods to locate the global minima of such complex potentials. Here we investigate an adapted version of the parallel tempering method as an efficient parallel stochastic optimization method for protein structure prediction. Using this approach we report the reproducible all-atom folding of the three-helix 40 amino acid HIV accessory protein from random conformations to within 2.4 A backbone RMS deviation from the experimental structure with modest computational resources.     

Review of the effects of within-patch scale structural complexity on seagrass fishes

Studies on the effects of within-patch scale structure of seagrass habitats on predator–prey fish interactions and abundance/habitat use patterns were reviewed. Most laboratory experiments have employed chase-and-attack predators, usually resulting in lower foraging efficiency in (denser) seagrass. However, a few laboratory procedures employed alternative foraging tactics, resulting in no differences in prey mortality rates. Field studies did not always result in lower prey mortality rates in seagrass habitats. Accordingly, it is premature to conclude that seagrass presence is almost always negatively related to predator foraging efficiency or that increasing seagrass abundance is usually associated with a decrease in predator efficiency. Because several categories of predator and prey fishes occur in seagrass habitats, further studies are needed with all of these predator–prey combinations, in order to fully clarify predator–prey fish interactions in association with seagrass structure. Seagrass fishes have been shown to respond to alterations in seagrass structure in various ways: seagrass height and/or density reduction or clearance resulted in decreased abundance of some species but increases or no change in others. Some explanations have been proposed, not all mutually exclusive, for these phenomena. Although within-patch scale processes have been well studied, room exists for improvement. For example, predator–prey fish interactions in relation to varying within-patch scale complexity is not yet fully understand. The relationships of patch size, edge effects and within-patch scale complexity also still remain unclear. Further studies, which add to the clarification of within-patch scale process, will in turn improve our understanding of larger spatial scale processes.     

Additive and interactive effects of plant genotypic diversity on arthropod communities and plant fitness

Recent research suggests that genetic diversity in plant populations can shape the diversity and abundance of consumer communities. We tested this hypothesis in a field experiment by manipulating patches of Evening Primrose ( Oenothera biennis ) to contain one, four or eight plant genotypes. We then surveyed 92 species of naturally colonizing arthropods. Genetically diverse plant patches had 18% more arthropod species, and a greater abundance of omnivorous and predacious arthropods, but not herbivores, compared with monocultures. The effects of genotypic diversity on arthropod communities were due to a combination of interactive and additive effects among genotypes within genetically diverse patches. Greater genetic diversity also led to a selective feedback, as mean genotype fitness was 27% higher in diverse patches than in monocultures. A comparison between our results and the literature reveals that genetic diversity and species diversity can have similar qualitative and quantitative effects on arthropod communities. Our findings also illustrate the benefit of preserving genetic variation to conserve species diversity and interactions within multitrophic communities.     

Interactions of intermediate filament protein synemin with dystrophin and utrophin

Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (alpha-synemin and beta-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).     

Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.