Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.     

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK₂) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK₂ complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V_max of 1.25 μmol P_i/min/mg and a K_m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA^Glc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.     

Molecular Identification and Hidden Diversity of Novel Daphnia Parasites from European Lakes

Parasites play important roles in local population dynamics and genetic structure. However, due to insufficient diagnostic tools, detailed host-parasite interactions may remain concealed by hidden parasite diversity in natural systems. Microscopic examination of 19 European lake Daphnia populations revealed the presence of three groups of parasites: fungi, microsporidia, and oomycetes. For most of these parasites no genetic markers have been described so far. Based on sequence similarities of the nuclear small-subunit and internal transcribed spacer (ITS) rRNA gene regions, one fungus, four microsporidian, and nine oomycete taxa were discovered in 147 infected Daphnia (and/or three other zooplankton crustaceans). Additionally, cloning of rRNA gene regions revealed parasite sequence variation within host individuals. This was most pronounced in the ITS region of one microsporidian taxon, where the within-host sequence variation ranged from 1.7% to 5.3% polymorphic sites for parasite isolates from 14 different geographical locations. Interestingly, the parasite isolates from close locations grouped together based on sequence similarities, suggesting that there was parasite dispersal. Taken together, the data obtained in this study revealed hidden diversity of parasite communities in Daphnia lake populations. Moreover, a higher level of resolution for identifying parasite strains makes it possible to test new hypotheses with respect to parasite dispersal, transmission routes, and coinfection.During the last decade, microparasites of Daphnia species, which are small zooplankton crustaceans, have become a popular study system in ecological and evolutionary research (for a review, see reference 15). It has been shown both in the field and under controlled laboratory conditions that parasites have a substantial impact on Daphnia fitness (7, 21, 52). Parasite-induced reductions in Daphnia population density (11, 12) or even population crashes (17) might result in disruptions of aquatic food webs, as daphnids play important roles as main phytoplankton grazers and as a major food of planktivorous fish (27). Moreover, as infections are often genotype specific (6, 8), they can lead to changes in the gene pool of a Daphnia population (7, 14), sometimes significantly increasing the genetic diversity of the host population (12, 54). Thus, Daphnia parasites cause not only ecological but also evolutionary changes in aquatic systems.Conclusions regarding the importance of parasites in natural systems require powerful tools to detect and properly identify parasite taxa. Thus far, few species-specific molecular markers have been developed for Daphnia parasites (33, 38, 39, 41) and then used in experimental studies (3). In surveys of natural Daphnia populations, parasite identification has been based primarily on microscopic examination (4, 5, 29, 52), with only one exception (32). The parasites recorded in natural populations of Daphnia are thus considered members of certain taxa, or even species, without genetic confirmation. The fact that molecular markers are not used to characterize Daphnia infections makes it difficult to compare epidemic patterns across different habitats and/or various field surveys, as parasites cannot be unambiguously identified by microscopic examination alone. Even if microscopic identification is theoretically possible (for example, by examining ultrastructural morphology by electron microscopy [37]), this approach is not feasible for routine analysis. Consequently, classification of parasites that actually belong to different taxa in the same group might introduce noise into field surveys, as parasite taxa differ widely in virulence and host range (for a review, see reference 15).Most of the known Daphnia parasites that have been described were obtained from small temporary ponds and rock pools (4, 16, 43). In permanent lakes, lower parasite diversity was assumed, mainly because increased fish predation reduces the population density of potential hosts (18), whereas high host density is a crucial determinant of epidemic spread (1, 2, 45). In addition, infected Daphnia spp. are more vulnerable to fish that hunt visually due to loss of their transparent appearance (11, 13). On the other hand, it was recently shown that even if Daphnia host density was reduced by selective fish predation, the prevalence of infection did not decline, probably due to the very high rates of transmission of the parasite that was observed (11). In contrast, we expected that the highly heterogeneous biotic and abiotic conditions in permanent lakes (27) would provide a variety of niches (for a review, see reference 47), which also favor a high level of parasite diversity. Therefore, Czech canyon-shaped reservoirs were chosen as our main study systems, because in these lakes environmental gradients are particularly pronounced in both the horizontal and vertical dimensions (42). Moreover, the Daphnia communities of these reservoirs are dominated by members of the Daphnia longispina complex (35), taxa which have previously been shown to be infected by a variety of parasites (52).The results of our study revealed a high level of diversity of Daphnia parasites in permanent lakes. Fourteen different parasite taxa were detected using nuclear small-subunit (SSU) and internal transcribed spacer (ITS) rRNA gene sequence information. In addition, a high level of sequence variation was observed in the ITS region of one microsporidian taxon. Thus, molecular markers are now available which allow discrimination with high resolution among and within parasite taxa and provide tools to address more detailed questions concerning lake Daphnia-microparasite systems.     

Magnetic Alignment in Carps: Evidence from the Czech Christmas Fish Market

While magnetoreception in birds has been studied intensively, the literature on magnetoreception in bony fish, and particularly in non-migratory fish, is quite scarce. We examined alignment of common carps (Cyprinus carpio) at traditional Christmas sale in the Czech Republic. The sample comprised measurements of the directional bearings in 14,537 individual fish, distributed among 80 large circular plastic tubs, at 25 localities in the Czech Republic, during 817 sampling sessions, on seven subsequent days in December 2011. We found that carps displayed a statistically highly significant spontaneous preference to align their bodies along the North-South axis. In the absence of any other common orientation cues which could explain this directional preference, we attribute the alignment of the fish to the geomagnetic field lines. It is apparent that the display of magnetic alignment is a simple experimental paradigm of great heuristic potential.     

Preparative use of isolated CYP102 monooxygenases -- a critical appraisal

Isolated P450 monooxygenases have for long been neglected catalysts in enzyme technology. This is surprising as they display a remarkable substrate specificity catalyzing reactions, which represent a challenge for classic organic chemistry. On the other hand, many P450 monooxygenases are membrane bound, depend on rather complicated electron transfer systems and require expensive cofactors such as NAD(P)H. Their activities are low, and stability leaves much to be desired. The use of bacterial P450 monooxygenases from CYP102 family allows overcoming some of these handicaps. They are soluble and their turnovers are high, presumably because their N-terminal heme monooxygenase and their C-terminal diflavin reductase domain are covalently linked. In recent years, protein engineering approaches have been successfully used to turn CYP102 monooxgenases into powerful biocatalysts.     

Rule-based modeling and simulations of the inner kinetochore structure

Background

Combinatorial complexity is a central problem when modeling biochemical reaction networks, since the association of a few components can give rise to a large variation of protein complexes. Available classical modeling approaches are often insufficient for the analysis of very large and complex networks in detail. Recently, we developed a new rule-based modeling approach that facilitates the analysis of spatial and combinatorially complex problems. Here, we explore for the first time how this approach can be applied to a specific biological system, the human kinetochore, which is a multi-protein complex involving over 100 proteins.

Results

Applying our freely available SRSim software to a large data set on kinetochore proteins in human cells, we construct a spatial rule-based simulation model of the human inner kinetochore. The model generates an estimation of the probability distribution of the inner kinetochore 3D architecture and we show how to analyze this distribution using information theory. In our model, the formation of a bridge between CenpA and an H3 containing nucleosome only occurs efficiently for higher protein concentration realized during S-phase but may be not in G1. Above a certain nucleosome distance the protein bridge barely formed pointing towards the importance of chromatin structure for kinetochore complex formation. We define a metric for the distance between structures that allow us to identify structural clusters. Using this modeling technique, we explore different hypothetical chromatin layouts.

Conclusions

Applying a rule-based network analysis to the spatial kinetochore complex geometry allowed us to integrate experimental data on kinetochore proteins, suggesting a 3D model of the human inner kinetochore architecture that is governed by a combinatorial algebraic reaction network. This reaction network can serve as bridge between multiple scales of modeling. Our approach can be applied to other systems beyond kinetochores.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.