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Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
Symbiotic nitrogen fixation (SNF) in root nodules of grain legumes such as chickpea is a highly complex process that drastically affects the gene expression patterns of both the prokaryotic as well as eukaryotic interacting cells. A successfully established symbiotic relationship requires mutual signaling mechanisms and a continuous adaptation of the metabolism of the involved cells to varying environmental conditions. Although some of these processes are well understood today many of the molecular mechanisms underlying SNF, especially in chickpea, remain unclear. Here, we reannotated our previously published transcriptome data generated by deepSuperSAGE (Serial Analysis of Gene Expression) to the recently published draft genome of chickpea to assess the root- and nodule-specific transcriptomes of the eukaryotic host cells. The identified gene expression patterns comprise up to 71 significantly differentially expressed genes and the expression of twenty of these was validated by quantitative real-time PCR with the tissues from five independent biological replicates. Many of the differentially expressed transcripts were found to encode proteins implicated in sugar metabolism, antioxidant defense as well as biotic and abiotic stress responses of the host cells, and some of them were already known to contribute to SNF in other legumes. The differentially expressed genes identified in this study represent candidates that can be used for further characterization of the complex molecular mechanisms underlying SNF in chickpea.
Altered microRNA (miRNA) expression is a hallmark of many cancer types. The combined analysis of miRNA and messenger RNA (mRNA) expression profiles is crucial to identifying links between deregulated miRNAs and oncogenic pathways. Therefore, we investigated the small non-coding (snc) transcriptomes of nine clear cell renal cell carcinomas (ccRCCs) and adjacent normal tissues for alterations in miRNA expression using a publicly available small RNA-Sequencing (sRNA-Seq) raw-dataset. We constructed a network of deregulated miRNAs and a set of differentially expressed genes publicly available from an independent study to in silico determine miRNAs that contribute to clear cell renal cell carcinogenesis. From a total of 1,672 sncRNAs, 61 were differentially expressed across all ccRCC tissue samples. Several with known implications in ccRCC development, like the upregulated miR-21-5p, miR-142-5p, as well as the downregulated miR-106a-5p, miR-135a-5p, or miR-206. Additionally, novel promising candidates like miR-3065, which i.a. targets NRP2 and FLT1, were detected in this study. Interaction network analysis revealed pivotal roles for miR-106a-5p, whose loss might contribute to the upregulation of 49 target mRNAs, miR-135a-5p (32 targets), miR-206 (28 targets), miR-363-3p (22 targets), and miR-216b (13 targets). Among these targets are the angiogenesis, metastasis, and motility promoting oncogenes c-MET, VEGFA, NRP2, and FLT1, the latter two coding for VEGFA receptors.
The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.
Noise-induced hearing loss is one of the most common auditory pathologies, resulting from overstimulation of the human cochlea, an exquisitely sensitive micromechanical device. At very low frequencies (less than 250 Hz), however, the sensitivity of human hearing, and therefore the perceived loudness is poor. The perceived loudness is mediated by the inner hair cells of the cochlea which are driven very inadequately at low frequencies. To assess the impact of low-frequency (LF) sound, we exploited a by-product of the active amplification of sound outer hair cells (OHCs) perform, so-called spontaneous otoacoustic emissions. These are faint sounds produced by the inner ear that can be used to detect changes of cochlear physiology. We show that a short exposure to perceptually unobtrusive, LF sounds significantly affects OHCs: a 90 s, 80 dB(A) LF sound induced slow, concordant and positively correlated frequency and level oscillations of spontaneous otoacoustic emissions that lasted for about 2 min after LF sound offset. LF sounds, contrary to their unobtrusive perception, strongly stimulate the human cochlea and affect amplification processes in the most sensitive and important frequency range of human hearing.
Biodiversity is unevenly distributed on Earth and hotspots of biodiversity are often associated with areas that have undergone orogenic activity during recent geological history (i.e. tens of millions of years). Understanding the underlying processes that have driven the accumulation of species in some areas and not in others may help guide prioritization in conservation and may facilitate forecasts on ecosystem services under future climate conditions. Consequently, the study of the origin and evolution of biodiversity in mountain systems has motivated growing scientific interest. Despite an increasing number of studies, the origin and evolution of diversity hotspots associated with the Qinghai-Tibetan Plateau (QTP) remains poorly understood. We review literature related to the diversification of organisms linked to the uplift of the QTP. To promote hypothesis-based research, we provide a geological and palaeoclimatic scenario for the region of the QTP and argue that further studies would benefit from providing a complete set of complementary analyses (molecular dating, biogeographic, and diversification rates analyses) to test for a link between organismic diversification and past geological and climatic changes in this region. In general, we found that the contribution of biological interchange between the QTP and other hotspots of biodiversity has not been sufficiently studied to date. Finally, we suggest that the biological consequences of the uplift of the QTP would be best understood using a meta-analysis approach, encompassing studies on a variety of organisms (plants and animals) from diverse habitats (forests, meadows, rivers), and thermal belts (montane, subalpine, alpine, nival). Since the species diversity in the QTP region is better documented for some organismic groups than for others, we suggest that baseline taxonomic work should be promoted.
The LPJ-GUESS dynamic vegetation model uniquely combines an individual- and patch-based representation of vegetation dynamics with ecosystem biogeochemical cycling from regional to global scales. We present an updated version that includes plant and soil N dynamics, analysing the implications of accounting for C–N interactions on predictions and performance of the model. Stand structural dynamics and allometric scaling of tree growth suggested by global databases of forest stand structure and development were well reproduced by the model in comparison to an earlier multi-model study. Accounting for N cycle dynamics improved the goodness of fit for broadleaved forests. N limitation associated with low N-mineralisation rates reduces productivity of cold-climate and dry-climate ecosystems relative to mesic temperate and tropical ecosystems. In a model experiment emulating free-air CO2 enrichment (FACE) treatment for forests globally, N limitation associated with low N-mineralisation rates of colder soils reduces CO2 enhancement of net primary production (NPP) for boreal forests, while some temperate and tropical forests exhibit increased NPP enhancement. Under a business-as-usual future climate and emissions scenario, ecosystem C storage globally was projected to increase by ca. 10%; additional N requirements to match this increasing ecosystem C were within the high N supply limit estimated on stoichiometric grounds in an earlier study. Our results highlight the importance of accounting for C–N interactions in studies of global terrestrial N cycling, and as a basis for understanding mechanisms on local scales and in different regional contexts.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in Northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still 5 not well explored. By incubating intact soil cores from four sites (3 savanna, 1 pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emission were very low (< 7.0± 5.0 μgNO-Nm−2 h−1; < 0.0± 1.4 μgN2O-Nm−2 h−1) or in case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μgNm−2 h−1) and relatively small N2O pulse emissions (max: 5.8±5.0 μgNm−2 h−1) were recorded following soil wetting, but these pulses were short-lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% at 50% SM and 30 °C ST. N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. Emissions were controlled by SM and ST for N2O and CO2, ST and pH for NO, and SM and pH for N2.
Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still not well explored. By incubating intact soil cores from four sites (three savanna, one pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emissions were very low (<7.0 ± 5.0 μg NO-N m−2 h−1; <0.0 ± 1.4 μg N2O-N m−2 h−1) or in the case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μg N m−2 h−1) and relatively small N2O pulse emissions (max: 5.8 ± 5.0 μg N m−2 h−1) were recorded following soil wetting, but these pulses were short lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was generally dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% to the total atmospheric nitrogen loss at 50% SM and 30 °C ST incubation settings (the contribution of N2 at these soil conditions was only 53.2%). N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. By using a conservative upscale approach we estimate total annual emissions from savanna soils to average 0.12 kg N ha−1 yr−1 (N2O), 0.68 kg N ha−1 yr−1 (NO) and 6.65 kg N ha−1 yr−1 (N2). The analysis of long-term SM and ST records makes it clear that extreme soil saturation that can lead to high N2O and N2 emissions only occurs a few days per year and thus has little impact on the annual total. The potential contribution of nitrogen released due to pulse events compared to the total annual emissions was found to be of importance for NO emissions (contribution to total: 5–22%), but not for N2O emissions. Our results indicate that the total gaseous release of nitrogen from these soils is low and clearly dominated by loss in the form of inert nitrogen. Effects of seasonally varying soil temperature and moisture were detected, but were found to be low due to the small amounts of available nitrogen in the soils (total nitrogen <0.1%).
In old and heavily weathered soils, the availability of P might be so small that the primary production of plants is limited. However, plants have evolved several mechanisms to actively take up P from the soil or mine it to overcome this limitation. These mechanisms involve the active uptake of P mediated by mycorrhiza, biotic de-occlusion through root clusters, and the biotic enhancement of weathering through root exudation. The objective of this paper is to investigate how and where these processes contribute to alleviate P limitation on primary productivity. To do so, we propose a process-based model accounting for the major processes of the carbon, water, and P cycles including chemical weathering at the global scale. Implementing P limitation on biomass synthesis allows the assessment of the efficiencies of biomass production across different ecosystems. We use simulation experiments to assess the relative importance of the different uptake mechanisms to alleviate P limitation on biomass production. We find that active P uptake is an essential mechanism for sustaining P availability on long timescales, whereas biotic de-occlusion might serve as a buffer on timescales shorter than 10 000 yr. Although active P uptake is essential for reducing P losses by leaching, humid lowland soils reach P limitation after around 100 000 yr of soil evolution. Given the generalized modelling framework, our model results compare reasonably with observed or independently estimated patterns and ranges of P concentrations in soils and vegetation. Furthermore, our simulations suggest that P limitation might be an important driver of biomass production efficiency (the fraction of the gross primary productivity used for biomass growth), and that vegetation on old soils has a smaller biomass production rate when P becomes limiting. With this study, we provide a theoretical basis for investigating the responses of terrestrial ecosystems to P availability linking geological and ecological timescales under different environmental settings.
Cryptochrome 1a, located in the UV/violet-sensitive cones in the avian retina, is discussed as receptor molecule for the magnetic compass of birds. Our previous immunohistochemical studies of chicken retinae with an antiserum that labelled only activated cryptochrome 1a had shown activation of cryptochrome 1a under 373 nm UV, 424 nm blue, 502 nm turquoise and 565 nm green light. Green light, however, does not allow the first step of photoreduction of oxidized cryptochromes to the semiquinone. As the chickens had been kept under ‘white’ light before, we suggested that there was a supply of the semiquinone present at the beginning of the exposure to green light, which could be further reduced and then re-oxidized. To test this hypothesis, we exposed chickens to various wavelengths (1) for 30 min after being kept in daylight, (2) for 30 min after a 30 min pre-exposure to total darkness, and (3) for 1 h after being kept in daylight. In the first case, we found activated cryptochrome 1a under UV, blue, turquoise and green light; in the second two cases we found activated cryptochrome 1a only under UV to turquoise light, where the complete redox cycle of cryptochrome can run, but not under green light. This observation is in agreement with the hypothesis that activated cryptochrome 1a is found as long as there is some of the semiquinone left, but not when the supply is depleted. It supports the idea that the crucial radical pair for magnetoreception is generated during re-oxidation.
Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.
Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.
Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1-3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.
The knowledge of phenotypic variation in the European range of the highly allergenic Ambrosia artemisiifolia L. (common ragweed) is not entirely complete, even though it is an invasive species of utmost concern. We hypothesized the prevalence of phenotypic differentiations between common ragweed populations in the introduced range, and we assumed that those differentiations were related to environmental conditions at the points of origin. Using a common garden experiment, we investigated biomass allocation, growth rates, and flowering phenology of 38 European common ragweed populations originating from a major geographical gradient. We observed considerable phenotypic variation in growth parameters and flowering phenology, e.g. mean aboveground biomass varied from 23.3 to 47.3 g between the populations. We were able to relate most measured traits with environmental parameters prevailing at the points of origin. For example, early growth of ruderal populations was highly correlated with temperature and precipitation at the point of origin. Late growth and flowering phenology were highly correlated with latitude, i.e. individuals from northern populations grew smaller and flowered and dispersed their pollen and seeds up to 5 weeks earlier than individuals from southern populations. We also found a longitudinal gradient in flowering phenology which has not yet been described. The existence of such a high variability in the introduced range may facilitate further range expansion. We suggest that the correlation with environmental variables rests upon genetic variation possibly due to adaptations to the respective environment. To clarify if such adaptation results from multiple events of introduction or as evolutionary response after introduction, genetic investigations are needed.
We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology.
Bacteria communicate via small diffusible molecules to mediate group-coordinated behavior, a process designated as quorum sensing. The basic molecular quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase producing acyl-homoserine lactones (AHLs) as signaling molecules, and a LuxR-type receptor detecting the AHLs to control expression of specific genes. However, many proteobacteria possess one or more unpaired LuxR-type receptors that lack a cognate LuxI-like synthase, referred to as LuxR solos. The enteric and insect pathogenic bacteria of the genus Photorhabdus harbor an extraordinarily high number of LuxR solos, more than any other known bacteria, and all lack a LuxI-like synthase. Here, we focus on the presence and the different types of LuxR solos in the three known Photorhabdus species using bioinformatics analyses. Generally, the N-terminal signal-binding domain (SBD) of LuxR-type receptors sensing AHLs have a motif of six conserved amino acids that is important for binding and specificity of the signaling molecule. However, this motif is altered in the majority of the Photorhabdus-specific LuxR solos, suggesting the use of other signaling molecules than AHLs. Furthermore, all Photorhabdus species contain at least one LuxR solo with an intact AHL-binding motif, which might allow the ability to sense AHLs of other bacteria. Moreover, all three species have high AHL-degrading activity caused by the presence of different AHL-lactonases and AHL-acylases, revealing a high quorum quenching activity against other bacteria. However, the majority of the other LuxR solos in Photorhabdus have a N-terminal so-called PAS4-domain instead of an AHL-binding domain, containing different amino acid motifs than the AHL-sensors, which potentially allows the recognition of a highly variable range of signaling molecules that can be sensed apart from AHLs. These PAS4-LuxR solos are proposed to be involved in host sensing, and therefore in inter-kingdom signaling. Overall, Photorhabdus species are perfect model organisms to study bacterial communication via LuxR solos and their role for a symbiotic and pathogenic life style.