Charakterisierung des Rho GDI Rdi1 in Saccharomyces cerevisiae

Rho GTPasen besitzen in allen Organismen eine herausragende Rolle bei der Etablierung und Aufrechterhaltung von Zellpolarität. Sie werden u.a. von Rho GDIs (Rho Guaninnukleotid- Dissoziationsinhibitor) reguliert, welche die Fähigkeit besitzen, den hydrophoben Membrananker von Rho GTPasen zu binden. Dadurch können sie Rho Proteine von Membranen extrahieren. In der Bäckerhefe Saccharomyces cerevisiae wird mit Rdi1 ein einziger, für Rho GTPasen spezifischer GDI exprimiert. Das Ziel der Arbeit war die umfassende Charakterisierung von Rdi1 und die Analyse neuer Regulationsmechanismen an denen Rdi1 beteiligt ist. Es zeigte sich zunächst, dass Rdi1 spezifisch an Cdc42, Rho1 und Rho4 binden konnte und diese Rho GTPasen von Membranen extrahierte. Die anderen drei Rho GTPasen Rho2, Rho3 und Rho5 interagierten nicht mit Rdi1. Es wurde deutlich, dass die Bindung von Rdi1 an Cdc42 und Rho1 von der PAK (p21-aktivierten Kinase) Cla4 abhängig war. Da Cla4 ein direktes Effektormolekül von Cdc42 ist, handelte es sich wahrscheinlich um einen positiven Rückkopplungsprozess, der die Rdi1-Cdc42/Rho1 Komplexe reguliert. Für die Regulation von Rho4 konnte ein neuer Mechanismus identifiziert werden. Die Extraktion von Rho4 durch Rdi1 führte hierbei zum selektiven Abbau des Rho Proteins durch das Proteasom und die Vakuole. Der Abbauprozess hing zusätzlich von der GSK-3β Glykogen-Synthase-Kinase Ygk3 ab. Durch Fluoreszenzmikroskopieexperimente wurde deutlich, dass Rho4, wie Cdc42 und Rho1, an Orten polarisierten Wachstums lokalisierte. Dies beinhaltete beispielsweise die Knospenhalsregion und den Ort des zukünftigen Knospenwachstums. Ausgenommen war jedoch die Spitze der Paarungsprojektion. In weiteren Studien wurde unter Zuhilfenahme von verschiedenen Screening-Techniken das Hefegenom hinsichtlich neuer Interaktoren von Rdi1 analysiert. In einem Split-Ubiquitin-Screen konnte, neben verschiedenen anderen putativen Interaktoren, die Exocystkomponente Sec6 als ein mutmaßlicher Interaktor von Rdi1 identifiziert werden. Die Interaktion von Rdi1 mit Sec6 konnte in einem in vitro Bindungsexperiment bestätigt werden. Unabhängig von der Charakterisierung des Rho GDI Rdi1, sollten die in einem Split-Ubiquitin-Screen identifizierten potentiellen Interaktionen der PAK Ste20 mit Proteinen, die eine Rolle bei der Ergosterolsynthese in S. cerevisiae spielen, in in vitro Bindungsexperimenten bestätigt werden. Dabei konnten die Interaktionen von rekombinantem Ste20 mit Erg4 und Cbr1 dargestellt werden. Die Ergebnisse dieser Arbeit tragen zu einem besseren Verständnis der molekularen Grundlagen der Regulation von Rho GTPasen und allgemeinen Zellpolaritätsprozessen bei

Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil.

Summer-growing perennial grasses such as Panicum coloratum L. cv. Bambatsi (Bambatsi panic), Chloris gayana Kunth cv. Katambora (Rhodes grass) and Digitaria eriantha Steud. cv. Premier (Premier digit grass) growing in the poor fertility sandy soils in the Mediterranean regions of southern Australia and western Australia mainly depend upon soil N and biological N inputs through diazotrophic (free living or associative) N fixation. We investigated the community composition and diversity ( nifH -amplicon sequencing), abundance (qPCR) and functional capacity (15N incubation assay) of the endophytic diazotrophic community in the below and above ground plant parts of field grown and unfertilized grasses. Results showed a diverse and abundant diazotrophic community inside plant both above and below-ground and there was a distinct diazotrophic assemblage in the different plant parts in all the three grasses. There was a limited difference in the diversity between leaves, stems and roots except that Panicum grass roots harbored greater species richness. Nitrogen fixation potentials ranged between 0.24 and 5.9 mg N kg-1 day-1 and N fixation capacity was found in both the above and below ground plant parts. Results confirmed previous reports of plant species-based variation and that Alpha-Proteobacteria were the dominant group of nifH -harboring taxa both in the belowground and aboveground parts of the three grass species. Results also showed a well-structured nifH -harboring community in all plant parts, an example for a functional endophytic community. Overall, the variation in the number and identity of module hubs and connectors among the different plant parts suggests that co-occurrence patterns within the nifH-harboring community specific to individual compartments and local environments of the niches within each plant part may dictate the overall composition of diazotrophs within a plant

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation.

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response

A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community

Abstract Background Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research. Results In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community. Conclusions The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases.http://deepblue.lib.umich.edu/bitstream/2027.42/112465/1/40168_2012_Article_9.pd

Microbial functional diversity covaries with permafrost thaw-induced environmental heterogeneity in tundra soil.

Permafrost soil in high latitude tundra is one of the largest terrestrial carbon (C) stocks and is highly sensitive to climate warming. Understanding microbial responses to warming-induced environmental changes is critical to evaluating their influences on soil biogeochemical cycles. In this study, a functional gene array (i.e., geochip 4.2) was used to analyze the functional capacities of soil microbial communities collected from a naturally degrading permafrost region in Central Alaska. Varied thaw history was reported to be the main driver of soil and plant differences across a gradient of minimally, moderately, and extensively thawed sites. Compared with the minimally thawed site, the number of detected functional gene probes across the 15-65 cm depth profile at the moderately and extensively thawed sites decreased by 25% and 5%, while the community functional gene β-diversity increased by 34% and 45%, respectively, revealing decreased functional gene richness but increased community heterogeneity along the thaw progression. Particularly, the moderately thawed site contained microbial communities with the highest abundances of many genes involved in prokaryotic C degradation, ammonification, and nitrification processes, but lower abundances of fungal C decomposition and anaerobic-related genes. Significant correlations were observed between functional gene abundance and vascular plant primary productivity, suggesting that plant growth and species composition could be co-evolving traits together with microbial community composition. Altogether, this study reveals the complex responses of microbial functional potentials to thaw-related soil and plant changes and provides information on potential microbially mediated biogeochemical cycles in tundra ecosystems

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf

Global Change Could Amplify Fire Effects on Soil Greenhouse Gas Emissions

Background: Little is known about the combined impacts of global environmental changes and ecological disturbances on ecosystem functioning, even though such combined impacts might play critical roles in shaping ecosystem processes that can in turn feed back to climate change, such as soil emissions of greenhouse gases.[br/] Methodology/Principal Findings: We took advantage of an accidental, low-severity wildfire that burned part of a long-term global change experiment to investigate the interactive effects of a fire disturbance and increases in CO(2) concentration, precipitation and nitrogen supply on soil nitrous oxide (N(2)O) emissions in a grassland ecosystem. We examined the responses of soil N(2)O emissions, as well as the responses of the two main microbial processes contributing to soil N(2)O production - nitrification and denitrification - and of their main drivers. We show that the fire disturbance greatly increased soil N(2)O emissions over a three-year period, and that elevated CO(2) and enhanced nitrogen supply amplified fire effects on soil N(2)O emissions: emissions increased by a factor of two with fire alone and by a factor of six under the combined influence of fire, elevated CO(2) and nitrogen. We also provide evidence that this response was caused by increased microbial denitrification, resulting from increased soil moisture and soil carbon and nitrogen availability in the burned and fertilized plots. [br/] Conclusions/Significance: Our results indicate that the combined effects of fire and global environmental changes can exceed their effects in isolation, thereby creating unexpected feedbacks to soil greenhouse gas emissions. These findings highlight the need to further explore the impacts of ecological disturbances on ecosystem functioning in the context of global change if we wish to be able to model future soil greenhouse gas emissions with greater confidence