Hexamerization and thermostability emerged very early during geranylgeranylglyceryl phosphate synthase evolution

A large number of archaea live in hyperthermophilic environments. In consequence, their proteins need to adopt to these harsh conditions, including the enzymes that catalyze the synthesis of their membrane ether lipids. The enzyme that catalyzes the formation of the first ether bond in these lipids, geranylgeranylglyceryl phosphate synthase (GGGPS), exists as a hexamer in many hyperthermophilic archaea, and a recent study suggested that hexamerization serves for a fine‐tuning of the flexibility – stability trade‐off under hyperthermophilic conditions. We have recently reconstructed the sequences of ancestral group II GGGPS enzymes and now present a detailed biochemical characterization of nine of these predecessors, which allowed us to trace back the evolution of hexameric GGGPS and to draw conclusions about the properties of extant GGGPS branches that were not accessible to experiments up to now. Almost all ancestral GGGPS proteins formed hexamers, which demonstrates that hexamerization is even more widespread among the GGGPS family than previously assumed. Furthermore, all experimentally studied ancestral proteins showed high thermostability. Our results indicate that the hexameric oligomerization state and thermostability were present very early during the evolution of group II GGGPS, while the fine tuning of the flexibility – stability trade‐off developed very late, independent of the emergence of hexamerization

Identification of acetylated diether lipids in halophilic Archaea

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects

Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli

An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur

Different paths to the modern state in Europe: the interaction between domestic political economy and interstate competition

Theoretical work on state formation and capacity has focused mostly on early modern Europe and on the experience of western European states during this period. While a number of European states monopolized domestic tax collection and achieved gains in state capacity during the early modern era, for others revenues stagnated or even declined, and these variations motivated alternative hypotheses for determinants of fiscal and state capacity. In this study we test the basic hypotheses in the existing literature making use of the large date set we have compiled for all of the leading states across the continent. We find strong empirical support for two prevailing threads in the literature, arguing respectively that interstate wars and changes in economic structure towards an urbanized economy had positive fiscal impact. Regarding the main point of contention in the theoretical literature, whether it was representative or authoritarian political regimes that facilitated the gains in fiscal capacity, we do not find conclusive evidence that one performed better than the other. Instead, the empirical evidence we have gathered lends supports to the hypothesis that when under pressure of war, the fiscal performance of representative regimes was better in the more urbanized-commercial economies and the fiscal performance of authoritarian regimes was better in rural-agrarian economie

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion

Silencing von Transgenen und Transposons durch DNA-Methylierung bei Volvox

Die vielzellige Kugelalge Volvox carteri ist mit einer vollständigen Arbeitsteilung zwischen nur zwei Zelltypen - somatischen und reproduktiven - ein idealer Modellorganismus für die Analyse grundlegender Mechanismen von Entwicklung und Zelldifferenzierung. Jedoch wird die Untersuchung von V. carteri oft durch die nicht kontrollierbare Stillegung von eingeschleusten Transgenen durch DNA-Methylierung erschwert. Ziel dieser Arbeit war es daher, die Art, den Vorgang und die Funktion der DNA-Methylierung bei Volvox aufzuklären, um einerseits Methodisches zu verbessern, andererseits und vor allem aber den Stellenwert dieses �epigenetischen Phänomens� bei Grünalgen besser zu verstehen. Mit dünnschichtchromatographischen Methoden wurde der Gehalt an modifizierten Basen in verschiedenen DNA-Präparationen von V. carteri bestimmt. Nukleäre DNA enthält demnach 1,1 % 5-Methylcytosin (5mC) und 0,3 % N6-Methyladenin (6mA). Das Gen einer DNA-Cytosin-Methyltransferase, met1, wurde kloniert und fast vollständig sequenziert. Vergleiche der abgeleiteten Proteinsequenz mit Sequenzen bekannter Methyltransferasen identifizierten Met1 als CpG-spezifische Erhaltungs-Methyltransferase. Das silencing eines funktionell intakten C-ars-Transgens bei Volvox korreliert mit CpG-Methylierung, die sich über das gesamte Transgen erstreckt. In vitro CpG-methylierte Gene werden nach Transformation von V. carteri nicht exprimiert und bleiben auch nach vielen Generationszyklen methyliert. Die Cytosin-Methylierung ist bei V. carteri - wie bei Tieren - auf CpG-Dinukleotide beschränkt, eine Methylierung von CpNpG- oder asymmetrischen Sequenzmotiven wie bei vielen Pflanzen wurde nicht beobachtet. Als eines der natürlichen Ziele der Cytosin-Methylierung bei V. carteri wurden Transposons identifiziert: Das nicht-mobile Retrotransposon Osser ist zu 20-30 % CpG-methyliert. Auffallend waren häufige C nach T � Austausche im CpG-Kontext, die auf Desaminierung von 5mC zurückgeführt wurden. Das führt zur Annahme, dass die Inaktivität von Retrotransposons wie Osser primär aus CpG-Methylierungen resultiert; die Integrität dieser Elemente geht dann sekundär durch Mutationen verloren

Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis.

The exclusive presence of glycerol-1-phosphate dehydrogenases (G1PDH) has been postulated to be a key feature that distinguishes archaea from bacteria. However, homologues of G1PDH genes can be found in several bacterial species, among them the hitherto uncharacterized open reading frame araM from Bacillus subtilis. We produced recombinant AraM in Escherichia coli and demonstrate that the purified protein forms a homodimer that reversibly reduces dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P) in a NADH-dependent manner. AraM, which constitutes the first identified G1PDH from bacteria, has a similar catalytic efficiency as its archaeal homologues, but its activity is dependent on the presence of Ni (2+) instead of Zn (2+). On the basis of these findings and the analysis of an araM knockout mutant, we propose that AraM generates G1P for the synthesis of phosphoglycerolipids in Gram-positive bacterial species

Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control

Selective switching of enzymatic activity has been a longstanding goal in synthetic biology. Drastic changes in activity upon mutational manipulation of the oligomerization state of enzymes have frequently been reported in the literature, but scarcely exploited for switching. Using geranylgeranylglyceryl phosphate synthase as a model, we demonstrate that catalytic activity can be efficiently controlled by exogenous modulation of the association state. We introduced a lysine-to-cysteine mutation, leading to the breakdown of the active hexamer into dimers with impaired catalytic efficiency. Addition of bromoethylamine chemically rescued the enzyme by restoring hexamerization and activity. As an alternative method, we incorporated the photosensitive unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into the hexamerization interface. This again led to inactive dimers, but the hexameric state and activity could be recovered by UV-light induced cleavage of ONBY. For both approaches, we obtained switching factors greater than 350-fold, which compares favorably with previously reported activity changes that were caused by site-directed mutagenesis

Data archive of "Investigating the prevalence of RNA binding metabolic enzymes in E. coli"

raw sequencing dat