Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein

ABCA1, an ATP-binding cassette transporter mutated in Tangier disease, promotes cellular phospholipid and cholesterol efflux by loading free apoA-I with these lipids. This process involves binding of apoA-I to the cell surface and phospholipid translocation by ABCA1. The goals of this study were to examine the relationship between ABCA1-mediated lipid efflux and apolipoprotein binding and to determine whether phospholipid and cholesterol efflux are coupled. Inhibition of lipid efflux by glybenclamide treatment or by mutation of the ATP-binding cassette of ABCA1 showed a close correlation between lipid efflux, the binding of apoA-I to cells, and cross-linking of apoA-I to ABCA1. The data suggest that a functionally important apoA-I binding site exists on ABCA1 and that the binding site could also involve lipids. After using cyclodextrin preincubation to deplete cellular cholesterol, ABCA1-mediated cholesterol efflux was abolished but phospholipid efflux and the binding of apoA-I were unaffected. The conditioned media from cyclodextrin-pretreated, ABCA1-expressing cells readily promoted cholesterol efflux when added to fresh cells not expressing ABCA1, indicating that cholesterol efflux can be dissociated from phospholipid efflux. Further, using a photoactivatable cholesterol analog, we showed that ABCA1 did not bind cholesterol directly, even though several other cholesterol-binding proteins specifically bound the cholesterol analog. The data suggest that the binding of apoA-I to ABCA1 leads to the formation of phospholipid-apoA-I complexes, which subsequently promote cholesterol efflux in an autocrine or paracrine fashion.     

A putative replicative form of the abutilon mosaic virus (gemini group) in a chromatin-like structure

Summary The putative replicative form of the abutilon mosaic virus (AbMV), a geminivirus, was purified from infected plants. It was shown to consist of a bipartite genome of 2660 and 2640 bp. This double-stranded DNA has a closed or relaxed circular conformation and part of it is packed in nucleoprotein complexes with a chromatin-like structure. Similarities between the geminiviruses and the animal simian virus 40 are discussed against this back-ground.Cloning was performed under L2/B1 conditions according to the licence of the ZKBS 1526/1This article is based on a doctoral study by A.A. and T.F. in the Faculty of Biology, University of Hamburg     

Cell recovery by continuous flotation

Summary Cell recovery by means of continuous flotation of the Hansenula polymorpha cultivation medium without additives was investigated as a function of the cultivation conditions as well as of the flotation equipment construction and flotation operational parameters. The cell enrichment and separation is improved at high liquid residence times, high aeration rates, small bubble sizes, increasing height of the aerated column, and diameter of the foam column. Increasing cell age and cultivation with nitrogen limitation reduce the cell separation.Symbols C_P cell mass concentration in medium g·l^–1 - C_R cell mass concentration in residue g·l^–1 - C_S cell mass concentration in foam liquid g·l^–1 - V equilibrium foam volume cm³ - V gas flow rate through the aerated liquid column cm³·s^–1 - V_F feed rate to the flotation column ml/min - ¹ V _S/V foaminess s - mean liquid residence time in the column s     

Macroecology in the age of Big Data – Where to go from here?

Recent years have seen an exponential increase in the amount of data available in all sciences and application domains. Macroecology is part of this “Big Data” trend, with a strong rise in the volume of data that we are using for our research. Here, we summarize the most recent developments in macroecology in the age of Big Data that were presented at the 2018 annual meeting of the Specialist Group Macroecology of the Ecological Society of Germany, Austria and Switzerland (GfÖ). Supported by computational advances, macroecology has been a rapidly developing field over recent years. Our meeting highlighted important avenues for further progress in terms of standardized data collection, data integration, method development and process integration. In particular, we focus on (a) important data gaps and new initiatives to close them, for example through space- and airborne sensors, (b) how various data sources and types can be integrated, (c) how uncertainty can be assessed in data-driven analyses and (d) how Big Data and machine learning approaches have opened new ways of investigating processes rather than simply describing patterns. We discuss how Big Data opens up new opportunities, but also poses new challenges to macroecological research. In the future, it will be essential to carefully assess data quality, the reproducibility of data compilation and analytical methods, and the communication of uncertainties. Major progress in the field will depend on the definition of data standards and workflows for macroecology, such that scientific quality and integrity are guaranteed, and collaboration in research projects is made easier.     

On the ATP-Dependent Activation of the Radical Enzyme (R)-2-Hydroxyisocaproyl-CoA Dehydratase

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 ? resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.     

Allergic airway inflammation inhibits pulmonary antibacterial host defense

The innate immune system of the lung is a multicomponent host defense system and in addition has an instructing role in regulating the quality and quantity of the adaptive immune response. When the interaction between innate and adaptive immunity is disturbed, pathological conditions such as asthma can develop. It was the aim of the study to investigate the effect of the allergic inflammation of the lung on the innate host defense during bacterial infection. Human bronchial epithelial cells were preincubated with Th2 cytokines and infected with Pseudomonas aeruginosa. The effect of the Th2 cytokines on the mRNA levels of antimicrobial peptides and the antimicrobial activity of HBEC was determined. To investigate the influence of an allergic inflammation on pulmonary host defense in vivo, mice sensitized and challenged with OVA were infected with P. aeruginosa, and the number of viable bacteria in the lungs was determined together with markers of inflammation like cytokines and antimicrobial peptides. Exposure of airway epithelial cells to Th2 cytokines resulted in a significantly decreased antimicrobial activity of the cells and in suppressed mRNA levels of the antimicrobial peptide human beta-defensin 2. Furthermore, mice with allergic airway inflammation had significantly more viable bacteria in their lungs after infection. This was consistent with reduced levels of proinflammatory cytokines and of the antimicrobial peptide cathelin-related antimicrobial peptide. These results show that an allergic airway inflammation suppresses the innate antimicrobial host defense. The adaptive immune system modulates the functions of the pulmonary innate immune system.     

Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins

The intracellular pathogen Legionella pneumophila has evolved sophisticated mechanisms that enable it to subvert host functions, enter, survive and replicate in amoebae or alveolar macrophages, and to finally evade these hosts. Protozoa are essential for the growth of Legionella and the interaction with amoeba seems to be the driving force in the evolution of its pathogenicity. This is reflected in the genome of this pathogen, which encodes a high number and variety of eukaryotic-like proteins that are able to interfere in the various steps of the infectious cycle by mimicking functions of eukaryotic proteins. Central to the pathogenicity of L. pneumophila are the many secretion systems delivering these and other effectors to the host cell. Recent studies have highlighted the multi-functional role of these factors secreted by L. pneumophila, in host-pathogen interactions.     

Assembly of natural and recombinant prion protein into fibrils

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.