The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy

Biotin (vitamin B₇) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world’s oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*10⁶ molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.Subject terms: Water microbiology, Ecosystem ecology, Marine microbiology     

Separation of human lymphocyte subclasses by rosettes with latex-lectin particles

The specificity and affinity of eight lectins (concanavalin A, L. culinaris, P. sativum, phytohemagglutinin P, D. biflorus, soybean agglutinin, T. purpureus, and T. vulgaris) to B, T, T gamma, and T mu lymphocytes from the blood of normal subjects were determined. Lectins attached to latex particles were used to evaluate the binding of each lectin to individual cells. The rosette percentage found in each lymphocyte population expresses the specificity index and the specific sugar concentrations needed to decrease the rosette percentage by 50% is taken as the affinity index. B Lymphocytes showed a major subclass, with respect to T lymphocytes, with receptors for WGA, SBA, D. biflorus, L. culinaris, and P. sativum lectins. In contrast, T lymphocytes exhibit a greater number of cells with specific receptors for Con A, T. purpureus, and PHA lectins than B lymphocytes, the T gamma subpopulation being responsible for the specificity of the first two lectins and the T mu subpopulation for the PHA lectin.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

The molecular basis of potassium nutrition in plants

Over the last five years, the cloning and characterization of K⁺ transport genes corresponding to K⁺ channels (KAT1, AKT1, KST1, AKT2), associated subunits (KAB1) and a high-affinity transporter (HKT1) has opened up important new avenues for research on plant K⁺ nutrition. With the abundance of molecular data now available it seems timely to link this information with the wealth of data previously accumulated on the physiology of plant K⁺ acquisition. The ultimate goal of all this research is to gain a better understanding of K⁺ transport and nutrition in the intact plant. Thus it is important to begin to integrate the molecular research with results from biochemical and physiological research conducted at the cellular, root and whole plant levels. This article will focus on describing the features of the cloned K⁺ transporters and their possible roles in mediating high- and low-affinity K⁺ uptake from the soil, as well as how K⁺ acquisition may be regulated.Abbreviations NEM N-ethyl maleimide - PCMBS p-chloromercuribenzene sulphonic acid     

Ecological restoration of rich fens in Europe and North America: from trial and error to an evidence‐based approach

Fens represent a large array of ecosystem services, including the highest biodiversity found among wetlands, hydrological services, water purification and carbon sequestration. Land‐use change and drainage has severely damaged or annihilated these services in many parts of North America and Europe; restoration plans are urgently needed at the landscape level. We review the major constraints on the restoration of rich fens and fen water bodies in agricultural areas in Europe and disturbed landscapes in North America: (i) habitat quality problems: drought, eutrophication, acidification, and toxicity, and (ii) recolonization problems: species pools, ecosystem fragmentation and connectivity, genetic variability, and invasive species; and here provide possible solutions. We discuss both positive and negative consequences of restoration measures, and their causes. The restoration of wetland ecosystem functioning and services has, for a long time, been based on a trial‐and‐error approach. By presenting research and practice on the restoration of rich fen ecosystems within agricultural areas, we demonstrate the importance of biogeochemical and ecological knowledge at different spatial scales for the management and restoration of biodiversity, water quality, carbon sequestration and other ecosystem services, especially in a changing climate. We define target processes that enable scientists, nature managers, water managers and policy makers to choose between different measures and to predict restoration prospects for different types of deteriorated fens and their starting conditions.     

Microbial metabolomics: toward a platform with full metabolome coverage

Achieving metabolome data with satisfactory coverage is a formidable challenge in metabolomics because metabolites are a chemically highly diverse group of compounds. Here we present a strategy for the development of an advanced analytical platform that allows the comprehensive analysis of microbial metabolomes. Our approach started with in silico metabolome information from three microorganisms-Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae-and resulted in a list of 905 different metabolites. Subsequently, these metabolites were classified based on their physicochemical properties, followed by the development of complementary gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods, each of which analyzes different metabolite classes. This metabolomics platform, consisting of six different analytical methods, was applied for the analysis of the metabolites for which commercial standards could be purchased (399 compounds). Of these 399 metabolites, 380 could be analyzed with the platform. To demonstrate the potential of this metabolomics platform, we report on its application to the analysis of the metabolome composition of mid-logarithmic E. coli cells grown on a mineral salts medium using glucose as the carbon source. Of the 431 peaks detected, 235 (=176 unique metabolites) could be identified. These include 61 metabolites that were not previously identified or annotated in existing E. coli databases.     

Hydrogen Peroxide Stimulates Salicylic Acid Biosynthesis in Tobacco

Hydrogen peroxide induced the accumulation of free benzoic acid (BA) and salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. Six hours after infiltration with 300 mM H2O2, the levels of BA and SA in leaves increased 5-fold over the levels detected in control leaves. The accumulation of BA and SA was preceded by the rapid activation of benzoic acid 2-hydroxylase (BA2H) in the H2O2-infiltrated tissues. This enzyme catalyzes the formation of SA from BA. Enzyme activation could be reproduced in vitro by addition of H2O2 or cumene hydroperoxide to the assay mixture. H2O2 was most effective in vitro when applied at 6 mM. In vitro activation of BA2H by peroxides was inhibited by the catalase inhibitor 3-amino-1,2,4-triazole. We suggest that H2O2 activates SA biosynthesis via two mechanisms. First, H2O2 stimulates BA2H activity directly or via the formation of its substrate, molecular oxygen, in a catalase-mediated reaction. Second, higher BA levels induce the accumulation of BA2H protein in the cells and provide more substrate for this enzyme.     

Critical evaluation of organic acid mediated iron dissolution in the rhizosphere and its potential role in root iron uptake

Both experimental extractions and theoretical calculations were undertaken to assess whether organic acid-mediated Fe dissolution could play a significant role in elevating the concentration of Fe-complexes in the rhizosphere, and further, whether this could satisfy the Fe demands of a plant utilizing ferric reduction to acquire Fe. Using a mathematical computer model, it was predicted that organic acids released from and diffusing away from the root would result in a solution organic acid concentration at the root surface of between 1 to 50 M. Over 99% of the organic acids lost by the root were predicted to remain within 1 mm of the root surface. The experimental results indicated that citrate-mediated Fe dissolution of amorphous Fe(OH)₃, was rapid in comparison with citrate dissolution of the Fe-oxides, Fe₂O₃ and Fe₃O₄. The rate of citrate and malate mediated Fe-dissolution was dependent on many factors such as pH, metal cations and phosphate saturation of the Fe(OH)₃ surface. At pH values 6.8, citrate formed stable complexes with Fe and dissolution proceeded rapidly. Under optimal growth conditions for a plant utilizing a reductive-bound mechanism of Fe acquisition (dicots and non-grass monocots), it can be expected that citrate and malate may be able to satisfy a significant proportion of the plant's Fe demand through the formation of plant-available organic-Fe³⁺ complexes in the rhizosphere. In high pH soils (pH7.0), the plant must rely on other sources of Fe, as citrate-mediated Fe dissolution is slow and Fe-citrate complexes are unstable. Alternatively, the root acidification of the rhizosphere could allow the formation of stable Fe-organic complexes. ei]H Marschner     

Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence

Ion channels are allosteric membrane proteins that open and close an ion-permeable pore in response to various stimuli. This gating process provides the regulation that underlies electrical signaling events such as action potentials, postsynaptic potentials, and sensory receptor potentials. Recently, the molecular structures of a number of ion channels and channel domains have been solved by x-ray crystallography. These structures have highlighted a gap in our understanding of the relationship between a channel's function and its structure. Here we introduce a new technique to fill this gap by simultaneously measuring the channel function with the inside-out patch-clamp technique and the channel structure with fluorescence spectroscopy. The structure and dynamics of short-range interactions in the channel can be measured by the presence of quenching of a covalently attached bimane fluorophore by a nearby tryptophan residue in the channel. This approach was applied to study the gating rearrangements in the bovine rod cyclic nucleotide-gated ion channel CNGA1 where it was found that C481 moves towards A461 during the opening allosteric transition induced by cyclic nucleotide. The approach offers new hope for elucidating the gating rearrangements in channels of known structure.     

Comparative structural analysis of glycoprotein gD of herpes simplex virus types 1 and 2.

We studied the synthesis and processing of the type-common glycoprotein gD in herpes simplex virus type 2 (HSV-2) and compared it structurally to glycoprotein gD of herpes simplex virus type 1 (HSV-1). We demonstrated that in HSV-2, gD undergoes posttranslational processing from a lower-molecular-weight precursor (pgD51) to a higher-molecular-weight product (gD56). Tryptic peptide analysis by cation-exchange chromatography indicated that this processing step altered neither the methionine nor the arginine tryptic peptide profile of gD of HSV-2. Comparative tryptic peptide analysis of gD of HSV-1 and HSV-2 showed that the methionine and arginine tryptic peptide profiles of these two proteins were very similar, but not identical. Some of the resolved peptides coeluted from the cation-exchange column, suggesting that some amino acid sequences of the two proteins might be very similar. However, each protein also appeared to possess several type-specific tryptic peptides. The structural similarity of these two glycoproteins correlates well with their antigenic cross-reactivity since monoprecipitin antibody to gD of HSV-1 also immunoprecipitates gD of HSV-2 and neutralizes the infectivity of both viruses to approximately the same extent.