A β-d-fructofuranosidase from Claviceps purpurea

Evidence suggests that sucrose is the main carbon source for growth of Claviceps spp. in the parasitic condition. The sucrose acts as substrate for an active beta-fructofuranosidase, produced by the fungus, which in the first instance converts the disaccharide into glucose and an oligofructoside. In this way, 50% of the glucose, supplied as sucrose, is made available to the parasite for assimilation. Subsequent action of the enzyme on both sucrose and the oligofructoside leads to the release of more glucose and the formation of additional oligosaccharides. The structures of the main oligosaccharides formed have been elucidated and the interactions of each compound studied. In experiments with purified enzyme in vitro the interaction of the oligosaccharides is rapid but in culture they are assimilated only slowly; in each case some free fructose is liberated. Free fructose is not assimilated in the presence of glucose and, further, inhibits growth at concentrations which might be expected to occur in the parasitic condition. A dual role has been suggested for the enzyme, with sucrose as substrate, in which glucose is made available to the growing parasite, while at the same time transfer of the fructose to form oligosaccharides prevents it from accumulating at inhibitory concentrations. Ultimately, when glucose becomes limiting, the fungus will adapt to fructose assimilation.     

A New Sesquiterpenoid from Russula lepida

A new aristolane sesquiterpenoid named rulepidol was isolated from the fruiting body ofRussula lepida Fr. Its structure was elucidated as (1aa,5a,7a,7aa,7b)-1,1a,4,5,6,7,7a,7b-octahydro-5-hydroxy-1,1,7,7a-tetramethyl-5H-cyclopropa［α］naphthalen-2-one mainly by 1D and 2D-NMR techniques.     

Myosotis margaritae—A new species from Bulgaria

A new species ofMyosotis ser.Palustres M. Pop.,Myosotis margaritae, related to theM. caespitosa group, is described from Bulgaria. Diploid chromosome number (2n=20) is given for the new species, and notes on its distribution, ecology and taxonomic relationships are presented.     

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.     

Kinetics of Extracellular ATP from Goldfish Hepatocytes: A Lesson from Mathematical Modeling

In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220–R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J _L ) and nonlytic mechanisms (J _NL ), ATPe diffusion (J _D ), and ATPe consumption by ectonucleotidases (J _V ). Particular focus was given to J _NL , defined as the product of a time function (J _R ) and a positive feedback mechanism whereby ATPe amplifies J _NL . Several J _R functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that:

Locustoside A — A new purine alkaloid glucoside from seeds of Gleditsia japonica

A new purine alkaloid glucoside designated locustoside A has been isolated from the seeds of Gleditsia japonica, which has been used in oriental traditional medicine. The structure was determined as 7-β-d-glucopyranosyl-3-(3-methyl-2-butenyl)-isoguanine by interpretation of spectroscopic data and was confirmed by X-ray crystallographic analysis.     

Enzymatic total synthesis of gibberellin A₄ from acetate

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. 13C-MVA was synthesized from 13C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from 13C-acetate and 13C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA? biosynthesis from ent-kaurene via GA?? were prepared in yeast and Escherichia coli. All intermediates and the final product GA? were uniformly labeled with 13C without dilution by natural abundance when [U-13C?] acetate was used. The 13C-NMR and MS data for [U-13C??] ent-kaurene confirmed 13C-13C coupling, and no dilution with the 12C atom was observed.     

Regulating cell–cell junctions from A to Z

Epithelial sheets often present a “cobblestone” appearance, but the mechanisms underlying the dynamics of this arrangement are unclear. In this issue, Choi et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506115) show that afadin and ZO-1 regulate tension and maintain zonula adherens architecture in response to changes in contractility.The textbook view of epithelial cells is that once such cells adopt a close, hexagonal packing, their “honeycomb” or “cobblestone” arrangement is static. This fixed appearance is misleading, as these cells are more like players in a rugby scrum, locked in a tussle in which the forces exerted by each of the players on the others maintains their seemingly static arrangement, but by a very dynamic force balance. How such balance is maintained in epithelia is a subject of substantial interest. A crucial role is played by F-actin and nonmuscle myosin II isoforms, which are deployed in contractile networks that transiently attach to cell–cell junctions to generate tensile forces along cell–cell boundaries (Lecuit and Yap, 2015). Contractile arrays of actomyosin are regulated by the monomeric G protein Rho, its upstream regulators, including Rho guanine nucleotide exchange factors (Quiros and Nusrat, 2014), and its effectors ROCK/Rho kinase and Shroom3 (Nishimura and Takeichi, 2008), but also by tension-mediated feedback between the myosin network and the junction (Lecuit and Yap, 2015). Cell–cell adhesion, including cadherin-dependent adhesion, also plays a crucial role in this process. As cells engage with one another via interactions of the extracellular domains of their cadherin complexes, they transduce forces to the actomyosin cytoskeleton through catenins. β-Catenin binds to the cytoplasmic domain of classical cadherins and recruits α-catenin, which binds F-actin.Given the dynamic nature of epithelia, the attachment of contractile actomyosin networks to junctions are also subject to regulation. One aspect of epithelial architecture that has received relatively little attention is that a typical epithelial monolayer (Fig. 1 A) displays two main types of cell–cell interfaces: bilateral junctions (BCJs), in which two cells establish a relatively long stretch of contact, and cellular vertices, which represent a confluence of three or more cell edges to form tricellular junctions (TCJs) or multicellular junctions. TCJs are not well understood, but are known to contain unique molecular components (Furuse et al., 2014; Flores-Benitez and Knust, 2015). In this issue, Choi et al. show that the multivalent scaffolding proteins afadin and ZO-1/2 regulate the spacing of and tension along lateral contacts in cultured cells, thereby shedding light on how contractile arrays containing bilateral and tri- or multicellular contact points are regulated in epithelia.Open in a separate windowFigure 1.ZO proteins and afadin regulate junctional tension and organization in cultured cells. (A) Untreated MDCK cells have sinuous cell boundaries, whereas ZO KD cells show extremely straight boundaries. When ZO proteins and afadin are knocked down, cells adopt contact zones of irregular length with other cells, sometimes clustering into foci (asterisks). Images courtesy of Mark Peifer (Choi et al., 2016). (B) A model for actomyosin organization at adherens junctions (adapted from Choi et al., 2016). Contractile actomyosin arrays run parallel to bicellular junctions and are anchored by side-on attachments (pink circles). At TCJs, end-on binding of actin, likely stabilized by afadin, anchors actomyosin filaments. In ZO KD cells, contractile elements and cadherin complexes collapse toward TCJs, and myosin minifilaments adopt a regularly spaced arrangement.Afadin and ZO-1/2 are far from new players at junctions. Afadin binds α-catenin, actin, and other cytoskeletal and junctional proteins and associates with the transmembrane protein nectin, which appears to form an alternative adhesion system at adherens junctions (Mandai et al., 2013). The zonula occludens proteins ZO-1 and ZO-2 are tight junction proteins that bind claudins and are required for tight junction formation (Itoh et al., 1999; Balda and Matter, 2008). In addition, ZO proteins also bind to α-catenin (Itoh et al., 1997), are involved in establishing the zonula adherens (ZA; Ikenouchi et al., 2007), and potentiate cadherin-dependent adhesion in Caenorhabditis elegans (Lockwood et al., 2008) and Drosophila melanogaster (Choi et al., 2011). Knockdown of ZO-1 and ZO-2 (ZO KD) in MDCK cells has previously been shown (Fanning et al., 2012) to lead to dramatic alterations of the ZA: F-actin and myosin IIs assemble into striking apical arrays at the ZA, spaced at regular intervals. In addition, the normally sinuous boundaries between cells give way to very straight borders (Fig. 1 A).Using superresolution microscopy, diffraction-limited junctional laser ablation, cell morphometry, kinetic analysis, and a whole-monolayer approach to contractility, Choi et al. (2016) now extend this story. To test whether contractility is increased after ZO KD, the authors first measured the recoil after laser ablation of ZO KD cells; an increase in recoil velocity indicated that the straight junctional boundaries between ZO-depleted cells are under tension. Imaging analysis of BCJs showed that the increase in contractility in ZO KD cells is associated with a strikingly dynamic behavior of the BCJs. Individual BCJs were found to undergo periods of shortening and elongation, whereas neighboring BCJs underwent compensatory, opposite changes in length. These changes in contractility have effects on the entire tissue sheet as well: whereas control cell sheets remained flat when detached from the substratum, ZO KD cells contracted into a cup-like shape. This constriction was blocked by the myosin inhibitor blebbistatin. Overall, these experiments indicated that ZO proteins regulate myosin assembly and contractility across the cellular sheet.To dissect the protein network mediating increased contractility in ZO KD cells, Choi et al. (2016) examined the role of ROCK and found that ROCK inhibitors abolished the straight BCJs, which became curvilinear. Additionally, Shroom3, which is known to recruit ROCK (Nishimura and Takeichi, 2008), was cytoplasmic in control cells but junctional in ZO KD cells. Transient Shroom3 overexpression led to ROCK recruitment to the ZA and drove formation of an actomyosin network similar to that in ZO KD cells. Conversely, Shroom3 knockdown resulted in loss of the actomyosin arrays in ZO KD cells. Collectively, these data indicated that Shroom3 is an effector of increased apical contractility in ZO KD cells.The researchers used ZO KD cells to test how tissue integrity is maintained despite elevated contractibility and how junctions are remodeled to maintain integrity when increased tension is present. Afadin is a good candidate: the Drosophila homologue of afadin, Canoe, plays roles in convergent extension and collective cell migration; in its absence, actomyosin networks at the apex of constricting epithelial cells in the embryo contract in a catastrophic, uncontrolled fashion (Sawyer et al., 2009), suggesting a potential role for afadin in the maintenance of tissue integrity during morphogenetic movements. Choi et al. (2016) therefore turned their attention to afadin. ZO KD cells have significantly more afadin at their adherens junctions and TCJs, a pattern reminiscent of the normal distribution of Canoe in Drosophila (Sawyer et al., 2009). Knocking down afadin by shRNA in ZO KD cells led to further defects in cell–cell boundary maintenance. In addition to the taut appearance of bicellular borders, cell boundary length became much more irregular, with occasional foci of highly contracted cells (Fig. 1 A). Velocimetry analysis and live-cell imaging indicated that loss of both ZO proteins and afadin led to large-scale cell movements within the monolayer not seen after ZO KD alone.New imaging techniques used by Choi et al. (2016) revealed further details about the changes in actomyosin arrays in ZO KD cells. Superresolution imaging of myosin light chain kinase staining via structured illumination showed that myosin II assembles into arrays of myosin minifilaments spaced ∼415 nm apart along bicellular contacts. Superresolution and transmission electron microscopy also revealed reorganization of F-actin and E-cadherin at TCJs in ZO KD cells. Lateral F-actin bundles appeared to terminate end-on at TCJs at sites where E-cadherin was present. ZO KD therefore induces assembly of a remarkably ordered actomyosin array along BCJs, and these arrays appear to be separate contractile units that anchor end-on at the ZA. Moreover, based on staining for vinculin and a specific epitope in αE-catenin that serve as markers for regions under high tension (Yonemura et al., 2010), the end-on attachments of actin cables to the ZA at TCJs experience significant tensile stress. Strikingly, although vinculin and αE-catenin accumulation at TCJs was relatively uniform after ZO KD, their distribution was more heterogeneous after ZO/afadin KD. Differences in staining paralleled differences in cell border length and correlated with the level of tension measured at BCJs after laser cutting, suggesting that afadin contributes to the ability of cells to distribute forces at TCJ/multicellular junctions throughout the monolayer. Lastly, the researchers investigated whether internal cues downstream of ZO KD are sufficient for myosin recruitment or whether such recruitment depends on mechanical cues exerted by neighboring cells. They designed an assay mixing small islands of wild-type cells surrounded by ZO KD cells (or vice versa) and found that the development of the contractile array at the ZA depends on the contractility of neighboring cells; however, afadin recruitment to the ZA was less dependent on the sustained contractility of neighboring cells. Taking these data together, Choi et al. (2016) propose that cells respond to elevated contractility by increasing junctional afadin; because combined ZO/afadin knockdown dramatically alters cell shape and barrier function in response to elevated contractility, afadin acts as a robust scaffold that maintains ZA architecture most crucially at TCJs.Although many aspects of the model proposed by Choi et al. (2016) remain to be tested, their data suggest new features regarding the detailed assembly of actomyosin contractile arrays in confluent cells (Fig. 1 B). In control cells, actomyosin arrays presumably extend parallel to individual BCJs. Choi et al. (2016) propose that these actomyosin bundles act as separate contractile units that terminate near TCJs, allowing the generation of tension along BCJs. In ZO KD cells, excessive assembly of actomyosin filaments, perhaps exacerbated by the tendency of F-actin/myosin minifilament arrays to self-assemble, somehow leads to regularly spaced actomyosin arrays, and perhaps collapse of cadherin complexes and other components toward TCJs. There is a precedent for such lateral collapse of cadherin-dependent attachments: it is a prominent feature of cadherin complexes at sites of high tension in the epidermis of the C. elegans embryo (Choi et al., 2015). If the new model of Choi et al. (2016) is correct, then the foci seen in ZO KD/afadin KD cells may be similar to what happens in a game of tug of war when one team stops pulling. If some end-on attachments (assisted by afadin) fail, filaments might be expected to collapse along BCJs as the other, still tethered end of a set of filaments contracts toward the remaining attachment at the opposite cell vertex.Several other interesting questions remain. First, what is the relationship of the striking, regularly spaced bipolar myosin II minifilaments that form in ZO KD cells to myosin arrays in normal cells? It is clear that untreated cells have junctional actomyosin networks, but not with this strict periodicity. One possibility is that this spacing is simply an epiphenomenon; when not appropriately anchored along junctions, actomyosin networks may self-organize as they are known to do in other systems, such as in the contractile ring and in migrating cells (Srivastava et al., 2015; Fenix et al., 2016). More optimistically, the spacing may represent an intensified version of processes that operate in normal cells at bicellular and multicellular contact sites. If so, components of the model of Choi et al. (2016) will require further investigation. For example, the organization of F-actin along BCJs remains unclear, as are the proteins that mediate the side-on binding envisioned in this model. It is also uncertain whether proteins assist bundling of filaments and what role dynamic growth and shrinkage of actin filaments plays in end-on binding. In some contexts, junctions are capable of seeding polymerization of F-actin (Brieher and Yap, 2013), and it may be that actin dynamics are important in the processes studied here.A second question has to do with the community events within monolayers that Choi et al. (2016) describe. The neighbor effects on ZA morphology that they document are intriguing, as are the long-range accelerated movements of cells lacking both ZO proteins and afadin. Collective properties of monolayers are only beginning to be explored; connecting these properties with subcellullar events is an exciting future challenge. Whatever the answers to these new questions, the work of Choi et al. (2016) refines our understanding of the roles of key scaffolding proteins in organizing and anchoring junctions in epithelia.     

A natively unfolded βγ-crystallin domain from Hahella chejuensis

To date, very few βγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative βγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a βγ-crystallin fold is not known. We describe here a βγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other βγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical βγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial βγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded βγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.     

Archakebia—A New Genus of Lardizabalaceae from China

The monotypic genus Archakebia from China is described as new．The genus shares many characters with members of the genus Akebia,except sepals 6,lanceolate or linear,which are common in members of the genus Stauntonia．     

A highly conserved α-tubulin sequence from Prunus amygdalus

The sequence of an -tubulin from Prunus amygdalus has been obtained by cDNA cloning. When this sequence is compared to that of the Tub1 gene from maize it shows a very high degree of similarity, much higher than any of the -tubulin sequences reported so far from plants. The expression of this gene is high in the stages of seed development where a high divisional activity is present. It is preferentially expressed in the radicular tissues as it is gene Tub1 in maize. Southern analysis indicates that this gene may from a subfamily of -tubulin genes having similar sequence and tissue specificity and existing at least in maize and in Prunus.     

Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

Dihydroconiferyl alcohol — A cell division factor from Acer species

A compound that stimulated growth of soybean callus was isolated from spring sap of sycamore (Acer pseudoplatanus L.). Insufficient compound was isolated to permit it to be characterised. A compound with identical properties was isolated from commercial maple syrup, the concentrated spring sap of Acer saccharum L. The compound was identified as 3-(3-methoxy-4-hydroxyphenyl)-propan-1-ol (dihydroconiferyl alcohol, DCA). DCA was also active in the tobacco callus and radish leaf senescence assays, but was inactive in four other tests for cytokinin activity. DCA acted synergistically with kinetin to promote soybean callus growth. It is concluded that DCA has properties distinct from those of purine cytokinins.Abbreviation DCA dihydroconiferyl acohol - GC gas chromatography - IAA indole-3-acetic acid - iP isopentenyladenine - [9R]iP isopentenyladenosine - LC liquid chromatography - MS mass spectrometry - NAA 1-napthylacetic acid - TLC thinlayer chromatography - TMSi trimethylsilyl     

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

A strain of Enterobacter cloacae B5 producing β-galactosidase with transglycosylation activity was isolated from the soil. Its freeze-thawed cells synthesized galacto-oligosaccharides with a high yield of 55% from 275 g/L lactose at 50 °C for 12 h. A novel β-galactosidase capable of glycosyl transfer was purified from this strain. It was a homotetramer with molecular mass of about 442 kDa. The optimal pH and temperature for hydrolysis activity on o-nitrophenyl-β-d-galactopyranoside (oNPGal) were 6.5–10.5 and 35 °C, respectively. The enzyme showed a wide range of acceptor specificity for transglycosylation and catalyzed glycosyl transfer from oNPGal to various chemicals such as galactose, glucose, fructose, arabinose, mannose, sorbose, rhamnose, xylose, cellobiose, sucrose, trehalose, melibiose, inositol, mannitol, sorbitol and salicin, resulting in novel saccharide yields ranging from 0.8% to 23.5%. A gene encoding the enzyme was cloned and the recombinant enzyme from Escherichia coli had similar transglycosylation activity to the natural enzyme.     

A New Pregnane from Munronia delavayi Franc （Meliaceae）

To search for pharmacologically active constituents of folk medicine, a new pregnane, 2α,3α,15β-trihydroxy-20（S）-tigloyl-pregnane （compound 1）, and nine known compounds, geranylgeraniol （compound 2）, β-daucosterol （compound 3）, 6-hydroxystigmast-4oen-3-one （compound 4）, sitoindoside Ⅰ （compound 5）, sitoindoside Ⅱ （compound 6）, β-sitosterol （compound 7）, kaempferol （compound 8）, quercetin （compound 9）, and rutin （compound 10）, were isolated from the ethanol extract of whole plants of Munronia delavayi Franch using chromatographic methods. The structures of compounds 1-10 were elucidated on the basis of spectral data.     

A new thermostable α-l-arabinofuranosidase from a novel thermophilic bacterium

An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.     

Antimony-Oxidizing Bacteria Isolated from Antimony-Contaminated Sediment – A Phylogenetic Study

Twelve antimony-resistant bacteria were isolated from sediment collected in the vicinity of an antimony oxide-producing factory in Korea. Eight of these strains were heterotrophic Sb(III)-oxidizing bacteria. Phylogenetic study showed that the Sb(III)-oxidizing bacteria fell within two subdivisions of Proteobacteria. Cupriavidus sp. NL4 and Comamonas sp. NL11 belong to the subdivision β-Proteobacteria. Acinetobacter sp. NL1, Acinetobacter sp. NL12, Pseudomonas sp. NL2, Pseudomonas sp. NL5, Pseudomonas sp. NL6, and Pseudomonas sp. NL10 are the members of the γ-subdivision of the Proteobacteria. Among them, Cupriavidus sp. NL4 completely oxidized 100 μmoles of Sb(III) per liter of medium in 500 h, while the other strains were not able to oxidize all of the Sb(III) in the medium, even with longer incubation. The results imply that diverse bacterial lineages are able to detoxify sites polluted with Sb(III) by oxidizing it to Sb(V), and to contribute to antimony cycling in natural environments.