Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.     

Biosynthesis of Silver Nanoparticles Using Brown Marine Macroalga,Sargassum Muticum Aqueous Extract

Biological synthesis of nanoparticles is a relatively new emerging field of nanotechnology which has economic and eco-friendly benefits over chemical and physical processes of synthesis. In the present work, for the first time, the brown marine algae Sargassum muticum (S. muticum) aqueous extract was used as a reducing agent for the synthesis of nanostructure silver particles (Ag-NPs). Structural, morphological and optical properties of the synthesized nanoparticles have been characterized systematically by using FTIR, XRD, TEM and UV–Vis spectroscopy. The formation of Ag-NPs was confirmed through the presence of an intense absorption peak at 420 nm using a UV–visible spectrophotometer. A TEM image showed that the particles are spherical in shape with size ranging from 5 to 15 nm. The nanoparticles were crystalline in nature. This was confirmed by the XRD pattern. From the FTIR results, it can be seen that the reduction has mostly been carried out by sulphated polysaccharides present in S. muticum.     

Biosynthesis of the Caenorhabditis elegans dauer pheromone

To sense its population density and to trigger entry into the stress-resistant dauer larval stage, Caenorhabditis elegans uses the dauer pheromone, which consists of ascaroside derivatives with short, fatty acid-like side chains. Although the dauer pheromone has been studied for 25 years, its biosynthesis is completely uncharacterized. The daf-22 mutant is the only known mutant defective in dauer pheromone production. Here, we show that daf-22 encodes a homolog of human sterol carrier protein SCPx, which catalyzes the final step in peroxisomal fatty acid β-oxidation. We also show that dhs-28, which encodes a homolog of the human d-bifunctional protein that acts just upstream of SCPx, is also required for pheromone production. Long-term daf-22 and dhs-28 cultures develop dauer-inducing activity by accumulating less active, long-chain fatty acid ascaroside derivatives. Thus, daf-22 and dhs-28 are required for the biosynthesis of the short-chain fatty acid-derived side chains of the dauer pheromone and link dauer pheromone production to metabolic state.     

Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

Carbohydrate moieties are important components of natural products, which are often imperative for the solubility and biological activity of the compounds. The aromatic polyketide alnumycin A contains an extraordinary sugar-like 4'-hydroxy-5'-hydroxymethyl-2',7'-dioxane moiety attached via a carbon-carbon bond to the aglycone. Here we have extensively investigated the biosynthesis of the dioxane unit through (13)C labeling studies, gene inactivation experiments and enzymatic synthesis. We show that AlnA and AlnB, members of the pseudouridine glycosidase and haloacid dehalogenase enzyme families, respectively, catalyze C-ribosylation conceivably through Michael-type addition of d-ribose-5-phosphate and dephosphorylation. The ribose moiety may be attached both in furanose (alnumycin C) and pyranose (alnumycin D) forms. The C(1')-C(2') bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cis-bicyclo[3.3.0]-2',4',6'-trioxaoctan-3'β-ol) structure present in alnumycin B. The reaction is catalyzed by Aln6, which belongs to a previously uncharacterized enzyme family. The conversion was accompanied with consumption of O(2) and formation of H(2)O(2), which allowed us to propose that the reaction may proceed via hydroxylation of C1' followed by retro-aldol cleavage and acetal formation. Interestingly, no cofactors could be detected and the reaction was also conducted in the presence of metal chelating agents. The last step is the conversion of alnumycin B into the final end-product alnumycin A catalyzed by Aln4, an NADPH-dependent aldo-keto reductase. This characterization of the dioxane biosynthetic pathway sets the basis for the utilization of C-C bound ribose, dioxolane and dioxane moieties in the generation of improved biologically active compounds.     

Beta-caryophyllene is a dietary cannabinoid

The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.     

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys¹¹⁸, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys¹¹⁸ contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.     

Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase

The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in anisotropic cell expansion. However, mutations to E at residues surrounding T166 (i.e., S162, T165, and S167) caused opposite effects. Live-cell imaging of fluorescently labeled CESA showed close correlations between tissue or cell morphology and patterns of bidirectional motility of CESA complexes in the plasma membrane. In the WT, CESA complexes moved at similar velocities in both directions along microtubule tracks. By contrast, the rate of movement of CESA particles was directionally asymmetric in mutant lines that exhibited abnormal tissue or cell expansion, and the asymmetry was removed upon depolymerizing microtubules with oryzalin. This suggests that phosphorylation of CESA differentially affects a polar interaction with microtubules that may regulate the length or quantity of a subset of cellulose microfibrils and that this, in turn, alters microfibril structure in the primary cell wall resulting in or contributing to the observed defect in anisotropic cell expansion.     

Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate

The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. Here, we identify phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. We propose that PIP2, through the control of Crag’s subcellular localization, restricts the secretion of BM proteins to the basal side.Epithelial cells are characterized by their polarized architecture, which enables them to exert their varied functions in embryonic and adult organisms. Epithelia exhibit a profound apical–basal polarity that is manifested in the cytoplasmic and surface organization of individual cells (1–3). Loss of apical–basal cell polarity is often associated with carcinoma progression and tumor metastasis (4, 5). The establishment and maintenance of cell polarity relies on the transport of newly synthesized and recycled proteins to their correct destinations (6, 7). The lipid composition of the transport vesicles and of the plasma membrane is crucial for the establishment and maintenance of cell polarity (6–9). In particular, in 3D in vitro culture of Madin–Darby canine kidney (MDCK) cells, phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two phosphoinositides (PtdIns) have been shown to play critical roles in polarized vesicle trafficking by mediating the recruitment of proteins to these different domains (10, 11).To set up a correct cell polarity, membrane asymmetry needs to be established. In cell culture, and likely during development of many tissues in multicellular organisms, this process is achieved by two external cues: one provided by the adjacent cells via cadherin-dependent adhesion and the other by interaction with the basement membrane (BM), a specialized sheet of the ECM secreted basally by the epithelial cells (12, 13). The main components of the BM are secreted glycoproteins, such as collagen IV (Coll IV), laminin, and the heparan sulfate proteoglycan perlecan (Pcan) (14), which interact with different membrane receptors, including integrin and dystroglycan (14, 15). Previous studies in model organisms and 3D culture models have shown that BM secreted by the epithelial cells at their basal side plays a role as an initial determinant in basal polarity (13, 16, 17). Moreover, Pcan, through its cellular receptor dystroglycan, is involved in the maintenance of epithelial polarity (18). Despite its important roles in the establishment and maintenance of polarity, it is not well understood how the BM achieves its own polarized accumulation on the basal side of the cells.Recently, two new factors have been shown to be critical for the polarized deposition of BM proteins: Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein) and Rab10 (19, 20). The loss of either Crag or Rab10 leads to anomalous accumulation of BM components on both the apical as well as the basal side of epithelial cells without directly disrupting the distribution of apical or basolateral proteins (19, 20). This finding indicates that these two factors are specifically required for the restriction of the BM to the basal side and that this process is independently regulated from apical and basolateral secretion. It has been shown that Crag can act as a GTP exchange factor (GEF) for Rab10 in Drosophila (21). In addition, in Drosophila embryos, Scarface, a protease-like protein, has been shown to act as a specific regulator of laminin A-polarized deposition (22).To further elucidate the molecular mechanism leading to the polarized secretion of BM components, we identified additional genes involved in this process using the Drosophila follicular epithelium (FE) as a model system. The FE is composed of a monolayer of somatic cells, the follicle cells (FCs), which surround the central germ-line cells during Drosophila oogenesis (Fig. 1 A and B) (23). The FE is a classical epithelium, with a distinct apical–basal polarity where the apical domain of the FCs faces the germ line and the BM is secreted at the basal side (Fig. 1 A and B). We identified Phosphatidylinositol synthase (Pis), an evolutionarily conserved enzyme involved in the synthesis of PtdIns, as a factor critical for restricted secretion of BM proteins. In addition, we show that the level of PIP2 is critical for this process. FCs with a reduced level of PIP2 allow an aberrant accumulation of BM components on both their apical and basal sides. Interestingly, a decrease of PIP2 significantly reduces the localization of Crag at the apical and lateral plasma membrane, indicating that the level of PIP2 also controls the subcellular distribution of Crag.Open in a separate windowFig. 1.Pis mutant FCs disrupt the polarized distribution of BM proteins. (A) Schematic representation of the FE with the different polarity domains indicated. AJ, adherens junction. (B–B′′′′) Longitudinal (Lg) section through a WT ovariole containing different egg chambers (B) and through the FC layer of a WT egg chamber (magnification of B indicated with the rectangular area, B′–B′′′′) expressing Coll IV–GFP (a α2–Coll IV–GFP protein-trap; green), a major component of the BM, which accumulates at the basal side of the cell. The egg chambers are stained with aPKC (an apical domain marker; red) and Discs Large (Dlg; a lateral domain marker; white), revealing the polarized structure of the epithelium, and DNA (blue). (B′) Apical (a) and basal (b) sides of FCs are marked. (C and D) Lg section through egg chambers containing Crag^CJ101 (C) and Pis^FM18 (D) mutant FC clones expressing Coll IV–GFP (green) and stained for F-Actin (red) and DNA (blue). (C, C′) In Crag mutant FCs, marked by the absence of intracellular GFP (green), the polarized distribution of BM is disrupted, as revealed by the strong accumulation of Coll IV–GFP at the apical side of the FCs (arrows). (D, D′) The same phenotype is observed in Pis mutant FCs, where Coll IV–GFP accumulates apically in aggregates (arrows). (E and F) Lg section through the FC layer of an egg chamber containing Pis clones, marked by the absence of intracellular GFP (green; clonal boundary indicated by a dashed line; WT and −/− homozygous mutant FCs are specified), coexpressing Coll IV–GFP (green, E) or Pcan–GFP (a Pcan–GFP protein-trap; green F), and stained for α1–Coll IV (red, E, E″), F-Actin (red, F and F″), and DNA (blue). In Pis mutant FCs, Coll IV (E–E″) and Pcan (F, F′) accumulate apically, indicating that Pis is required for restriction of BM deposition to the basal side. (Bars, 10 μm.)     

A single amino acid converts a repressor to an activator of flowering

Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.     

A purple acidophilic di-ferric DNA ligase from Ferroplasma

We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two Fe(3+)-tyrosinate centers and lacks any requirement for either Mg(2+) or K(+) for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require Mg(2+)/K(+) for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the Fe(3+) to Fe(2+) results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH.     

Pseudomonas fluorescens-like bacteria from the stomach:A microbiological and molecular study

AIM:To characterize oxidase-and urease-producing bacterial isolates,grown aerobically,that originated from antral biopsies of patients suffering from acid peptic diseases.METHODS:A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production,acid tolerance and aerobic growth.The selected isolates were further characterized by molecular techniques viz.amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers.The amplicons were subjected to restriction analysis and partial sequencing.A phylogenetic tree was generated using unweighted pair group method with arithmetic mean(UPGMA) from evolutionary distance computed with bootstrap test of phylogeny.Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10-7 mol/L to 10-1 mol/L.RESULTS:Of the 258 antral biopsy specimens collected from patients,179(69.4%) were positive for urease production by rapid urease test and 31%(80/258) yielded typical Helicobacter pylori(H.pylori) after 5-7 d of incubation under a microaerophilic environment.A total of 240(93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation.The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species.A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens(P.fluorescens).On the basis ofHSP60 sequences applied to the UPGMA phylogenetic tree,it was observed that isolated strains in an aerobic environment were likely to be P.fluorescens,and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences.Interestingly,this bacterium was acid tolerant for hours at low pH.Further,a total of 250(96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P.fluorescens-specific conserved putative out     

Introduced rats indirectly change marine rocky intertidal communities from algae- to invertebrate-dominated

It is widely recognized that trophic interactions structure ecological communities, but their effects are usually only demonstrated on a small scale. As a result, landscape-level documentations of trophic cascades that alter entire communities are scarce. Islands invaded by animals provide natural experiment opportunities both to measure general trophic effects across large spatial scales and to determine the trophic roles of invasive species within native ecosystems. Studies addressing the trophic interactions of invasive species most often focus on their direct effects. To investigate both the presence of a landscape-level trophic cascade and the direct and indirect effects of an invasive species, we examined the impacts of Norway rats (Rattus norvegicus) introduced to the Aleutian Islands on marine bird densities and marine rocky intertidal community structures through surveys conducted on invaded and rat-free islands throughout the entire 1,900-km archipelago. Densities of birds that forage in the intertidal were higher on islands without rats. Marine intertidal invertebrates were more abundant on islands with rats, whereas fleshy algal cover was reduced. Our results demonstrate that invasive rats directly reduce bird densities through predation and significantly affect invertebrate and marine algal abundance in the rocky intertidal indirectly via a cross-community trophic cascade, unexpectedly changing the intertidal community structure from an algae- to an invertebrate-dominated system.     

Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.     

A new flea from Iran

Fleas are obligatory ectoparsites of humans and animals. These tiny insects are hematophagous and they can transmit a wide varity of disease agents to humans and domesticated animals. Indeed, this pest causes a considerable economic damages and health dangers particularly in tropical and subtropical. During an investigation on ectoparasites of five Mus muscuuls in Semnan province, Iran, 15 fleas (8 males and 7 females) were collected. The extracted fleas mounted using clearing, dehydrating, mounting process and preserved with Canada balsam. After precise study, all of examined specimens were recognized as Leptopsylla aethiopicus aethiopicus using available systematic keys. This is the first report of this genus and species in Iran. And this country is new locality for Leptopsylla aethiopicus aethiopicus.     

Arabidopsis semidwarfs evolved from independent mutations in GA20ox1, ortholog to green revolution dwarf alleles in rice and barley

Understanding the genetic bases of natural variation for developmental and stress-related traits is a major goal of current plant biology. Variation in plant hormone levels and signaling might underlie such phenotypic variation occurring even within the same species. Here we report the genetic and molecular basis of semidwarf individuals found in natural Arabidopsis thaliana populations. Allelism tests demonstrate that independent loss-of-function mutations at GA locus 5 (GA5), which encodes gibberellin 20-oxidase 1 (GA20ox1) involved in the last steps of gibberellin biosynthesis, are found in different populations from southern, western, and northern Europe; central Asia; and Japan. Sequencing of GA5 identified 21 different loss-of-function alleles causing semidwarfness without any obvious general tradeoff affecting plant performance traits. GA5 shows signatures of purifying selection, whereas GA5 loss-of-function alleles can also exhibit patterns of positive selection in specific populations as shown by Fay and Wu’s H statistics. These results suggest that antagonistic pleiotropy might underlie the occurrence of GA5 loss-of-function mutations in nature. Furthermore, because GA5 is the ortholog of rice SD1 and barley Sdw1/Denso green revolution genes, this study illustrates the occurrence of conserved adaptive evolution between wild A.thaliana and domesticated plants.Bioactive gibberellins (GAs) are plant growth regulators involved in important traits such as seed germination, flowering time, flower development, and elongation growth (1). GA biosynthesis and signaling pathways are well defined (1, 2) and have been targeted in crop breeding. Modification of GA pathways was crucial in the green revolution because it conferred semidwarfness, thus reducing lodging and increasing crop yields (3–6). Green revolution semidwarf varieties in wheat are due to mutations in DELLA genes, whereas many short straw rice varieties carry a mutation in the Semi-Dwarf-1 (SD1) locus. This locus codes for GA 20-oxidase-2, a GA biosynthesis gene that is also mutated in most modern barley varieties in which the gene was called Denso or Semi-dwarf 1 (Sdw1) (7).GA 20-oxidases are involved in the later steps of GA biosynthesis and belong to the group of 2-oxoglutarate–dependent dioxygenases that, together with GA 3-oxidases, form biologically active GA (8). Arabidopsis thaliana has five GA20ox paralogous genes. AtGA20ox-1, AtGA20ox-2, AtGA20ox-3, and AtGA20ox-4 can catalyze the in vitro conversion of GA₁₂ to GA₉. Therefore, GA20ox paralogs might have partial redundant functions (9). However, among paralog genes, only AtGA20ox-1 (GA5), which was cloned on the basis of the ga5 mutant (10), affected plant height (8).Natural variation for GA biosynthesis has been previously described in A. thaliana because the Bur-0 accession carries a loss-of-function allele at GA20ox4 (9), which does not result in a semidwarf phenotype. In addition, genetic variation in GA1 has been associated with variation in floral morphology (11). Furthermore, the semidwarf phenotype (here defined as a plant height shorter than half the size of genetically related individuals) observed in the Kas-2 accession is due to a recessive allele at the GA5 locus (12). The latter finding led to the questions of whether green revolution alleles, artificially selected in cereals, could also occur in natural populations of the wild species A. thaliana, and if so, how many different GA5 loss-of-function alleles exist, how they are distributed, and why they occur in some populations.     

Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination

Manipulation of the host’s ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeA_Dub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Ų of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8.Ubiquitin, a small, 76-aa protein modifier, is involved in a wide array of eukaryotic cellular processes. The functionality of ubiquitin depends on the precise timing of the conjugation/deconjugation of the C terminus of ubiquitin to the ε-amino group of a lysine residue of a target protein. At the heart of this process are ligases (responsible for the covalent attachment of ubiquitin) and deubiquitinases (DUBs), which function to cleave isopeptide bonds between ubiquitin and substrates or within polyubiquitin chains (1). Even though many eukaryotic DUBs have already been characterized, little is known of these enzymes in prokaryotes (1–3).Given the essential role of ubiquitination in eukaryotic cells, it is not surprising that infectious agents have evolved numerous elegant strategies to exploit host signaling mediated by ubiquitination. Many bacterial pathogens use virulence factors to hijack the host ubiquitin pathway to establish successful infections (4). Even though E3 ubiquitin ligases of bacterial or viral origin have been relatively well characterized, bacterial DUBs have not, despite their importance in the life cycles and pathogenicity of several microbial species, including Salmonella typhimurium (SseL), Chlamydia trachomatis (ChlaDub1 and ChlaDub2), and ElaD (Escherichia coli) (5–7). These bacterial DUBs belong to a larger group of peptidases called the CE clan (the MEROPS database), which comprises eukaryotic, bacterial, and viral representatives (7, 8). Although quite different from eukaryotic DUBs (<10% identity), bacterial DUBs are phylogenetically related to mammalian desumoylating enzymes (SENP1, 2, and 3) and the deneddylase Den1 (NEDP1 or SENP8). In light of a large divergence from their eukaryotic counterparts, the overall structure of these proteases, as well as how they function and recognize ubiquitin to act as DUBs, remains to be structurally analyzed.Modulation of host ubiquitination pathways has emerged as an important theme in the pathogenesis of the opportunistic pathogen Legionella pneumophila responsible for Legionnaires’ disease (4, 9). Ubiquitinated species are enriched on the L. pneumophila-containing vacuole (LCV), and interference with such association disturbs bacterial intracellular replication (10). Among the approximately 300 effectors injected into host cells by L. pneumophila via the Dot/Icm type IV secretion system (11), eight proteins appear to possess F-box or U-box domains typical of some E3 ligases (12–16). This ligase activity has been demonstrated for LegU1, LegAU13/AnkB, and LubX (14, 17). A recent study revealed that SidC and SdcA are E3 ligases that catalyze the ligation reaction with a unique mechanism, and are required for efficient enrichment of ubiquitinated species on the bacterial phagosome (18).Because balanced regulation of host cell processes is critical for the virulence of L. pneumophila (19), we initiated experiments to identify L. pneumophila proteins with DUB activity. Our efforts revealed that members of the SidE family contain a DUB domain, which catalyzes the reaction with a Cys-His-Asp (CHD) catalytic triad showing a preference for Lys63-linked polyubiquitin chains. Structural analysis of the DUB domain and its complex with the mechanism-based inhibitor, ubiquitin vinyl methyl ester (Ub-VME), revealed a canonical core ubiquitin-like protease (Ulp) fold with a ubiquitin interface that is quite different from those used by structurally characterized eukaryotic DUBs. We also found that although the DUB activity is dispensable for the SidE family’s role in intracellular bacterial replication, it is important for the dynamics of the association of ubiquitinated species with the bacterial phagosome.     

Beneficial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress

Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases the expression of oxidation-sensitive responsive genes (such as ELK-1 and p-JUN) in the endothelium. Evidence suggests that polyphenolic antioxidants contained in the juice derived from the pomegranate can contribute to the reduction of oxidative stress and atherogenesis. The aim of the present study was to evaluate the effects of intervention with pomegranate juice (PJ) on oxidation-sensitive genes and endothelial NO synthase (eNOS) expression induced by high shear stress in vitro and in vivo. Cultured human coronary artery endothelial cells (EC) exposed to high shear stress in vitro and hypercholesterolemic mice were used in this study. PJ concentrate reduced the activation of redox-sensitive genes (ELK-1 and p-JUN) and increased eNOS expression (which was decreased by perturbed shear stress) in cultured EC and in atherosclerosis-prone areas of hypercholesterolemic mice. Moreover, oral administration of PJ to hypercholesterolemic mice at various stages of disease reduced significantly the progression of atherosclerosis. This experimental study indicates that the proatherogenic effects induced by perturbed shear stress can be reversed by chronic administration of PJ. This approach may have implications for the prevention or treatment of atherosclerosis and its clinical manifestations.