Sample records for acyl-homoserine lactone hsl

  1. N-acyl-homoserine lactone-mediated quorum-sensing in Azospirillum: an exception rather than a rule.

    PubMed

    Vial, Ludovic; Cuny, Caroline; Gluchoff-Fiasson, Katia; Comte, Gilles; Oger, Phil M; Faure, Denis; Dessaux, Yves; Bally, René; Wisniewski-Dyé, Florence

    2006-11-01

    Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.

  2. Long Chain N-acyl Homoserine Lactone Production by Enterobacter sp. Isolated from Human Tongue Surfaces

    PubMed Central

    Yin, Wai-Fong; Purmal, Kathiravan; Chin, Shenyang; Chan, Xin-Yue; Chan, Kok-Gan

    2012-01-01

    We report the isolation of N-acyl homoserine lactone-producing Enterobacter sp. isolate T1-1 from the posterior dorsal surfaces of the tongue of a healthy individual. Spent supernatants extract from Enterobacter sp. isolate T1-1 activated the biosensor Agrobacterium tumefaciens NTL4(pZLR4), suggesting production of long chain AHLs by these isolates. High resolution mass spectrometry analysis of these extracts confirmed that Enterobacter sp. isolate T1-1 produced a long chain N-acyl homoserine lactone, namely N-dodecanoyl-homoserine lactone (C12-HSL). To the best of our knowledge, this is the first isolation of Enterobacter sp., strain T1-1 from the posterior dorsal surface of the human tongue and N-acyl homoserine lactones production by this bacterium. PMID:23202161

  3. Pseudomonas cremoricolorata Strain ND07 Produces N-acyl Homoserine Lactones as Quorum Sensing Molecules

    PubMed Central

    Yunos, Nina Yusrina Muhamad; Tan, Wen-Si; Koh, Chong-Lek; Sam, Choon-Kook; Mohamad, Nur Izzati; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Quorum sensing (QS) is a bacterial cell-to-cell communication system controlling QS-mediated genes which is synchronized with the population density. The regulation of specific gene activity is dependent on the signaling molecules produced, namely N-acyl homoserine lactones (AHLs). We report here the identification and characterization of AHLs produced by bacterial strain ND07 isolated from a Malaysian fresh water sample. Molecular identification showed that strain ND07 is clustered closely to Pseudomonas cremoricolorata. Spent culture supernatant extract of P. cremoricolorata strain ND07 activated the AHL biosensor Chromobacterium violaceum CV026. Using high resolution triple quadrupole liquid chromatography-mass spectrometry, it was confirmed that P. cremoricolorata strain ND07 produced N-octanoyl-l-homoserine lactone (C8-HSL) and N-decanoyl-l-homoserine lactone (C10-HSL). To the best of our knowledge, this is the first documentation on the production of C10-HSL in P. cremoricolorata strain ND07. PMID:24984061

  4. Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions.

    PubMed

    Nieto Penalver, Carlos G; Morin, Danièle; Cantet, Franck; Saurel, Olivier; Milon, Alain; Vorholt, Julia A

    2006-01-23

    Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

  5. N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

    PubMed

    Welch, M; Todd, D E; Whitehead, N A; McGowan, S J; Bycroft, B W; Salmond, G P

    2000-02-15

    Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

  6. Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium.

    PubMed

    Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Sa, Tongmin

    2007-02-01

    Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).

  7. Rapid Acyl-Homoserine Lactone Quorum Signal Biodegradation in Diverse Soils†

    PubMed Central

    Wang, Ya-Juan; Leadbetter, Jared Renton

    2005-01-01

    Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 μM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol · h−1 · g of fresh weight soil−1. Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 × 105 cells · g of turf soil−1 degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems. PMID:15746331

  8. Identification, synthesis and regulatory function of the N-acylated homoserine lactone signals produced by Pseudomonas chlororaphis HT66.

    PubMed

    Peng, Huasong; Ouyang, Yi; Bilal, Muhammad; Wang, Wei; Hu, Hongbo; Zhang, Xuehong

    2018-01-22

    Pseudomonas chlororaphis HT66 isolated from the rice rhizosphere is an important plant growth-promoting rhizobacteria that produce phenazine-1-carboxamide (PCN) in high yield. Phenazine production is regulated by a quorum sensing (QS) system that involves the N-acylated homoserine lactones (AHLs)-a prevalent type of QS molecule. Three QS signals were detected by thin layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS), which identified to be N-(3-hydroxy hexanoyl)-L-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy octanoyl)-L-homoserine lactone (3-OH-C8-HSL) and N-(3-hydroxy decanoyl)-L-homoserine lactone (3-OH-C10-HSL). The signal types and methods of synthesis were different from that in other phenazine-producing Pseudomonas strains. By non-scar deletion and heterologous expression techniques, the biosynthesis of the AHL-signals was confirmed to be only catalyzed by PhzI, while other AHLs synthases i.e., CsaI and HdtS were not involved in strain HT66. In comparison to wild-type HT66, PCN production was 2.3-folds improved by over-expression of phzI, however, phzI or phzR mutant did not produce PCN. The cell growth of HT66∆phzI mutant was significantly decreased, and the biofilm formation in phzI or phzR inactivated strains of HT66 decreased to various extents. In conclusion, the results demonstrate that PhzI-PhzR system plays a critical role in numerous biological processes including phenazine production.

  9. Pantoea sp. Isolated from Tropical Fresh Water Exhibiting N-Acyl Homoserine Lactone Production

    PubMed Central

    Tan, Wen-Si; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry. PMID:25197715

  10. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography

    PubMed Central

    Shaw, Paul D.; Ping, Gao; Daly, Sean L.; Cha, Chung; Cronan, John E.; Rinehart, Kenneth L.; Farrand, Stephen K.

    1997-01-01

    Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-l-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-l-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-l-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules. PMID:9177164

  11. The algicidal activity of Aeromonas sp. strain GLY-2107 against bloom-forming Microcystis aeruginosa is regulated by N-acyl homoserine lactone-mediated quorum sensing.

    PubMed

    Guo, Xingliang; Liu, Xianglong; Wu, Lishuang; Pan, Jianliang; Yang, Hong

    2016-11-01

    Cyanobacterial blooms have disrupted the efficient utilization of freshwater worldwide. A new freshwater bacterial strain with strong algicidal activity, GLY-2107, was isolated from Lake Taihu and identified as Aeromonas sp. It produced two algicidal compounds: 2107-A (3-benzyl-piperazine-2,5-dione) and 2107-B (3-methylindole). Both compounds exhibited potent algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. The EC 50 values (concentration for 50% maximal effect) of 3-benzyl-piperazine-2,5-dione and 3-methylindole were 4.72 and 1.10 μg ml -1 respectively. Based on a thin-layer chromatography biosensor assay and ultra-performance liquid chromatography-coupled high resolution-tandem mass spectrometry (UPLC-HRMS/MS), the N-acyl homoserine lactone (AHL) profile of strain GLY-2107 was identified as two short side-chain AHLs: N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL). The production of the two algicidal compounds was controlled by AHL-mediated quorum sensing (QS), and C4-HSL was the key QS signal for the algicidal activity of the strain GLY-2107. Moreover, 3-methylindole was found to be positively regulated by C4-HSL-mediated QS, whereas 3-benzyl-piperazine-2,5-dione might be negatively controlled by C4-HSL-mediated QS. This study suggests that a QS-regulated algicidal system may have potential use for the development of a novel control strategy for harmful cyanobacterial blooms. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants.

    PubMed

    Götz, Christine; Fekete, Agnes; Gebefuegi, Istvan; Forczek, Sándor T; Fuksová, Kvetoslava; Li, Xiaojing; Englmann, Matthias; Gryndler, Milan; Hartmann, Anton; Matucha, Miroslav; Schmitt-Kopplin, Philippe; Schröder, Peter

    2007-11-01

    Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.

  13. N-Acyl-Homoserine Lactone Primes Plants for Cell Wall Reinforcement and Induces Resistance to Bacterial Pathogens via the Salicylic Acid/Oxylipin Pathway[C][W][OPEN

    PubMed Central

    Schenk, Sebastian T.; Hernández-Reyes, Casandra; Samans, Birgit; Stein, Elke; Neumann, Christina; Schikora, Marek; Reichelt, Michael; Mithöfer, Axel; Becker, Annette; Kogel, Karl-Heinz; Schikora, Adam

    2014-01-01

    The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection. PMID:24963057

  14. Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

    PubMed

    Mellbye, Brett L; Spieck, Eva; Bottomley, Peter J; Sayavedra-Soto, Luis A

    2017-11-15

    The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl-l-homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl-l-homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with

  15. Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.

    PubMed

    de Almeida, Felipe Alves; Pimentel-Filho, Natan de Jesus; Carrijo, Lanna Clícia; Bento, Cláudia Braga Pereira; Baracat-Pereira, Maria Cristina; Pinto, Uelinton Manoel; de Oliveira, Leandro Licursi; Vanetti, Maria Cristina Dantas

    2017-01-01

    Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Paraoxonase 2 modulates a proapoptotic function in LS174T cells in response to quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone

    PubMed Central

    Tao, Shiyu; Luo, Yanwen; Bin He; Liu, Jie; Qian, Xi; Ni, Yingdong; Zhao, Ruqian

    2016-01-01

    A mucus layer coats the gastrointestinal tract and serves as the first line of intestinal defense against infection. N-acyl-homoserine lactone (AHL) quorum-sensing molecules produced by gram-negative bacteria in the gut can influence the homeostasis of intestinal epithelium. In this study, we investigated the effects of two representative long- and short-chain AHLs, N-3-(oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl homoserine lactone (C4-HSL), on cell viability and mucus secretion in LS174T cells. C12-HSL but not C4-HSL significantly decreased cell viability by inducing mitochondrial dysfunction and activating cell apoptosis which led to a decrease in mucin expression. Pretreatment with lipid raft disruptor (Methyl-β-cyclodextrin, MβCD) and oxidative stress inhibitor (N-acetyl-L-cysteine, NAC) slightly rescued the viability of cells damaged by C12-HSL exposure, while the paraoxonase 2 (PON2) inhibitor (Triazolo[4,3-a]quinolone, TQ416) significantly affected recovering cells viability and mucin secretion. When LS174T cells were treated with C12-HSL and TQ416 simultaneously, TQ416 showed the maximal positive effect on cells viability. However, if cells were first treated with C12-HSL for 40 mins, and then TQ46 was added, the TQ416 had no effect on cell viability. These results suggest that the C12-HSL-acid process acts at an early step to activate apoptosis as part of C12-HSL’s effect on intestinal mucus barrier function. PMID:27364593

  17. N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens.

    PubMed

    Hernández-Reyes, Casandra; Schenk, Sebastian T; Neumann, Christina; Kogel, Karl-Heinz; Schikora, Adam

    2014-11-01

    The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance-inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S. meliloti to induce resistance depends on the production of the quorum-sensing molecule, oxo-C14-HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis-Salmonella model, we also demonstrate that the application of N-acyl-homoserine lactones-producing bacteria could be a successful strategy to prevent plant-originated infections with human pathogens. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean.

    PubMed

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants' pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  19. Influence of bacterial N-acyl-homoserine lactones on growth parameters, pigments, antioxidative capacities and the xenobiotic phase II detoxification enzymes in barley and yam bean

    PubMed Central

    Götz-Rösch, Christine; Sieper, Tina; Fekete, Agnes; Schmitt-Kopplin, Philippe; Hartmann, Anton; Schröder, Peter

    2015-01-01

    Bacteria are able to communicate with each other and sense their environment in a population density dependent mechanism known as quorum sensing (QS). N-acyl-homoserine lactones (AHLs) are the QS signaling compounds of Gram-negative bacteria which are frequent colonizers of rhizospheres. While cross-kingdom signaling and AHL-dependent gene expression in plants has been confirmed, the responses of enzyme activities in the eukaryotic host upon AHLs are unknown. Since AHL are thought to be used as so-called plant boosters or strengthening agents, which might change their resistance toward radiation and/or xenobiotic stress, we have examined the plants’ pigment status and their antioxidative and detoxifying capacities upon AHL treatment. Because the yield of a crop plant should not be negatively influenced, we have also checked for growth and root parameters. We investigated the influence of three different AHLs, namely N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL), and N-decanoyl- homoserine lactone (C10-HSL) on two agricultural crop plants. The AHL-effects on Hordeum vulgare (L.) as an example of a monocotyledonous crop and on the tropical leguminous crop plant Pachyrhizus erosus (L.) were compared. While plant growth and pigment contents in both plants showed only small responses to the applied AHLs, AHL treatment triggered tissue- and compound-specific changes in the activity of important detoxification enzymes. The activity of dehydroascorbate reductase in barley shoots after C10-HSL treatment for instance increased up to 384% of control plant levels, whereas superoxide dismutase activity in barley roots was decreased down to 23% of control levels upon C6-HSL treatment. Other detoxification enzymes reacted similarly within this range, with interesting clusters of positive or negative answers toward AHL treatment. In general the changes on the enzyme level were more severe in barley than in yam bean which might be due to the different abilities of the plants to

  20. Changes in the Salmonella enterica Enteritidis phenotypes in presence of acyl homoserine lactone quorum sensing signals.

    PubMed

    Campos-Galvão, Maria Emilene Martino; Ribon, Andrea Oliveira Barros; Araújo, Elza Fernandes; Vanetti, Maria Cristina Dantas

    2016-05-01

    Quorum sensing is used by bacteria to coordinate gene expression in response to population density and involves the production, detection and response to extracellular signaling molecules known as autoinducers (AIs). Salmonella does not synthesize the AI-1, acyl homoserine lactone (AHL) common to gram-negative bacteria; however, it has a receptor for AI-1, the SdiA protein. The effect of SdiA in modulating phenotypes of Salmonella has not been elucidated. In this report, we provide evidence that the AIs-1 affect Salmonella enterica serovar Enteritidis behavior by enhancing the biofilm formation and expression of virulence genes under anaerobic conditions. Biofilm formation by Salmonella was detected by the crystal violet method and by scanning electron microscopy. The presence of AHLs, particularly C12-HSL, increased biofilm formation and promoted expression of biofilm formation genes (lpfA, fimF, fliF, glgC) and virulence genes (hilA, invA, invF). Our results demonstrated that AHLs produced by other organisms played an important role in virulence phenotypes of Salmonella Enteritidis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Profile of Citrobacter freundii ST2, a Multi-acyl-homoserine Lactone Producer Associated with Marine Dinoflagellates.

    PubMed

    Huang, Xinqi; Gao, Yan; Ma, Zhiping; Lin, Guanghui; Cai, Zhonghua; Zhou, Jin

    2017-01-01

    Marine algae provide a unique niche termed the phycosphere for microorganism inhabitation. The phycosphere environment is an important niche for mutualistic and competitive interactions between algae and bacteria. Quorum sensing (QS) serves as a gene regulatory system in the microbial biosphere that allows bacteria to sense the population density with signaling molecules, such as acyl-homoserine lactone (AHL), and adapt their physiological activities to their surroundings. Understanding the QS system is important to elucidate the interactions between algal-associated microbial communities in the phycosphere condition. In this study, we isolated an epidermal bacterium (ST2) from the marine dinoflagellate Scrippsiella trochoidea and evaluated its AHL production profile. Strain ST2 was classified as a member of the genus Citrobacter closely related to Citrobacter freundii by 16S rRNA gene sequence analysis. Thin-layer chromatography revealed that C. freundii ST2 secreted three active AHL compounds into the culture supernatant. Specific compounds, such as N-butyryl-L-homoserine lactone (C4-AHL), N-octanoyl-DL-homoserine lactone (C8-AHL), and N-decanoyl-DL-homoserine lactone (C10-AHL), were identified by high-resolution tandem mass spectrometry. Carbon metabolic profiling with Biolog EcoPlate™ indicated that C. freundii ST2 was widely used as a carbon source and preferred carbohydrates, amino acids, and carboxylic acids as carbon substrates. Our results demonstrated that C. freundii ST2 is a multi-AHL producer that participates in the phycosphere carbon cycle.

  2. Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

    PubMed

    Nieto-Peñalver, Carlos G; Bertini, Elisa V; de Figueroa, Lucía I C

    2012-07-01

    The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

  3. O-Succinyl-L-homoserine-based C4-chemical production: succinic acid, homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives, and 1,4-butanediol.

    PubMed

    Hong, Kuk-Ki; Kim, Jeong Hyun; Yoon, Jong Hyun; Park, Hye-Min; Choi, Su Jin; Song, Gyu Hyeon; Lee, Jea Chun; Yang, Young-Lyeol; Shin, Hyun Kwan; Kim, Ju Nam; Cho, Kyung Ho; Lee, Jung Ho

    2014-10-01

    There has been a significant global interest to produce bulk chemicals from renewable resources using engineered microorganisms. Large research programs have been launched by academia and industry towards this goal. Particularly, C4 chemicals such as succinic acid (SA) and 1,4-butanediol have been leading the path towards the commercialization of biobased technology with the effort of replacing chemical production. Here we present O-Succinyl-L-homoserine (SH) as a new, potentially important platform biochemical and demonstrate its central role as an intermediate in the production of SA, homoserine lactone (HSL), γ-butyrolactone (GBL) and its derivatives, and 1,4-butanediol (BDO). This technology encompasses (1) the genetic manipulation of Escherichia coli to produce SH with high productivity, (2) hydrolysis into SA and homoserine (HS) or homoserine lactone hydrochloride, and (3) chemical conversion of either HS or homoserine lactone HCL (HSL·HCl) into drop-in chemicals in polymer industry. This production strategy with environmental benefits is discussed in the perspective of targeting of fermented product and a process direction compared to petroleum-based chemical conversion, which may reduce the overall manufacturing cost.

  4. Acyl-homoserine lactone quorum sensing: from evolution to application.

    PubMed

    Schuster, Martin; Sexton, D Joseph; Diggle, Stephen P; Greenberg, E Peter

    2013-01-01

    Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

  5. The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase

    PubMed Central

    Kim, Myung Hee; Choi, Won-Chan; Kang, Hye Ok; Lee, Jong Suk; Kang, Beom Sik; Kim, Kyung-Jin; Derewenda, Zygmunt S.; Oh, Tae-Kwang; Lee, Choong Hwan; Lee, Jung-Kee

    2005-01-01

    In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-l-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-Å resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-Å crystal structure of its complex with l-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-β-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases. PMID:16314577

  6. A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

    PubMed

    Veliz-Vallejos, Debora F; van Noorden, Giel E; Yuan, Mengqi; Mathesius, Ulrike

    2014-01-01

    N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

  7. Activity of the Rhodopseudomonas palustris p-coumaroyl-homoserine lactone-responsive transcription factor RpaR.

    PubMed

    Hirakawa, Hidetada; Oda, Yasuhiro; Phattarasukol, Somsak; Armour, Christopher D; Castle, John C; Raymond, Christopher K; Lappala, Colin R; Schaefer, Amy L; Harwood, Caroline S; Greenberg, E Peter

    2011-05-01

    The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

  8. Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

    PubMed Central

    Gram, Lone; Christensen, Allan Beck; Ravn, Lars; Molin, Søren; Givskov, Michael

    1999-01-01

    Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 106 CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N2 atmosphere when mean cell densities increased to 106 CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 106 CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity. PMID:10427034

  9. Acyl-homoserine-lactone autoinducer (AHL) in the gastrointestinal tract of feedlot cattle and correlation to season, E. coli 0157:H7 prevalence and diet

    USDA-ARS?s Scientific Manuscript database

    Acyl-homoserine-lactone autoinducer (AHL) produced by non-enterohemorrhagic E. coli (EHEC) species in cattle appears to be required for EHEC colonization of the gastro¬intestinal tract (GIT). The objectives of the current research were to examine the effect of season, diet, EHEC shedding, and locat...

  10. Diverse profiles of N-acyl-homoserine lactones in biofilm forming strains of Cronobacter sakazakii.

    PubMed

    Singh, Niharika; Patil, Amrita; Prabhune, Asmita A; Raghav, Mamta; Goel, Gunjan

    2017-04-03

    The present study investigates the role of quorum sensing (QS) molecules expressed by C. sakazakii in biofilm formation and extracellular polysaccharide expression. The QS signaling was detected using Chromobacterium violaceum 026 and Agrobacterium tumefaciens NTL4(pZLR4) based bioassay. Long chain N-acyl-homoserine lactones (AHLs) with C6- C18 chain length were identified using High Performance Liquid Chromatography and Liquid Chromatography-High Resolution Mass Spectrometry. A higher Specific Biofilm Formation (SBF) index (p < 0.05) with the presence of genes associated with cellulose biosynthesis (bcsA, bcsC and bcsG) was observed in the strains. AHLs and their mechanisms can serve as novel targets for developing technologies to eradicate and prevent biofilm formation by C. sakazakii.

  11. Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR ▿ †

    PubMed Central

    Hirakawa, Hidetada; Oda, Yasuhiro; Phattarasukol, Somsak; Armour, Christopher D.; Castle, John C.; Raymond, Christopher K.; Lappala, Colin R.; Schaefer, Amy L.; Harwood, Caroline S.; Greenberg, E. Peter

    2011-01-01

    The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His6-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon. PMID:21378182

  12. Global and Phylogenetic Distribution of Quorum Sensing Signals, Acyl Homoserine Lactones, in the Family of Vibrionaceae

    PubMed Central

    Barker Rasmussen, Bastian; Fog Nielsen, Kristian; Machado, Henrique; Melchiorsen, Jette; Gram, Lone; Sonnenschein, Eva C.

    2014-01-01

    Bacterial quorum sensing (QS) and the corresponding signals, acyl homoserine lactones (AHLs), were first described for a luminescent Vibrio species. Since then, detailed knowledge has been gained on the functional level of QS; however, the abundance of AHLs in the family of Vibrionaceae in the environment has remained unclear. Three hundred and one Vibrionaceae strains were collected on a global research cruise and the prevalence and profile of AHL signals in this global collection were determined. AHLs were detected in 32 of the 301 strains using Agrobacterium tumefaciens and Chromobacterium violaceum reporter strains. Ethyl acetate extracts of the cultures were analysed by ultra-high performance liquid chromatography-high resolution mass spectrometry (MS) with automated tandem MS confirmation for AHLs. N-(3-hydroxy-hexanoyl) (OH-C6) and N-(3-hydroxy-decanoyl) (OH-C10) homoserine lactones were the most common AHLs found in 17 and 12 strains, respectively. Several strains produced a diversity of different AHLs, including N-heptanoyl (C7) HL. AHL-producing Vibrionaceae were found in polar, temperate and tropical waters. The AHL profiles correlated with strain phylogeny based on gene sequence homology, however not with geographical location. In conclusion, a wide range of AHL signals are produced by a number of clades in the Vibrionaceae family and these results will allow future investigations of inter- and intra-species interactions within this cosmopolitan family of marine bacteria. PMID:25419995

  13. Acyl-homoserine lactones produced by Pantoea sp. isolated from the "maize white spot" foliar disease.

    PubMed

    Pomini, Armando M; Paccola-Meirelles, Luzia D; Marsaioli, Anita J

    2007-02-21

    The "maize white spot" foliar disease is a problem of increasing importance to Brazilian maize crops. A bacterium isolated from water-soaked lesions from infected maize leaves was pathogenic in biological assays in vivo. It was identified as a Gram-negative, nonsporulating, facultative anaerobic bacterium, belonging to the genus Pantoea. Chemical study of the extracts from bacterial cultivation media allowed the identification of (S)-(-)-N-butanoyl-homoserine lactone and trace amounts of N-hexanoyl-homoserine lactone, widely recognized quorum-sensing signaling substances employed in cell-to-cell communication systems. The absolute configuration of natural (S)-(-)-N-butanoyl-homoserine lactone was determined by gas chromatography-flame ionization detection with a chiral stationary phase and by comparison of circular dichroism spectroscopic data with enantiopure synthetic substances. Biological evaluations with reporter Agrobacterium tumefaciens NTL4(pZLR4) were carried out with synthetic and natural products and also with extracts from maize leaves contaminated with the isolated bacterium, as well as from healthy leaves.

  14. A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

    PubMed

    Jiang, Donglei; Liu, Yan; Jiang, Hui; Rao, Shengqi; Fang, Wu; Wu, Mangang; Yuan, Limin; Fang, Weiming

    2018-04-15

    A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). Experimental results show that 3OC 12 -HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC 12 -HSL in the range of 0.1-1μM, and the detection limit for 3OC 12 -HSL was calculated to be 0.094μM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

    PubMed Central

    See-Too, Wah Seng; Ee, Robson; Lim, Yan-Lue; Convey, Peter; Pearce, David A.; Yin, Wai-Fong; Chan, Kok-Gan

    2017-01-01

    Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions. PMID:28225085

  16. Preliminary study on an innovative, simple mast cell-based electrochemical method for detecting foodborne pathogenic bacterial quorum signaling molecules (N-acyl-homoserine-lactones).

    PubMed

    Jiang, Donglei; Feng, Dongdong; Jiang, Hui; Yuan, Limin; Yongqi, Yin; Xu, Xin; Fang, Weiming

    2017-04-15

    This paper reports the a novel and simple mast cell-based electrochemical method for detecting of bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs), which can be utilized to preliminarily evaluate the toxicity of food-borne pathogenic bacteria. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide hydrogel were immobilized on a gold electrode, while mast cells as recognition elements were cultured in a 3D cell culture system. Electrochemical impedance spectroscopy (EIS) was utilized to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). The results indicated that cellular activities such as cell viability, apoptosis, intracellular calcium, and degranulation were markedly influenced by the AHLs. Importantly, the exposure of 3OC 12 -HSL to mast cells induced a marked decrease in the electrochemical impedance signal in a dose-dependent manner. The detection limit for 3OC 12 -HSL was 0.034μM with a linear range of 0.1-1μM. These results were confirmed via conventional cell assay and transmission electron microscope (TEM) analysis. Altogether, the proposed method appears to be an innovative and effective approach to the quantitative measurement of Gram-negative bacterial quorum signaling molecules; to this effect, it also may serve as a primary evaluation of the cytotoxicity of food-borne pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Characterization of N-Acylhomoserine Lactones Produced by Bacteria Isolated from Industrial Cooling Water Systems.

    PubMed

    Okutsu, Noriya; Morohoshi, Tomohiro; Xie, Xiaonan; Kato, Norihiro; Ikeda, Tsukasa

    2015-12-30

    The cooling water systems are used to remove heat generated in the various industries. Biofouling of the cooling water systems causes blocking of condenser pipes and the heat exchanger tubes. In many Gram-negative bacteria, N-acylhomoserine lactone (AHL) are used as quorum-sensing signal molecule and associated with biofilm formation. To investigate the relationship between quorum sensing and biofouling in the cooling water system, we isolated a total of 192 bacterial strains from the five cooling water systems, and screened for AHL production. Seven isolates stimulated AHL-mediated purple pigment production in AHL reporter strain Chromobacterium violaceum CV026 or VIR07. Based on their 16S rRNA gene sequences, AHL-producing isolates were assigned to Aeromonas hydrophila, Lysobacter sp., Methylobacterium oryzae, and Bosea massiliensis. To the best of our knowledge, B. massiliensis and Lysobacter sp. have not been reported as AHL-producing species in the previous researches. AHLs extracted from the culture supernatants of B. massiliensis and Lysobacter sp. were identified by liquid chromatography-mass spectrometry. AHLs produced by B. massiliensis were assigned as N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), and N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL). AHLs produced by Lysobacter sp. were assigned as N-decanoyl-L-homoserine lactone (C10-HSL) and N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL). This is the first report of identification of AHLs produced by B. massiliensis and Lysobacter sp. isolated from the cooling water system.

  18. Cellular effects of bacterial N-3-Oxo-dodecanoyl-L-Homoserine lactone on the sponge Suberites domuncula (Olivi, 1792): insights into an intimate inter-kingdom dialogue.

    PubMed

    Gardères, Johan; Henry, Joël; Bernay, Benoit; Ritter, Andrès; Zatylny-Gaudin, Céline; Wiens, Matthias; Müller, Werner E G; Le Pennec, Gaël

    2014-01-01

    Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on

  19. Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family

    PubMed Central

    Purohit, AA; Johansen, J A; Hansen, H; Leiros, H-KS; Kashulin, A; Karlsen, C; Smalås, A; Haugen, P; Willassen, NP

    2013-01-01

    Aims The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny. Methods and Results We used a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio,Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25–5000 nmol l−1. Conclusions This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles. Significance and Impact of the Study The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains. PMID:23725044

  20. Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

    PubMed

    Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

    2015-06-01

    N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

  1. A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

    PubMed Central

    2009-01-01

    Background The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria. Results An aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity. Conclusion This is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum

  2. Regulation of acylated homoserine lactones (AHLs) in beef by spice marination.

    PubMed

    Gopu, Venkadesaperumal; Shetty, Prathapkumar Halady

    2016-06-01

    Quorum sensing (QS) is a signaling mechanism used by bacteria to communicate each other through the release of auto-inducing signaling molecules. Despite the fact that bacteria regulate its phenotypes by QS mechanism, their potential role in meat spoilage is not yet elucidated. In the current study, beef samples were analyzed for its microbial association and for the presence of N-acyl-homoserine-lactone (AHLs) throughout the storage experiments. Isolates were screened for AHLs production and selected spices were screened for their quorum sensing inhibitory (QSI) activity. In addition, effect of spices on AHLs production of Y. enterocolitica was quantified through high performance thin layer chromatography (HP-TLC). Outcome showed that microbial association of beef mainly consists of lactic acid bacteria (LAB) and Enterobacteriaceae. Samples stored at both aerobic and modified atmospheric packaging (MAP) exhibited higher counts whereas; marinated samples stored at MAP exhibited the lowest. It was found that out of 35 isolates Y. enterocolitica induced reporter strain CV026 and its cell-free supernatant contained 26.36 nM/100 ml of AHLs when compared to standard. Among the tested spices, C. cyminum exhibited pronounced results by significantly reducing the AHLs concentration up to 47.75 %. Findings revealed the presence of quorum molecules (AHLs) in beef meat throughout the spoilage process and spices can acts as quorum quenchers to influence the spoilage rate by reducing AHLs production.

  3. Possible Quorum Sensing in Marine Snow Bacteria: Production of Acylated Homoserine Lactones by Roseobacter Strains Isolated from Marine Snow

    PubMed Central

    Gram, Lone; Grossart, Hans-Peter; Schlingloff, Andrea; Kiørboe, Thomas

    2002-01-01

    We report here, for the first time, that bacteria associated with marine snow produce communication signals involved in quorum sensing in gram-negative bacteria. Four of 43 marine microorganisms isolated from marine snow were found to produce acylated homoserine lactones (AHLs) in well diffusion and thin-layer chromatographic assays based on the Agrobacterium tumefaciens reporter system. Three of the AHL-producing strains were identified by 16S ribosomal DNA gene sequence analysis as Roseobacter spp., and this is the first report of AHL production by these α-Proteobacteria. It is likely that AHLs in Roseobacter species and other marine snow bacteria govern phenotypic traits (biofilm formation, exoenzyme production, and antibiotic production) which are required mainly when the population reaches high densities, e.g., in the marine snow community. PMID:12147515

  4. N-(3-oxododecanoyl)-l-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells.

    PubMed

    Tao, Shiyu; Niu, Liqiong; Cai, Liuping; Geng, Yali; Hua, Canfeng; Ni, Yingdong; Zhao, Ruqian

    2018-05-15

    The quorum-sensing molecule N‑(3‑oxododecanoyl)‑l‑homoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T goblet cells. In this study, the effects of 100 μM C12-HSL on mitochondrial function and cell proliferation in LS174T cells treated for 4 h were evaluated by real-time PCR, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (P < 0.05) in LS174T cells after C12-HSL treatment, with elevated intracellular ATP generation (P < 0.05). Flow cytometry analysis revealed significantly increased intracellular Ca 2+ levels (P < 0.05), as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (P < 0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1 μM) remarkably reversed the above C12-HSL associated effects in LS174T cells. These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Impact of phenolic compounds in the acyl homoserine lactone-mediated quorum sensing regulatory pathways.

    PubMed

    Hossain, Md Akil; Lee, Seung-Jin; Park, Na-Hye; Mechesso, Abraham Fikru; Birhanu, Biruk Tesfaye; Kang, JeongWoo; Reza, Md Ahsanur; Suh, Joo-Won; Park, Seung-Chun

    2017-09-06

    Quorum sensing (QS) is a cell density-dependent regulation of virulent bacterial gene expression by autoinducers that potentially pertains in the epidemic of bacterial virulence. This study was initially designed to evaluate the effect of 5 phenolic compounds in the modulation of QS and virulence factors of Chromobacterium violaceum and Pseudomonas aeruginosa, and to determine the mechanisms of their effects. Biosensor strains were used to assess antibacterial and anti-QS effect of these compounds. Only methyl gallate (MG) among these compounds demonstrated profound anti-QS effect in the preliminary study, and thus only MG was utilized further to evaluate the effects on the synthesis and activity of acyl homoserine lactone (AHL) in C. violaceum and on the modulation of biofilm, motility, proteolytic, elastase, pyocyanin, and rhamnolipid activity in P. aeruginosa. Finally, the effect of MG on the expression of QS-regulated genes of P. aeruginosa was verified. MG suppressed both the synthesis and activity of AHL in C. violaceum. It also restricted the biofilm formation and other QS-associated virulence factor of P. aeruginosa. MG concentration-dependently suppressed the expression of lasI/R, rhlI/R, and pqsA of P. aeruginosa and was non-toxic in in vitro study. This is the first report of the anti-QS mechanism of MG.

  6. Promotion of acute-phase skin wound healing by Pseudomonas aeruginosa C4 -HSL.

    PubMed

    Kanno, Emi; Kawakami, Kazuyoshi; Miyairi, Shinichi; Tanno, Hiromasa; Suzuki, Aiko; Kamimatsuno, Rina; Takagi, Naoyuki; Miyasaka, Tomomitsu; Ishii, Keiko; Gotoh, Naomasa; Maruyama, Ryoko; Tachi, Masahiro

    2016-12-01

    A Pseudomonas aeruginosa quorum-sensing system, which produces N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-l-homoserine lactone (C 4 -HSL), regulates the virulence factors. In our previous study, 3-oxo-C 12 -HSL, encoded by lasI gene, was shown to promote wound healing. However, the effect of C 4 -HSL, encoded by rhlI gene, remains to be elucidated. We addressed the effect of C 4 -HSL on wounds in P. aeruginosa infection. Wounds were created on the backs of Sprague-Dawley SD rats, and P. aeruginosa PAO1 (PAO1) or its rhlI deletion mutant (ΔrhlI) or lasI deletion mutant (ΔlasI) was inoculated onto the wound. Rats were injected intraperitoneally with anti-C 4 -HSL antiserum or treated with C 4 -HSL at the wound surface. PAO1 inoculation led to significant acceleration of wound healing, which was associated with neutrophil infiltration and TNF-α synthesis. These responses were reversed, except for TNF-α production, when ΔrhlI was inoculated instead of PAO1 or when rats were co-treated with PAO1 and anti-C 4 -HSL antiserum. In contrast, the healing process and neutrophil infiltration, but not TNF-α synthesis, were accelerated when C 4 -HSL was administered in the absence of PAO1. This acceleration was not affected by anti-TNF-α antibody. These results suggest that C 4 -HSL may be involved in the acceleration of acute wound healing in P. aeruginosa infection by modifying the neutrophilic inflammation. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  7. Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

    PubMed Central

    Saroj, Sunil D.; Holmer, Linda; Berengueras, Júlia M.; Jonsson, Ann-Beth

    2017-01-01

    Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence. PMID:28303956

  8. Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165.

    PubMed

    Saroj, Sunil D; Holmer, Linda; Berengueras, Júlia M; Jonsson, Ann-Beth

    2017-03-17

    Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence.

  9. High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

    PubMed Central

    Downham, Christina; Broadbent, Ian; Charlton, Keith; Porter, Andrew J.

    2014-01-01

    A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections. PMID:24185854

  10. Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

    PubMed Central

    Hao, Guixia; Burr, Thomas J.

    2006-01-01

    Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N-acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI, ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI. The addition of synthetic AHLs (C16:1 and 3-O-C16:1) complemented grape necrosis in the avsR, avsI, ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR, two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI::lacZ fusion construct. PMID:16513747

  11. Non-native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability

    PubMed Central

    Ho, Jessica S.; Geske, Grant D.; Blackwell, Helen E.; Ruby, Edward G.

    2014-01-01

    SUMMARY Quorum sensing, a group behavior coordinated by a diffusible pheromone signal and a cognate receptor, is typical of bacteria that form symbioses with plants and animals. LuxIR-type acyl homoserine-lactone (AHL) quorum sensing is common in Gram-negative proteobacteria, and many members of this group have additional quorum-sensing networks. The bioluminescent symbiont Vibrio fischeri encodes two AHL signal synthases: AinS and LuxI. AinS-dependent quorum sensing converges with LuxI-dependent quorum sensing at the LuxR regulatory element. Both AinS- and LuxI-mediated signaling are required for efficient and persistent colonization of the squid host, Euprymna scolopes. The basis of the mutualism is symbiont bioluminescence, which is regulated by both LuxI- and AinS-dependent quorum sensing, and is essential for maintaining a colonization of the host. Here, we used chemical and genetic approaches to probe the dynamics of LuxI- and AinS-mediated regulation of bioluminescence during symbiosis. We demonstrate that both native AHLs and non-native AHL analogs can be used to non-invasively and specifically modulate induction of symbiotic bioluminescence via LuxI-dependent quorum sensing. Our data suggest that the first day of colonization, during which symbiont bioluminescence is induced by LuxIR, is a critical period that determines the stability of the V. fischeri population once symbiosis is established. PMID:24191970

  12. MomL, a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

    PubMed Central

    Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom

    2014-01-01

    Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105 s−1 M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. PMID:25398866

  13. Characteristics of N-Acylhomoserine Lactones Produced by Hafnia alvei H4 Isolated from Spoiled Instant Sea Cucumber

    PubMed Central

    Hou, Hong-Man; Zhu, Yao-Lei; Wang, Jia-Ying; Jiang, Feng; Qu, Wen-Yan; Zhang, Gong-Liang; Hao, Hong-Shun

    2017-01-01

    This study aimed to identify N-acylhomoserine lactone (AHL) produced by Hafnia alvei H4, which was isolated from spoiled instant sea cucumber, and to investigate the effect of AHLs on biofilm formation. Two biosensor strains, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens KYC55, were used to detect the quorum sensing (QS) activity of H. alvei H4 and to confirm the existence of AHL-mediated QS system. Thin layer chromatography (TLC) and high resolution triple quadrupole liquid chromatography/mass spectrometry (LC/MS) analysis of the AHLs extracted from the culture supernatant of H. alvei H4 revealed the existence of at least three AHLs: N-hexanoyl-l-homoserine lactone (C6-HSL), N-(3-oxo-octanoyl)-l-homoserine lactone (3-oxo-C8-HSL), and N-butyryl-l-homoserine lactone (C4-HSL). This is the first report of the production of C4-HSL by H. alvei. In order to determine the relationship between the production of AHL by H. alvei H4 and bacterial growth, the β-galactosidase assay was employed to monitor AHL activity during a 48-h growth phase. AHLs production reached a maximum level of 134.6 Miller unites at late log phase (after 18 h) and then decreased to a stable level of about 100 Miller unites. AHL production and bacterial growth displayed a similar trend, suggesting that growth of H. alvei H4 might be regulated by QS. The effect of AHLs on biofilm formation of H. alvei H4 was investigated by adding exogenous AHLs (C4-HSL, C6-HSL and 3-oxo-C8-HSL) to H. alvei H4 culture. Biofilm formation was significantly promoted (p < 0.05) by 5 and 10 µM C6-HSL, inhibited (p < 0.05) by C4-HSL (5 and 10 µM) and 5 µM 3-oxo-C8-HSL, suggesting that QS may have a regulatory role in the biofilm formation of H. alvei H4. PMID:28379194

  14. Agent-based model of diffusion of N-acyl homoserine lactones in a multicellular environment of Pseudomonas aeruginosa and Candida albicans.

    PubMed

    Pérez-Rodríguez, Gael; Dias, Sónia; Pérez-Pérez, Martín; Fdez-Riverola, Florentino; Azevedo, Nuno F; Lourenço, Anália

    2018-03-08

    Experimental incapacity to track microbe-microbe interactions in structures like biofilms, and the complexity inherent to the mathematical modelling of those interactions, raises the need for feasible, alternative modelling approaches. This work proposes an agent-based representation of the diffusion of N-acyl homoserine lactones (AHL) in a multicellular environment formed by Pseudomonas aeruginosa and Candida albicans. Depending on the spatial location, C. albicans cells were variably exposed to AHLs, an observation that might help explain why phenotypic switching of individual cells in biofilms occurred at different time points. The simulation and algebraic results were similar for simpler scenarios, although some statistical differences could be observed (p < 0.05). The model was also successfully applied to a more complex scenario representing a small multicellular environment containing C. albicans and P. aeruginosa cells encased in a 3-D matrix. Further development of this model may help create a predictive tool to depict biofilm heterogeneity at the single-cell level.

  15. MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia.

    PubMed

    Tang, Kaihao; Su, Ying; Brackman, Gilles; Cui, Fangyuan; Zhang, Yunhui; Shi, Xiaochong; Coenye, Tom; Zhang, Xiao-Hua

    2015-01-01

    Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 10(5) s(-1) M(-1). Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the "HXHXDH" motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group.

    PubMed

    Schikora, Adam; Schenk, Sebastian T; Hartmann, Anton

    2016-04-01

    Bacterial quorum sensing (QS) mechanisms play a crucial role in the proper performance and ecological fitness of bacterial populations. Many key physiological processes are regulated in a QS-dependent manner by auto-inducers, like the N-acyl homoserine lactones (AHLs) in numerous Gram-negative bacteria. In addition, also the interaction between bacteria and eukaryotic hosts can be regulated by AHLs. Those mechanisms gained much attention, because of the positive effects of different AHL molecules on plants. This positive impact ranges from growth promotion to induced resistance and is quite contrasting to the rather negative effects observed in the interactions between bacterial AHL molecules and animals. Only very recently, we began to understand the molecular mechanisms underpinning plant responses to AHL molecules. In this review, we gathered the latest information in this research field. The first part gives an overview of the bacterial aspects of quorum sensing. Later we focus on the impact of AHLs on plant growth and AHL-priming, as one of the most understood phenomena in respect to the inter-kingdom interactions based on AHL-quorum sensing molecules. Finally, we discuss the potential benefits of the understanding of bacteria-plant interaction for the future agricultural applications.

  17. The first report on Listeria monocytogenes producing siderophores and responds positively to N-acyl homoserine lactone (AHL) molecules by enhanced biofilm formation.

    PubMed

    Naik, Milind Mohan; Bhangui, Purva; Bhat, Chinmay

    2017-12-01

    Listeria monocytogenes are Gram-positive well-known emerging food-borne pathogens causing listeriosis in humans. In the present study, we have isolated biofilm-forming Listeria sp. from utensils used by a local milk collection dairy society at Usgao Goa, which collects milk for Goa dairy. Through biochemical tests and 16S rRNA sequence analysis, the bacterium was confirmed to be L. monocytogenes and designated as strain BN3, having GenBank accession number MF095110. We report for the first time Gram-positive L. monocytogenes strain BN3 producing iron-chelating siderophores by chrome azurol S (CAS) agar test. Also, this is a first report which reveals that L. monocytogenes strain BN3 responds to N-hexanoyl-homoserine lactone molecule (C 6 -HSL) by gradual increase in their biofilm-forming potential with a gradual increase in AHL (C 6 -HSL) concentration (250, 500 nM-1 μM) as compared to control revealed by crystal violet assay (CV) in microtiter plate. These results were further confirmed by scanning electron microscopy (SEM). A significant decrease in biofilm formation was observed when L. monocytogenes strain BN3 was treated with 10 µg/ml (R)-2-(2-hydroxynaphthalen-1-yl)thiazolidine-4-carboxylic acid, but when 250 and 500 nM AHL molecules were added, biofilm formation in strain BN3 was found to be enhanced as compared to control even in the presence of antibacterial compound, (R)-2-(2-hydroxynaphthalen-1-yl)thiazolidine-4-carboxylic acid. These results revealed that AHL molecules nullify the effect of antimicrobial compound and promote biofilm formation in L. monocytogenes strain BN3.

  18. Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer.

    PubMed

    Siddiqui, Muhammad Faisal; Sakinah, Mimi; Singh, Lakhveer; Zularisam, A W

    2012-10-31

    Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. [Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

    PubMed

    Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

    2010-01-01

    Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

  20. Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

    PubMed

    Mäe, A; Montesano, M; Koiv, V; Palva, E T

    2001-09-01

    Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

  1. N-Acyl Homoserine Lactone-Mediated Quorum Sensing with Special Reference to Use of Quorum Quenching Bacteria in Membrane Biofouling Control

    PubMed Central

    Paul, Diby

    2014-01-01

    Membrane biofouling remains a severe problem to be addressed in wastewater treatment systems affecting reactor performance and economy. The finding that many wastewater bacteria rely on N-acyl homoserine lactone-mediated quorum sensing to synchronize their activities essential for biofilm formations; the quenching bacterial quorum sensing suggests a promising approach for control of membrane biofouling. A variety of quorum quenching compounds of both synthetic and natural origin have been identified and found effective in inhibition of membrane biofouling with much less environmental impact than traditional antimicrobials. Work over the past few years has demonstrated that enzymatic quorum quenching mechanisms are widely conserved in several prokaryotic organisms and can be utilized as a potent tool for inhibition of membrane biofouling. Such naturally occurring bacterial quorum quenching mechanisms also play important roles in microbe-microbe interactions and have been used to develop sustainable nonantibiotic antifouling strategies. Advances in membrane fabrication and bacteria entrapment techniques have allowed the implication of such quorum quenching bacteria for better design of membrane bioreactor with improved antibiofouling efficacies. In view of this, the present paper is designed to review and discuss the recent developments in control of membrane biofouling with special emphasis on quorum quenching bacteria that are applied in membrane bioreactors. PMID:25147787

  2. [Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria].

    PubMed

    Muliukin, A L; Filippova, S N; Kozlova, A N; Surgucheva, N A; Bogdanova, T I; Tsaplina, I A; El'-Registan, G I

    2006-01-01

    We conducted a comparative study of the effects of alpha-amino-gamma-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of gram-positive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10(-5)-10(-3) M HSL or 10(-5)-10(-4) M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10(-4)-10(-3) M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10(-4) to (1.5-2.5) 10(-4) M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10(-4) M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075-0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest

  3. N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

    PubMed

    Tinh, Nguyen Thi Ngoc; Asanka Gunasekara, R A Y S; Boon, Nico; Dierckens, Kristof; Sorgeloos, Patrick; Bossier, Peter

    2007-10-01

    Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

  4. Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine the performance of biological wastewater treatments: An overview.

    PubMed

    Huang, Jinhui; Shi, Yahui; Zeng, Guangming; Gu, Yanling; Chen, Guiqiu; Shi, Lixiu; Hu, Yi; Tang, Bi; Zhou, Jianxin

    2016-08-01

    Quorum sensing (QS) is a communication process between cells, in which bacteria secrete and sense the specific chemicals, and regulate gene expression in response to population density. Quorum quenching (QQ) blocks QS system, and inhibits gene expression mediating bacterial behaviors. Given the extensive research of acyl-homoserine lactone (AHL) signals, existences and effects of AHL-based QS and QQ in biological wastewater treatments are being subject to high concern. This review summarizes AHL structure, synthesis mode, degradation mechanisms, analytical methods, environmental factors, AHL-based QS and QQ mechanisms. The existences and roles of AHL-based QS and QQ in biomembrane processes, activated sludge processes and membrane bioreactors are summarized and discussed, and corresponding exogenous regulation strategy by selective enhancement of AHL-based QS or QQ coexisting in biological wastewater treatments is suggested. Such strategies including the addition of AHL signals, AHL-producing bacteria as well as quorum quenching enzyme or bacteria can effectively improve wastewater treatment performance without killing or limiting bacterial survival and growth. This review will present the theoretical and practical cognition for bacterial AHL-based QS and QQ, suggest the feasibility of exogenous regulation strategies in biological wastewater treatments, and provide useful information to scientists and engineers who work in this field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Rhodotorula mucilaginosa, a quorum quenching yeast exhibiting lactonase activity isolated from a tropical shoreline.

    PubMed

    Ghani, Norshazliza Ab; Sulaiman, Joanita; Ismail, Zahidah; Chan, Xin-Yue; Yin, Wai-Fong; Chan, Kok-Gan

    2014-04-09

    Two microbial isolates from a Malaysian shoreline were found to be capable of degrading N-acylhomoserine lactones. Both Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry and 18S rDNA phylogenetic analyses confirmed that these isolates are Rhodotorula mucilaginosa. Quorum quenching activities were detected by a series of bioassays and rapid resolution liquid chromatography analysis. The isolates were able to degrade various quorum sensing molecules namely N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Using a relactonisation assay to verify the quorum quenching mechanism, it is confirmed that Rh. mucilaginosa degrades the quorum sensing molecules via lactonase activity. To the best of our knowledge, this is the first documentation of the fact that Rh. mucilaginosa has activity against a broad range of AHLs namely C6-HSL, 3-oxo-C6-HSL and 3-hydroxy-C6-HSL.

  6. Rhodotorula Mucilaginosa, a Quorum Quenching Yeast Exhibiting Lactonase Activity Isolated from a Tropical Shoreline

    PubMed Central

    Ghani, Norshazliza Ab; Sulaiman, Joanita; Ismail, Zahidah; Chan, Xin-Yue; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Two microbial isolates from a Malaysian shoreline were found to be capable of degrading N-acylhomoserine lactones. Both Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry and 18S rDNA phylogenetic analyses confirmed that these isolates are Rhodotorula mucilaginosa. Quorum quenching activities were detected by a series of bioassays and rapid resolution liquid chromatography analysis. The isolates were able to degrade various quorum sensing molecules namely N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). Using a relactonisation assay to verify the quorum quenching mechanism, it is confirmed that Rh. mucilaginosa degrades the quorum sensing molecules via lactonase activity. To the best of our knowledge, this is the first documentation of the fact that Rh. mucilaginosa has activity against a broad range of AHLs namely C6-HSL, 3-oxo-C6-HSL and 3-hydroxy-C6-HSL. PMID:24721765

  7. Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone.

    PubMed

    Flodgaard, L R; Dalgaard, P; Andersen, J B; Nielsen, K F; Givskov, M; Gram, L

    2005-04-01

    Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.

  8. Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque.

    PubMed

    Goh, Share-Yuan; Khan, Saad Ahmed; Tee, Kok Keng; Abu Kasim, Noor Hayaty; Yin, Wai-Fong; Chan, Kok-Gan

    2016-02-10

    Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.

  9. Quorum sensing activity of Citrobacter amalonaticus L8A, a bacterium isolated from dental plaque

    PubMed Central

    Goh, Share-Yuan; Khan, Saad Ahmed; Tee, Kok Keng; Abu Kasim, Noor Hayaty; Yin, Wai-Fong; Chan, Kok-Gan

    2016-01-01

    Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium. PMID:26860259

  10. Complete Genome Sequence of Pseudomonas chlororaphis subsp. aurantiaca Reveals a Triplicate Quorum-Sensing Mechanism for Regulation of Phenazine Production

    PubMed Central

    Morohoshi, Tomohiro; Yamaguchi, Takahito; Xie, Xiaonan; Wang, Wen-zhao; Takeuchi, Kasumi; Someya, Nobutaka

    2017-01-01

    Pseudomonas chlororaphis subsp. aurantiaca StFRB508 regulates phenazine production through N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing. Two sets of AHL-synthase and AHL-receptor genes, phzI/phzR and aurI/aurR, have been identified from the incomplete draft genome of StFRB508. In the present study, the complete genome of StFRB508, comprising a single chromosome of 6,997,933 bp, was sequenced. The complete genome sequence revealed the presence of a third quorum-sensing gene set, designated as csaI/csaR. An LC-MS/MS analysis revealed that StFRB508 produced six types of AHLs, with the most important AHL being N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-OH-C6-HSL). PhzI mainly catalyzed the biosynthesis of 3-OH-C6-HSL, while AurI and CsaI catalyzed that of N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, respectively. A mutation in phzI decreased phenazine production, whereas that in aurI or csaI did not. A phzI aurI csaI triple mutant (508ΔPACI) did not produce phenazine. Phenazine production by 508ΔPACI was stimulated by exogenous AHLs and 3-OH-C6-HSL exerted the strongest effects on phenazine production at the lowest concentration tested (0.1 μM). The plant protection efficacy of 508ΔPACI against an oomycete pathogen was lower than that of wild-type StFRB508. These results demonstrate that the triplicate quorum-sensing system plays an important role in phenazine production by and the biocontrol activity of StFRB508. PMID:28239068

  11. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

    PubMed

    Zhao, Guoping; Neely, Aaron M; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G; Stivers, Nicole S; Burlison, Joseph A; White, Carl; Machen, Terry E; Li, Chi

    2016-02-02

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells.

  12. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

    PubMed Central

    Zhao, Guoping; Neely, Aaron M.; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G.; Stivers, Nicole S.; Burlison, Joseph A.; White, Carl; Machen, Terry E.; Li, Chi

    2016-01-01

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

  13. Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples.

    PubMed

    Haslan, Ezgi; Kimiran-Erdem, Ayten

    2013-09-01

    In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p < 0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.

  14. N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts.

    PubMed

    Neely, Aaron M; Zhao, Guoping; Schwarzer, Christian; Stivers, Nicole S; Whitt, Aaron G; Meng, Shuhan; Burlison, Joseph A; Machen, Terry E; Li, Chi

    2018-01-01

    N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis. © 2017 John Wiley & Sons Ltd.

  15. Estimation of spatial distribution of quorum sensing signaling in sequencing batch biofilm reactor (SBBR) biofilms.

    PubMed

    Wang, Jinfeng; Ding, Lili; Li, Kan; Huang, Hui; Hu, Haidong; Geng, Jinju; Xu, Ke; Ren, Hongqiang

    2018-01-15

    Quorum sensing (QS) signaling, plays a significant role in regulating formation of biofilms in the nature; however, little information about the occurrence and distribution of quorum sensing molecular in the biofilm of carriers has been reported. In this study, distribution of QS signaling molecules (the acylated homoserine lactones-AHLs, and AI-2), extracellular polymeric substances (EPS) and the mechanical properties in sequencing batch biofilm reactor (SBBR) biofilms have been investigated. Using increased centrifugal force, the biofilms were detached into different fractions. The AHLs ranged from 5.2ng/g to 98.3ng/g in different fractions of biofilms, and N-decanoyl-dl-homoserine lactone (C10-HSL) and N-dodecanoyl-dl-homoserine lactone (C12-HSL) in the biofilms obtained at various centrifugal forces displayed significant differences (p<0.01). Interspecies communication signal autoinducer-2(AI-2) in the biofilms ranged from 79.2ng/g to 98.3ng/g. Soluble EPS and loosely bound EPS content in the different fractions of biofilms displayed significant positive relationship with the distribution of C12-HSL (r=0.86, p<0.05). Furthermore, 49.62% of bacteria in the biofilms were positively related with AHLs with 22.76% was significantly positively (p<0.05) related with AHLs. Biofilm adhesion and compliance was the strongest in the tightly-bound biofilm, the weakest in the supernatant/surface biofilm, which was in accordance with the distribution of C12 HSL(r=0.77, p<0.05) and C10-HSL(r=0.75, p<0.05), respectively. This study addressed on better understanding of possible methods for the improvement of wastewater bio-treatment through biofilm application. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. [The Effect of Introduction of the Heterologous Gene Encoding the N-acyl-homoserine Lactonase (aiiA) on the Properties of Burkholderia cenocepacia 370].

    PubMed

    Plyuta, V A; Lipasova, V A; Koksharova, O A; Veselova, M A; Kuznetsov, A E; Khmel, I A

    2015-08-01

    To study the role of Quorum Sensing (QS) regulation in the control of the cellular processes of Burkholderia cenocepacia 370, plasmid pME6863 was transferred into its cells. The plasmid contains a heterologous gene encoding for AiiA N-acyl-homoserine lactonase, which degrades the signaling molecules of the QS system of N-acyl-homoserine lactones (AHL). An absence or reduction of AHL in the culture was revealed with the biosensors Chromobacterium violaceum CV026 and Agrobacterium tumifaciens NT1/pZLR4, respectively. The presence of the aiiA gene, which was cloned from Bacillus sp. A24 in the cells of B. cenocepacia 370, resulted in a lack of hemolytic activity, which reduced the extracellular proteolytic activity and decreased the cells' ability to migration in swarms on the surface of the agar medium. The introduction of the aiiA gene did not affect lipase activity, fatty acids synthesis, HCN synthesis, or biofilm formation. Hydrogen peroxide was shown to stimulate biofilm formation by B. cenocepacia 370 in concentrations that inhibited or weakly suppressed bacterial growth. The introduction of the aiiA gene into the cells did not eliminate this effect but it did reduce it.

  17. Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family

    PubMed Central

    Gan, Han Ming; Dailey, Lucas K.; Halliday, Nigel; Williams, Paul; Hudson, André O.

    2016-01-01

    Background Members of the genus Novosphingobium have been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated. Methods In this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by the N. subterraneum DSM 12447 strain. Results Three predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference. Discussion This study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae. PMID:27635318

  18. Evaluation of anti-quorum-sensing activity of edible plants and fruits through inhibition of the N-acyl-homoserine lactone system in Chromobacterium violaceum and Pseudomonas aeruginosa.

    PubMed

    Musthafa, K Syed; Ravi, A Veera; Annapoorani, A; Packiavathy, I Sybiya Vasantha; Pandian, S Karutha

    2010-01-01

    To find out an alternative strategy to antibiotic usage against bacterial infection. The purpose of this study is to describe the quorum-sensing (QS) inhibitory activity of edible plants and fruits against N-acyl-homoserine lactone (AHL)-mediated violacein production in Chromobacterium violaceum and virulence factor expression in Pseudomonas aeruginosa PAO1. Aqueous extracts of Ananas comosus (Bromeliaceae), Musa paradiciaca (Musaceae), Manilkara zapota (Sapotaceae) and Ocimum sanctum (Lamiaceae) were prepared and anti-QS activity of each extract was tested against AHL-mediated phenotypic expressions of C. violaceum and PAO1. Most of these extracts showed significant reduction in AHL-mediated violacein production in C. violaceum as well as pyocyanin pigment, staphylolytic protease, elastase production and biofilm formation in PAO1. However, these extracts were not inhibitory to bacterial growth, revealing that the QS inhibition by the extracts is not related to static or killing effects on the bacteria. The present study identified the anti-QS activity of A. comosus, M. paradiciaca, M. zapota and O. sanctum. An AHL-inactivating compound from these plant sources can be used as an alternative to antibiotic compounds to prevent AHL-mediated bacterial infection in higher organisms. Copyright © 2010 S. Karger AG, Basel.

  19. Atomic Force Microscopy Reveals a Morphological Differentiation of Chromobacterium violaceum Cells Associated with Biofilm Development and Directed by N-Hexanoyl-L-Homoserine Lactone

    PubMed Central

    Kamaeva, Anara A.; Vasilchenko, Alexey S.; Deryabin, Dmitry G.

    2014-01-01

    Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure. PMID:25111599

  20. Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

    PubMed

    Kamaeva, Anara A; Vasilchenko, Alexey S; Deryabin, Dmitry G

    2014-01-01

    Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

  1. The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: Novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules

    PubMed Central

    Romano, Ariel A.; Hahn, Tobias; Davis, Nicole; Lowery, Colin A.; Struss, Anjali K.; Janda, Kim D.; Böttger, Lars H.; Matzanke, Berthold F.; Carrano, Carl J.

    2011-01-01

    Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C12-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding. PMID:22178671

  2. Identification and Quantification of N-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

    PubMed

    Leipert, Jan; Treitz, Christian; Leippe, Matthias; Tholey, Andreas

    2017-12-01

    N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. Graphical abstract ᅟ.

  3. Identification and Quantification of N-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Leipert, Jan; Treitz, Christian; Leippe, Matthias; Tholey, Andreas

    2017-12-01

    N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. [Figure not available: see fulltext.

  4. Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae.

    PubMed

    Toth, I K; Newton, J A; Hyman, L J; Lees, A K; Daykin, M; Ortori, C; Williams, P; Fray, R G

    2004-08-01

    Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

  5. Quorum Sensing Activity of Serratia fonticola Strain RB-25 Isolated from an Ex-landfill Site

    PubMed Central

    Ee, Robson; Lim, Yan-Lue; Tee, Kok-Keng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Quorum sensing is a unique bacterial communication system which permits bacteria to synchronize their behaviour in accordance with the population density. The operation of this communication network involves the use of diffusible autoinducer molecules, termed N-acylhomoserine lactones (AHLs). Serratia spp. are well known for their use of quorum sensing to regulate the expression of various genes. In this study, we aimed to characterized the AHL production of a bacterium designated as strain RB-25 isolated from a former domestic waste landfill site. It was identified as Serratia fonticola using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis and this was confirmed by 16S ribosomal DNA sequencing. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis of S. fonticola strain RB-25 spent culture supernatant indicated the existence of three AHLs namely: N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine-lactone (3-oxo-C6 HSL). This is the first report of the production of these AHLs in S. fonticola. PMID:24625739

  6. Insights into the Quorum Sensing Regulon of the Acidophilic Acidithiobacillus ferrooxidans Revealed by Transcriptomic in the Presence of an Acyl Homoserine Lactone Superagonist Analog

    PubMed Central

    Mamani, Sigde; Moinier, Danielle; Denis, Yann; Soulère, Laurent; Queneau, Yves; Talla, Emmanuel; Bonnefoy, Violaine; Guiliani, Nicolas

    2016-01-01

    While a functional quorum sensing system has been identified in the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270T and shown to modulate cell adhesion to solid substrates, nothing is known about the genes it regulates. To address the question of how quorum sensing controls biofilm formation in A. ferrooxidansT, the transcriptome of this organism in conditions in which quorum sensing response is stimulated by a synthetic superagonist AHL (N-acyl homoserine lactones) analog has been studied. First, the effect on biofilm formation of a synthetic AHL tetrazolic analog, tetrazole 9c, known for its agonistic QS activity, was assessed by fluorescence and electron microscopy. A fast adherence of A. ferrooxidansT cells on sulfur coupons was observed. Then, tetrazole 9c was used in DNA microarray experiments that allowed the identification of genes regulated by quorum sensing signaling, and more particularly, those involved in early biofilm formation. Interestingly, afeI gene, encoding the AHL synthase, but not the A. ferrooxidans quorum sensing transcriptional regulator AfeR encoding gene, was shown to be regulated by quorum sensing. Data indicated that quorum sensing network represents at least 4.5% (141 genes) of the ATCC 23270T genome of which 42.5% (60 genes) are related to biofilm formation. Finally, AfeR was shown to bind specifically to the regulatory region of the afeI gene at the level of the palindromic sequence predicted to be the AfeR binding site. Our results give new insights on the response of A. ferrooxidans to quorum sensing and on biofilm biogenesis. PMID:27683573

  7. Insights into the Quorum Sensing Regulon of the Acidophilic Acidithiobacillus ferrooxidans Revealed by Transcriptomic in the Presence of an Acyl Homoserine Lactone Superagonist Analog.

    PubMed

    Mamani, Sigde; Moinier, Danielle; Denis, Yann; Soulère, Laurent; Queneau, Yves; Talla, Emmanuel; Bonnefoy, Violaine; Guiliani, Nicolas

    2016-01-01

    While a functional quorum sensing system has been identified in the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270(T) and shown to modulate cell adhesion to solid substrates, nothing is known about the genes it regulates. To address the question of how quorum sensing controls biofilm formation in A. ferrooxidans (T), the transcriptome of this organism in conditions in which quorum sensing response is stimulated by a synthetic superagonist AHL (N-acyl homoserine lactones) analog has been studied. First, the effect on biofilm formation of a synthetic AHL tetrazolic analog, tetrazole 9c, known for its agonistic QS activity, was assessed by fluorescence and electron microscopy. A fast adherence of A. ferrooxidans (T) cells on sulfur coupons was observed. Then, tetrazole 9c was used in DNA microarray experiments that allowed the identification of genes regulated by quorum sensing signaling, and more particularly, those involved in early biofilm formation. Interestingly, afeI gene, encoding the AHL synthase, but not the A. ferrooxidans quorum sensing transcriptional regulator AfeR encoding gene, was shown to be regulated by quorum sensing. Data indicated that quorum sensing network represents at least 4.5% (141 genes) of the ATCC 23270(T) genome of which 42.5% (60 genes) are related to biofilm formation. Finally, AfeR was shown to bind specifically to the regulatory region of the afeI gene at the level of the palindromic sequence predicted to be the AfeR binding site. Our results give new insights on the response of A. ferrooxidans to quorum sensing and on biofilm biogenesis.

  8. Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation.

    PubMed

    Dixit, Swarnita; Dubey, Ramesh Chand; Maheshwari, Dinesh Kumar; Seth, Prahlad Kishore; Bajpai, Vivek K

    Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C 8 -HSL and 3-OH-C 14 -HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C 8 -HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C 8 -HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C 8 -HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  9. Identification and characterization of a novel N-acyl-homoserine lactonase gene in Sphingomonas ursincola isolated from industrial cooling water systems.

    PubMed

    Morohoshi, Tomohiro; Sato, Niina; Iizumi, Taro; Tanaka, Airi; Ikeda, Tsukasa

    2017-05-01

    Biofilm formation by bacteria is one of the main causes of fouling in industrial cooling water systems. In many gram-negative bacteria, biofilm formation is regulated by N-acyl-homoserine lactone (AHL)-mediated quorum sensing. In this study, we isolated three AHL-degrading bacteria from cooling water systems and identified them as Sphingomonas ursincola. The draft genome sequence of S. ursincola A1 revealed the presence of an AHL-degrading gene homolog, designated qsdS. The qsdS region was also amplified by PCR from the genomes of the other two S. ursincola strains, SF1 and SF8. Escherichia coli DH5α harboring a QsdS-expressing plasmid showed high degradative activity against AHLs with short and 3-oxo-substituted acyl chains. High-performance liquid chromatography analysis revealed that QsdS is an AHL lactonase, an enzyme that catalyzes AHL ring opening. Furthermore, heterologous expression of QsdS in Pseudomonas aeruginosa PAO1 resulted in degradation of endogenous AHLs and interfered with the quorum-sensing-regulated phenotype. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Development of a dry, stable and inhalable acyl-homoserine-lactone-acylase powder formulation for the treatment of pulmonary Pseudomonas aeruginosa infections.

    PubMed

    Wahjudi, Mariana; Murugappan, Senthil; van Merkerk, Ronald; Eissens, Anko C; Visser, Marinella R; Hinrichs, Wouter L J; Quax, Wim J

    2013-03-12

    In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl-homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20°C and 55°C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55°C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 μm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation. Copyright © 2013 Elsevier B.V. All

  11. Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

    PubMed

    Chu, Weihua; Vattem, Dhiraj A; Maitin, Vatsala; Barnes, Mary B; McLean, Robert J C

    2011-01-01

    In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

  12. N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora.

    PubMed Central

    Bainton, N J; Stead, P; Chhabra, S R; Bycroft, B W; Salmond, G P; Stewart, G S; Williams, P

    1992-01-01

    Erwinia carotovora A.T.C.C. 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid. A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis. Cross-feeding studies revealed that the mutants fell into two discrete groups. Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants. This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c. M.s. and n.m.r. spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone. Both D- and L-isomers were synthesized, and subsequent analysis by c.d. established that the natural product has the L-configuration. Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml. N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate. This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri. It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species. Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes. PMID:1335238

  13. Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

    PubMed

    Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

    2008-01-01

    The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

  14. Theoretical and structural analysis of the active site of the transcriptional regulators LasR and TraR, using molecular docking methodology for identifying potential analogues of acyl homoserine lactones (AHLs) with anti-quorum sensing activity.

    PubMed

    Ahumedo, Maicol; Díaz, Antonio; Vivas-Reyes, Ricardo

    2010-02-01

    In the present study the homology of transcriptional receptors LuxR type were evaluated using as point of reference the receptors TraR and LasR of the bacterial types Agrobacterium tumefaciens and Pseudomonas aureginosa respectively. A series of alignments were performed in order to demonstrate that the active site of the protein is conserved in wide range of gram negative bacteria. Moreover, some docking calculations were carried out for analogs of the acyl homoserin lactones (AHLs) and regulatory proteins LasR and TraR, to understand the complex microenvironment in which the ligands are exposed. The molecular alignments show clearly that there are preserved motifs in the residues (Y53, Y61, W57, D70, W85 to TraR, Y56, Y64, W60, D73, W88 to LasR) analyzed, which may serve as site-specific targets for the development of potential antagonists. In this study was found that the anti-quorum sensing activity of the AHLs molecular analogs appears to depend on; the structure of the lactone ring and on appropriate combination of absolute and relative stereochemistry of the carbonyl (C=O) and amide (NH(2)) groups of the side chain of these AHLs molecular analogs, in combination with the interactions with the conserved amino acids (D73, W60, Y56, S129 to LasR and D70, W57, Y53 to TraR) of the LuxR type protein family. Copyright 2009 Elsevier Masson SAS. All rights reserved.

  15. Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays.

    PubMed

    Baldrich, Eva; Muñoz, Francesc Xavier; García-Aljaro, Cristina

    2011-03-15

    n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a β-galactosidase (β-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl β-D-galactopyranoside was used to detect β-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by β-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.

  16. The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations.

    PubMed

    Whitehead, Neil A; Byers, Joseph T; Commander, Paul; Corbett, Mark J; Coulthurst, Sarah J; Everson, Lee; Harris, Abigail K P; Pemberton, Clare L; Simpson, Natalie J L; Slater, Holly; Smith, Debra S; Welch, Martin; Williamson, Neil; Salmond, George P C

    2002-08-01

    Erwinia carotovora is a Gram-negative bacterial phytopathogen that causes soft-rot disease and potato blackleg. The organism is environmentally widespread and exhibits an opportunistic plant pathogenesis. The ability to secrete multiple plant cell wall-degrading enzymes is a key virulence trait and exoenzyme production is responsive to multiple environmental and physiological cues. One important cue is the cell population density of the pathogen. Cell density is monitored via an acylated homoserine lactone (acyl HSL) signalling molecule, which is thought to diffuse between Erwinia cells in a process now commonly known as 'quorum sensing'. This molecule also acts as the chemical communication signal controlling production of a broad-spectrum beta-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem) synthesised in concert with exoenzyme elaboration, possibly for niche defence. In antibiotic production control, quorum sensing acts at the level of transcriptional activation of the antibiotic biosynthetic cluster. This is achieved via a dedicated LuxR-type protein, CarR that is bound to the signalling molecule. The molecular relay connecting acyl HSL production and exoenzyme induction is not clear, despite the identification of a multitude of global regulatory genes, including those of the RsmA/rsmB system, impinging on enzyme synthesis. Quorum sensing control mediated by acyl HSLs is widespread in Gram-negative bacteria and is responsible for the regulation of diverse phenotypes. Although there is still a paucity of meaningful information on acyl HSL availability and in-situ biological function, there is growing evidence that such molecules play significant roles in microbial ecology.

  17. Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene

    PubMed Central

    How, Kah Yan; Hong, Kar-Wai; Sam, Choon-Kook; Koh, Chong-Lek; Yin, Wai-Fong; Chan, Kok-Gan

    2015-01-01

    Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production, and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homologue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was ∼20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high-resolution liquid chromatography–mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl-homoserine lactone and 3-hydroxy-dodecanoyl-homoserine lactone from induced E. coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium. PMID:25926817

  18. Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura

    PubMed Central

    Singh, Ravindra Pal; Baghel, Ravi S.; Reddy, C. R. K.; Jha, Bhavanath

    2015-01-01

    Epiphytic and endophytic bacteria associated with green macroalgae Ulva (U. fasciata and U. lactuca) and red macroalgae Gracilaria (G. corticata and G. dura) have been identified from three different seasons to evaluate the effect of quorum sensing (QS) molecules on carpospores liberation from Gracilaria dura. The bacterial isolates belonging to the orders Bacillales, Pseudomonadales, Alteromonadales, and Vibrionales were present in all seasons, whereas Actinomycetales and Enterobacteriales were confined to pre-monsoon and post-monsoon seasons, respectively. Among all the Gram-negative bacteria, seven isolates were found to produce different types of N-acyl homoserine lactones (AHLs). Interestingly, Shewanella algae produced five types of AHL: C4-HSL, HC4-HSL, C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL. Subsequently, the AHLs producing bacterial isolates were screened for carpospore liberation from G. dura and these isolates were found to positively induce carpospore liberation over the control. Also, observed that carpospore liberation increased significantly in C4- and C6-HSL treated cystocarps. Sodium dodecyl sulfate and native polyacrylamide gel electrophoresis of the total protein of the C4- and C6-HSL treated cystocarps showed two specific peptide bands of different molecular weights (50 kDa and 60 kDa) as compared to the control, confirming their indirect effect on carpospore liberation. PMID:25788899

  19. Octanoyl-Homoserine Lactone Is the Cognate Signal for Burkholderia mallei BmaR1-BmaI1 Quorum Sensing

    DTIC Science & Technology

    2007-07-01

    of genes. Acyl-HSL sig- naling was first identified in the luminescent marine bacterium Vibrio fischeri, which produces blue light at high cell...with shaking at 16°C, cells were harvested by centrifugation at 2,750 g for 20 min. Cell pellets were frozen, thawed at room temperature, suspended in...the Vibrio fischeri strain ATCC7744. Proc. Natl. Acad. Sci. USA 86:5688–5692. 11. Engebrecht, J., and M. Silverman. 1984. Identification of genes and

  20. Virulence of Burkholderia mallei Quorum-Sensing Mutants

    PubMed Central

    Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard

    2013-01-01

    Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved. PMID:23429539

  1. Plant Pathogenic Microbial Communication Affected by Elevated Temperature in Pectobacterium carotovorum subsp. carotovorum.

    PubMed

    Saha, N D; Chaudhary, A; Singh, S D; Singh, D; Walia, S; Das, T K

    2015-11-01

    Gram-negative plant pathogenic bacteria regulate specific gene expression in a population density-dependent manner by sensing level of Acyl-Homoserine Lactone (HSL) molecules which they produce and liberate to the environment, called Quorum Sensing (QS). The production of virulence factors (extracellular enzyme viz. cellulase, pectinase, etc.) in Pectobacterium carotovorum subsp. carotovorum (Pcc) is under strong regulation of QS. The QS signal molecule, N-(3-oxohexanoyl)-L-Homoserine Lactone (OHHL) was found as the central regulatory system for the virulence factor production in Pcc and is also under strict regulation of external environmental temperature. Under seven different incubation temperatures (24, 26, 28, 30, 33, 35, and 37 °C) in laboratory condition, highest amount of OHHL (804 violacein unit) and highest (79 %) Disease Severity Index (DSI) were measured at 33 °C. The OHHL production kinetics showed accumulation of highest concentration of OHHL at late log phase of the growth but diminution in the concentration occurred during stationary phase onwards to death phase. At higher temperature (35 and 37 °C) exposure, OHHL was not at detectable range. The effect of temperature on virulence factor production is the concomitant effect of HSL production and degradation which justifies less disease severity index in cross-inoculated tomato fruits incubated at 35 and 37 °C. The nondetection of the OHHL in the elevated temperature may because of degradation as these signal molecules are quite sensitive and prone to get degraded under different physical factors. This result provides the rationale behind the highest disease severity up to certain elevated temperature and leaves opportunities for investigation on mutation, co-evolution of superior plant pathogen with more stable HSL signals-mediated pathogenesis under global warming context.

  2. The BlcC (AttM) lactonase of Agrobacterium tumefaciens does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer.

    PubMed

    Khan, Sharik R; Farrand, Stephen K

    2009-02-01

    The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

  3. Identification of the Quorum-Sensing Target DNA Sequence and N-Acyl Homoserine Lactone Responsiveness of the Brucella abortus virB promoter▿

    PubMed Central

    Arocena, Gastón M.; Sieira, Rodrigo; Comerci, Diego J.; Ugalde, Rodolfo A.

    2010-01-01

    VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (PvirB) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in PvirB by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in PvirB increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of PvirB. Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella. PMID:20400542

  4. Freshwater-Borne Bacteria Isolated from a Malaysian Rainforest Waterfall Exhibiting Quorum Sensing Properties

    PubMed Central

    Tan, Wen-Si; Yunos, Nina Yusrina Muhamad; Tan, Pui-Wan; Mohamad, Nur Izzati; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    One obvious requirement for concerted action by a bacterial population is for an individual to be aware of and respond to the other individuals of the same species in order to form a response in unison. The term “quorum sensing” (QS) was coined to describe bacterial communication that is able to stimulate expression of a series of genes when the concentration of the signaling molecules has reached a threshold level. Here we report the isolation from aquatic environment of a bacterium that was later identified as Enterobacter sp.. Chromobacterium violaceum CV026 and Escherichia coli [pSB401] were used for preliminary screening of N-acyl homoserine lactone (AHL) production. The Enterobacter sp. isolated was shown to produce two types of AHLs as confirmed by analysis using high resolution tandem mass spectrometry. To the best of our knowledge, this is the first documentation of an Enterobacter sp. that produced both 3-oxo-C6-HSL and 3-oxo-C8-HSL as QS signaling molecules. PMID:24932870

  5. Autoinducer Sensing Microarrays by Reporter Bacteria Encapsulated in Hybrid Supramolecular-Polysaccharide Hydrogels.

    PubMed

    Li, Ping; Dou, Xiaoqiu; Müller, Mareike; Feng, Chuanliang; Chang, Matthew Wook; Frettlöh, Martin; Schönherr, Holger

    2017-11-01

    A generally applicable strategy to obtain mechanically robust hydrogels for the incorporation and containment of functional reporter bacteria for the microarray and microparticle-based detection and signaling of N-acyl homoserine lactone autoinducers (3OC 12 HSL) at relevant concentrations is reported. For reinforcing hydrogels of 1,4-bi(phenylalanine-diglycol)-benzene (PDB), a hybrid hydrogel is formed by the combination of PDB self-assembly with Ca 2+ mediated alginate crosslinking. The different assembly mechanisms are shown not to interfere with each other and despite the more than four-fold increased moduli of the hydrogels, diffusion of autoinducers into the gels remains efficient and Escherichia coli pLuxR-green fluorescent protein (GFP) reporter bacteria are proliferating. Templating affords reporter bacteria-loaded hydrogels with controllable shape and size. Upon exposure to 3OC 12 HSL, the embedded bacteria exhibit an up to 12 ± 3 times increase in fluorescence intensity due to autoinducer-triggered GFP expression. This approach can serve as a potentially generally applicable strategy to sensitively detect bacteria via their secreted autoinducers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease.

    PubMed

    Takaya, Akiko; Tabuchi, Fumiaki; Tsuchiya, Hiroko; Isogai, Emiko; Yamamoto, Tomoko

    2008-06-01

    Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

  7. Mutational studies on HslU and its docking mode with HslV.

    PubMed

    Song, H K; Hartmann, C; Ramachandran, R; Bochtler, M; Behrendt, R; Moroder, L; Huber, R

    2000-12-19

    HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the "sensor arginine" (R393) and the "arginine finger" (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

  8. Role of gallic and p-coumaric acids in the AHL-dependent expression of flgA gene and in the process of biofilm formation in food-associated Pseudomonas fluorescens KM120.

    PubMed

    Myszka, Kamila; Schmidt, Marcin T; Białas, Wojciech; Olkowicz, Mariola; Leja, Katarzyna; Czaczyk, Katarzyna

    2016-09-01

    In the process of Pseudomonas fluorescens biofilm formation, N-acyl-l-homoserine lactone (AHL)-mediated flagella synthesis plays a key role. Inhibition of AHL production may attenuate P. fluorescens biofilm on solid surfaces. This work validated the anti-biofilm properties of p-coumaric and gallic acids via the ability of phenolics to suppress AHL synthesis in P. fluorescens KM120. The dependence between synthesis of AHL molecules, expression of flagella gene (flgA) and the ability of biofilm formation by P. fluorescens KM120 on a stainless steel surface (type 304L) was also investigated. Research was carried out in a purpose-built flow cell device. Limitations on AHL synthesis in P. fluorescens KM120 were observed at concentrations of 120 and 240 µmol L(-1) of phenolic acids in medium. At such levels of gallic and p-coumaric acids the ability of P. fluorescens KM120 to synthesize 3-oxo-C6-homoserine lactone (HSL) was not observed. These concentrations caused decreased expression of flgA gene in P. fluorescens KM120. The changes in expression of AHL-dependent flgA gene significantly decreased the rate of microorganism colonization on the stainless steel surface. Phenolic acids are able to inhibit biofilm formation. The results obtained in the work may help to develop alternative techniques for anti-biofilm treatment in the food industry. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  9. Quorum sensing controls the synthesis of virulence factors by modulating rsmA gene expression in Erwinia carotovora subsp. carotovora.

    PubMed

    Kõiv, V; Mäe, A

    2001-04-01

    The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora (Ecc) causes disease mainly by means of a number of extracellular plant cell wall-degrading enzymes (PCWDEs), also referred to as virulence factors. The production of PCWDEs is coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone (HSL) in a population density-dependent manner ("quorum sensing"). ExpI is the enzyme responsible for the synthesis of HSL. The Rsm system negatively regulates the production of PCWDEs. It includes three components: RsmA is an RNA-binding protein which promotes mRNA decay; rsmB is a unique regulator RNA, and RsmC regulates expression of rsmA positively and of rsmB negatively. We report here that in an expI knockout mutant of Ecc strain SCC3193, the levels of rsmA and rsmB RNA are remarkably enhanced in comparison to the wild-type strain, while the level of the rsmC transcript is not affected. The increase in transcription of rsmA in the expI strain represses production of PCWDEs, which in turn leads to the avirulent phenotype of this mutant. In the expI- mutant, addition of exogenous HSL caused repression of rsmA and rsmB transcription to the wild-type level, whereas the expression of rsmC was not affected. Taken together, these data suggest that HSL affects the expression of rsmA, and that this effect is not mediated by RsmC. This specific effect and the previous demonstration that HSL is required for PCWDE production in Ecc support the hypothesis that regulation by quorum sensing in Ecc, in contrast to most other systems already described, requires HSL to repress rsmA transcription, which in turn leads to the activation of PCWDE production. A model is presented that explains how HSL controls the production of PCWDEs by modulating the expression of rsmA.

  10. Crystal Structures of a Quorum-Quenching Antibody

    PubMed Central

    Debler, Erik W.; Kaufmann, Gunnar F.; Kirchdoerfer, Robert N.; Mee, Jenny M.; Janda, Kim D.; Wilson, Ian A.

    2007-01-01

    Summary A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely-related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short and narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow for entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody combining site with a closed binding pocket, suggestive of an induced fit mechanism for ligand binding. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will now aid in further development of an antibody

  11. N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection.

    PubMed

    Vinoj, Gopalakrishnan; Jayakumar, Rengarajan; Chen, Jiann-Chu; Withyachumnarnkul, Boonsirm; Shanthi, Sathappan; Vaseeharan, Baskaralingam

    2015-01-01

    Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Using Surface Enhanced Raman Scattering to Analyze the Interactions of Protein Receptors with Bacterial Quorum Sensing Modulators

    PubMed Central

    2015-01-01

    Many members of the LuxR family of quorum sensing (QS) transcriptional activators, including LasR of Pseudomonas aeruginosa, are believed to require appropriate acyl-homoserine lactone (acyl-HSL) ligands to fold into an active conformation. The failure to purify ligand-free LuxR homologues in nonaggregated form at the high concentrations required for their structural characterization has limited the understanding of the mechanisms by which QS receptors are activated. Surface-enhanced Raman scattering (SERS) is a vibrational spectroscopy technique that can be applied to study proteins at extremely low concentrations in their active state. The high sensitivity of SERS has allowed us to detect molecular interactions between the ligand-binding domain of LasR (LasRLBD) as a soluble apoprotein and modulators of P. aeruginosa QS. We found that QS activators and inhibitors produce differential SERS fingerprints in LasRLBD, and in combination with molecular docking analysis provide insight into the relevant interaction mechanism. This study reveals signal-specific structural changes in LasR upon ligand binding, thereby confirming the applicability of SERS to analyze ligand-induced conformational changes in proteins. PMID:25927541

  13. Hypersensitive response and acyl‐homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae

    PubMed Central

    Jakovljevic, Vladimir; Jock, Susanne; Du, Zhiqiang; Geider, Klaus

    2008-01-01

    Summary Fire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight. PMID:21261861

  14. Regulon Studies and In Planta Role of the BraI/R Quorum-Sensing System in the Plant-Beneficial Burkholderia Cluster

    PubMed Central

    Coutinho, Bruna G.; Mitter, Birgit; Talbi, Chouhra; Sessitsch, Angela; Bedmar, Eulogio J.; Halliday, Nigel; James, Euan K.; Cámara, Miguel

    2013-01-01

    The genus Burkholderia is composed of functionally diverse species, and it can be divided into several clusters. One of these, designated the plant-beneficial-environmental (PBE) Burkholderia cluster, is formed by nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that members of the PBE group share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system, designated BraI/R, that produces and responds to 3-oxo-C14-HSL (OC14-HSL). Moreover, some of them also possess a second AHL QS system, designated XenI2/R2, producing and responding to 3-hydroxy-C8-HSL (OHC8-HSL). In the present study, we performed liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis to determine which AHL molecules are produced by each QS system of this group of bacteria. The results showed that XenI2/R2 is mainly responsible for the production of OHC8-HSL and that the BraI/R system is involved in the production of several different AHLs. This analysis also revealed that Burkholderia phymatum STM815 produces greater amounts of AHLs than the other species tested. Further studies showed that the BraR protein of B. phymatum is more promiscuous than other BraR proteins, responding equally well to several different AHL molecules, even at low concentrations. Transcriptome studies with Burkholderia xenovorans LB400 and B. phymatum STM815 revealed that the BraI/R regulon is species specific, with exopolysaccharide production being the only common phenotype regulated by this system in the PBE cluster. In addition, BraI/R was shown not to be important for plant nodulation by B. phymatum strains or for endophytic colonization and growth promotion of maize by B. phytofirmans PsJN. PMID:23686262

  15. The Systematic Investigation of the Quorum Sensing System of the Biocontrol Strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 Unveils aurI to Be a Biosynthetic Origin for 3-Oxo-Homoserine Lactones

    PubMed Central

    Bauer, Judith S.; Hauck, Nils; Christof, Lisa; Mehnaz, Samina; Gust, Bertolt; Gross, Harald

    2016-01-01

    The shoot endophytic biocontrol strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 produces a wide range of exoproducts, including enzymes and antibiotics. The production of exoproducts is commonly tightly regulated. In order to get a deeper insight into the regulatory network of PB-St2, the strain was systematically investigated regarding its quorum sensing systems, both on the genetic and metabolic level. The genome analysis of PB-St2 revealed the presence of four putative acyl homoserine lactone (AHL) biosynthesis genes: phzI, csaI, aurI, and hdtS. LC-MS/MS analyses of the crude supernatant extracts demonstrated that PB-St2 produces eight AHLs. In addition, the concentration of all AHL derivatives was quantified time-resolved in parallel over a period of 42 h during the growth of P. aurantiaca PB-St2, resulting in production curves, which showed differences regarding the maximum levels of the AHLs (14.6 nM– 1.75 μM) and the production period. Cloning and heterologous overexpression of all identified AHL synthase genes in Escherichia coli proved the functionality of the resulting synthases PhzI, CsaI, and AurI. A clear AHL production pattern was assigned to each of these three AHL synthases, while the HdtS synthase did not lead to any AHL production. Furthermore, the heterologous expression study demonstrated unequivocally and for the first time that AurI directs the synthesis of two 3-oxo-AHLs. PMID:27861617

  16. Thiourea-Catalyzed Aminolysis of N-acyl Homoserine Lactones

    DTIC Science & Technology

    2013-01-01

    D. Geske , R. J. Wezeman, A. P. Siegel and H. E. Blackwell, J. Am. Chem. Soc., 2005, 127, 12762–12763; T. B. Rasmussen and M. Givskov, Microbiology...2006, 152, 895–904; G. D. Geske , J. C. O’Neill, D. M. Miller, M. E. Mattmann and H. E. Blackwell, J. Am. Chem. Soc., 2007, 129, 13613–13625. 6 Enzyme

  17. Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

    PubMed Central

    Nasuno, Eri; Fujita, Masaki J.; Nakatsu, Cindy H.; Kamagata, Yoichi; Hanada, Satoshi

    2012-01-01

    A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-l-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-l-homoserine lactone (C12-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria. PMID:22983963

  18. Influence of glucose concentrations on biofilm formation, motility, exoprotease production, and quorum sensing in Aeromonas hydrophila.

    PubMed

    Jahid, Iqbal Kabir; Lee, Na-Young; Kim, Anna; Ha, Sang-Do

    2013-02-01

    Aeromonas hydrophila recently has received increased attention because it is opportunistic and a primary human pathogen. A. hydrophila biofilm formation and its control are a major concern for food safety because biofilms are related to virulence. Therefore, we investigated biofilm formation, motility inhibition, quorum sensing, and exoprotease production of this opportunistic pathogen in response to various glucose concentrations from 0.05 to 2.5% (wt/vol). More than 0.05% glucose significantly impaired (P < 0.05) quorum sensing, biofilm formation, protease production, and swarming and swimming motility, whereas bacteria treated with 0.05% glucose had activity similar to that of the control (0% glucose). A stage shift biofilm assay revealed that the addition of glucose (2.5%) inhibited initial biofilm formation but not later stages. However, addition of quorum sensing molecules N-3-butanoyl-DL-homoserine lactone and N-3-hexanoyl homoserine lactone partially restored protease production, indicating that quorum sensing is controlled by glucose concentrations. Thus, glucose present in food or added as a preservative could regulate acyl-homoserine lactone quorum sensing molecules, which mediate biofilm formation and virulence in A. hydrophila.

  19. Characterisation of two quorum sensing systems in the endophytic Serratia plymuthica strain G3: differential control of motility and biofilm formation according to life-style.

    PubMed

    Liu, Xiaoguang; Jia, Jinli; Popat, Roman; Ortori, Catherine A; Li, Jun; Diggle, Stephen P; Gao, Kexiang; Cámara, Miguel

    2011-02-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) systems have been described in many plant-associated Gram-negative bacteria to control certain beneficial phenotypic traits, such as production of biocontrol factors and plant growth promotion. However, the role of AHL-mediated signalling in the endophytic strains of plant-associated Serratia is still poorly understood. An endophytic Serratia sp. G3 with biocontrol potential and high levels of AHL signal production was isolated from the stems of wheat and the role of QS in this isolate was determined. Strain G3 classified as Serratia plymuthica based on 16S rRNA was subjected to phylogenetic analysis. Using primers to conserved sequences of luxIR homologues from the Serratia genus, splIR and spsIR from the chromosome of strain G3 were cloned and sequenced. AHL profiles from strain G3 and Escherichia coli DH5α expressing splI or spsI from recombinant plasmids were identified by liquid chromatography-tandem mass spectrometry. This revealed that the most abundant AHL signals produced by SplI in E. coli were N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), N-3-oxo-heptanoylhomoserine lactone (3-oxo-C7-HSL), N-3-hydroxy-hexanoylhomoserine lactone (3-hydroxy-C6-HSL), N-hexanoylhomoserine lactone (C6-HSL), and N-heptanoyl homoserine lactone (C7-HSL); whereas SpsI was primarily responsible for the synthesis of N-butyrylhomoserine lactone (C4-HSL) and N-pentanoylhomoserine lactone (C5-HSL). Furthermore, a quorum quenching analysis by heterologous expression of the Bacillus A24 AiiA lactonase in strain G3 enabled the identification of the AHL-regulated biocontrol-related traits. Depletion of AHLs with this lactonase resulted in altered adhesion and biofilm formation using a microtiter plate assay and flow cells coupled with confocal laser scanning microscopy respectively. This was different from the closely related S. plymuthica strains HRO-C48 and RVH1, where biofilm formation for both strains is AHL

  20. Characterisation of two quorum sensing systems in the endophytic Serratia plymuthica strain G3: differential control of motility and biofilm formation according to life-style

    PubMed Central

    2011-01-01

    Background N-acylhomoserine lactone (AHL)-based quorum sensing (QS) systems have been described in many plant-associated Gram-negative bacteria to control certain beneficial phenotypic traits, such as production of biocontrol factors and plant growth promotion. However, the role of AHL-mediated signalling in the endophytic strains of plant-associated Serratia is still poorly understood. An endophytic Serratia sp. G3 with biocontrol potential and high levels of AHL signal production was isolated from the stems of wheat and the role of QS in this isolate was determined. Results Strain G3 classified as Serratia plymuthica based on 16S rRNA was subjected to phylogenetic analysis. Using primers to conserved sequences of luxIR homologues from the Serratia genus, splIR and spsIR from the chromosome of strain G3 were cloned and sequenced. AHL profiles from strain G3 and Escherichia coli DH5α expressing splI or spsI from recombinant plasmids were identified by liquid chromatography-tandem mass spectrometry. This revealed that the most abundant AHL signals produced by SplI in E. coli were N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), N-3-oxo-heptanoylhomoserine lactone (3-oxo-C7-HSL), N-3-hydroxy-hexanoylhomoserine lactone (3-hydroxy-C6-HSL), N-hexanoylhomoserine lactone (C6-HSL), and N-heptanoyl homoserine lactone (C7-HSL); whereas SpsI was primarily responsible for the synthesis of N-butyrylhomoserine lactone (C4-HSL) and N-pentanoylhomoserine lactone (C5-HSL). Furthermore, a quorum quenching analysis by heterologous expression of the Bacillus A24 AiiA lactonase in strain G3 enabled the identification of the AHL-regulated biocontrol-related traits. Depletion of AHLs with this lactonase resulted in altered adhesion and biofilm formation using a microtiter plate assay and flow cells coupled with confocal laser scanning microscopy respectively. This was different from the closely related S. plymuthica strains HRO-C48 and RVH1, where biofilm formation for both strains is

  1. Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves

    USDA-ARS?s Scientific Manuscript database

    Quorum sensing transcriptional regulator SdiA has been shown to enhance the survival of Escherichia coli O157:H7 (O157) in the acidic compartment of bovine rumen in response to N-acyl-L-homoserine lactones (AHLs) produced by the rumen bacteria. Bacteria that survive the rumen environment subsequentl...

  2. Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

    PubMed Central

    Schwarzer, Christian; Fu, Zhu; Patanwala, Maria; Hum, Lauren; Lopez-Guzman, Mirielle; Illek, Beate; Kong, Weidong; Lynch, Susan V.; Machen, Terry E.

    2014-01-01

    Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis CF) patients, a process regulated by quorum sensing molecules including N-(3-oxododecanoyl)-L-homoserine lactone, C12. C12 (10–100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψmito and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψmito and increases in Cacyto like 10–50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed. PMID:22233488

  3. Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species.

    PubMed

    Kimura, Soichiro; Tateda, Kazuhiro; Ishii, Yoshikazu; Horikawa, Manabu; Miyairi, Shinichi; Gotoh, Naomasa; Ishiguro, Masaji; Yamaguchi, Keizo

    2009-06-01

    Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12)-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C(12)-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C(12)-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C(12)-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 microM 3-oxo-C(12)-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C(12)-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C(12)-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.

  4. Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon.

    PubMed

    Schuster, Martin; Greenberg, E Peter

    2007-08-22

    Quorum-sensing regulation of gene expression in Pseudomonas aeruginosa is complex. Two interconnected acyl-homoserine lactone (acyl-HSL) signal-receptor pairs, 3-oxo-dodecanoyl-HSL-LasR and butanoyl-HSL-RhlR, regulate more than 300 genes. The induction of most of the genes is delayed during growth of P. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. Many of these late genes can be induced by addition of signals early by using specific media conditions. While several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally. To better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors (in the presence of their cognate signals) and another component that affects quorum sensing, the stationary phase sigma factor RpoS, early in growth. We determined the effect on target gene expression by microarray and real-time PCR analysis. Our results show that many target genes (e.g. lasB and hcnABC) are directly responsive to receptor protein levels. Most genes (e.g. lasA, lecA, and phnAB), however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. The majority of promoters advanced by RhlR appeared to be regulated directly, which allowed us to build a RhlR consensus sequence. The direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. The observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is controlled

  5. The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

    PubMed Central

    Jones, S; Yu, B; Bainton, N J; Birdsall, M; Bycroft, B W; Chhabra, S R; Cox, A J; Golby, P; Reeves, P J; Stephens, S

    1993-01-01

    Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes. Images PMID:8508773

  6. Quorum sensing of microalgae associated marine Ponticoccus sp. PD-2 and its algicidal function regulation.

    PubMed

    Chi, Wendan; Zheng, Li; He, Changfei; Han, Bin; Zheng, Minggang; Gao, Wei; Sun, Chengjun; Zhou, Gefei; Gao, Xiangxing

    2017-12-01

    Quorum sensing (QS) systems play important roles in regulating many physiological functions of microorganisms, such as biofilm formation, bioluminescence, and antibiotic production. One marine algicidal bacterium, Ponticoccus sp. PD-2, was isolated from the microalga Prorocentrum donghaiense, and its N-acyl-homoserine lactone (AHL)-mediated QS system was verified. In this study, we analyzed the AHLs profile of strain PD-2. Two AHLs, 3-oxo-C8-HSL and 3-oxo-C10-HSL, were detected using a biosensor overlay assay and GC-MS methods. Two complete AHL-QS systems (designated zlaI/R and zlbI/R) were identified in the genome of strain PD-2. When expressed in Escherichia coli, both zlaI and zlbI genes could each produce 3-oxo-C8-HSL and 3-oxo-C10-HSL. Algicidal activity was investigated by evaluating the inhibitory rate (IR) of microalgae growth by measuring the fluorescence of viable cells. We found that the metabolites of strain PD-2 had algicidal activity against its host P. donghaiense (IR 84.81%) and two other red tide microalgae, Phaeocystis globosa (IR 78.91%) and Alexandrium tamarense (IR 67.14%). β-cyclodextrin which binds to AHLs and inhibits the QS system reduced the algicidal activity more than 50%. This indicates that inhibiting the QS system may affect the algicidal metabolites production of strain PD-2. Our study indicated that a QS-regulated algicidal system may play a potential role in the process of red tides disintegration. QS might be a potential way to control red tides.

  7. Investigation of acyl migration in mono- and dicaffeoylquinic acids under aqueous basic, aqueous acidic, and dry roasting conditions.

    PubMed

    Deshpande, Sagar; Jaiswal, Rakesh; Matei, Marius Febi; Kuhnert, Nikolai

    2014-09-17

    Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties from one quinic acid alcohol group to another, thus interconverting chlorogenic acid regioisomers. It therefore constitutes a special case of transesterification reaction. Acyl migration constitutes an important reaction pathway in both coffee roasting and brewing, altering the structure of chlorogenic acid initially present in the green coffee bean. In this contribution we describe detailed and comprehensive mechanistic studies comparing inter- and intramolecular acyl migration involving the seven most common chlorogenic acids in coffee. We employe aqueous acidic and basic conditions mimicking the brewing of coffee along with dry roasting conditions. We show that under aqueous basic conditions intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl migration. 3-Caffeoylquinic acid was shown to be most labile to basic hydrolysis. We additionally show that the acyl migration process is strongly pH dependent with increased transesterification taking place at basic pH. Under dry roasting conditions acyl migration competes with dehydration to form lactones. We argue that acyl migration precedes lactonization, with 3-caffeoylquinic acid lactone being the predominant product.

  8. Marine-Derived Metabolites of S-Adenosylmethionine as Templates for New Anti-Infectives

    PubMed Central

    Sufrin, Janice R.; Finckbeiner, Steven; Oliver, Colin M.

    2009-01-01

    S-Adenosylmethionine (AdoMet) is a key biochemical co-factor whose proximate metabolites include methylated macromolecules (e.g., nucleic acids, proteins, phospholipids), methylated small molecules (e.g., sterols, biogenic amines), polyamines (e.g., spermidine, spermine), ethylene, and N-acyl-homoserine lactones. Marine organisms produce numerous AdoMet metabolites whose novel structures can be regarded as lead compounds for anti-infective drug design. PMID:19841722

  9. Chemoselective synthesis of sialic acid 1,7-lactones.

    PubMed

    Allevi, Pietro; Rota, Paola; Scaringi, Raffaella; Colombo, Raffaele; Anastasia, Mario

    2010-08-20

    The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.

  10. Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence.

    PubMed

    Flynn, Padrig B; Busetti, Alessandro; Wielogorska, Ewa; Chevallier, Olivier P; Elliott, Christopher T; Laverty, Garry; Gorman, Sean P; Graham, William G; Gilmore, Brendan F

    2016-05-31

    The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30-60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.

  11. Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence

    PubMed Central

    Flynn, Padrig B.; Busetti, Alessandro; Wielogorska, Ewa; Chevallier, Olivier P.; Elliott, Christopher T.; Laverty, Garry; Gorman, Sean P.; Graham, William G.; Gilmore, Brendan F.

    2016-01-01

    The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30–60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa. PMID:27242335

  12. Metabolites with Gram-negative bacteria quorum sensing inhibitory activity from the marine animal endogenic fungus Penicillium sp. SCS-KFD08.

    PubMed

    Kong, Fan Dong; Zhou, Li Man; Ma, Qing Yun; Huang, Sheng Zhuo; Wang, Pei; Dai, Hao Fu; Zhao, You Xing

    2017-01-01

    Three new compounds named penicitor A, aculene E and penicitor B, as well as four known compounds, were isolated from the fermentation broth of Penicillium sp. SCS-KFD08 associated with a marine animal Sipunculus nudus from the Haikou bay of China. Their planar structures and absolute configurations were unambiguously elucidated by spectroscopic data, Mosher's method, CD spectrum analysis along with quantum ECD calculation. Among them, compounds 2-7 showed quorum sensing inhibitory activity against Chromobacterium violaceum CV026, and could significantly reduce violacein production in N-hexanoyl-l-homoserine lactone (C6-HSL) induced C. violaceum CV026 cultures at sub-inhibitory concentrations.

  13. Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50

    PubMed Central

    Garge, Sneha S.; Nerurkar, Anuradha S.

    2016-01-01

    Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved “HXHXDH” motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent. PMID:27911925

  14. A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

    NASA Astrophysics Data System (ADS)

    Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

    2004-03-01

    Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

  15. Biofilm formation, communication and interactions of leaching bacteria during colonization of pyrite and sulfur surfaces.

    PubMed

    Bellenberg, Sören; Díaz, Mauricio; Noël, Nanni; Sand, Wolfgang; Poetsch, Ansgar; Guiliani, Nicolas; Vera, Mario

    2014-11-01

    Bioleaching of metal sulfides is an interfacial process where biofilm formation is considered to be important in the initial steps of this process. Among the factors regulating biofilm formation, molecular cell-to-cell communication such as quorum sensing is involved. A functional LuxIR-type I quorum sensing system is present in Acidithiobacillus ferrooxidans. However, cell-to-cell communication among different species of acidophilic mineral-oxidizing bacteria has not been studied in detail. These aspects were the scope of this study with emphasis on the effects exerted by the external addition of mixtures of synthetic N-acyl-homoserine-lactones on pure and binary cultures. Results revealed that some mixtures had inhibitory effects on pyrite leaching. Some of them correlated with changes in biofilm formation patterns on pyrite coupons. We also provide evidence that A. thiooxidans and Acidiferrobacter spp. produce N-acyl-homoserine-lactones. In addition, the observation that A. thiooxidans cells attached more readily to pyrite pre-colonized by living iron-oxidizing acidophiles than to heat-inactivated or biofilm-free pyrite grains suggests that other interactions also occur. Our experiments show that pre-cultivation conditions influence A. ferrooxidans attachment to pre-colonized pyrite surfaces. The understanding of cell-to-cell communication may consequently be used to develop attempts to influence biomining/bioremediation processes. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Enantioselective N-Heterocyclic Carbene Catalysis via the Dienyl Acyl Azolium.

    PubMed

    Gillard, Rachel M; Fernando, Jared E M; Lupton, David W

    2018-04-16

    Herein we report the enantioselective N-heterocyclic carbene catalyzed (4+2) annulation of the dienyl acyl azolium with enolates. The reaction exploits readily accessible acyl fluorides and TMS enol ethers to give a range of highly enantio- and diastereo-enriched cyclohexenes (most >97:3 er and >20:1 dr). The reaction was found to require high nucleophilicity NHC catalysts with mechanistic studies supporting a stepwise 1,6-addition/β-lactonization. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.

    PubMed

    Tizzano, Marco; Gulbransen, Brian D; Vandenbeuch, Aurelie; Clapp, Tod R; Herman, Jake P; Sibhatu, Hiruy M; Churchill, Mair E A; Silver, Wayne L; Kinnamon, Sue C; Finger, Thomas E

    2010-02-16

    The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

  18. Structural and biochemical characterization of AidC, a quorum-quenching lactonase with atypical selectivity

    DOE PAGES

    Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun -Xiang; ...

    2015-06-26

    Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a k cat/K M value of approximately 2 × 10 6 M -1 s -1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typicalmore » AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. Lastly, the structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. We discuss implications for the catalytic mechanism of AHL lactonases.« less

  19. Structural and biochemical characterization of AidC, a quorum-quenching lactonase with atypical selectivity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun -Xiang

    Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a k cat/K M value of approximately 2 × 10 6 M -1 s -1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typicalmore » AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. Lastly, the structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. We discuss implications for the catalytic mechanism of AHL lactonases.« less

  20. The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Paes, Camila, E-mail: camilaquinetti@gmail.com; Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp; Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalianmore » cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.« less

  1. The enzymes of bacterial census and censorship

    PubMed Central

    Fast, Walter; Tipton, Peter A.

    2011-01-01

    N-Acyl-l-homoserine lactones (AHLs) are a major class of quorum sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide detailed insight into the chemistry and enzymology of bacterial communication. PMID:22099187

  2. The presence and role of bacterial quorum sensing in activated sludge

    PubMed Central

    Chong, Grace; Kimyon, Onder; Rice, Scott A.; Kjelleberg, Staffan; Manefield, Mike

    2012-01-01

    Summary Activated sludge used for wastewater treatment globally is composed of a high‐density microbial community of great biotechnological significance. In this study the presence and purpose of quorum sensing via N‐acylated‐l‐homoserine lactones (AHLs) in activated sludge was explored. The presence of N‐heptanoyl‐l‐homoserine lactone in organic extracts of sludge was demonstrated along with activation of a LuxR‐based AHL monitor strain deployed in sludge, indicating AHL‐mediated gene expression is active in sludge flocculates but not in the bulk aqueous phase. Bacterial isolates from activated sludge were screened for AHL production and expression of phenotypes commonly but not exclusively regulated by AHL‐mediated gene transcription. N‐acylated‐l‐homoserine lactone and exoenzyme production were frequently observed among the isolates. N‐acylated‐l‐homoserine lactone addition to sludge upregulated chitinase activity and an AHL‐ and chitinase‐producing isolate closely related to Aeromonas hydrophila was shown to respond to AHL addition with upregulation of chitinase activity. N‐acylated‐l‐homoserine lactones produced by this strain were identified and genes ahyI/R and chiA, encoding AHL production and response and chitinase activity respectively, were sequenced. These experiments provide insight into the relationship between AHL‐mediated gene expression and exoenzyme activity in activated sludge and may ultimately create opportunities to improve sludge performance. PMID:22583685

  3. The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

    PubMed Central

    Sundar, Shankar; Baker, Tania A; Sauer, Robert T

    2012-01-01

    In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase KM and decrease Vmax for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine. PMID:22102327

  4. Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

    PubMed Central

    Sayegh, Joyce; Clarke, Steven G.

    2008-01-01

    The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase. PMID:18515076

  5. Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer

    PubMed Central

    Zimmer, Beth L.; May, Amanda L.; Bhedi, Chinmayee D.; Dearth, Stephen P.; Prevatte, Carson W.; Pratte, Zoe; Campagna, Shawn R.; Richardson, Laurie L.

    2014-01-01

    Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome. PMID:25268348

  6. Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone*

    PubMed Central

    Schwarzer, Christian; Fu, Zhu; Morita, Takeshi; Whitt, Aaron G.; Neely, Aaron M.; Li, Chi; Machen, Terry E.

    2015-01-01

    Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca2+ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca2+] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca2+ release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12. PMID:25627690

  7. The enzymes of bacterial census and censorship.

    PubMed

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. The hydrocarbon-degrading marine bacterium Cobetia sp. strain MM1IDA2H-1 produces a biosurfactant that interferes with quorum sensing of fish pathogens by signal hijacking.

    PubMed

    Ibacache-Quiroga, C; Ojeda, J; Espinoza-Vergara, G; Olivero, P; Cuellar, M; Dinamarca, M A

    2013-07-01

    Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra- or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish aquaculture. © 2013

  9. Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia

    PubMed Central

    Hiblot, Julien; Bzdrenga, Janek; Champion, Charlotte; Chabriere, Eric; Elias, Mikael

    2015-01-01

    A new representative of the Phosphotriesterase-Like Lactonases (PLLs) family from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia has been characterized and crystallized. VmoLac is a native, proficient lactonase with promiscuous, low phosphotriesterase activity. VmoLac therefore represents an interesting candidate for engineering studies, with the aim of developing an efficient bacterial quorum-quenching agent. Here, we provide an extensive biochemical and kinetic characterization of VmoLac and describe the X-ray structures of the enzyme bound to a fatty acid and to its cognate substrate 3-oxo-C10 AHL (Acyl-Homoserine Lactone). The structures highlight possible structural determinants that may be involved in its extreme thermal stability (Tm = 128°C). Moreover, the structure reveals that the substrate binding mode of VmoLac significantly differs from those of its close homologues, possibly explaining the substrate specificity of the enzyme. Finally, we describe the specific interactions between the enzyme and its substrate, and discuss the possible lactone hydrolysis mechanism of VmoLac. PMID:25670483

  10. Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts.

    PubMed

    Leguina, Ana Carolina Del V; Nieto, Carolina; Pajot, Hipólito M; Bertini, Elisa V; Mac Cormack, Walter; Castellanos de Figueroa, Lucía I; Nieto-Peñalver, Carlos G

    2018-01-01

    The inactivation of quorum sensing signals, a phenomenon known as quorum quenching, has been described in diverse microorganisms, though it remains almost unexplored in yeasts. Beyond the well-known properties of these microorganisms for the industry or as eukaryotic models, the role of yeasts in soil or in the inner tissues of a plant is largely unknown. In this report, the wider survey of quorum quenching activities in yeasts isolated from Antarctic soil and the inner tissues of sugarcane, a tropical crop, is presented. Results show that, independently of their niche, quorum quenching activities are broadly present in unicellular fungi. Although yeasts showing a broad range of quorum quenching activity are present in the two niches, at the same time specific AHL inactivation profiles can also be found. Furthermore, yeasts from both sampling sites show quorum quenching activities compatible with lactonase-like and acylase-like inactivations of AHLs. Interestingly, the characterization of Rhodotorula mucilaginosa 7Apo1 showed that the presence of a particular AHL does not interfere with the quenching of a second molecule. Evidence suggests that yeasts could play a role in the modulation of the quorum sensing activity of bacteria. The relationship among phylogeny, sampling sites and yeast quorum quenching activities of the isolates is analyzed. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Imidazolines as Non-Classical Bioisosteres of N-Acyl Homoserine Lactones and Quorum Sensing Inhibitors

    PubMed Central

    Reyes-Arellano, Alicia; Bucio-Cano, Alejandro; Montenegro-Sustaita, Mabel; Curiel-Quesada, Everardo; Salgado-Zamora, Héctor

    2012-01-01

    A series of selected 2-substituted imidazolines were synthesized in moderate to excellent yields by a modification of protocols reported in the literature. They were evaluated as potential non-classical bioisosteres of AHL with the aim of counteracting bacterial pathogenicity. Imidazolines 18a, 18e and 18f at various concentrations reduced the violacein production by Chromobacterium violaceum, suggesting an anti-quorum sensing profile against Gram-negative bacteria. Imidazoline 18b did not affect the production of violacein, but had a bacteriostatic effect at 100 μM and a bactericidal effect at 1 mM. Imidazoline 18a bearing a hexyl phenoxy moiety was the most active compound of the series, rendering a 72% inhibitory effect of quorum sensing at 100 μM. Imidazoline 18f bearing a phenyl nonamide substituent presented an inhibitory effect on quorum sensing at a very low concentration (1 nM), with a reduction percentage of 28%. This compound showed an irregular performance, decreasing inhibition at concentrations higher than 10 μM, until reaching 100 μM, at which concentration it increased the inhibitory effect with a 49% reduction percentage. When evaluated on Serratia marcescens, compound 18f inhibited the production of prodigiosin by 40% at 100 μM. PMID:22408391

  12. Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

    PubMed

    Tanigawa, Kazunari; Degang, Yang; Kawashima, Akira; Akama, Takeshi; Yoshihara, Aya; Ishido, Yuko; Makino, Masahiko; Ishii, Norihisa; Suzuki, Koichi

    2012-05-01

    Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.

  13. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

    PubMed

    Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2017-06-01

    Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Quorum Sensing in Chromobacterium violaceum: DNA Recognition and Gene Regulation by the CviR Receptor ▿ †

    PubMed Central

    Stauff, Devin L.; Bassler, Bonnie L.

    2011-01-01

    The bacterial pathogen Chromobacterium violaceum uses a LuxIR-type quorum-sensing system to detect and respond to changes in cell population density. CviI synthesizes the autoinducer C10-homoserine lactone (C10-HSL), and CviR is a cytoplasmic DNA binding transcription factor that activates gene expression following binding to C10-HSL. A number of behaviors are controlled by quorum sensing in C. violaceum. However, few genes have been shown to be directly controlled by CviR, in part because the DNA motif bound by CviR is not well characterized. Here, we define the DNA sequence required for promoter recognition by CviR. Using in vivo data generated from a library of point mutations in a CviR-regulated promoter, we find that CviR binds to a palindrome with the ideal sequence CTGNCCNNNNGGNCAG. We constructed a position weight matrix using these in vivo data and scanned the C. violaceum genome to predict CviR binding sites. We measured direct activation of the identified promoters by CviR and found that CviR controls the expression of the promoter for a chitinase, a type VI secretion-related gene, a transcriptional regulator gene, a guanine deaminase gene, and cviI. Indeed, regulation of cviI expression by CviR generates a canonical quorum-sensing positive-feedback loop. PMID:21622734

  15. Quorum sensing in Chromobacterium violaceum: DNA recognition and gene regulation by the CviR receptor.

    PubMed

    Stauff, Devin L; Bassler, Bonnie L

    2011-08-01

    The bacterial pathogen Chromobacterium violaceum uses a LuxIR-type quorum-sensing system to detect and respond to changes in cell population density. CviI synthesizes the autoinducer C(10)-homoserine lactone (C(10)-HSL), and CviR is a cytoplasmic DNA binding transcription factor that activates gene expression following binding to C(10)-HSL. A number of behaviors are controlled by quorum sensing in C. violaceum. However, few genes have been shown to be directly controlled by CviR, in part because the DNA motif bound by CviR is not well characterized. Here, we define the DNA sequence required for promoter recognition by CviR. Using in vivo data generated from a library of point mutations in a CviR-regulated promoter, we find that CviR binds to a palindrome with the ideal sequence CTGNCCNNNNGGNCAG. We constructed a position weight matrix using these in vivo data and scanned the C. violaceum genome to predict CviR binding sites. We measured direct activation of the identified promoters by CviR and found that CviR controls the expression of the promoter for a chitinase, a type VI secretion-related gene, a transcriptional regulator gene, a guanine deaminase gene, and cviI. Indeed, regulation of cviI expression by CviR generates a canonical quorum-sensing positive-feedback loop.

  16. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

    PubMed Central

    Hong, Kar Wai; Chan, Kok-Gan

    2015-01-01

    Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity. PMID:26290785

  17. Assembly interdependence among the S. cerevisiae bud neck ring proteins Elm1p, Hsl1p and Cdc12p.

    PubMed

    Thomas, Courtney L; Blacketer, Melissa J; Edgington, Nicholas P; Myers, Alan M

    2003-07-15

    In Saccharomyces cerevisiae, a complex comprising more than 20 different polypeptides assembles in a ring at the neck between the mother cell and the bud. This complex functions to coordinate cell morphology with cell division. Relatively little is known about this control system, including the physical relationships between the components of the neck ring. This study addressed the assembly interactions of three components of the ring, specifically the protein kinases Elm1p and Hsl1p and the septin Cdc12p. Specific amino acid substitutions in each of these three proteins were identified that either cause or suppress a characteristic phenotype of abnormally elongated cells and delay in the G(2)-M transition. Each protein was fused to green fluorescent protein, and its ability to localize at the neck was monitored in vivo in cells of various genotypes. Localization of Hsl1p to the neck requires Elm1p function. Elm1p localized normally in the absence of Hsl1p, although a specific point mutation in Hsl1p clearly affected Elm1p localization. The cdc12-122 mutation prevented assembly of Elm1p or Hsl1p into the neck ring. Normal assembly of Cdc12p at the neck was dependent upon Elm1p and also, to a smaller extent, on Hsl1p. Ectopic localization of Cdc12p at the bud tip was observed frequently in elm1 mutants and also, to a lesser extent, in hsl1 mutants. Thus, Elm1p is a key factor in the assembly and/or maintenance of Hsl1p, as well as at least one septin, into the bud neck ring. Copyright 2003 John Wiley & Sons, Ltd.

  18. The quorum-quenching lactonase from Alicyclobacter acidoterrestris : purification, kinetic characterization, crystallization and crystallographic analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bergonzi, Celine; Schwab, Michael; Chabriere, Eric

    Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacteriumAlicyclobacter acidoterrestris. Kinetic characterization proves AaL to be a proficient lactonase, with catalytic efficiencies (k cat/K m) against AHLs in the region of 10 5Mmore » -1s -1. AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65Å resolution are reported.« less

  19. Novel linear polymers able to inhibit bacterial quorum sensing.

    PubMed

    Cavaleiro, Eliana; Duarte, Ana Sofia; Esteves, Ana Cristina; Correia, António; Whitcombe, Michael J; Piletska, Elena V; Piletsky, Sergey A; Chianella, Iva

    2015-05-01

    Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

    PubMed

    Fukami, Josiane; Abrantes, Julia Laura Fernandes; Del Cerro, Pablo; Nogueira, Marco Antonio; Ollero, Francisco Javier; Megías, Manuel; Hungria, Mariangela

    2018-01-01

    Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

  1. C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa

    PubMed Central

    Blier, Anne-Sophie; Veron, Wilfried; Bazire, Alexis; Gerault, Eloïse; Taupin, Laure; Vieillard, Julien; Rehel, Karine; Dufour, Alain; Le Derf, Franck; Orange, Nicole; Hulen, Christian; Feuilloley, Marc G. J.

    2011-01-01

    Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during

  2. Conservation of the abscission signaling peptide IDA during Angiosperm evolution: withstanding genome duplications and gain and loss of the receptors HAE/HSL2

    PubMed Central

    Stø, Ida M.; Orr, Russell J. S.; Fooyontphanich, Kim; Jin, Xu; Knutsen, Jonfinn M. B.; Fischer, Urs; Tranbarger, Timothy J.; Nordal, Inger; Aalen, Reidunn B.

    2015-01-01

    The peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2), controls different cell separation events in Arabidopsis thaliana. We hypothesize the involvement of this signaling module in abscission processes in other plant species even though they may shed other organs than A. thaliana. As the first step toward testing this hypothesis from an evolutionarily perspective we have identified genes encoding putative orthologs of IDA and its receptors by BLAST searches of publically available protein, nucleotide and genome databases for angiosperms. Genes encoding IDA or IDA-LIKE (IDL) peptides and HSL proteins were found in all investigated species, which were selected as to represent each angiosperm order with available genomic sequences. The 12 amino acids representing the bioactive peptide in A. thaliana have virtually been unchanged throughout the evolution of the angiosperms; however, the number of IDL and HSL genes varies between different orders and species. The phylogenetic analyses suggest that IDA, HSL2, and the related HSL1 gene, were present in the species that gave rise to the angiosperms. HAE has arisen from HSL1 after a genome duplication that took place after the monocot—eudicots split. HSL1 has also independently been duplicated in the monocots, while HSL2 has been lost in gingers (Zingiberales) and grasses (Poales). IDA has been duplicated in eudicots to give rise to functionally divergent IDL peptides. We postulate that the high number of IDL homologs present in the core eudicots is a result of multiple whole genome duplications (WGD). We substantiate the involvement of IDA and HAE/HSL2 homologs in abscission by providing gene expression data of different organ separation events from various species. PMID:26579174

  3. Inhibition of Vibrio biofilm formation by a marine actinomycete strain A66.

    PubMed

    You, JianLan; Xue, XiaoLi; Cao, LiXiang; Lu, Xin; Wang, Jian; Zhang, LiXin; Zhou, ShiNing

    2007-10-01

    China remains by far the largest aquaculture producer in the world. However, biofilms formed by pathogenic Vibrio strains pose serious problems to marine aquaculture. To provide a strategy for biofilm prevention, control, and eradication, extracts from 88 marine actinomycetes were screened. Thirty-five inhibited the biofilm formation of Vibrio harveyi, Vibrio vulnificus, and Vibrio anguillarum at a concentration of 2.5% (v/v). Thirty-three of the actinomycete extracts dispersed the mature biofilm. Six extracts inhibited the quorum-sensing system of V. harveyi by attenuating the signal molecules N-acylated homoserine lactones' activity. Strain A66, which was identified as Streptomyces albus, both attenuated the biofilms and inhibited their quorum-sensing system. It is suggested that strain A66 is a promising candidate to be used in future marine aquaculture.

  4. Biochar and microbial signaling: production conditions determine effects on microbial communication.

    PubMed

    Masiello, Caroline A; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R; Rudgers, Jennifer A; Wagner, Daniel S; Zygourakis, Kyriacos; Silberg, Jonathan J

    2013-10-15

    Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell-cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m(2)/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m(2)/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.

  5. Biochar and microbial signaling: production conditions determine effects on microbial communication

    PubMed Central

    Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.

    2013-01-01

    Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell-cell communication varied, with the biochar prepared at 700°C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300°C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops. PMID:24066613

  6. Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat.

    PubMed

    Abed, Raeid M M; Dobretsov, Sergey; Al-Fori, Marwan; Gunasekera, Sarath P; Sudesh, Kumar; Paul, Valerie J

    2013-07-01

    In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22-25 % and a maximum temperature of 45-60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-isoLeu), and cyclo(L-Pro-D-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(L-Pro-L-Phe) and cyclo(L-Pro-L-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.

  7. Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase With Atypical Selectivity

    PubMed Central

    Mascarenhas, Romila; Thomas, Pei W.; Wu, Chun-Xiang; Nocek, Boguslaw P.; Hoang, Quyen Q.; Liu, Dali; Fast, Walter

    2015-01-01

    Quorum-quenching catalysts are of interest for potential application as biochemical tools to interrogate interbacterial communication pathways, as anti-biofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-L-homoserine (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is one of the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M−1s−1 for N-heptanoyl-L-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) than typical AHL lactonases (ca. > 1 mM). X-ray crystal structures of AidC alone, and with the product N-hexanoyl-L-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multi-angle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a ‘kinked’ α-helix that forms part of a closed binding pocket which provides affinity and enforces selectivity for AHL substrates, and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed. PMID:26115006

  8. [Formation of the Pseudomonas aeruginosa PAO1 biofilms in the presence of hydrogen peroxide; the effect of the AiiA gene].

    PubMed

    Pliuta, V A; Andreenko, Iu V; Kuznetsov, A E; Khmel', I A

    2013-01-01

    In the natural ecosystems, most bacteria exist as specifically organized biofilms attached to various surfaces; the biofilms have a complex architecture and are surrounded by an exopolymeric matrix. The bacteria in the biofilms are extremely resistant to antibacterial agents. The ability of the pathogenic bacteria to produce biofilms causes serious problems in medicine. Therefore, the study of the action of different compounds with antibacterial activity is of great interest. In this work, we studied the effect of the hydrogen peroxide (H2O2) on the formation of biofilms by Pseudomonas aeruginosa PAO1. It was shown that H2O2 in concentrations that do not suppress bacterial growth (or suppress it only weakly) stimulates the formation of the biofilms. At higher concentrations, H2O2 inhibits the formation of the biofilms. In order to determine if the stimulation of the biofilm formation depends on Quorum Sensing (QS) regulation, the plasmid pME6863 containing the heterologous gene aiiA encoding the N-acyl-homoserine lactonase AiiA was introduced into P. aeruginosa PAO1. The synthesis by cells of this enzyme degrading N-acyl-homoserine lactones (AHL), signaling molecules of the QS systems, led to the absence of the stimulation of the biofilm formation by the action of H2O2. This fact indicates that the stimulation of the biofilm formation in the presence of H2O2 depends on the functioning of the QS systems of the gene expression regulation of P. aeruginosa PAO1.

  9. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Sterling, Sarah M.; Duvalyan, Angela; Liao, Elizabeth N.; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-01-01

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611–950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611–950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379–1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. PMID:27193302

  10. The quorum-quenching lactonase from Geobacillus caldoxylosilyticus : purification, characterization, crystallization and crystallographic analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bergonzi, Celine; Schwab, Michael; Elias, Mikael

    Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-β-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacteriumGeobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidatemore » for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (k cat/K m) against AHLs of greater than 10 6 M $-$1 s $-$1. The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Å resolution of GcL are reported.« less

  11. 21 CFR 184.1318 - Glucono delta-lactone.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Glucono delta-lactone. 184.1318 Section 184.1318... Listing of Specific Substances Affirmed as GRAS § 184.1318 Glucono delta-lactone. (a) Glucono delta-lactone (C6H10O6, CAS Reg. No. 90-80-2), also called D-gluconic acid delta-lactone or D-glucono-1,5...

  12. 21 CFR 184.1318 - Glucono delta-lactone.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Glucono delta-lactone. 184.1318 Section 184.1318... Listing of Specific Substances Affirmed as GRAS § 184.1318 Glucono delta-lactone. (a) Glucono delta-lactone (C6H10O6, CAS Reg. No. 90-80-2), also called D-gluconic acid delta-lactone or D-glucono-1,5...

  13. Strain identification and quorum sensing inhibition characterization of marine-derived Rhizobium sp. NAO1

    NASA Astrophysics Data System (ADS)

    Chang, Hong; Zhou, Jin; Zhu, Xiaoshan; Yu, Shenchen; Chen, Lu; Jin, Hui; Cai, Zhonghua

    2017-03-01

    A novel strategy for combating pathogens is through the ongoing development and use of anti-quorum sensing (QS) treatments such as therapeutic bacteria or their anti-QS substances. Relatively little is known about the bacteria that inhabit the open ocean and of their potential anti-pathogenic attributes; thus, in an initiative to identify these types of therapeutic bacteria, planktonic microbes from the North Atlantic Ocean were collected, isolated, cultured and screened for anti-QS activity. Screening analysis identified one such strain, Rhizobium sp. NAO1. Extracts of Rhizobium sp. NAO1 were identified via ultra-performance liquid chromatography (UPLC) analysis. They were shown to contain N-acyl homoserine lactone (AHL)-based QS analogues (in particular, the N-butyryl homoserine lactone (C4-AHL) analogue) and could disrupt biofilm formation by Pseudomonas aeruginosa PAO1. QS inhibition was confirmed using confocal scanning laser microscopy and growth curves, and it was shown to occur in a dose-dependent manner without affecting bacterial growth. Secondary metabolites of Rhizobium sp. NAO1 inhibited PAO1 pathogenicity by downregulating AHL-mediated virulence factors such as elastase activity and siderophore production. Furthermore, as a result of biofilm structure damage, the secondary metabolite products of Rhizobium sp. NAO1 significantly increased the sensitivity of PAO1 to aminoglycoside antibiotics. Our results demonstrated that Rhizobium sp. strain NAO1 has the ability to disrupt P. aeruginosa PAO1 biofilm architecture, in addition to attenuating P. aeruginosa PAO1 virulence factor production and pathogenicity. Therefore, the newly identified ocean-derived Rhizobium sp. NAO1 has the potential to serve as a QS inhibitor and may be a new microbial resource for drug development.

  14. Crystal Structure of Homoserine Transacetylase from Haemophilus Influenzae Reveals a New Family of alpha/beta-Hydrolases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mirza,I.; Nazi, I.; Korczynska, M.

    2005-01-01

    Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the propertiesmore » of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs. hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.« less

  15. Charcoal disrupts cell-cell communication through multiple mechanisms

    NASA Astrophysics Data System (ADS)

    Gao, X.; Cheng, H. Y.; Liu, S.; Masiello, C. A.; Silberg, J. J.; Del Valle, I.

    2016-12-01

    Microbial cell-cell communication through the release and detection of small signaling molecules is employed by soil microbes to manage many biogeochemically relevant processes including production of biofilms, priming effects on native SOM, and management of methanogenesis and denitrification. Charcoal is a ubiquitous component of soil, entering soil either from natural fire or intentionally amended as biochar. Charcoal's presence in soil introduces spatial and temporal heterogeneity in nutrients and habitats for soil microbes and may trigger a range of biological effects not yet predictable, in part because it interferes with microbial cell-cell communication. We hypothesized that charcoal's alkalinity and large active surface area could affect the lifetime of some chemical compounds that microbes use for cell-cell signaling on times scales relevant to growth and communication. To test this idea, we examined the extent and rate of charcoal quenching of cell-cell communication caused by ten charcoals with a wide range of physicochemical properties. Our measurements focused on signaling mediated by an acyl-homoserine lactone (AHL), N-3-oxo-dodecanoyl-L-homoserine lactone, which is used by many gram-negative bacteria for quorum sensing. Our results from a bioassay and chemical sorption experiments revealed that charcoal can decrease the bioavailable level of AHL through a combination of sorption and pH-dependent hydrolysis of the lactone ring. We found that the kinetics of hydrolysis can exceed those of sorption. These findings implicate charcoal surface area and alkalinity as properties that could be tuned to regulate the degradation rates of cell-cell signaling molecules in soils. We then built a quantitative model that predicts the half-lives of different microbial signaling compounds in the presence of charcoals varying in pH and surface area. Our model results suggest that the effects of charcoal on pH-sensitive bacterial AHL signals will be fundamentally

  16. Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

    PubMed Central

    Crépin, Alexandre; Barbey, Corinne; Beury-Cirou, Amélie; Hélias, Valérie; Taupin, Laure; Reverchon, Sylvie; Nasser, William; Faure, Denis; Dufour, Alain; Orange, Nicole; Feuilloley, Marc; Heurlier, Karin; Burini, Jean-François; Latour, Xavier

    2012-01-01

    Background Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. Methodology/Principal Findings Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. Conclusions/Significance Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the

  17. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

    PubMed

    Finnigan, Gregory C; Sterling, Sarah M; Duvalyan, Angela; Liao, Elizabeth N; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-07-15

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. © 2016 Finnigan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two

  18. CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ †

    PubMed Central

    Yakhnin, Helen; Baker, Carol S.; Berezin, Igor; Evangelista, Michael A.; Rassin, Alisa; Romeo, Tony; Babitzke, Paul

    2011-01-01

    The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target. PMID:21908661

  19. Untapped Resources: Biotechnological Potential of Peptides and Secondary Metabolites in Archaea

    PubMed Central

    Charlesworth, James C.; Burns, Brendan P.

    2015-01-01

    Archaea are an understudied domain of life often found in “extreme” environments in terms of temperature, salinity, and a range of other factors. Archaeal proteins, such as a wide range of enzymes, have adapted to function under these extreme conditions, providing biotechnology with interesting activities to exploit. In addition to producing structural and enzymatic proteins, archaea also produce a range of small peptide molecules (such as archaeocins) and other novel secondary metabolites such as those putatively involved in cell communication (acyl homoserine lactones), which can be exploited for biotechnological purposes. Due to the wide array of metabolites produced there is a great deal of biotechnological potential from antimicrobials such as diketopiperazines and archaeocins, as well as roles in the cosmetics and food industry. In this review we will discuss the diversity of small molecules, both peptide and nonpeptide, produced by archaea and their potential biotechnological applications. PMID:26504428

  20. Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects.

    PubMed

    Wang, Yan; Song, Qinghao; Zhang, Xiao-Hua

    2016-09-22

    Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.

  1. Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

    PubMed

    Pan, Jieru; Huang, Tianpei; Yao, Fan; Huang, Zhipeng; Powell, Charles A; Qiu, Sixin; Guan, Xiong

    2008-01-01

    AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

  2. Secondary metabolites produced by the marine bacterium Halobacillus salinus that inhibit quorum sensing-controlled phenotypes in gram-negative bacteria.

    PubMed

    Teasdale, Margaret E; Liu, Jiayuan; Wallace, Joselynn; Akhlaghi, Fatemeh; Rowley, David C

    2009-02-01

    Certain bacteria use cell-to-cell chemical communication to coordinate community-wide phenotypic expression, including swarming motility, antibiotic biosynthesis, and biofilm production. Here we present a marine gram-positive bacterium that secretes secondary metabolites capable of quenching quorum sensing-controlled behaviors in several gram-negative reporter strains. Isolate C42, a Halobacillus salinus strain obtained from a sea grass sample, inhibits bioluminescence production by Vibrio harveyi in cocultivation experiments. With the use of bioassay-guided fractionation, two phenethylamide metabolites were identified as the active agents. The compounds additionally inhibit quorum sensing-regulated violacein biosynthesis by Chromobacterium violaceum CV026 and green fluorescent protein production by Escherichia coli JB525. Bacterial growth was unaffected at concentrations below 200 microg/ml. Evidence is presented that these nontoxic metabolites may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding.

  3. Quorum quenching properties of Actinobacteria isolated from Malaysian tropical soils.

    PubMed

    Devaraj, Kavimalar; Tan, Geok Yuan Annie; Chan, Kok-Gan

    2017-08-01

    In this study, a total of 147 soil actinobacterial strains were screened for their ability to inhibit response of Chromobacterium violaceum CV026 to short chain N-acyl homoserine lactone (AHL) which is a quorum sensing molecule. Of these, three actinobacterial strains showed positive for violacein inhibition. We further tested these strains for the inhibition of Pseudomonas aeruginosa PAO1 quorum sensing-regulated phenotypes, namely, swarming and pyocyanin production. The three strains were found to inhibit at least one of the quorum sensing-regulated phenotypes of PAO1. Phylogenetic analysis of the 16S rRNA gene sequences indicated that these strains belong to the genera Micromonospora, Rhodococcus and Streptomyces. This is the first report presenting quorum quenching activity by a species of the genus Micromonospora. Our data suggest that Actinobacteria may be a rich source of active compounds that can act against bacterial quorum sensing system.

  4. Discovery of new lactones in sweet cream butter oil.

    PubMed

    Sarrazin, Elise; Frerot, Eric; Bagnoud, Alain; Aeberhardt, Kasia; Rubin, Mark

    2011-06-22

    Sweet cream butter oil was analyzed to identify new volatile compounds that may contribute to its flavor, with an emphasis on lactones. The volatile part of butter oil was obtained by using short-path distillation. As some previously unknown lactones were detected in this first extract, it was fractionated further. The fatty acids were removed, and the extract was fractionated by flash chromatography. Three lactonic fractions possessing a creamy, buttery, and fatty character were investigated in depth by gas chromatography (GC) and mass spectrometry (MS) (EI and CI) and high-resolution GC-time-of-flight MS. Many lactones were identified by their mass fragmentation and by comparison with reference materials synthesized during this work. Six γ-lactones, five δ-lactones, and one ε-lactone were identified for the first time in butter oil, seven of them for the first time in a natural product. The possible contribution of these new lactones to the aroma of butter oil is briefly discussed.

  5. Inactivation of gacS Does Not Affect the Competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana Rhizosphere

    PubMed Central

    Schmidt-Eisenlohr, Heike; Gast, Astrid; Baron, Christian

    2003-01-01

    Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac− phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in

  6. Stenotrophomonas maltophilia responds to exogenous AHL signals through the LuxR solo SmoR (Smlt1839).

    PubMed

    Martínez, Paula; Huedo, Pol; Martinez-Servat, Sònia; Planell, Raquel; Ferrer-Navarro, Mario; Daura, Xavier; Yero, Daniel; Gibert, Isidre

    2015-01-01

    Quorum Sensing (QS) mediated by Acyl Homoserine Lactone (AHL) molecules are probably the most widespread and studied among Gram-negative bacteria. Canonical AHL systems are composed by a synthase (LuxI family) and a regulator element (LuxR family), whose genes are usually adjacent in the genome. However, incomplete AHL-QS machinery lacking the synthase LuxI is frequently observed in Proteobacteria, and the regulator element is then referred as LuxR solo. It has been shown that certain LuxR solos participate in interspecific communication by detecting signals produced by different organisms. In the case of Stenotrophomonas maltophilia, a preliminary genome sequence analysis revealed numerous putative luxR genes, none of them associated to a luxI gene. From these, the hypothetical LuxR solo Smlt1839, here designated SmoR, presents a conserved AHL binding domain and a helix-turn-helix DNA binding motif. Its genomic organization-adjacent to hchA gene-indicate that SmoR belongs to the new family "LuxR regulator chaperone HchA-associated." AHL-binding assays revealed that SmoR binds to AHLs in-vitro, at least to oxo-C8-homoserine lactone, and it regulates operon transcription, likely by recognizing a conserved palindromic regulatory box in the hchA upstream region. Supplementation with concentrated supernatants from Pseudomonas aeruginosa, which contain significant amounts of AHLs, promoted swarming motility in S. maltophilia. Contrarily, no swarming stimulation was observed when the P. aeruginosa supernatant was treated with the lactonase AiiA from Bacillus subtilis, confirming that AHL contributes to enhance the swarming ability of S. maltophilia. Finally, mutation of smoR resulted in a swarming alteration and an apparent insensitivity to the exogenous AHLs provided by P. aeruginosa. In conclusion, our results demonstrate that S. maltophilia senses AHLs produced by neighboring bacteria through the LuxR solo SmoR, regulating population behaviors such as swarming

  7. Sesquiterpenoids Lactones: Benefits to Plants and People

    PubMed Central

    Chadwick, Martin; Trewin, Harriet; Gawthrop, Frances; Wagstaff, Carol

    2013-01-01

    Sesquiterpenoids, and specifically sesquiterpene lactones from Asteraceae, may play a highly significant role in human health, both as part of a balanced diet and as pharmaceutical agents, due to their potential for the treatment of cardiovascular disease and cancer. This review highlights the role of sesquiterpene lactones endogenously in the plants that produce them, and explores mechanisms by which they interact in animal and human consumers of these plants. Several mechanisms are proposed for the reduction of inflammation and tumorigenesis at potentially achievable levels in humans. Plants can be classified by their specific array of produced sesquiterpene lactones, showing high levels of translational control. Studies of folk medicines implicate sesquiterpene lactones as the active ingredient in many treatments for other ailments such as diarrhea, burns, influenza, and neurodegradation. In addition to the anti-inflammatory response, sesquiterpene lactones have been found to sensitize tumor cells to conventional drug treatments. This review explores the varied ecological roles of sesquiterpenes in the plant producer, depending upon the plant and the compound. These include allelopathy with other plants, insects, and microbes, thereby causing behavioural or developmental modification to these secondary organisms to the benefit of the sesquiterpenoid producer. Some sesquiterpenoid lactones are antimicrobial, disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stresses that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers. The implications of sesquiterpenoid lactone qualities for future crop production are discussed. PMID:23783276

  8. Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

    PubMed

    Wysoczynski-Horita, Christina L; Boursier, Michelle E; Hill, Ryan; Hansen, Kirk; Blackwell, Helen E; Churchill, Mair E A

    2018-05-01

    Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems. © 2018 John Wiley & Sons Ltd.

  9. Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

    PubMed Central

    Majerczyk, Charlotte D.; Brittnacher, Mitchell J.; Jacobs, Michael A.; Armour, Christopher D.; Radey, Matthew C.; Bunt, Richard; Hayden, Hillary S.; Bydalek, Ryland

    2014-01-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei. PMID:25182491

  10. Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase.

    PubMed

    Kisch, Johannes Martin; Utpatel, Christian; Hilterhaus, Lutz; Streit, Wolfgang R; Liese, Andreas

    2014-09-05

    Biofilms are matrix-encapsulated cell aggregates that cause problems in technical and health-related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilm-forming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria. We applied horse liver esterase, porcine kidney acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Quorum Sensing in Marine Microbial Environments.

    PubMed

    Hmelo, Laura R

    2017-01-03

    Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surface-attached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures.

  12. Quorum Sensing in Marine Microbial Environments

    NASA Astrophysics Data System (ADS)

    Hmelo, Laura R.

    2017-01-01

    Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surface-attached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures.

  13. Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

    PubMed

    Majerczyk, Charlotte D; Brittnacher, Mitchell J; Jacobs, Michael A; Armour, Christopher D; Radey, Matthew C; Bunt, Richard; Hayden, Hillary S; Bydalek, Ryland; Greenberg, E Peter

    2014-11-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. Purification and antibiofilm activity of AHL-lactonase from endophytic Enterobacter aerogenes VT66.

    PubMed

    Rajesh, P S; Rai, V Ravishankar

    2015-11-01

    The opportunistic pathogen Pseudomonas aeruginosa uses biofilm lifestyle to resist antibiotic treatment. In our study, endophytic bacterium Enterobacter aerogenes VT66 quenched the N-acyl homoserine lactone (AHL) molecules produced by P. aeruginosa PAO1. The quorum quenching activity was attributed to the presence of AHL-lactonase. The AHL-lactonase was purified using column chromatography and purified AHL-lactonase was applied for the control of biofilm formation in P. aeruginosa PAO1. The results showed that purified AHL-lactonase obtained with a molecular weight about 30kDa was able to inhibit more than 70% of biofilm in P. aeruginosa PAO1 (P<0.001). Antibiofilm activity of AHL-lactonase was correlated well with results from staining technique used to determine inhibition of biomass and viable cell activity. Therefore, results unambiguously confirm that the AHL-lactonase from E. aerogenes VT66 could be used as antibiofilm therapeutics in P. aeruginosa associated biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Possible Correlation Between Bile Salt Hydrolysis and AHL Deamidation: Staphylococcus epidermidis RM1, a Potent Quorum Quencher and Bile Salt Hydrolase Producer.

    PubMed

    Mukherji, Ruchira; Prabhune, Asmita

    2015-05-01

    The aim of the present work was to isolate a bile salt hydrolase (BSH) producer from fermented soy curd and explore the ability of the BSH produced to cleave bacterial quorum sensing signals. Bacterial isolates with possible ability to deconjugate bile salts were enriched and isolated on De Man, Rogosa and Sharpe (MRS) medium containing 0.2% bile salts. BSH-producing positive isolate with orange-pink-pigmented colonies was isolated and was identified as a strain of Staphylococcus epidermidis using biochemical and phylogenetic tools. S. epidermidis RM1 was shown to possess both potent BSH and N-acyl homoserine lactone (AHL) cleavage activity. Genetic basis of this dual-enzyme activity was explored by means of specific primers designed using S. epidermidis ATCC 12228 genome as template. It was observed that a single enzyme was not responsible for both the activity. Two different genetic elements corresponding to each of the enzymatic activity were successfully amplified from the genomic DNA of the isolate.

  16. Mass spectrometry analysis of terpene lactones in Ginkgo biloba.

    PubMed

    Ding, Shujing; Dudley, Ed; Song, Qingbao; Plummer, Sue; Tang, Jiandong; Newton, Russell P; Brenton, A Gareth

    2008-01-01

    Terpene lactones are a family of compounds with unique chemical structures, first recognised in an extract of Ginkgo biloba. The discovery of terpene lactone derivatives has recently been reported in more and more plant extracts and even food products. In this study, mass spectrometric characteristics of the standard terpene lactones in Ginkgo biloba were comprehensively studied using both an ion trap and a quadrupole time-of-flight (QTOF) mass spectrometer. The mass spectral fragmentation data from both techniques was compared to obtain the mass spectrometric fragmentation pathways of the terpene lactones with high confidence. The data obtained will facilitate the analysis and identification of terpene lactones in future plant research via the fragmentation knowledge reported here.

  17. Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily*

    PubMed Central

    Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C.; Yew, Wen Shan

    2010-01-01

    A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of kcat/Km of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis. PMID:20980257

  18. Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

    PubMed

    Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C; Yew, Wen Shan

    2010-12-24

    A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

  19. Fragrance material review on 16-hydroxy-7-hexadecenoic acid lactone.

    PubMed

    McGinty, D; Letizia, C S; Api, A M

    2011-12-01

    A toxicologic and dermatologic review of 16-hydroxy-7-hexadecenoic acid lactone when used as a fragrance ingredient is presented. 16-Hydroxy-7-hexadecenoic acid lactone is a member of the fragrance structural group macrocyclic lactone and lactide derivatives. The fragrance ingredient described herein is one of 12 structurally diverse C14, C15 and C16 compounds that include (1) saturated mono-and (2) saturated di-ester lactones and (3) unsaturated lactones. For the latter, the double bond is not adjacent to (in conjugation with) the ester group. This review contains a detailed summary of all available toxicology and dermatology papers that are related to 16-hydroxy-7-hexadecenoic acid lactone and is not intended as a stand-alone document. Available data were evaluated, then summarized, and include physical properties data. A safety assessment of the entire macrocyclic lactone and lactide derivatives will be published simultaneously with this document. Please refer to Belsito et al., 2011 for an overall assessment of the safe use of this material and all macrocyclic lactone and lactide derivatives in fragrances. Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Hanifin, J.H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2011. A toxicologic and dermatologic assessment of macrocylic lactones and lactide derivatives when used as fragrance ingredients. Copyright © 2011. Published by Elsevier Ltd.

  20. Effects of 14-Alpha-Lipoyl Andrographolide on Quorum Sensing in Pseudomonas aeruginosa

    PubMed Central

    Ma, Li; Liu, Xiangyang; Liang, Haihua; Che, Yizhou; Chen, Caixia; Dai, Huanqin; Yu, Ke; Liu, Mei; Ma, Luyan; Yang, Ching-Hong; Song, Fuhang

    2012-01-01

    In Pseudomonas aeruginosa, the quorum-sensing (QS) system is closely related to biofilm formation. We previously demonstrated that 14-alpha-lipoyl andrographolide (AL-1) has synergistic effects on antibiofilm and antivirulence factors (pyocyanin and exopolysaccharide) of P. aeruginosa when combined with conventional antibiotics, while it has little inhibitory effect on its growth. However, its molecular mechanism remains elusive. Here we investigated the effect of AL-1 on QS systems, especially the Las and Rhl systems. This investigation showed that AL-1 can inhibit LasR–3-oxo-C12-homoserine lactone (HSL) interactions and repress the transcriptional level of QS-regulated genes. Reverse transcription (RT)-PCR data showed that AL-1 significantly reduced the expression levels of lasR, lasI, rhlR, and rhlI in a dose-dependent manner. AL-1 not only decreased the expression level of Psl, which is positively regulated by the Las system, but also increased the level of secretion of ExoS, which is negatively regulated by the Rhl system, indicating that AL-1 has multiple effects on both the Las and Rhl systems. It is no wonder that AL-1 showed synergistic effects with other antimicrobial agents in the treatment of P. aeruginosa infections. PMID:22802260

  1. New sources and antifungal activity of sesquiterpene lactones.

    PubMed

    Barrero, A F; Oltra, J E; Alvarez, M; Raslan, D S; Saúde, D A; Akssira, M

    2000-02-01

    In the search for new sources of sesquiterpene lactones, six Centaurea species have been analyzed. The activity against the fungus Cunninghamella echinulata of (+)-cnicin (1) and (+)-salonitenolide (2), isolated from the Centaurea plants, as well as that of (+)-costunolide (3), (-)-dehydrocostuslactone (4), (-)-lychnopholide (5) and (-)-eremantholide C (6), has been evaluated. Compounds 3 and 4 showed noticeable EC50 values, whilst more polar lactones were inactive. These results suggest that a relatively low polarity is one of the molecular requirements for the antifungal activity of sesquiterpene lactones.

  2. Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation.

    PubMed

    Sarkar, Ratul; Mondal, Chaitali; Bera, Rammohan; Chakraborty, Sumon; Barik, Rajib; Roy, Paramita; Kumar, Alekh; Yadav, Kirendra K; Choudhury, Jayanta; Chaudhary, Sushil K; Samanta, Samir K; Karmakar, Sanmoy; Das, Satadal; Mukherjee, Pulok K; Mukherjee, Joydeep; Sen, Tuhinadri

    2015-07-01

    This study attempts to investigate the antimicrobial properties of Kalanchoe blossfeldiana with a particular reference to quorum sensing (QS)-mediated biofilm formation. The methanol extract of K. blossfeldiana leaves (MEKB) was evaluated for antimicrobial properties including QS-controlled production of biofilm (including virulence factor, motility and lactone formation) in Pseudomonas aeruginosa. Methanol extract of K. blossfeldiana was also evaluated for anti-cytokine (tumour necrosis factor-alpha, interleukin-6 and interleukin-1 beta) properties in peripheral blood mononuclear cells (PBMC). Methanol extract of K. blossfeldiana exhibited antimicrobial effect on clinical isolates, as well as standard reference strains. Pseudomonas aeruginosa exposed to MEKB (subminimum inhibitory concentration (MIC)) displayed reduced biofilm formation, whereas supra-MIC produced destruction of preformed biofilms. Methanol extract of K. blossfeldiana reduced the secretion of virulence factors (protease and pyoverdin) along with generation of acyl homoserine lactone (AHL). Confocal laser scanning microscopy images indicate reduction of biofilm thickness. The extract also reduced cytokine formation in lipopolysaccharide-stimulated PBMC. Kalanchoe blossfeldiana was found to interfere with AHL production, which in turn may be responsible for downregulating QS-mediated production of biofilm and virulence. This first report on the antibiofilm and anticytokine properties of this plant may open up new vistas for future exploration of this plant for combating biofilm-related resistant infections. © 2015 Royal Pharmaceutical Society.

  3. Two new aliphatic lactones from the fruits of Coriandrum sativum L.

    PubMed Central

    2012-01-01

    Background The present paper describes the isolation and characterization of two new aliphatic δ-lactones along with three glycerides and n-nonadecanyl cetoleate from the fruits of Coriandrum sativum L. (Apiaceae). The structures of all the isolated phytoconstituents have been established on the basis of spectral data analysis and chemical reactions. Results Phytochemical investigation of the methanolic extract of C. sativum L. (Apiaceae) fruits resulted in the isolation of two new aliphatic δ-lactones characterized as 2α-n-heptatriacont-(Z)-3-en-1,5-olide (1) (coriander lactone) and 2α-n-tetracont-(Z,Z)-3,26-dien-18α-ol-1,5-olide (2) (hydroxy coriander lactone) together with glyceryl-1,2-dioctadec-9,12-dienoate-3-octadec-9-enoate (3); glyceryl-1,2,3-trioctadecanoate (4); n-nonadecanyl-n-docos-11-enoate (5) and oleiyl glucoside (6). Conclusions Phytochemical investigation of the methanolic extract of C. sativum gave coriander lactone and hydroxy coriander lactone as the new phytoconstituents. PMID:22800677

  4. Construction and phenotypic characterization of M68, an RruI quorum sensing knockout mutant of the photosynthetic alphaproteobacterium Rhodospirillum rubrum.

    PubMed

    Condori, Sandra; Atkinson, Steve; Leys, Natalie; Wattiez, Ruddy; Mastroleo, Felice

    2016-06-01

    Many bacterial species communicate using a complex system known as quorum sensing (QS) in which gene expression is controlled in response to cell density. In this study an N-acylhomoserine lactone (AHL) synthase (Rru_A3396) knockout mutant (M68) of Rhodospirillum rubrum S1H (WT) was constructed and characterized phenotypically under light anaerobic conditions. Results showed that R. rubrum WT produces unsubstituted, 3-OH and 3-oxo-substituted AHLs with acyl chains ranging from 4 to 14 carbons, with 3-OH-C8 being the most abundant. Growth, pigment content and swimming motility were found to be under the control of this LuxI-type QS system. In addition, cultivation in a low shear environment put forward the aggregative phenotype of M68 and linked biofilm formation to QS in R. rubrum S1H. Interestingly, QS-mutant M68 continued to produce decreased levels of 3-OH-C8-HSL, probably due to the presence of an extra HdtS-type AHL synthase. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Characteristic gamma-lactone odor production of the genus Pityrosporum.

    PubMed Central

    Labows, J N; McGinley, K J; Leyden, J J; Webster, G F

    1979-01-01

    Mass spectrometric-gas chromatographic analysis of culture headspaces revealed that members of the genous Pityrosporum produce volatile gamma-lactones during growth on lipid-containing media. Representative members of other yeast genera found on humans failed to produce these compounds. Addition of lecithin, oleic acids, triolein, or human sebum to the culture media stimulated gamma-lactone production by Pityrosporum species. All yeasts tested produced isopentanol and phenylethanol. Production of gamma-lactones may serve as a valuable characteristic in the identification of organisms of the genus Pityrosporum. PMID:533274

  6. Biosynthesis of the antifungal haterumalide, oocydin A, in Serratia, and its regulation by quorum sensing, RpoS and Hfq

    PubMed Central

    Matilla, Miguel A; Leeper, Finian J; Salmond, George P C

    2015-01-01

    Polyketides represent an important class of bioactive natural products with a broad range of biological activities. We identified recently a large trans-acyltransferase (AT) polyketide synthase gene cluster responsible for the biosynthesis of the antifungal, anti-oomycete and antitumor haterumalide, oocydin A (ooc). Using genome sequencing and comparative genomics, we show that the ooc gene cluster is widespread within biocontrol and phytopathogenic strains of the enterobacteria, Serratia and Dickeya. The analysis of in frame deletion mutants confirmed the role of a hydroxymethylglutaryl-coenzyme A synthase cassette, three flavin-dependent tailoring enzymes, a free-standing acyl carrier protein and two hypothetical proteins in oocydin A biosynthesis. The requirement of the three trans-acting AT domains for the biosynthesis of the macrolide was also demonstrated. Expression of the ooc gene cluster was shown to be positively regulated by an N-acyl-L-homoserine lactone-based quorum sensing system, but operating in a strain-dependent manner. At a post-transcriptional level, the RNA chaperone, Hfq, plays a key role in oocydin A biosynthesis. The Hfq-dependent regulation is partially mediated by the stationary phase sigma factor, RpoS, which was also shown to positively regulate the synthesis of the macrolide. Our results reveal differential regulation of the divergently transcribed ooc transcriptional units, highlighting the complexity of oocydin A production. PMID:25753587

  7. Treatment of MDR1 Mutant Dogs with Macrocyclic Lactones

    PubMed Central

    Geyer, Joachim; Janko, Christina

    2012-01-01

    P-glycoprotein, encoded by the multidrug resistance gene MDR1, is an ATP-driven drug efflux pump which is highly expressed at the blood-brain barrier of vertebrates. Drug efflux of macrocyclic lactones by P-glycoprotein is highly relevant for the therapeutic safety of macrocyclic lactones, as thereby GABA-gated chloride channels, which are confined to the central nervous system in vertebrates, are protected from high drug concentrations that otherwise would induce neurological toxicity. A 4-bp deletion mutation exists in the MDR1 gene of many dog breeds such as the Collie and the Australian Shepherd, which results in the expression of a non-functional P-glycoprotein and is associated with multiple drug sensitivity. Accordingly, dogs with homozygous MDR1 mutation are in general prone to neurotoxicity by macrocyclic lactones due to their increased brain penetration. Nevertheless, treatment of these dogs with macrocyclic lactones does not inevitably result in neurological symptoms, since, the safety of treatment highly depends on the treatment indication, dosage, route of application, and the individual compound used as outlined in this review. Whereas all available macrocyclic lactones can safely be administered to MDR1 mutant dogs at doses usually used for heartworm prevention, these dogs will experience neurological toxicity following a high dose regimen which is common for mange treatment in dogs. Here, we review and discuss the neurotoxicological potential of different macrocyclic lactones as well as their treatment options in MDR1 mutant dogs. PMID:22039792

  8. Statin Lactonization by Uridine 5'-Diphospho-glucuronosyltransferases (UGTs).

    PubMed

    Schirris, Tom J J; Ritschel, Tina; Bilos, Albert; Smeitink, Jan A M; Russel, Frans G M

    2015-11-02

    Statins are cholesterol-lowering drugs that have proven to be effective in lowering the risk of major cardiovascular events. Although well tolerated, statin-induced myopathies are the most common side effects. Compared to their pharmacologically active acid form, statin lactones are more potent inducers of toxicity. They can be formed by glucuronidation mediated by uridine 5'-diphospho-glucuronosyltransferases (UGTs), but a systematic characterization of subtype specificity and kinetics of lactonization is lacking. Here, we demonstrate for six clinically relevant statins that only UGT1A1, 1A3, and 2B7 contribute significantly to their lactonization. UGT1A3 appeared to have the highest lactonization capacity with marked differences in statin conversion rates: pitavastatin ≫ atorvastatin > cerivastatin > lovastatin > rosuvastatin (simvastatin not converted). Using in silico modeling we could identify a probable statin interaction region in the UGT binding pocket. Polymorphisms in these regions of UGT1A1, 1A3, and 2B7 may be a contributing factor in statin-induced myopathies, which could be used in personalization of statin therapy with improved safety.

  9. A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors And Controls Bacterial Pathogenicity

    PubMed Central

    Swem, Lee R.; Swem, Danielle L.; O’Loughlin, Colleen T.; Gatmaitan, Raleene; Zhao, Bixiao; Ulrich, Scott M.; Bassler, Bonnie L.

    2009-01-01

    Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development. PMID:19647512

  10. A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria

    PubMed Central

    Chu, Ya-Yun; Nega, Mulugeta; Wölfle, Martina; Plener, Laure; Grond, Stephanie; Jung, Kirsten; Götz, Friedrich

    2013-01-01

    The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community. PMID:24098134

  11. The Organization of the Quorum Sensing luxI/R Family Genes in Burkholderia

    PubMed Central

    Choudhary, Kumari Sonal; Hudaiberdiev, Sanjarbek; Gelencsér, Zsolt; Coutinho, Bruna Gonçalves; Venturi, Vittorio; Pongor, Sándor

    2013-01-01

    Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS) is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology) of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/. PMID:23820583

  12. Global analysis of the Burkholderia thailandensis quorum sensing-controlled regulon.

    PubMed

    Majerczyk, Charlotte; Brittnacher, Mitchell; Jacobs, Michael; Armour, Christopher D; Radey, Mathew; Schneider, Emily; Phattarasokul, Somsak; Bunt, Richard; Greenberg, E Peter

    2014-04-01

    Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei.

  13. Global Analysis of the Burkholderia thailandensis Quorum Sensing-Controlled Regulon

    PubMed Central

    Majerczyk, Charlotte; Brittnacher, Mitchell; Jacobs, Michael; Armour, Christopher D.; Radey, Mathew; Schneider, Emily; Phattarasokul, Somsak; Bunt, Richard

    2014-01-01

    Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei. PMID:24464461

  14. In silico and experimental methods revealed highly diverse bacteria with quorum sensing and aromatics biodegradation systems--a potential broad application on bioremediation.

    PubMed

    Huang, Yili; Zeng, Yanhua; Yu, Zhiliang; Zhang, Jing; Feng, Hao; Lin, Xiuchun

    2013-11-01

    Phylogenetic overlaps between aromatics-degrading bacteria and acyl-homoserine-lactone (AHL) or autoinducer (AI) based quorum-sensing (QS) bacteria were evident in literatures; however, the diversity of bacteria with both activities had never been finely described. In-silico searching in NCBI genome database revealed that more than 11% of investigated population harbored both aromatic ring-hydroxylating-dioxygenase (RHD) gene and AHL/AI-synthetase gene. These bacteria were distributed in 10 orders, 15 families, 42 genus and 78 species. Horizontal transfers of both genes were common among them. Using enrichment and culture dependent method, 6 Sphingomonadales and 4 Rhizobiales with phenanthrene- or pyrene-degrading ability and AHL-production were isolated from marine, wetland and soil samples. Thin-layer-chromatography and gas-chromatography-mass-spectrum revealed that these Sphingomonads produced various AHL molecules. This is the first report of highly diverse bacteria that harbored both aromatics-degrading and QS systems. QS regulation may have broad impacts on aromatics biodegradation, and would be a new angle for developing bioremediation technology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 quorum sensing in Vibrio harveyi

    PubMed Central

    Swem, Lee R.; Swem, Danielle L.; Wingreen, Ned S.; Bassler, Bonnie L.

    2008-01-01

    Summary Quorum sensing, a process of bacterial cell-cell communication, relies on production, detection, and response to autoinducer signaling molecules. Here we focus on LuxN, a nine transmembrane domain protein from Vibrio harveyi, and the founding example of membrane-bound receptors for acyl-homoserine lactone (AHL) autoinducers. Previously, nothing was known about signal recognition by membrane-bound AHL receptors. We used mutagenesis and suppressor analyses to identify the AHL-binding domain of LuxN, and discovered LuxN mutants that confer decreased and increased AHL sensitivity. Our analysis of dose-response curves of multiple LuxN mutants pins these inverse phenotypes on quantifiable opposing shifts in the free-energy bias of LuxN for its kinase and phosphatase states. To extract signaling parameters, we exploited a strong LuxN antagonist, one of fifteen small-molecule antagonists we identified. We find that quorum-sensing-mediated communication can be manipulated positively and negatively to control bacterial behavior, and that signaling parameters can be deduced from in vivo data. PMID:18692469

  16. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl-galactose acyl composition varies with the plant species and applied stress

    PubMed Central

    Vu, Hieu Sy; Roth, Mary R.; Tamura, Pamela; Samarakoon, Thilani; Shiva, Sunitha; Honey, Samuel; Lowe, Kaleb; Schmelz, Eric A.; Williams, Todd D.; Welti, Ruth

    2014-01-01

    Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection, and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses. PMID:24286212

  17. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takayama, Yuriko; CREST, Japan Science and Technology Agency, 7-1-2 Yoto, Utsunomiya, Tochigi 321-8585; Kato, Norihiro, E-mail: katon@cc.utsunomiya-u.ac.jp

    The opportunistic human pathogen Serratia marcescens AS-1 produces the N-hexanoylhomoserine lactone (C6HSL) receptor SpnR, a homologue of LuxR from Vibrio fischeri, which activates pig clusters to produce the antibacterial prodigiosin. In this study, we attempted to artificially regulate quorum sensing (QS) by changing the role of SpnR in N-acylhomoserine lactone (AHL)-mediated QS. SpnR was obtained as a fusion protein tagged with maltose-binding protein (MBP) from overexpression in Escherichia coli, and its specific affinity to C6HSL was demonstrated by quartz crystal microbalance analysis and AHL-bioassay with Chromobacterium violaceum CV026. Prodigiosin production was effectively inhibited by externally added MBP-SpnR in both wild-typemore » AS-1 and the AHL synthase-defective mutant AS-1(ΔspnI). For the mutant, the induced amount of prodigiosin was drastically reduced to approximately 4% with the addition of 18 μM MBP-SpnR to the liquid medium, indicating 81% trapping of C6HSL. A system for inhibiting QS can be constructed by adding exogenous AHL receptor to the culture broth to keep the concentration of free AHL low, whereas intracellular SpnR naturally functions as the activator in response to QS. - Highlights: • Quorum sensing (QS) regulates the expression of some bacterial genes. • We added an AHL receptor to culture media to inhibit QS in Serratia marcescens AS-1. • The exogenous receptor effectively bound C6HSL and inhibited QS. • This approach can be used to artificially regulate AHL-mediated QS.« less

  18. Oxidative acylation using thioacids

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.

    1997-01-01

    Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

  19. Two-Way Chemical Communication between Artificial and Natural Cells

    PubMed Central

    2017-01-01

    Artificial cells capable of both sensing and sending chemical messages to bacteria have yet to be built. Here we show that artificial cells that are able to sense and synthesize quorum signaling molecules can chemically communicate with V. fischeri, V. harveyi, E. coli, and P. aeruginosa. Activity was assessed by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications for this technology were demonstrated. First, the extent to which artificial cells could imitate natural cells was quantified by a type of cellular Turing test. Artificial cells capable of sensing and in response synthesizing and releasing N-3-(oxohexanoyl)homoserine lactone showed a high degree of likeness to natural V. fischeri under specific test conditions. Second, artificial cells that sensed V. fischeri and in response degraded a quorum signaling molecule of P. aeruginosa (N-(3-oxododecanoyl)homoserine lactone) were constructed, laying the foundation for future technologies that control complex networks of natural cells. PMID:28280778

  20. Acylation of Ferrocene: A Greener Approach

    ERIC Educational Resources Information Center

    Birdwhistell, Kurt R.; Nguyen, Andy; Ramos, Eric J.; Kobelja, Robert

    2008-01-01

    The acylation of ferrocene is a common reaction used in organic laboratories to demonstrate Friedel-Crafts acylation and the purification of compounds using column chromatography. This article describes an acylation of ferrocene experiment that is more eco-friendly than the conventional acylation experiment. The traditional experiment was modified…

  1. Potential Use of Dimethyl Sulfoxide in Treatment of Infections Caused by Pseudomonas aeruginosa

    PubMed Central

    Guo, Qiao; Wu, Qiaolian; Bai, Dangdang; Liu, Yang; Chen, Lin; Jin, Sheng; Wu, Yuting

    2016-01-01

    Dimethyl sulfoxide (DMSO) is commonly used as a solvent to dissolve water-insoluble drugs or other test samples in both in vivo and in vitro experiments. It was observed during our experiment that DMSO at noninhibitory concentrations could significantly inhibit pyocyanin production in the human pathogen Pseudomonas aeruginosa. Pyocyanin is an important pathogenic factor whose production is controlled by a cell density-dependent quorum-sensing (QS) system. Investigation of the effect of DMSO on QS showed that DMSO has significant QS antagonistic activities and concentrations of DMSO in the micromolar range attenuated a battery of QS-controlled virulence factors, including rhamnolipid, elastase, and LasA protease production and biofilm formation. Further study indicated that DMSO inhibition of biofilm formation and pyocyanin production was attained by reducing the level of production of an autoinducer molecule of the rhl QS system, N-butanoyl-l-homoserine lactone (C4-HSL). In a mouse model of a burn wound infection with P. aeruginosa, treatment with DMSO significantly decreased mouse mortality compared with that for mice in the control group. The capacity of DMSO to attenuate the pathogenicity of P. aeruginosa points to the potential use of DMSO as an antipathogenic agent for the treatment of P. aeruginosa infection. As a commonly used solvent, however, DMSO's impact on bacterial virulence calls for cautionary attention in its usage in biological, medicinal, and clinical studies. PMID:27645245

  2. Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

    PubMed

    Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

    2009-11-01

    Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

  3. Diterpene lactones with labdane, halimane and clerodane frameworks.

    PubMed

    Silva, Lúcia; Gomes, Arlindo C; Rodilla, Jesus M L

    2011-04-01

    The labdane, halimane and clerodane type diterpenoids are compounds that have been isolated in plants of several families. These molecules and their derivatives with a lactone group on the side chain or on the decaline system, have a great interest because of their biological properties as insect antifeedant, antiviral, cytotoxic and trypanocidal. The scope of this review is lactones diterpenoids with labdane, halimane and clerodane frameworks.

  4. Herbicidal and Fungicidal Activities of Lactones in Kava (Piper methysticum).

    PubMed

    Xuan, T D; Elzaawely, A A; Fukuta, M; Tawata, S

    2006-02-08

    This is the first report showing that kava lactones are plant and plant fungus growth inhibitors. Aqueous extract of kava roots showed high allelopathic potential and strongly suppressed germination and growth of lettuce, radish, barnyardgrass, and monochoria. Nine kava lactones were detected using GC-MS including desmethoxyyagonin, kavain, 7,8-dihydrokavain, hydroxykavain, yagonin, 5,6,7,8-tetrahydroxyyagonin, methysticin, dihydromethysticin, and 11-hydroxy-12-methoxydihydrokavain. Quantities of desmethoxyyagonin, kavain, 7,8-dihydrokavain, yagonin, methysticin, and dihydromethysticin detected were 4.3, 6.9, 18.6, 5.7, 1.4, and 5.4 mg/g of dry weight, respectively. These six major lactones in kava roots showed great herbicidal and antifungal activities. Growth of lettuce and barnyardgrass were significantly inhibited at 1-10 ppm, and four plant fungi including Colletotrichum gloeosporides, Fusarium solani, Fusarium oxysporum, and Trichoderma viride were significantly inhibited at 10-50 ppm. The biological activities of kava lactones were characterized by different double-bond linkage patterns in positions 5,6 and 7,8. The findings of this study suggest that kava lactones may be useful for the development of bioactive herbicides and fungicides.

  5. The Physiology of Protein S-acylation

    PubMed Central

    Chamberlain, Luke H.; Shipston, Michael J.

    2015-01-01

    Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field. PMID:25834228

  6. Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein.

    PubMed

    Dörmann, P; Spener, F; Ohlrogge, J B

    1993-03-01

    Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.

  7. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge

    PubMed Central

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-01-01

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge. PMID:27490553

  8. Relationship between Menthiafolic Acid and Wine Lactone in Wine.

    PubMed

    Giaccio, Joanne; Curtin, Chris D; Sefton, Mark A; Taylor, Dennis K

    2015-09-23

    Menthiafolic acid (6-hydroxy-2,6-dimethylocta-2,7-dienoic acid, 2a) was quantified by GC-MS in 28 white wines, 4 Shiraz wines, and for the first time in 6 white grape juice samples. Menthiafolic acid was detected in all but one of the wine samples at concentrations ranging from 26 to 342 μg/L and in the juice samples from 16 to 236 μg/L. Various model fermentation experiments showed that some menthiafolic acid in wine could be generated from the grape-derived menthiafolic acid glucose ester (2b) during alcoholic and malolactic fermentation. Samples containing high concentrations of menthiafolic acid were also analyzed by enantioselective GC-MS and were shown to contain this compound in predominantly the (S)-configuration. Enantioselective analysis of wine lactone (1) in one of these samples, a four-year-old Chardonnay wine showed, for the first time, the presence of the 3R,3aR,7aS isomer of wine lactone (1b), which is the enantiomer of the form previously reported as the sole isomer present in young wine samples. The weakly odorous 3R,3aR,7aS 1b form comprised 69% of the total wine lactone in the sample. On the basis of the enantioselectivity of the hydrolytic conversion of menthiafolic acid to wine lactone at pH 3.0 determined previously and the relative proportions of (R)- and (S)-menthiafolic acid in the Chardonnay wine, the predicted ratio of wine lactone enantiomers that would be formed from hydrolysis at ambient temperature of the menthiafolic acid present in this wine was close to the ratio measured, which was consistent with menthiafolic acid being the major or sole precursor to wine lactone in this sample.

  9. Signal Integration in Quorum Sensing Enables Cross-Species Induction of Virulence in Pectobacterium wasabiae.

    PubMed

    Valente, Rita S; Nadal-Jimenez, Pol; Carvalho, André F P; Vieira, Filipe J D; Xavier, Karina B

    2017-05-23

    Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora ), two signaling networks-the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway-control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone

  10. Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator

    DOE PAGES

    Nguyen, Y.; Nguyen, Nam X.; Rogers, Jamie L.; ...

    2015-05-19

    Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not inmore » an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan Lux

  11. Homoserine as an Aspartic Acid Precursor for Synthesis of Proteoglycan Glycopeptide Containing Aspartic Acid and a Sulfated Glycan Chain.

    PubMed

    Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei

    2016-12-02

    Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is the first time that a glycopeptide containing aspartic acid and an O-sulfated glycan was synthesized.

  12. Sesquiterpene lactone stereochemistry influences herbivore resistance and plant fitness in the field.

    PubMed

    Ahern, Jeffrey R; Whitney, Kenneth D

    2014-03-01

    Stereochemical variation is widely known to influence the bioactivity of compounds in the context of pharmacology and pesticide science, but our understanding of its importance in mediating plant-herbivore interactions is limited, particularly in field settings. Similarly, sesquiterpene lactones are a broadly distributed class of putative defensive compounds, but little is known about their activities in the field. Natural variation in sesquiterpene lactones of the common cocklebur, Xanthium strumarium (Asteraceae), was used in conjunction with a series of common garden experiments to examine relationships between stereochemical variation, herbivore damage and plant fitness. The stereochemistry of sesquiterpene lactone ring junctions helped to explain variation in plant herbivore resistance. Plants producing cis-fused sesquiterpene lactones experienced significantly higher damage than plants producing trans-fused sesquiterpene lactones. Experiments manipulating herbivore damage above and below ambient levels found that herbivore damage was negatively correlated with plant fitness. This pattern translated into significant fitness differences between chemotypes under ambient levels of herbivore attack, but not when attack was experimentally reduced via pesticide. To our knowledge, this work represents only the second study to examine sesquiterpene lactones as defensive compounds in the field, the first to document herbivore-mediated natural selection on sesquiterpene lactone variation and the first to investigate the ecological significance of the stereochemistry of the lactone ring junction. The results indicate that subtle differences in stereochemistry may be a major determinant of the protective role of secondary metabolites and thus of plant fitness. As stereochemical variation is widespread in many groups of secondary metabolites, these findings suggest the possibility of dynamic evolutionary histories within the Asteraceae and other plant families showing

  13. The level of circulating octanoate does not predict ghrelin O-acyl transferase (GOAT)-mediated acylation of ghrelin during fasting.

    PubMed

    Nass, Ralf; Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S; Farhy, Leon S; Patrie, James; Gaylinn, Bruce D; Heiman, Mark; Thorner, Michael O

    2015-01-01

    Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

  14. Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

    USDA-ARS?s Scientific Manuscript database

    Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the mol...

  15. Sesquiterpene lactones from Centaurea tweediei.

    PubMed

    Fortuna, A M.; de Riscala, E C.; Catalan, C A.N.; Gedris, T E.; Herz, W

    2001-10-01

    Aerial parts of Centaurea tweediei from Argentina afforded as the main constituent the sesquiterpene lactone onopordopicrin and minor amounts of a new heliangolide, a new guaianolide, a new eudesmanolide, a new eudesmane acid and the lignans arctigenin and matairesinol.

  16. Concurrent treatment with a macrocyclic lactone and benzimidazole provides season long performance advantages in grazing cattle harboring macrocyclic lactone resistant nematodes.

    PubMed

    Edmonds, M D; Vatta, A F; Marchiondo, A A; Vanimisetti, H B; Edmonds, J D

    2018-03-15

    In 2013, a 118-day study was initiated to investigate the efficacy of concurrent treatment at pasture turnout with an injectable macrocyclic lactone with activity up to 28 days and an oral benzimidazole, referred to as "conventional" anthelmintics, when compared to treatment with conventional macrocyclic lactone alone or an injectable macrocyclic lactone with extended activity of 100 days or longer. A group of 210 steers were obtained from a ranch in California and transported to Idaho, USA. A total of 176 steers with the highest fecal egg counts were blocked by pre-treatment body weights and pasture location. A total of 44 pasture paddocks were assigned with 4 steers per paddock with 12 paddocks per therapeutic treatment group and 8 paddocks per controls. The four treatments were injectable doramectin (Dectomax ® , Zoetis Inc., 0.2 mg kg -1 BW, SC), injectable doramectin concurrently with oral albendazole (Valbazen ® , Zoetis Inc., 10 mg kg -1 BW, PO), extended release injectable eprinomectin (LongRange™, Merial Limited, 1 mg kg -1 BW, SC) or saline. Cattle were individually weighed and sampled for fecal egg count on Days 0, 31/32, 61, 88, and 117/118 with an additional fecal sample on Day 14. At conclusion, one steer per paddock was euthanized for nematode recovery. The results from the first 32 days found evidence of macrocyclic lactone resistance against injectable doramectin and extended release eprinomectin. During this period the concurrent therapy provided nearly 100% efficacy based on fecal egg count reduction and a 19.98% improvement in total weight gain compared to controls (P = 0.039). At the conclusion of the 118-day study and past the approved efficacy for the conventional anthelmintics, the concurrent therapy with conventional anthelmintics provided a 22.98% improvement in total weight gain compared to controls (P = 0.004). The 118-day improvement in weight gain for the extended release eprinomectin group (29.06% compared to

  17. Trimethylated homoserine functions as the major compatible solute in the globally significant oceanic cyanobacterium Trichodesmium.

    PubMed

    Pade, Nadin; Michalik, Dirk; Ruth, Wolfgang; Belkin, Natalia; Hess, Wolfgang R; Berman-Frank, Ilana; Hagemann, Martin

    2016-11-15

    The oceanic N 2 -fixing cyanobacterium Trichodesmium spp. form extensive surface blooms and contribute significantly to marine carbon and nitrogen cycles in the oligotrophic subtropical and tropical oceans. Trichodesmium grows in salinities from 27 to 43 parts per thousand (ppt), yet its salt acclimation strategy remains enigmatic because the genome of Trichodesmium erythraeum strain IMS101 lacks all genes for the biosynthesis of any known compatible solute. Using NMR and liquid chromatography coupled to mass spectroscopy, we identified the main compatible solute in T. erythraeum strain IMS101 as the quaternary ammonium compound N,N,N-trimethyl homoserine (or homoserine betaine) and elucidated its biosynthetic pathway. The identification of this compatible solute explains how Trichodesmium spp. can thrive in the marine system at varying salinities and provides further insight into the diversity of microbial salt acclimation.

  18. Sesquiterpene lactone stereochemistry influences herbivore resistance and plant fitness in the field

    PubMed Central

    Ahern, Jeffrey R.; Whitney, Kenneth D.

    2014-01-01

    Background and Aims Stereochemical variation is widely known to influence the bioactivity of compounds in the context of pharmacology and pesticide science, but our understanding of its importance in mediating plant–herbivore interactions is limited, particularly in field settings. Similarly, sesquiterpene lactones are a broadly distributed class of putative defensive compounds, but little is known about their activities in the field. Methods Natural variation in sesquiterpene lactones of the common cocklebur, Xanthium strumarium (Asteraceae), was used in conjunction with a series of common garden experiments to examine relationships between stereochemical variation, herbivore damage and plant fitness. Key Results The stereochemistry of sesquiterpene lactone ring junctions helped to explain variation in plant herbivore resistance. Plants producing cis-fused sesquiterpene lactones experienced significantly higher damage than plants producing trans-fused sesquiterpene lactones. Experiments manipulating herbivore damage above and below ambient levels found that herbivore damage was negatively correlated with plant fitness. This pattern translated into significant fitness differences between chemotypes under ambient levels of herbivore attack, but not when attack was experimentally reduced via pesticide. Conclusions To our knowledge, this work represents only the second study to examine sesquiterpene lactones as defensive compounds in the field, the first to document herbivore-mediated natural selection on sesquiterpene lactone variation and the first to investigate the ecological significance of the stereochemistry of the lactone ring junction. The results indicate that subtle differences in stereochemistry may be a major determinant of the protective role of secondary metabolites and thus of plant fitness. As stereochemical variation is widespread in many groups of secondary metabolites, these findings suggest the possibility of dynamic evolutionary histories

  19. Effects of dynamic exercise intensity on the activation of hormone-sensitive lipase in human skeletal muscle

    PubMed Central

    Watt, Matthew J; Heigenhauser, George J F; Spriet, Lawrence L

    2003-01-01

    It has been proposed that hormone-sensitive lipase (HSL) regulates intramuscular triacylglycerol hydrolysis in skeletal muscle. The primary purpose of this study was to examine the early activation of HSL and the changes in the putative intramuscular and hormonal regulators of HSL activity at various aerobic exercise intensities. Eight male subjects cycled for 10 min at power outputs corresponding to 30, 60 and 90 % peak oxygen uptake (V̇O2,peak). Muscle samples were obtained at rest and following 1 and 10 min of exercise. Intramuscular triacylglycerol (mean ±s.e.m.: 24.3 ± 2.3 mmol (kg dry mass (DM))-1), long-chain fatty acyl CoA (13.9 ± 1.4 µmol (kg DM)-1) and HSL activity (1.87 ± 0.07 mmol min-1 (kg DM)-1)) were not different between trials at rest. HSL activity increased at 1 min of exercise at 30 and 60 % V̇O2,peak, and to a greater extent at 90 % V̇O2,peak. HSL activity remained elevated after 10 min of exercise at 30 and 60 % V̇O2,peak, and decreased at 90 % V̇O2,peak from the rates observed at 1 min (1 min: 3.41 ± 0.3 mmol min-1 (kg DM)-1; 10 min: 2.92 ± 0.26 mmol min-1 (kg DM)-1), P < 0.05). There were no effects of exercise power output or time on long-chain fatty acyl CoA content. At 90 % V̇O2,peak, skeletal muscle contents of ATP and phosphocreatine were decreased (P < 0.05), and free ADP and free AMP were increased (P < 0.05) during exercise. No changes in these metabolites occurred at 30 % V̇O2,peak and only modest changes were observed at 60 % V̇O2,peak. Plasma adrenaline increased (P < 0.05) during exercise at 90 % V̇O2,peak only. These data suggest that a factor related to the onset of exercise (e.g. Ca2+) activates HSL early in exercise. Given the activation of HSL early in exercise, at a time when intramuscular triacylglycerol hydrolysis and fat oxidation are considered to be negligible, we propose that the control of intramuscular triacylglycerol hydrolysis is not solely related to the level of HSL activation, but must also be

  20. A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

    PubMed Central

    Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

    2017-01-01

    SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

  1. Gas Phase Dissociation Behavior of Acyl-Arginine Peptides.

    PubMed

    McGee, William M; McLuckey, Scott A

    2013-11-15

    The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N -hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed "ornithine effect". Path 2 involves the coordinated losses of H 2 O and NH 3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

  2. Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays

    NASA Technical Reports Server (NTRS)

    White, D. H.; Kennedy, R. M.; Macklin, J.

    1984-01-01

    Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. The proposed mechanism has been confirmed by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespead, geologically realistic setting for prebiotic peptide formation via in situ activation.

  3. The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

    PubMed Central

    Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

    2009-01-01

    The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

  4. Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164.

    PubMed

    Bruns, Hilke; Herrmann, Jennifer; Müller, Rolf; Wang, Hui; Wagner Döbler, Irene; Schulz, Stefan

    2018-01-26

    The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.

  5. Sesquiterpene Lactones from Artemisia Genus: Biological Activities and Methods of Analysis

    PubMed Central

    Miron, Anca; Corciova, Andreia

    2015-01-01

    Sesquiterpene lactones are a large group of natural compounds, found primarily in plants of Asteraceae family, with over 5000 structures reported to date. Within this family, genus Artemisia is very well represented, having approximately 500 species characterized by the presence of eudesmanolides and guaianolides, especially highly oxygenated ones, and rarely of germacranolides. Sesquiterpene lactones exhibit a wide range of biological activities, such as antitumor, anti-inflammatory, analgesic, antiulcer, antibacterial, antifungal, antiviral, antiparasitic, and insect deterrent. Many of the biological activities are attributed to the α-methylene-γ-lactone group in their molecule which reacts through a Michael-addition with free sulfhydryl or amino groups in proteins and alkylates them. Due to the fact that most sesquiterpene lactones are thermolabile, less volatile compounds, they present no specific chromophores in the molecule and are sensitive to acidic and basic mediums, and their identification and quantification represent a difficult task for the analyst. Another problematic aspect is represented by the complexity of vegetal samples, which may contain compounds that can interfere with the analysis. Therefore, this paper proposes an overview of the methods used for the identification and quantification of sesquiterpene lactones found in Artemisia genus, as well as the optimal conditions for their extraction and separation. PMID:26495156

  6. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    PubMed

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-03-01

    Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

  7. Antimalarial activity of sesquiterpene lactones from Vernonia cinerea.

    PubMed

    Chea, Aun; Hout, Sotheara; Long, Christophe; Marcourt, Laurence; Faure, Robert; Azas, Nadine; Elias, Riad

    2006-10-01

    Two new sesquiterpene lactones, vernolides C and D as well as six known ones were isolated from the dichloromethane fraction of an aqueous extract from Vernonia cinerea. Their structures were elucidated by spectroscopic methods. Among the known sesquiterpene lactones, three of them were described in this plant for the first time. In vitro antiplasmodial evaluation showed that the three major compounds 1, 7 and 8 were active against chloroquine resistant Plasmodium falciparum strain (W2) with IC(50) 3.9, 3.7 and 3.5 microM, respectively.

  8. X-ray crystal structures of the pheromone-binding domains of two quorum-hindered transcription factors, YenR of Yersinia enterocolitica and CepR2 of Burkholderia cenocepacia: KIM et al.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Youngchang; Chhor, Gekleng; Tsai, Ching-Sung

    The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and some of them require AHLs for folding and stability, and for protease-resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. One such protein is YenR of Yersinia enterocolitica, which is antagonized by N-3-oxohexanoyl-l-homoserine lactone (OHHL). This pheromone is produced by the OHHL synthase, a product of the adjacent yenI gene. Anothermore » example is CepR2 of Burkholderia cenocepacia, which is antagonized by N-octanoyl-l-homoserine lactone (OHL), whose synthesis is directed by the cepI gene of the same bacterium. Here, we describe the high-resolution crystal structures of the AHL binding domains of YenR and CepR2. YenR was crystallized in the presence and absence of OHHL. While this ligand does not cause large scale changes in the YenR structure, it does alter the orientation of several highly conserved YenR residues within and near the pheromone-binding pocket, which in turn caused a significant movement of a surface-exposed loop.« less

  9. Analysis of the microbial communities on corroded concrete sewer pipes--a case study.

    PubMed

    Vincke, E; Boon, N; Verstraete, W

    2001-12-01

    Conventional as well as molecular techniques have been used to determine the microbial communities present on the concrete walls of sewer pipes. The genetic fingerprint of the microbiota on corroded concrete sewer pipes was obtained by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The DGGE profiles of the bacterial communities present on the concrete surface changed as observed by shifts occurring at the level of the dominance of bands from non-corroded places to the most severely corroded places. By means of statistical tools, it was possible to distinguish two different groups, corresponding to the microbial communities on corroded and non-corroded surfaces, respectively. Characterization of the microbial communities indicated that the sequences of typical bands showed the highest level of identity to sequences from the bacterial strains Thiobacillus thiooxidans, Acidithiobacillus sp., Mycobacterium sp. and different heterotrophs belonging to the alpha-, beta- and gamma-Proteobacteria, Acidobacteria and Actinobacteria. In addition, the presence of N-acyl-homoserine lactone signal molecules was shown by two bio-assays of the biofilm on the concrete under the water level and at the most severely corroded places on the concrete surface of the sewer pipe.

  10. Quorum sensing control of Type VI secretion factors restricts the proliferation of quorum-sensing mutants

    PubMed Central

    Majerczyk, Charlotte; Schneider, Emily; Greenberg, E Peter

    2016-01-01

    Burkholderia thailandensis uses acyl-homoserine lactone-mediated quorum sensing systems to regulate hundreds of genes. Here we show that cell-cell contact-dependent type VI secretion (T6S) toxin-immunity systems are among those activated by quorum sensing in B. thailandensis. We also demonstrate that T6S is required to constrain proliferation of quorum sensing mutants in colony cocultures of a BtaR1 quorum-sensing signal receptor mutant and its parent. However, the BtaR1 mutant is not constrained by and outcompetes its parent in broth coculture, presumably because no cell contact occurs and there is a metabolic cost associated with quorum sensing gene activation. The increased fitness of the wild type over the BtaR1 mutant during agar surface growth is dependent on an intact T6SS-1 apparatus. Thus, quorum sensing activates B. thailandensis T6SS-1 growth inhibition and this control serves to police and constrain quorum-sensing mutants. This work defines a novel role for T6SSs in intraspecies mutant control. DOI: http://dx.doi.org/10.7554/eLife.14712.001 PMID:27183270

  11. Microbiota on spoiled vegetables and their characterization.

    PubMed

    Lee, Dong Hwan; Kim, Jin-Beom; Kim, Mihyun; Roh, Eunjung; Jung, Kyusuk; Choi, Minseon; Oh, Changsik; Choi, Jaehyuk; Yun, Jongchul; Heu, Sunggi

    2013-08-01

    Spoilage causes vegetables to deteriorate and develop unpleasant characteristics. Approximately 30 % of fresh vegetables are lost to spoilage, mainly due to colonization by bacteria. In the present study, a total of 44 bacterial isolates were obtained from a number of spoiled vegetables. The isolates were identified and classified into 20 different species of 14 genera based on fatty acid composition, biochemical tests, and 16S rDNA sequence analyses. Pseudomonas spp. were the species most frequently isolated from the spoiled vegetables. To evaluate the spoilage ability of each species, a variety of fresh vegetables were treated with each isolate and their degree of maceration was observed. In addition, the production of plant cell wall-degrading enzymes (PCWDEs), such as cellulase, xylanase, pectate lyase, and polygalacturonase, was compared among isolates to investigate their potential associations with spoilage. Strains that produce more PCWDEs cause spoilage on more diverse plants, and pectinase may be the most important enzyme among PCWDEs for vegetable spoilage. Most gram-negative spoilage bacteria produced acylated homoserine lactone, a quorum-sensing signal molecule, suggesting that it may be possible to use this compound effectively to prevent or slow down the spoilage of vegetables contaminated with diverse bacteria.

  12. The Burkholderia cepacia rpoE gene is not involved in exopolysaccharide production and onion pathogenicity.

    PubMed

    Devescovi, Giulia; Venturi, Vittorio

    2006-03-01

    Burkholderia cepacia was originally described as the causative agent of bacterial rot of onions, and it has now emerged as an important opportunistic pathogen causing severe chronic lung infections in patients having cystic fibrosis. Burkholderia cepacia is now classified into nine very closely related species (previously designated as genomovars), all of which have been isolated from both environmental and clinical sources and are collectively known as the B. cepacia complex. The alternative extracytoplasmic function sigma factor, sigmaE, has been determined in several bacterial species as making substantial contributions to bacterial survival under stress conditions. Here, we report the identification and characterization of the rpoE gene, encoding sigmaE, of B. cepacia. It is highly similar to sigmaE of other bacteria, including Escherichia coli and Pseudomonas aeruginosa. Studies using an rpoE knockout mutant of B. cepacia revealed that many stress adaptations, including osmotic, oxidative, desiccation, carbon, and nitrogen stress, were independent of sigmaE. Similarly, biofilm formation; production of exopolysaccharides, N-acyl homoserine lactones, and several exoenzymes; and onion pathogenicity were not affected by the absence of sigmaE. In contrast, sigmaE contributed to the adaptation to heat stress and phosphate starvation.

  13. Crosstalk Regulates the Capacity for Robust Collective Decision Making in Heterogeneous Microbial Communities

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir; Boedicker, James

    Microbial communities frequently communicate via quorum sensing (QS), where cells produce, secrete, and respond to a threshold level of an autoinducer (AI) molecule, thereby modulating density-dependent gene expression. However, the biology of QS remains incompletely understood in heterogeneous communities, where crosstalk between distinct QS systems leads to novel effects. Such knowledge is necessary both for understanding signaling in real microbial communities, and for the rational design of synthetic communities with designer properties. As a step towards this goal, we investigate the effects of crosstalk between Gram-negative bacteria communicating via LuxI/LuxR-type QS systems, with acyl-homoserine lactone (AHL) AI molecules. After mapping QS in a heterogeneous community onto an artificial neural network model, we systematically analyze how heterogeneity regulates the community's capability for stable yet flexible decision making. We find that there are preferred distributions of interactions which provide optimal tradeoffs between capacity, or the number of different decisions a population can make, and robustness, or the tolerance of the community to disturbances. We compare our results to inferences made from experimental data, and critically discuss implications for the biological significance of crosstalk.

  14. Production of quorum-sensing signals by bacteria in the coral mucus layer

    NASA Astrophysics Data System (ADS)

    Li, Jie; Kuang, Weiqi; Long, Lijuan; Zhang, Si

    2017-12-01

    Quorum sensing is an integral part of bacterial communication and interaction, but has not been well characterized in coral mucus microbiota. In this study, of 61 coral mucus isolates, five alphaproteobacteria and one Vibrio species were found to produce N-acyl homoserine lactone (AHL), a quorum-sensing signal in bacteria. Eight gammaproteobacteria isolates were found to produce autoinducer-2 (AI-2) quorum-sensing signals along with two actinobacteria of the genus Rothia. Coral mucus is rich in the antioxidant dimethylsulfoniopropionate (DMSP), the concentration of which has been found to increase under heat stress. Neither AHL nor AI-2 activity was induced by DMSP in those coral mucus isolates that did not initially produce quorum-sensing signals. However, the AI-2 activities of one Rothia isolate (SCSIO 13017) from coral mucus and of Vibrio shilonii (DSM 13774 isolated from a bleached coral) were found to increase in response to 5 μM DMSP but decreased in response to 50 μM DMSP for the first time. These findings suggest that the production of quorum-sensing signals in the coral mucus microbiota may play a role in structuring the surface microbial community as they respond to environmental stress.

  15. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2013-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:23505340

  16. Acyl-Lipid Metabolism

    PubMed Central

    Li-Beisson, Yonghua; Shorrosh, Basil; Beisson, Fred; Andersson, Mats X.; Arondel, Vincent; Bates, Philip D.; Baud, Sébastien; Bird, David; DeBono, Allan; Durrett, Timothy P.; Franke, Rochus B.; Graham, Ian A.; Katayama, Kenta; Kelly, Amélie A.; Larson, Tony; Markham, Jonathan E.; Miquel, Martine; Molina, Isabel; Nishida, Ikuo; Rowland, Owen; Samuels, Lacey; Schmid, Katherine M.; Wada, Hajime; Welti, Ruth; Xu, Changcheng; Zallot, Rémi; Ohlrogge, John

    2010-01-01

    Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. PMID:22303259

  17. Fundamental role of the fostriecin unsaturated lactone and implications for selective protein phosphatase inhibition.

    PubMed

    Buck, Suzanne B; Hardouin, Christophe; Ichikawa, Satoshi; Soenen, Danielle R; Gauss, C-M; Hwang, Inkyu; Swingle, Mark R; Bonness, Kathy M; Honkanen, Richard E; Boger, Dale L

    2003-12-24

    Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP beta12-beta13 active site loop accounting for PP2A/4 potency and selectivity.

  18. Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans.

    PubMed

    Honda, Sari; Miura, Yukari; Masuda, Akiko; Masuda, Toshiya

    2014-01-01

    Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee.

  19. Friedel-Crafts Acylation with Amides

    PubMed Central

    Raja, Erum K.; DeSchepper, Daniel J.; Nilsson Lill, Sten O.; Klumpp, Douglas A.

    2012-01-01

    Friedel-Crafts acylation has been known since the 1870s and it is an important organic synthetic reaction leading to aromatic ketone products. Friedel-Crafts acylation is usually done with carboxylic acid chlorides or anhydrides while amides are generally not useful substrates in these reactions. Despite being the least reactive carboxylic acid derivative, we have found a series of amides capable of providing aromatic ketones in good yields (55–96%, 17 examples). We propose a mechanism involving diminished C-N resonance through superelectrophilic activation and subsequent cleavage to acyl cations. PMID:22690740

  20. Identification of N-Acyl Phosphatidylserine Molecules in Eukaryotic Cells

    PubMed Central

    Guan, Ziqiang; Li, Shengrong; Smith, Dale C.; Shaw, Walter A.; Raetz, Christian R. H.

    2008-01-01

    While profiling the lipidome of the mouse brain by mass spectrometry, we discovered a novel family of N-acyl phosphatidylserine (N-acyl-PS) molecules. These N-acyl-PS species were enriched by DEAE-cellulose column chromatography, and they were then characterized by accurate mass measurements, tandem mass spectrometry, liquid chromatography/mass spectrometry, and comparison to an authentic standard. Mouse brain N-acyl-PS molecules are heterogeneous and constitute about 0.1 % of the total lipid. In addition to various ester-linked fatty acyl chains on their glycerol backbones, the complexity of the N-acyl-PS series is further increased by the presence of diverse amide-linked N-acyl chains, which include saturated, mono-unsaturated and poly-unsaturated species. N-acyl-PS molecular species were also detected in the lipids of pig brain, mouse RAW264.7 macrophage tumor cells and yeast, but not E. coli. N-acyl-PSs may be biosynthetic precursors of N-acyl serine molecules, such as the recently reported signaling lipid N-arachidonoyl serine from bovine brain. We suggest that a phospholipase D might cleave N-acyl-PS to generate N-acyl serine, in analogy to the biosynthesis of the endocannabinoid N-arachidonoyl ethanolamine (anadamide) from N-arachidonoyl phosphatidylethanolamine. PMID:18031065

  1. Sesquiterpene lactones from Taraxacum obovatum.

    PubMed

    Michalska, Klaudia; Kisiel, Wanda

    2003-02-01

    Two new guaianolide glucosides, deacetylmatricarin 8-O-beta-glucopyranoside and 11beta-hydroxyleukodin 11-O-beta-glucopyranoside, were isolated from roots of Taraxacum obovatum, along with four known sesquiterpene lactones, deacetylmatricarin, sonchuside A, taraxinic acid beta-glucopyranosyl ester and its 11beta,13-dihydro derivative. Their structures were established by spectral methods.

  2. Potential Use of Dimethyl Sulfoxide in Treatment of Infections Caused by Pseudomonas aeruginosa.

    PubMed

    Guo, Qiao; Wu, Qiaolian; Bai, Dangdang; Liu, Yang; Chen, Lin; Jin, Sheng; Wu, Yuting; Duan, Kangmin

    2016-12-01

    Dimethyl sulfoxide (DMSO) is commonly used as a solvent to dissolve water-insoluble drugs or other test samples in both in vivo and in vitro experiments. It was observed during our experiment that DMSO at noninhibitory concentrations could significantly inhibit pyocyanin production in the human pathogen Pseudomonas aeruginosa Pyocyanin is an important pathogenic factor whose production is controlled by a cell density-dependent quorum-sensing (QS) system. Investigation of the effect of DMSO on QS showed that DMSO has significant QS antagonistic activities and concentrations of DMSO in the micromolar range attenuated a battery of QS-controlled virulence factors, including rhamnolipid, elastase, and LasA protease production and biofilm formation. Further study indicated that DMSO inhibition of biofilm formation and pyocyanin production was attained by reducing the level of production of an autoinducer molecule of the rhl QS system, N-butanoyl-l-homoserine lactone (C 4 -HSL). In a mouse model of a burn wound infection with P. aeruginosa, treatment with DMSO significantly decreased mouse mortality compared with that for mice in the control group. The capacity of DMSO to attenuate the pathogenicity of P. aeruginosa points to the potential use of DMSO as an antipathogenic agent for the treatment of P. aeruginosa infection. As a commonly used solvent, however, DMSO's impact on bacterial virulence calls for cautionary attention in its usage in biological, medicinal, and clinical studies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Biosynthesis of Costunolide, Dihydrocostunolide, and Leucodin. Demonstration of Cytochrome P450-Catalyzed Formation of the Lactone Ring Present in Sesquiterpene Lactones of Chicory

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C.R.; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J.

    2002-01-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme. PMID:12011356

  4. Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory.

    PubMed

    de Kraker, Jan-Willem; Franssen, Maurice C R; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J

    2002-05-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.

  5. The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets.

    PubMed Central

    Bakken, A M; Farstad, M

    1992-01-01

    The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium. PMID:1471991

  6. Enantiodifferentiation of whisky and cognac lactones using gas chromatography with different cyclodextrin chiral stationary phases.

    PubMed

    Schmarr, Hans-Georg; Mathes, Maximilian; Wall, Kristina; Metzner, Frank; Fraefel, Marius

    2017-09-22

    The chiral lactone 5-butyl-4-methyloxolan-2-one or 5-butyl-4-methyldihydro-2(3H)-furanone, often named whisky lactone, is found in oak wood, then contributing to the appreciated flavor of beverages stored in such wooden barrels. Its next higher homologue is named cognac lactone (5-pentyl-4-methyloxolan-2-one or 5-pentyl-4-methyldihydro-2(3H)-furanone), however is much less known, probably due to its minor concentration level. In order to study the direct enantioseparation of both lactones by gas chromatography on chiral stationary phases, individual enantiomers, particularly for cognac lactone were made available. This was achieved by baker's yeast reduction of synthesized ethyl 3-methyl-4-oxononanoate or, after hydrolysis, of the corresponding 4-ketoacid, that gave access to individual enantiomers of cognac lactone. Good enantioseparation was achieved for both whisky and cognac lactone with high values for the chiral resolution with 6-O-tert. butyl dimethylsilyl-2,3-dialkylated or 6-O-tert. butyl dimethylsilyl-2,3-diacylated cyclodextrin derivatives as chiral selectors. The influence of the nature and position of derivatization of the cyclodextrin moiety revealed a strong impact on the chiral recognition mechanism, as the investigated alkylated derivatives heptakis-(2,6-di-O-iso-pentyl-3-O-allyl)-β-cyclodextrin and octakis-(2,3-di-O-pentyl-6-O-methyl)-γ-cyclodextrin did not provide any or only minor chiral selectivity for the two lactones. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

    PubMed

    Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

    2018-02-28

    The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

  8. Controlled synthesis of phosphorylcholine derivatives of poly(serine) and poly(homoserine).

    PubMed

    Yakovlev, Ilya; Deming, Timothy J

    2015-04-01

    We report methods for the synthesis of polypeptides that are fully functionalized with desirable phosphorylcholine, PC, groups. Because of the inherent challenges in the direct incorporation of the PC group into α-amino acid N-carboxyanhydride (NCA) monomers, we developed a synthetic approach that combined functional NCA polymerization with efficient postpolymerization modification. While poly(L-phosphorylcholine serine) was found to be unstable upon synthesis, we successfully prepared poly(L-phosphorylcholine homoserine) with controlled chain lengths and found these to be water-soluble with disordered chain conformations.

  9. Strong inhibitory effect of furanoses and sugar lactones on beta-galactosidase Escherichia coli.

    PubMed

    Huber, R E; Brockbank, R L

    1987-03-24

    Various sugars and their lactones were tested for their inhibition of beta-galactosidase (Escherichia coli). L-Ribose, which in the furanose form has a hydroxyl configuration similar to that of D-galactose at positions equivalent to the 3- and 4-positions of D-galactose, was a very strong inhibitor, and D-lyxose, which in the furanose form also resembles D-galactose, was a much better inhibitor than expected. Structural comparisons prelude the pyranose forms of these sugars from being significant contributors to the inhibition, and inhibition at different temperatures (at which there are different furanose concentrations) strongly supported the conclusion that the furanose form is inhibitory. Studies with sugar derivatives that can only be in the furanose form also supported the conclusion. This is the first report of the inhibitory effect of furanose on beta-galactosidase. Lactones were also inhibitory. Every lactone tested was much more inhibitory than was its parent sugar. D-Galactonolactone was especially good. Experiments indicated that it was D-galactono-1,5-lactone rather than D-galactono-1,4-lactone which was inhibitory. Inhibition of beta-galactosidases from mammalian sources by lactones has been reported previously, but this is the first report of the effect of beta-galactosidase from E. coli. Since furanoses in the envelope form are analogous (in some ways) to half-chair or sofa conformations and since lactones with six-membered rings probably have half-chair or sofa conformations, the results indicate that beta-galactosidase probably destabilizes its substrate into a planar conformation of some type and that the galactose in the transition state may, therefore, also be quite planar.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids.

    PubMed

    Ardhaoui, M; Falcimaigne, A; Ognier, S; Engasser, J M; Moussou, P; Pauly, G; Ghoul, M

    2004-06-10

    Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and omega-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on omega-substituted fatty acid chain were unfavourable to these reactions. The 1H NMR and 13C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4"'-OH of the rhamnose residue of rutin. Copyright 2004 Elsevier B.V.

  11. Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

    PubMed Central

    Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

    2015-01-01

    The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

  12. Versatility of acyl-acyl carrier protein synthetases

    DOE PAGES

    Beld, Joris; Finzel, Kara; Burkart, Michael D.

    2014-10-09

    The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

  13. An Analysis of the Advantages and Disadvantages Associated with the Consolidation of the HS And HSL Missions and Communities

    DTIC Science & Technology

    1994-03-01

    DISADVANTAGES ASSOCIATED WITH THE CONSOLIDATION OF THE HS AND HSL MISSIONS AND COMMUNITIES by Raymond B. Roll March 1994 Thesis Advisor: Nancy C...REPORT TYPE AND DATES COVERED I March 1994 Master’s Thsis 4L. "M AND SUBTITLE S. FUNDING NUMBERS An Analysis of the Advantages and Disadvantages ...consolidation issue and proposes four combined community organizations. Additionally, the advantages and disadvantages of each new structure are reviewed to

  14. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    PubMed

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  15. Membrane-Surface Anchoring of Charged Diacylglycerol-Lactones Correlates with Biological Activities | Center for Cancer Research

    Cancer.gov

    The inside cover picture shows the molecular structure of a DAG lactone derivative on top of the inner leaflet of a DMPC bilayer. The confocal microscopy image illustrates DAG-lactone-stimulated membrane localization of PKCδ-ECFP in living cells, while the space-filling model shows the surface of the C1B domain of PKCδ, the target of the lactone.

  16. Structural and Sensory Characterization of Novel Sesquiterpene Lactones from Iceberg Lettuce.

    PubMed

    Mai, Franziska; Glomb, Marcus A

    2016-01-13

    Lactuca sativa var. capitate (iceberg lettuce) is a delicious vegetable and popular for its mild taste. Nevertheless, iceberg lettuce is a source of bitter substances, such as the sesquiterpene lactones. Chemical investigations on the n-butanol extract led to the isolation of three novel sesquiterpene lactones. All compounds were isolated by multilayer countercurrent chromatography followed by preparative high-performance liquid chromatography. The structures were verified by means of spectroscopic methods, including NMR and mass spectrometry techniques. For the first time 11ß,13-dihydrolactucin-8-O-sulfate (jaquinelin-8-O-sulfate) was structurally elucidated and identified in plants. In addition, the sesquiterpene lactones cichorioside B and 8-deacetylmatricarin-8-O-sulfate were identified as novel ingredients of iceberg lettuce. Further flowering plants in the daisy family Asteraceae were examined for the above three compounds. At least one of the compounds was identified in nine plants. The comparison between the lettuce butt end and the leaves of five types of the Cichorieae tribe showed an accumulation of the compounds in the butt end. Further experiments addressed the impact of sesquiterpene lactones on color formation and bitter taste.

  17. Cadmium ion inhibition of quorum signalling in Chromobacterium violaceum.

    PubMed

    Thornhill, Starla G; Kumar, Manish; Vega, Leticia M; McLean, Robert J C

    2017-10-01

    Single-celled bacteria are capable of acting as a community by sensing and responding to population density via quorum signalling. Quorum signalling in Chromobacterium violaceum, mediated by the luxI/R homologue, cviI/R, regulates a variety of phenotypes including violacein pigmentation, virulence and biofilm formation. A number of biological and organic molecules have been described as quorum signalling inhibitors but, to date, metal-based inhibitors have not been widely tested. In this study, we show that quorum sensing is inhibited in C. violaceum in the presence of sub-lethal concentrations of cadmium salts. Notable Cd 2+ -inhibition was seen against pigmentation, motility, chitinase production and biofilm formation. Cd-inhibition of quorum-signalling genes occurred at the level of transcription. There was no direct inhibition of chitinase activity by Cd 2+ at the concentrations tested. Addition of the cognate quorum signals, N-hexanoyl homoserine lactone or N-decanoyl homoserine lactone, even at concentrations in excess of physiological levels, did not reverse the inhibition, suggesting that Cd-inhibition of quorum signaling is irreversible. This study represents the first description of heavy metal-based quorum inhibition in C. violaceum.

  18. Isolation of AHL-degrading bacteria from micro-algal cultures and their impact on algal growth and on virulence of Vibrio campbellii to prawn larvae.

    PubMed

    Pande, Gde Sasmita Julyantoro; Natrah, Fatin Mohd Ikhsan; Flandez, Ace Vincent Bravo; Kumar, Uday; Niu, Yufeng; Bossier, Peter; Defoirdt, Tom

    2015-12-01

    Inactivation of quorum sensing (QS) signal molecules, such as acylhomoserine lactones (AHLs) of pathogenic bacteria, has been proposed as a novel method to combat bacterial diseases in aquaculture. Despite the importance of micro-algae for aquaculture, AHL degradation by bacteria associated with micro-algal cultures has thus far not been investigated. In this study, we isolated Pseudomonas sp. NFMI-T and Bacillus sp. NFMI-C from open cultures of the micro-algae Tetraselmis suecica and Chaetoceros muelleri, respectively. An AHL degradation assay showed that either monocultures or co-cultures of the isolates were able to degrade the AHL N-hexanoyl-L-homoserine lactone. In contrast, only Bacillus sp. NFMI-C was able to inactivate N-hydroxybutanoyl-L-homoserine lactone, the AHL produced by Vibrio campbellii. The isolated bacteria were able to persist for up to 3 weeks in conventionalized micro-algal cultures, indicating that they were able to establish and maintain themselves within open algal cultures. Using gnotobiotic algal cultures, we found that the isolates did not affect growth of the micro-algae from which they were isolated, whereas a mixture of both isolates increased the growth of Tetraselmis and decreased the growth of Chaetoceros. Finally, addition of Bacillus sp. NFMI-C to the rearing water of giant river prawn (Macrobrachium rosenbergii) larvae significantly improved survival of the larvae when challenged with pathogenic V. campbellii, whereas it had no effect on larval growth.

  19. Variability of sesquiterpene lactones in Neurolaena lobata of different origin.

    PubMed

    Passreiter, C M; Aldana, B E

    1998-06-01

    Leaves of Neurolaena lobata (L.) R. Br. originating from Guatemala, were analyzed using HPLC for their qualitative and quantitative sesquiterpene lactone contents. Significant differences in the individual amounts of neurolenins and furanoheliangolides were found between four natural populations. When plants were cultivated on proving fields at two different localities in Guatemala, their sesquiterpene lactone patterns matched the natural population, but differed quantitatively. The meaning of these differences for the use of N. lobata in traditional medicine and its cultivation is discussed.

  20. Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs.

    PubMed

    Li, Qingling; Zhang, Shenghui; Berthiaume, Jessica M; Simons, Brigitte; Zhang, Guo-Fang

    2014-03-01

    A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

  1. Developmental and Environmental Effects on Sesquiterpene Lactones in Cultivated Arnica montana L.

    PubMed

    Todorova, Milka; Trendafilova, Antoaneta; Vitkova, Antonina; Petrova, Maria; Zayova, Ely; Antonova, Daniela

    2016-08-01

    The amount of sesquiterpene lactones and the lactone profile of Arnica montana L. in flowering and seed formation stages in vitro and in vivo propagated from seeds of German, Ukrainian, and Austrian origin and grown in two experimental fields were studied. It was found that in vitro propagated 2-year plants in full flowering stage accumulated higher amount of lactones in comparison to in vivo propagated 3-year plants and to the seed formation stage, respectively. Helenalins predominated in in vivo propagated 2-year or in vitro propagated 3-year plants. 2-Methylbutyrate (2MeBu) was the principal ester in the samples with prevalence of helenalins, while isobutyrate (iBu) was the major one in the samples with predominance of 11,13-dihydrohelenalins. The results revealed that the environmental conditions on Vitosha Mt. are more suitable for cultivation of A. montana giving higher content of lactones. © 2016 Wiley-VHCA AG, Zürich.

  2. Sesquiterpene lactones from neurolaena oaxacana

    PubMed

    Passreiter; Sandoval-Ramirez; Wright

    1999-08-01

    Twelve sesquiterpene lactones, two new (1 and 2) and 10 known neurolenin-type germacranolides and furanoheliangolides (3-12) were isolated from Neurolaena oaxacana, and their structures were elucidated by NMR and GC-MS analysis. The chemotaxonomic importance of these findings is discussed. As N. lobata is used against dysenteries, neurolenin B (4) and a mixture of the neurolenins C (5) and D (6) were tested against Entamoeba histolytica and Giardia intestinalis.

  3. Age-dependent decline in acyl-ghrelin concentrations and reduced association of acyl-ghrelin and growth hormone in healthy older adults.

    PubMed

    Nass, Ralf; Farhy, Leon S; Liu, Jianhua; Pezzoli, Suzan S; Johnson, Michael L; Gaylinn, Bruce D; Thorner, Michael O

    2014-02-01

    Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001). These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

  4. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed Central

    Jones, A; Davies, H M; Voelker, T A

    1995-01-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

  5. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed

    Jones, A; Davies, H M; Voelker, T A

    1995-03-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase.

  6. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    USDA-ARS?s Scientific Manuscript database

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  7. Preferred Conformers of Non-Proteinogenic Amino Acids Homoserine and Homocysteine

    NASA Astrophysics Data System (ADS)

    Díez, Verónica; Rodríguez, Miguel A.; Mata, Santiago; Alonso, E. R.; Cabezas, Carlos; Alonso, José L.

    2016-06-01

    Vaporization of solid homoserine and homocysteine by laser ablation in combination with Fourier transform microwave spectroscopy techniques made possible the detection of their most stable structures in a supersonic expansion. All detected conformers have been identified through their rotational and 14N quadrupole coupling constants. They show hydrogen bonds linking the amino and carboxylic group through N-H\\cdot\\cdot\\cdotO=C (type I) or N\\cdot\\cdot\\cdotH-O (type II) interactions. In some of them there are additional hydrogen bonds established between the amino group and the hydroxyl/thiol groups in the gamma position. Entropic effects related to the side chain have been found to be significant in determining the most populated conformations.

  8. Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

    PubMed

    Yurchenko, Olga; Singer, Stacy D; Nykiforuk, Cory L; Gidda, Satinder; Mullen, Robert T; Moloney, Maurice M; Weselake, Randall J

    2014-06-01

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1 cisΔ11 ) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2 cisΔ9,12 ; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1 cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3 cisΔ9,12,15 ) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

  9. Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

    PubMed

    Namekawa, S; Suda, S; Uyama, H; Kobayashi, S

    1999-01-01

    Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.

  10. DNA Microarray-Based Identification of Genes Controlled by Autoinducer 2-Stimulated Quorum Sensing in Escherichia Coli

    DTIC Science & Technology

    2001-09-01

    and pathogenicity in Erwinia carotovora (rsmA) (12). Additionally, csrA has been documented to affect cell size and surface properties, which is in...machinery to cell wall 13.1 b1502 Putative adhesin; similar to FimH protein 13.0 tap Methyl-accepting chemotaxis protein IV, peptide sensor receptor...oxohexanoyl)-L-homoserine lactone 5246 DELISA ET AL. J. BACTERIOL. regulates carbapenem antibiotic production in Erwinia carotovora . Biochem. J. 288:997

  11. High Prevalence of Quorum-Sensing and Quorum-Quenching Activity among Cultivable Bacteria and Metagenomic Sequences in the Mediterranean Sea

    PubMed Central

    López-Pérez, Mario; Mayer, Celia; Parga, Ana; Amaro-Blanco, Jaime

    2018-01-01

    There is increasing evidence being accumulated regarding the importance of N-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15–2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment. PMID:29462892

  12. Bacterial quorum sensing and nitrogen cycling in rhizosphere soil

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DeAngelis, K.M.; Lindow, S.E.; Firestone, M.K.

    2008-10-01

    Plant photosynthate fuels carbon-limited microbial growth and activity, resulting in increased rhizosphere nitrogen (N)-mineralization. Most soil organic N is macromolecular (chitin, protein, nucleotides); enzymatic depolymerization is likely rate-limiting for plant N accumulation. Analyzing Avena (wild oat) planted in microcosms containing sieved field soil, we observed increased rhizosphere chitinase and protease specific activities, bacterial cell densities, and dissolved organic nitrogen (DON) compared to bulk soil. Low-molecular weight DON (<3000 Da) was undetectable in bulk soil but comprised 15% of rhizosphere DON. Extracellular enzyme production in many bacteria requires quorum sensing (QS), cell-density dependent group behavior. Because proteobacteria are considered major rhizospheremore » colonizers, we assayed the proteobacterial QS signals acyl-homoserine lactones (AHLs), which were significantly increased in the rhizosphere. To investigate the linkage between soil signaling and N cycling, we characterized 533 bacterial isolates from Avena rhizosphere: 24% had chitinase or protease activity and AHL production; disruption of QS in 7 of 8 eight isolates disrupted enzyme activity. Many {alpha}-Proteobacteria were newly found with QS-controlled extracellular enzyme activity. Enhanced specific activities of N-cycling enzymes accompanied by bacterial density-dependent behaviors in rhizosphere soil gives rise to the hypothesis that QS could be a control point in the complex process of rhizosphere N-mineralization.« less

  13. RETRACTED ARTICLE: Quorum-sensing of bacteria and its application

    NASA Astrophysics Data System (ADS)

    Jiang, Guoliang; Su, Mingxia

    2009-12-01

    Quorum sensing, or auto induction, as a cell density dependent signaling mechanism in many microorganisms, is triggered via auto inducers which passively diffuse across the bacterial envelope and therefore intracellulaly accumulate only at higher bacterial densities to regulate specialized processes such as genetic competence, bioluminescence, virulence and sporulation. N-acyl homoserine lactones are the most common type of signal molecules. Aquaculture is one of the fastest-growing food-producing industries, but disease outbreaks caused by pathogenic bacteria are a significant constraint on the development of the sector worldwide. Many of these pathogens have been found to be controlled by their quorum sensing systems. As there is relevance between the pathogenic bacteria's virulence factor expression and their auto inducers, quorum quenching is a new effective anti-infective strategy to control infections caused by bacterial pathogens in aquaculture. The techniques used to do this mainly include the following: (1) the inhibition of signal molecule biosynthesis, (2) blocking signal transduction, and (3) chemical inactivation and biodegradation of signal molecules. To provide a basis for finding alternative means of controlling aquatic diseases by quorum quenching instead of treatment by antibiotics and disinfectants, we will discuss the examination, purification and identification of auto inducers in this paper.

  14. Alkamides Activate Jasmonic Acid Biosynthesis and Signaling Pathways and Confer Resistance to Botrytis cinerea in Arabidopsis thaliana

    PubMed Central

    Méndez-Bravo, Alfonso; Calderón-Vázquez, Carlos; Ibarra-Laclette, Enrique; Raya-González, Javier; Ramírez-Chávez, Enrique; Molina-Torres, Jorge; Guevara-García, Angel A.; López-Bucio, José; Herrera-Estrella, Luis

    2011-01-01

    Alkamides are fatty acid amides of wide distribution in plants, structurally related to N-acyl-L-homoserine lactones (AHLs) from Gram-negative bacteria and to N- acylethanolamines (NAEs) from plants and mammals. Global analysis of gene expression changes in Arabidopsis thaliana in response to N-isobutyl decanamide, the most highly active alkamide identified to date, revealed an overrepresentation of defense-responsive transcriptional networks. In particular, genes encoding enzymes for jasmonic acid (JA) biosynthesis increased their expression, which occurred in parallel with JA, nitric oxide (NO) and H2O2 accumulation. The activity of the alkamide to confer resistance against the necrotizing fungus Botrytis cinerea was tested by inoculating Arabidopsis detached leaves with conidiospores and evaluating disease symptoms and fungal proliferation. N-isobutyl decanamide application significantly reduced necrosis caused by the pathogen and inhibited fungal proliferation. Arabidopsis mutants jar1 and coi1 altered in JA signaling and a MAP kinase mutant (mpk6), unlike salicylic acid- (SA) related mutant eds16/sid2-1, were unable to defend from fungal attack even when N-isobutyl decanamide was supplied, indicating that alkamides could modulate some necrotrophic-associated defense responses through JA-dependent and MPK6-regulated signaling pathways. Our results suggest a role of alkamides in plant immunity induction. PMID:22076141

  15. Characterisation of non-autoinducing tropodithietic Acid (TDA) production from marine sponge Pseudovibrio species.

    PubMed

    Harrington, Catriona; Reen, F Jerry; Mooij, Marlies J; Stewart, Fiona A; Chabot, Jean-Baptiste; Guerra, Antonio F; Glöckner, Frank O; Nielsen, Kristian F; Gram, Lone; Dobson, Alan D W; Adams, Claire; O'Gara, Fergal

    2014-12-10

    The search for new antimicrobial compounds has gained added momentum in recent years, paralleled by the exponential rise in resistance to most known classes of current antibiotics. While modifications of existing drugs have brought some limited clinical success, there remains a critical need for new classes of antimicrobial compound to which key clinical pathogens will be naive. This has provided the context and impetus to marine biodiscovery programmes that seek to isolate and characterize new activities from the aquatic ecosystem. One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule.

  16. Characterisation of Non-Autoinducing Tropodithietic Acid (TDA) Production from Marine Sponge Pseudovibrio Species

    PubMed Central

    Harrington, Catriona; Reen, F. Jerry; Mooij, Marlies J.; Stewart, Fiona A.; Chabot, Jean-Baptiste; Guerra, Antonio F.; Glöckner, Frank O.; Nielsen, Kristian F.; Gram, Lone; Dobson, Alan D. W.; Adams, Claire; O’Gara, Fergal

    2014-01-01

    The search for new antimicrobial compounds has gained added momentum in recent years, paralleled by the exponential rise in resistance to most known classes of current antibiotics. While modifications of existing drugs have brought some limited clinical success, there remains a critical need for new classes of antimicrobial compound to which key clinical pathogens will be naive. This has provided the context and impetus to marine biodiscovery programmes that seek to isolate and characterize new activities from the aquatic ecosystem. One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule. PMID:25513851

  17. Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste

    PubMed Central

    Arregui, Lucía; Arroyo, Miguel; Mendoza, José Antonio; Álvarez, Cristina; García-Vera, Cristina; Marquina, Domingo; Santos, Antonio; Serrano, Susana

    2018-01-01

    Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N-acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes. PMID:29783658

  18. Bacterial biofilms and quorum sensing: fidelity in bioremediation technology.

    PubMed

    Mangwani, Neelam; Kumari, Supriya; Das, Surajit

    Increased contamination of the environment with toxic pollutants has paved the way for efficient strategies which can be implemented for environmental restoration. The major problem with conventional methods used for cleaning of pollutants is inefficiency and high economic costs. Bioremediation is a growing technology having advanced potential of cleaning pollutants. Biofilm formed by various micro-organisms potentially provide a suitable microenvironment for efficient bioremediation processes. High cell density and stress resistance properties of the biofilm environment provide opportunities for efficient metabolism of number of hydrophobic and toxic compounds. Bacterial biofilm formation is often regulated by quorum sensing (QS) which is a population density-based cell-cell communication process via signaling molecules. Numerous signaling molecules such as acyl homoserine lactones, peptides, autoinducer-2, diffusion signaling factors, and α-hydroxyketones have been studied in bacteria. Genetic alteration of QS machinery can be useful to modulate vital characters valuable for environmental applications such as biofilm formation, biosurfactant production, exopolysaccharide synthesis, horizontal gene transfer, catabolic gene expression, motility, and chemotaxis. These qualities are imperative for bacteria during degradation or detoxification of any pollutant. QS signals can be used for the fabrication of engineered biofilms with enhanced degradation kinetics. This review discusses the connection between QS and biofilm formation by bacteria in relation to bioremediation technology.

  19. Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste.

    PubMed

    Soler, Albert; Arregui, Lucía; Arroyo, Miguel; Mendoza, José Antonio; Muras, Andrea; Álvarez, Cristina; García-Vera, Cristina; Marquina, Domingo; Santos, Antonio; Serrano, Susana

    2018-05-18

    Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N -acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains ( Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes.

  20. Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria.

    PubMed

    Ham, Jong Hyun

    2013-04-01

    Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  1. Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, D.; Thomas, P.W.; Momb, J.

    2009-06-03

    N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be amore » metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.« less

  2. Detection and inhibition of bacterial cell-cell communication.

    PubMed

    Rice, Scott A; McDougald, Diane; Givskov, Michael; Kjelleberg, Staffan

    2008-01-01

    Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling purposes, including fatty acids, peptides, N-acylated homoserine lactones, and the signals collectively called autoinducer 2 (AI-2). This chapter will focus on the latter two signaling systems as they are present in a range of medically relevant bacteria, and here we describe assays for determining whether an organism produces a particular signal and assays that can be used to identify inhibitors of the signaling cascade. Lastly, the signal detection and inhibition assays will be directly linked to the expression of virulence factors of specific pathogens.

  3. Antibiotic resistome promotion in drinking water during biological activated carbon treatment: Is it influenced by quorum sensing?

    PubMed

    Zheng, Ji; Chen, Tao; Chen, Hong

    2018-01-15

    The contamination of antibiotic resistance genes (ARGs) in drinking water may pose a direct threat to human health. This study applied high-throughput qPCR and sequencing to investigate the dynamics of ARGs and bacterial communities during the advanced treatment of drinking water using biological activated carbon. The promotion of ARGs was observed, and the normalized copy number of ARGs increased significantly after BAC treatment, raising the number of detected ARGs from 84 to 159. Twenty-nine ARGs were identified as biofilm-influencing sources in the BAC, and they persisted after chlorination. The shift of bacterial communities primarily had effects on the changes in resistome. Firmicutes, Cyanobacteria were related to persistent ARGs mostly in the BAC biofilm. Meanwhile, the Acyl-Homoserine Lactones (AHLs), quorum sensing molecules, and bacteria that produced AHLs were identified to understand the promotion of ARGs. The isolated AHL-producing bacteria belonged to the Proteobacteria, Firmicutes and Bacteroidetes phyla. Six detectable AHLs had an influence on plasmid-based horizontal gene transfer in the intragenus mating systems, indicating that the dynamics of ARGs were strongly affected by quorum sensing between specific bacteria in the biofilm. These results provide new insight into the mechanism of antibiotic resistome promotion in BAC biofilms. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. l-Canavanine Made by Medicago sativa Interferes with Quorum Sensing in Sinorhizobium meliloti

    PubMed Central

    Keshavan, Neela D.; Chowdhary, Puneet K.; Haines, Donovan C.; González, Juan E.

    2005-01-01

    Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes. PMID:16321947

  5. Inhibition of biofilm formation by T7 bacteriophages producing quorum-quenching enzymes.

    PubMed

    Pei, Ruoting; Lamas-Samanamud, Gisella R

    2014-09-01

    Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

    PubMed Central

    Lamas-Samanamud, Gisella R.

    2014-01-01

    Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings. PMID:24951790

  7. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  8. Macrocyclic lactones: A versatile source for omega radiohalogenated fatty acid analogs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dougan, A.H.; Lyster, D.M.; Robertson, K.A.

    For each omega halogenated fatty acid there exists a potential omega hydroxy fatty acid and the corresponding macrocyclic lactone. The authors have utilized such lactones as starting materials for omega /sup 123/I fatty acid analogs intended for myocardial imaging. Macrocyclic musk lactones are industrially available; 120 analogs are described in the literature. The preparation requires saponification, tosylation, and radio-iodide substitution. Iodo-fatty acids are readily separated from tosylate fatty acids on TLC. While providing a secure source of 16-iodo-hexadecanoic acid and 17-iodo-heptadecanoic acid, the scheme allows ready access to a large number of untried fatty acid analogs. Examples presented are 16-iodo-hexadecanoicmore » acid, 16-iodo-7-hexadecanoic acid, 16-iodo-12-oxa-hexadecanoic acid, 15-iodo-pentadecanoic acid, and 15-iodo-12-keto-pentadecanoic acid. Metabolic studies are in progress in mice and dogs to assess the utility of these analogs for myocardial imaging.« less

  9. Regioselective self-acylating cyclodextrins in organic solvent

    NASA Astrophysics Data System (ADS)

    Cho, Eunae; Yun, Deokgyu; Jeong, Daham; Im, Jieun; Kim, Hyunki; Dindulkar, Someshwar D.; Choi, Youngjin; Jung, Seunho

    2016-03-01

    Amphiphilic cyclodextrins have been synthesized with self-acylating reaction using vinyl esters in dimethylformamide. In the present study no base, catalyst, or enzyme was used, and the structural analyses using thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry show that the cyclodextrin is substituted preferentially by one acyl moiety at the C2 position of the glucose unit, suggesting that cyclodextrin functions as a regioselective catalytic carbohydrate in organic solvent. In the self-acylation, the most acidic OH group at the 2-position and the inclusion complexing ability of cyclodextrin were considered to be significant. The substrate preference was also observed in favor of the long-chain acyl group, which could be attributed to the inclusion ability of cyclodextrin cavity. Furthermore, using the model amphiphilic building block, 2-O-mono-lauryl β-cyclodextrin, the self-organized supramolecular architecture with nano-vesicular morphology in water was investigated by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The cavity-type nano-assembled vesicle and the novel synthetic methods for the preparation of mono-acylated cyclodextrin should be of great interest with regard to drug/gene delivery systems, functional surfactants, and carbohydrate derivatization methods.

  10. Triacetic acid lactone production from Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    Triacetic acid lactone (TAL) is a potential platform chemical produced from acetyl-CoA and malonyl-CoA by the Gerbera hybrida 2-pyrone synthase (2PS) gene. Studies are ongoing to optimize production, purification, and chemical modification of TAL, which can be used to create the commercial chemicals...

  11. Biosynthesis of Jasmine Lactone in Tea ( Camellia sinensis) Leaves and Its Formation in Response to Multiple Stresses.

    PubMed

    Zeng, Lanting; Zhou, Ying; Fu, Xiumin; Liao, Yinyin; Yuan, Yunfei; Jia, Yongxia; Dong, Fang; Yang, Ziyin

    2018-04-18

    Jasmine lactone has a potent odor that contributes to the fruity, sweet floral aroma of tea ( Camellia sinensis). Our previous study demonstrated that jasmine lactone was mostly accumulated at the turnover stage of the oolong tea manufacturing process. This study investigates the previously unknown mechanism of formation of jasmine lactone in tea leaves exposed to multiple stresses occurring during the growth and manufacturing processes. Both continuous mechanical damage and the dual stress of low temperature and mechanical damage enhanced jasmine lactone accumulation in tea leaves. In addition, only one pathway, via hydroperoxy fatty acids from unsaturated fatty acid, including linoleic acid and α-linolenic acid, under the action of lipoxygenases (LOXs), especially CsLOX1, was significantly affected by these stresses. This is the first evidence of the mechanism of jasmine lactone formation in tea leaves and is a characteristic example of plant volatile formation in response to dual stress.

  12. Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

    PubMed

    Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

    1977-02-01

    The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

  13. Progress toward Understanding Protein S-acylation: Prospective in Plants

    PubMed Central

    Li, Yaxiao; Qi, Baoxiu

    2017-01-01

    S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs). Since the discovery of the first PAT in yeast in 2002 research in S-acylation has accelerated in the mammalian system and followed by in plant. However, it is still a difficult field to study due to the large number of PATs and even larger number of putative S-acylated substrate proteins they modify in each genome. This is coupled with drawbacks in the techniques used to study S-acylation, leading to the slower progress in this field compared to protein phosphorylation, for example. In this review we will summarize the discoveries made so far based on knowledge learnt from the characterization of protein S-acyltransferases and the S-acylated proteins, the interaction mechanisms between PAT and its specific substrate protein(s) in yeast and mammals. Research in protein S-acylation and PATs in plants will also be covered although this area is currently less well studied in yeast and mammalian systems. PMID:28392791

  14. Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

    PubMed

    Smith, Dennis A; Hammond, Timothy; Baillie, Thomas A

    2018-06-01

    While simple O - (ether-linked) and N -glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Functional Amyloids Keep Quorum-sensing Molecules in Check*

    PubMed Central

    Seviour, Thomas; Hansen, Susan Hove; Yang, Liang; Yau, Yin Hoe; Wang, Victor Bochuan; Stenvang, Marcel R.; Christiansen, Gunna; Marsili, Enrico; Givskov, Michael; Chen, Yicai; Otzen, Daniel E.; Nielsen, Per Halkjær; Geifman-Shochat, Susana; Kjelleberg, Staffan; Dueholm, Morten S.

    2015-01-01

    The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats. PMID:25586180

  16. Novel synthesis of [11C]GVG (Vigabatgrin) for pharmacokinetic studies of addiction treatment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ding, Y.S.; Studenov, A.R.; Zhang, Z.

    2001-06-10

    We report here a novel synthetic route to prepare the precursor and to efficiently label GVG with C-11. 5-Bromo-3-(carbobenzyloxy)amino-1-pentene was synthesized in five steps from homoserine lactone. This was used in a two step radiosynthesis, displacement with [{sup 11}C]cyanide followed by acid hydrolysis to afford [{sup 11}C]GVG with high radiochemical yields (> 35%, not optimized) and high specific activity (2-5 Ci/{micro}mol). The [{sup 11}C]cyanide trapping was achieved at {minus}5 C with a mixture of Kryptofix and K{sub 2}CO{sub 3} without using conventional aqueous trapping procedure [7]. At this temperature, the excess NH{sub 3} from the target that may interfere withmore » the synthesis would not be trapped [8]. This procedure would be advantageous to any moisture sensitive radiosynthetic steps, as it was the case for our displacement reaction. When conventional aqueous trapping procedure was used, any trace amount of water left, even after prolonged heating, resulted in either no reaction or extremely low yields for the displacement reaction. The entire synthetic procedure should be extendible to the labeling of the pharmacologically active S- form of GVG when using S-homoserine lactone.« less

  17. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    PubMed Central

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase. PMID:12228351

  18. Plasma levels of acylated ghrelin in patients with functional dyspepsia

    PubMed Central

    Kim, Yeon Soo; Lee, Joon Seong; Lee, Tae Hee; Cho, Joo Young; Kim, Jin Oh; Kim, Wan Jung; Kim, Hyun Gun; Jeon, Seong Ran; Jeong, Hoe Su

    2012-01-01

    AIM: To investigate the relationship between plasma acylated ghrelin levels and the pathophysiology of functional dyspepsia. METHODS: Twenty-two female patients with functional dyspepsia and twelve healthy volunteers were recruited for the study. The functional dyspepsia patients were each diagnosed based on the Rome III criteria. Eligible patients completed a questionnaire concerning the severity of 10 symptoms. Plasma acylated ghrelin levels before and after a meal were determined in the study participants using a commercial human acylated enzyme immunoassay kit; electrogastrograms were performed for 50 min before and after a standardized 10-min meal containing 265 kcal. RESULTS: There were no significant differences in plasma acylated ghrelin levels between healthy volunteers and patients with functional dyspepsia. However, in patients with functional dyspepsia, there was a negative correlation between fasting plasma acylated ghrelin levels and the sum score of epigastric pain (r = -0.427, P = 0.047) and a positive correlation between the postprandial/fasting plasma acylated ghrelin ratio and the sum score of early satiety (r = 0.428, P =0.047). Additionally, there was a negative correlation between fasting acylated ghrelin plasma levels and fasting normogastria (%) (r = -0.522, P = 0.013). Interestingly, two functional dyspepsia patients showed paradoxically elevated plasma acylated ghrelin levels after the meal. CONCLUSION: Abnormal plasma acylated ghrelin levels before or after a meal may be related to several of the dyspeptic symptoms seen in patients with functional dyspepsia. PMID:22611317

  19. Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

    PubMed Central

    Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

    2016-01-01

    Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

  20. Oxonitriles: A Grignard Addition-Acylation Route to Enamides

    PubMed Central

    Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W.

    2008-01-01

    Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents, or a trialkyl zincate, effectively assembling highly substituted, cyclic enamides. PMID:17020332

  1. Antibacterial and cytotoxic activities of the sesquiterpene lactones cnicin and onopordopicrin.

    PubMed

    Bach, Sandra M; Fortuna, Mario A; Attarian, Rodgoun; de Trimarco, Juliana T; Catalán, César A N; Av-Gay, Yossef; Bach, Horacio

    2011-02-01

    The antimicrobial and cytotoxic activities of chloroform extracts from the weeds Centaurea tweediei and C. diffusa, and the main sesquiterpene lactones isolated from these species, onopordopicrin and cnicin, respectively, were assayed. Results show that the chloroform extracts from both Centaurea species possess antibacterial activities against a panel of Gram-positive and Gram-negative bacteria. Remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus was also measured. Both the extracts and the purified sesquiterpene lactones show high cytotoxicity against human-derived macrophages. Despite this cytotoxicity, C. diffusa chloroform extract and cnicin are attractive candidates for evaluation as antibiotics in topical preparations against skin-associated pathogens.

  2. Oxonitriles: a grignard addition-acylation route to enamides.

    PubMed

    Fleming, Fraser F; Wei, Guoqing; Zhang, Zhiyu; Steward, Omar W

    2006-10-12

    [reaction: see text] Sequential addition of three different Grignard reagents and pivaloyl chloride to 3-oxo-1-cyclohexene-1-carbonitrile installs four new bonds to generate a diverse array of cyclic enamides. Remarkably, formation of the C-magnesiated nitrile intermediate is followed by preferential acylation by pivaloyl chloride rather than consumption by an in situ Grignard reagent. Rapid N-acylation of the C-magnesiated nitrile generates an acyl ketenimine that reacts readily with Grignard reagents or a trialkylzincate, effectively assembling highly substituted, cyclic enamides.

  3. An Unusual Fatty Acyl:Adenylate Ligase (FAAL)-Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis.

    PubMed

    Hemmerling, Franziska; Lebe, Karen E; Wunderlich, Johannes; Hahn, Frank

    2018-03-08

    The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Selective enzymatic hydrolysis of chlorogenic acid lactones in a model system and in a coffee extract. Application to reduction of coffee bitterness.

    PubMed

    Kraehenbuehl, Karin; Page-Zoerkler, Nicole; Mauroux, Olivier; Gartenmann, Karin; Blank, Imre; Bel-Rhlid, Rachid

    2017-03-01

    Chlorogenic acid lactones have been identified as key contributors to coffee bitterness. These compounds are formed during roasting by dehydration and cyclization of their precursors, the chlorogenic acids (CGAs). In the present study, we investigated an approach to decompose these lactones in a selective way without affecting the positive coffee attributes developed during roasting. A model system composed of (3-caffeoylquinic acid lactone (3-CQAL), 4- caffeoyl quinic acid lactone (4-CQAL), and 4-feruloylquinic acid lactone (4-FQAL)) was used for the screening of enzymes before treatment of the coffee extracts. Hog liver esterase (HLE) hydrolyzed chlorogenic acid lactones (CQALs, FQALs) selectively, while chlorogenate esterase hydrolyzed all chlorogenic acids (CQAs, FQAs) and their corresponding lactones (CQALs, FQALs) in a non-selective way. Enzymatically treated coffee samples were evaluated for their bitterness by a trained sensory panel and were found significantly less bitter than the untreated samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. In silico prediction of acyl glucuronide reactivity

    NASA Astrophysics Data System (ADS)

    Potter, Tim; Lewis, Richard; Luker, Tim; Bonnert, Roger; Bernstein, Michael A.; Birkinshaw, Timothy N.; Thom, Stephen; Wenlock, Mark; Paine, Stuart

    2011-11-01

    Drugs and drug candidates containing a carboxylic acid moiety, including many widely used non-steroidal anti-inflammatory drugs (NSAIDs) are often metabolized to form acyl glucuronides (AGs). NSAIDs such as Ibuprofen are amongst the most widely used drugs on the market, whereas similar carboxylic acid drugs such as Suprofen have been withdrawn due to adverse events. Although the link between these AG metabolites and toxicity is not proven, there is circumstantial literature evidence to suggest that more reactive acyl glucuronides may, in some cases, present a greater risk of exhibiting toxic effects. We wished therefore to rank the reactivity of potential new carboxylate-containing drug candidates, and performed kinetic studies on synthetic acyl glucuronides to benchmark our key compounds. Driven by the desire to quickly rank the reactivity of compounds without the need for lengthy synthesis of the acyl glucuronide, a correlation was established between the degradation half-life of the acyl glucuronide and the half life for the hydrolysis of the more readily available methyl ester derivative. This finding enabled a considerable broadening of chemical property space to be investigated. The need for kinetic measurements was subsequently eliminated altogether by correlating the methyl ester hydrolysis half-life with the predicted 13C NMR chemical shift of the carbonyl carbon together with readily available steric descriptors in a PLS model. This completely in silico prediction of acyl glucuronide reactivity is applicable within the earliest stages of drug design with low cost and acceptable accuracy to guide intelligent molecular design. This reactivity data will be useful alongside the more complex additional pharmacokinetic exposure and distribution data that is generated later in the drug discovery process for assessing the overall toxicological risk of acidic drugs.

  6. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed

    Stahl, U.; Banas, A.; Stymne, S.

    1995-03-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

  7. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohlrogge, John B.

    2013-11-05

    Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

  8. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  9. Sialomucins are characteristically O-acylated in poorly differentiated and colloid prostatic adenocarcinomas.

    PubMed

    Sáez, C; Japón, M A; Conde, A F; Poveda, M A; Luna-Moré, S; Segura, D I

    1998-12-01

    Mucinous glycoproteins are secreted by prostatic adenocarcinomas and might play important roles in tumor invasion and metastasis. Their histochemical properties on routine biopsy specimens have not been fully characterized. We present a histochemical study of mucin in 21 prostatic adenocarcinomas, with particular focus on the demonstration of different types of sialomucins. We applied the following histochemical techniques to routinely processed, formalin-fixed, paraffin-embedded tissue sections: Alcian blue (pH 2.5) and periodic acid-Schiff to reveal both acidic and neutral mucins; high iron diamine and Alcian blue (pH 2.5) to show sulfated and acidic nonsulfated mucosubstances simultaneously; periodic acid borohydride, potassium hydroxide, and periodic acid-Schiff to demonstrate O-acylated sialic acids; periodic acid thionine-Schiff, potassium hydroxide, and periodic acid-Schiff to differentiate pre-existing glycols from those revealed after saponification procedures; and periodic acid borohydride and periodic acid-Schiff to show C9-O-acylated sialic acid. These techniques are useful tools for demonstrating neutral and acidic (sialo- and sulfo-) mucins and di(C8,C9- or C7,C9-)-O-acylated, tri(C7,C8,C9-)-O-acylated and mono(C9)-O-acylated sialomucins. Most prostatic adenocarcinomas showed acidic mucins, with sialomucins predominating over sulfomucins. Well-differentiated and moderately differentiated noncolloid tumors had non-O-acylated sialomucins. Poorly differentiated tumors contained mono-O-acylated (C9) sialomucins, and colloid-type tumors secreted mono-, di-, and tri-O-acylated sialoglycoproteins. Acidic mucins, mainly sialomucins, constitute the major secretory component in prostatic adenocarcinomas, and our results show that the O-acylation of these sialoglycoproteins inversely correlates with tumor differentiation. Well-differentiated and moderately differentiated tumors are not O-acylated, whereas the poorly differentiated ones characteristically have O-acylated

  10. Acyl donors for native chemical ligation.

    PubMed

    Yan, Bingjia; Shi, Weiwei; Ye, Linzhi; Liu, Lei

    2018-04-11

    Native chemical ligation (NCL) has become one of the most important methods in chemical syntheses of proteins. Recently, in order to expand its scope, considerable effort has been devoted to tuning the C-terminal acyl donor thioesters used in NCL. This article reviews the recent advances in the design of C-terminal acyl donors, their precursors and surrogates, and highlights some noteworthy progress that may lead the future direction of protein chemical synthesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Effect of different types of sesquiterpene lactones on the maturation of Rhinella arenarum oocytes.

    PubMed

    Sánchez-Toranzo, G; Zapata-Martínez, J; Catalán, C; Bühler, M I

    2015-06-01

    The sesquiterpene lactones (STLs) are a large class of plant secondary metabolites that are generally found in the Asteraceae family and that have high diversity with respect to chemical structure as well as biological activity. STLs have been classified into different groups, such as guaianolides, germacranolides, and melampolides etc., based on their carboxylic skeleton. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under the stimulus of progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. The purpose of this work was to determine whether sesquiterpene lactones from the germacranolide and melampolide groups act as inhibitor agents on the meiosis of amphibian oocytes in vitro. Results for germacranolides indicated that the addition of deoxyelephantopins caused a high degree of inhibition and that minimolide showed a moderate inhibitory effect, whereas glaucolide A was inactive. Furthermore, the addition of melampolides (uvedalin, enhydrin, polymatin A and polymatin B) showed inhibitory effects. For enhydrin and uvedalin, inhibitory effects were observed at the higher concentrations assayed. The results of this study suggest that the inhibitory activity of the tested sesquiterpene lactones on the meiosis of Rhinella arenarum oocytes is not dependent on the group to which they belong, i.e. not on the carboxylic skeleton, but probably due to the arrangement and type of function groups present in the molecules. All assayed lactones in the germacranolide group showed low toxicity. In contrast, important differences in toxicity were observed for lactones from the melampolide group: enhydrin and uvedalin showed low toxicity, but polymatin A and B were highly toxic.

  12. Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

    PubMed

    Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

    2013-04-02

    Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Capillary zone electrophorsis for the analysis of naturally occurring 2-hydroxycitric acids and their lactones.

    PubMed

    Abhijith, B L; Mohan, Moolya; Joseph, David; Haleema, Simimole; Aboul-Enein, Hassan Y; Ibnusaud, Ibrahim

    2017-08-01

    A simple capillary zone electrophoresis method with direct ultraviolet detection has been developed for the analysis of naturally occurring diastereomeric 2-hydroxycitric acid lactones. Using 50 mM sodium phosphate buffer of pH 7, a baseline resolution R s > 3.0 was observed for all organic acids selected for the present study. This method was employed for the quantitative determination of title acids present in the plant sources namely Garcinia cambogia fruit rinds and Hibiscus sabdariffa calyx. Conversion of 2-hydroxycitric acids to their lactones on heating the above plant sources is deliberated. The Hydrolysis of hydroxycitric acid lactones in aqueous solution is reported for the first time. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Sesquiterpene lactones of Vernonia - influence of glaucolide-A on the growth rate and survival of Lepidopterous larvae.

    PubMed

    Jones, Samuel B; Burnett, William C; Coile, Nancy C; Mabry, Tom J; Betkouski, M F

    1979-01-01

    Sesquiterpene lactone glaucolide-A from Vernonia, incorporated in the rearing diets of five species of Lepidoptera, significantly reduced the rate of growth of larvae of the southern armyworm, Spodoptera eridania; fall armyworm, S. frugiperda; and yellowstriped armyworm, S. ornithogalli. Quantitative feeding tests demonstrated that decreased feeding levels and reduced growth resulted from ingestion of a sesquiterpene lactone. Ingestion of glaucolide-A increased the number of days to pupation in four of the species. In the southern armyworm, it significantly reduced pupal weight. Glaucolide-A decidedly reduced percentage of survival of southern and fall armyworms. Yellow woollybear, Diacrisia virginica, and cabbage looper, Trichoplusia ni, larvae were essentially uneffected by the ingestion of the sesquiterpene lactone. Sesquiterpene lactones adversely affect growth rate and survival of certain insects that feed upon plants containing them. They apparently function as defensive products, screening out a portion of the potential herbivores.

  15. Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis1

    PubMed Central

    Jasieniecka-Gazarkiewicz, Katarzyna; Lager, Ida; Carlsson, Anders S.; Gutbrod, Katharina; Peisker, Helga; Dörmann, Peter; Stymne, Sten; Banaś, Antoni

    2017-01-01

    Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE. The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC. The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells. PMID:28408542

  16. Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals

    PubMed Central

    Lowery, Colin A.; Park, Junguk; Gloeckner, Christian; Meijler, Michael M.; Mueller, Ryan S.; Boshoff, Helena I.; Ulrich, Ricky L.; Barry, Clifton E.; Bartlett, Douglas H.; Kravchenko, Vladimir V.; Kaufmann, Gunnar F.; Janda, Kim D.

    2009-01-01

    In Nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in Nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated and C12-TA was found to dissipate both the membrane potential and pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species, and also to defend against both host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics. PMID:19807189

  17. Development of an Ex Vivo Porcine Lung Model for Studying Growth, Virulence, and Signaling of Pseudomonas aeruginosa

    PubMed Central

    Muruli, Aneesha; Higgins, Steven; Diggle, Stephen P.

    2014-01-01

    Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations. In vitro models cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed an ex vivo pig lung (EVPL) model for quantifying Pseudomonas aeruginosa growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show that lasR mutants, which do not respond to 3-oxo-C12-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show that lasR mutants grow as well as or better than a corresponding wild-type strain in EVPL. lasR mutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis that lasR mutants act as social “cheats” in the lung; rather, our results support the hypothesis that lasR mutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of microbial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment. PMID:24866798

  18. Neocosmospora sp.-derived resorcylic acid lactones with in vitro binding capacity for human opioid and cannabinoid receptors

    USDA-ARS?s Scientific Manuscript database

    Bioassay-guided fractionation of a fungus Neocosmospora sp. (UM-031509) resulted in the isolation of three new resorcyclic acid lactones, neocosmosin A (2), neocosmosin B (3) and neocosmosin C (4). Three known resorcylic acid lactones, monocillin IV (1), monocillin II (5) and monorden (6) were also ...

  19. Regioselective Acylation of Diols and Triols: The Cyanide Effect.

    PubMed

    Peng, Peng; Linseis, Michael; Winter, Rainer F; Schmidt, Richard R

    2016-05-11

    Central topics of carbohydrate chemistry embrace structural modifications of carbohydrates and oligosaccharide synthesis. Both require regioselectively protected building blocks that are mainly available via indirect multistep procedures. Hence, direct protection methods targeting a specific hydroxy group are demanded. Dual hydrogen bonding will eventually differentiate between differently positioned hydroxy groups. As cyanide is capable of various kinds of hydrogen bonding and as it is a quite strong sterically nondemanding base, regioselective O-acylations should be possible at low temperatures even at sterically congested positions, thus permitting formation and also isolation of the kinetic product. Indeed, 1,2-cis-diols, having an equatorial and an axial hydroxy group, benzoyl cyanide or acetyl cyanide as an acylating agent, and DMAP as a catalyst yield at -78 °C the thermodynamically unfavorable axial O-acylation product; acyl migration is not observed under these conditions. This phenomenon was substantiated with 3,4-O-unproteced galacto- and fucopyranosides and 2,3-O-unprotected mannopyranosides. Even for 3,4,6-O-unprotected galactopyranosides as triols, axial 4-O-acylation is appreciably faster than O-acylation of the primary 6-hydroxy group. The importance of hydrogen bonding for this unusual regioselectivity could be confirmed by NMR studies and DFT calculations, which indicate favorable hydrogen bonding of cyanide to the most acidic axial hydroxy group supported by hydrogen bonding of the equatorial hydroxy group to the axial oxygen. Thus, the "cyanide effect" is due to dual hydrogen bonding of the axial hydroxy group which enhances the nucleophilicity of the respective oxygen atom, permitting an even faster reaction for diols than for mono-ols. In contrast, fluoride as a counterion favors dual hydrogen bonding to both hydroxy groups leading to equatorial O-acylation.

  20. Use of Whole-Cell Bioassays for Screening Quorum Signaling, Quorum Interference, and Biofilm Dispersion.

    PubMed

    Thornhill, Starla G; McLean, Robert J C

    2018-01-01

    In most bacteria, a global level of regulation, termed quorum sensing (QS), exists involving intercellular communication via the production and response to cell density-dependent signal molecules. QS has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. N-acylated homoserine lactones (AHLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, commonly found in soil and water, produces the characteristic purple pigment violacein, regulated by AHL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of AHL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect AHL and QS inhibitors. Due to the inherent low susceptibility of biofilm bacteria to antimicrobial agents, biofilm dispersion, whereby bacteria reenter the planktonic community, is another increasingly important area of research. At least one signal, distinct from traditional QS, has been identified and there are a variety of other environmental factors that also trigger dispersion. We describe a microtiter-based experimental strategy whereby potential biofilm dispersion compounds can be screened.

  1. Inhibition of quorum sensing in Chromobacterium violaceum by Syzygium cumini L. and Pimenta dioica L.

    PubMed

    Vasavi, Halkare Suryanarayana; Arun, Ananthapadmanabha Bhagwath; Rekha, Punchapady Devasya

    2013-12-01

    To investigated into the anti-quorum sensing (QS) activity of Syzygium cumini L. (S. cumini) and Pimenta dioica L. (P. dioica) using Chromobacterium violaceum (C. violaceum) strains. In this study, anti-QS activity of ethanol extract of Syzygium cumini L. and Pimenta dioica L. were screened using C. violaceum CV026 biosensor bioassay. By bioassay guided fractionation of S. cumini and P. dioica, ethyl acetate fraction (EAF) with strong anti-QS activity was separated. Inhibition of QS regulated violacein production in C. violaceum ATCC12472 by EAF was assessed at different concentrations. The effect of EAF on the synthesis of autoinducer like N-acyl homoserine lactone (AHL) was studied in C. violaceum ATCC31532 using its mutant C. violaceum CV026 by standard methods. EAF inhibited violacein production in C. violaceum ATCC12472 in a concentration dependent manner without significant reduction in bacterial growth. Complete inhibition of violacein production was evidenced in 0.75-1.0 mg/mL concentration of EAF without inhibiting the synthesis of the AHL. TLC biosensor overlay profile of EAF revealed two translucent spots in S. cumini and P. dioica that inhibited C6-AHL mediated violacein production in C. violaceum CV026. This study indicates the anti-QS activity of the tested medicinal plants against C. violaceum. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

  2. Inhibition of quorum sensing in Chromobacterium violaceum by Syzygium cumini L. and Pimenta dioica L.

    PubMed Central

    Vasavi, Halkare Suryanarayana; Arun, Ananthapadmanabha Bhagwath; Rekha, Punchapady Devasya

    2013-01-01

    Objective To investigated into the anti-quorum sensing (QS) activity of Syzygium cumini L. (S. cumini) and Pimenta dioica L. (P. dioica) using Chromobacterium violaceum (C. violaceum) strains. Methods In this study, anti-QS activity of ethanol extract of Syzygium cumini L. and Pimenta dioica L. were screened using C. violaceum CV026 biosensor bioassay. By bioassay guided fractionation of S. cumini and P. dioica, ethyl acetate fraction (EAF) with strong anti-QS activity was separated. Inhibition of QS regulated violacein production in C. violaceum ATCC12472 by EAF was assessed at different concentrations. The effect of EAF on the synthesis of autoinducer like N-acyl homoserine lactone (AHL) was studied in C. violaceum ATCC31532 using its mutant C. violaceum CV026 by standard methods. Results EAF inhibited violacein production in C. violaceum ATCC12472 in a concentration dependent manner without significant reduction in bacterial growth. Complete inhibition of violacein production was evidenced in 0.75-1.0 mg/mL concentration of EAF without inhibiting the synthesis of the AHL. TLC biosensor overlay profile of EAF revealed two translucent spots in S. cumini and P. dioica that inhibited C6-AHL mediated violacein production in C. violaceum CV026. Conclusions This study indicates the anti-QS activity of the tested medicinal plants against C. violaceum. PMID:24093786

  3. Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications

    PubMed Central

    Rémy, Benjamin; Plener, Laure; Poirier, Laetitia; Elias, Mikael; Daudé, David; Chabrière, Eric

    2016-01-01

    Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures. PMID:27876889

  4. N-Acetylglucosamine Inhibits LuxR, LasR and CviR Based Quorum Sensing Regulated Gene Expression Levels

    PubMed Central

    Kimyon, Önder; Ulutürk, Zehra İ.; Nizalapur, Shashidhar; Lee, Matthew; Kutty, Samuel K.; Beckmann, Sabrina; Kumar, Naresh; Manefield, Mike

    2016-01-01

    N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50–80% reduction at low millimolar concentrations (0.25–5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities. PMID:27602027

  5. Bacterial communications in implant infections: a target for an intelligence war.

    PubMed

    Costerton, J W; Montanaro, L; Arciola, C R

    2007-09-01

    The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called "quorum-sensing". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

  6. Gas Replacements for GFP to Track Microbial Dynamics in Soils and Sediments

    NASA Astrophysics Data System (ADS)

    Cheng, Hsiao-Ying; Silberg, Jonathan; Masiello, Caroline

    2016-04-01

    Metagenomic analyses offer unprecedented views of soil microbial communities, and additionally provide a host of testable hypotheses about the biological mechanisms driving global biogeochemical fluxes. Outside the biogeosciences, hypotheses generated by metagenomics are often tested using biosensors, microbes programmed to respond in a detectable way to either changes in their metabolism or changes in the environment. A very large number of microbial behaviors can be monitored using biosensors, but these sensors typically report in ways that are undetectable in soils, e.g. by releasing green fluorescent protein (GFP). We are building a new class of biosensors that report by releasing easily-detected gases. We will provide an overview of the potential uses of gas-reporting biosensors in geobiology, and will report the current development these sensors. One goal in the development of these sensors is to make tractable the testing of gene expression hypotheses derived from metagenomics data. Examples of processes that could be tracked non-invasively with gas sensors include coordination of biofilm formation, nitrification, rhizobial infection of plant roots, and at least some forms of methanogenesis, all of which are managed by the easily-engineered acyl homoserine lactone cell-cell communication system. Another relatively simple process to track with gas sensors is horizontal gene transfer. We will report on the progress of these proof-of-concept examples.

  7. Indole-based novel small molecules for the modulation of bacterial signalling pathways.

    PubMed

    Biswas, Nripendra Nath; Kutty, Samuel K; Barraud, Nicolas; Iskander, George M; Griffith, Renate; Rice, Scott A; Willcox, Mark; Black, David StC; Kumar, Naresh

    2015-01-21

    Gram-negative bacteria such as Pseudomonas aeruginosa use N-acylated L-homoserine lactones (AHLs) as autoinducers (AIs) for quorum sensing (QS), a major regulatory and cell-to-cell communication system for social adaptation, virulence factor production, biofilm formation and antibiotic resistance. Some bacteria use indole moieties for intercellular signaling and as regulators of various bacterial phenotypes important for evading the innate host immune response and antimicrobial resistance. A range of natural and synthetic indole derivatives have been found to act as inhibitors of QS-dependent bacterial phenotypes, complementing the bactericidal ability of traditional antibiotics. In this work, various indole-based AHL mimics were designed and synthesized via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N'-dicyclohexylcarbodiimide (DCC) mediated coupling reactions of a variety of substituted or unsubstituted aminoindoles with different alkanoic acids. All synthesized compounds were tested for QS inhibition using a P. aeruginosa QS reporter strain by measuring the amount of green fluorescent protein (GFP) production. Docking studies were performed to examine their potential to bind and therefore inhibit the target QS receptor protein. The most potent compounds 11a, 11d and 16a showed 44 to 65% inhibition of QS activity at 250 μM concentration, and represent promising drug leads for the further development of anti-QS antimicrobial compounds.

  8. Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

    PubMed Central

    2015-01-01

    Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

  9. Natural synergism of acid and lactone type mixed sophorolipids in interfacial activities and cytotoxicities.

    PubMed

    Hirata, Yoshihiko; Ryu, Mizuyuki; Igarashi, Keisuke; Nagatsuka, Asami; Furuta, Taro; Kanaya, Shigenori; Sugiura, Masaki

    2009-01-01

    Sophorolipids (SLs) naturally produced from Candida bombicola are a mixture of lactonic (SL-lactone) and acidic (SL-acid) sophorosides of 17-L-hydroxydecanoic acid with an SL-lactone:SL-acid ratio of 72:28. SLs are biodegradable low-foaming surfactants with high detergency and hardness-tolerance properties. To analyze the effect of the SL-lactone:SL-acid ratio on these properties, SL-LXs containing X% SL-lactone, in which X varied from 0 to 100, were prepared and their interfacial activities and cytotoxicities examined. The minimum surface tension values for all SLs examined were comparable. The critical micelle concentration (CMC) was 680 mg/L for SL-L0 and 62-110 mg/L for the other SLs. Interestingly, natural SL (SL-L72) had the lowest surface tension and CMC among all of the SLs examined. The foaming ability and stability of the SLs were dependent on the SL-L content. SL-L0 and L17 had higher foaming values than the other SLs examined in 0-ppm hardness water. These values greatly reduced and became constant when the SL-L content increased over 55%. The detergencies of all of the SLs examined were comparable, except for those of SL-L0 and SL-L100, which were slightly lower than those of the other SLs. These results suggest that natural synergism between SLs creates a better balance for many interfacial activities. The cytotoxicity of SL-L72 was higher than that of SL-L0, but was comparable to that of surfactin, which is commercially available for cosmetic use. The low cytotoxicities and high interfacial properties of SLs increase their usefulness as biocompatible surface active agents for many applications.

  10. Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi

    PubMed Central

    Papademetrio, Daniela; Batlle, Alcira; Martino, Virginia S.; Frank, Fernanda M.; Lombardo, María E.

    2016-01-01

    Trypanosoma cruzi is the causative agent of Chagas’ disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin accomplished its

  11. Divergent synthesis and chemical reactivity of bicyclic lactone fragments of complex rearranged spongian diterpenes.

    PubMed

    Schnermann, Martin J; Beaudry, Christopher M; Genung, Nathan E; Canham, Stephen M; Untiedt, Nicholas L; Karanikolas, Breanne D W; Sütterlin, Christine; Overman, Larry E

    2011-11-02

    The synthesis and direct comparison of the chemical reactivity of the two highly oxidized bicyclic lactone fragments found in rearranged spongian diterpenes (8-substituted 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one and 6-substituted 7-acetoxy-2,8-dioxabicyclo[3.3.0]octan-3-one) are reported. Details of the first synthesis of the 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one ring system, including an examination of several possibilities for the key bridging cyclization reaction, are described. In addition, the first synthesis of 7-acetoxy-2,8-dioxabicyclo[3.3.0]octanones containing quaternary carbon substituents at C6 is disclosed. Aspects of the chemical reactivity and Golgi-modifying properties of these bicyclic lactone analogs of rearranged spongian diterpenes are also reported. Under both acidic and basic conditions, 8-substituted 2,7-dioxabicyclo[3.2.1]octanones are converted to 6-substituted-2,8-dioxabicyclo[3.3.0]octanones. Moreover, these dioxabicyclic lactones react with primary amines and lysine side chains of lysozyme to form substituted pyrroles, a conjugation that could be responsible for the unique biological properties of these compounds. These studies demonstrate that acetoxylation adjacent to the lactone carbonyl group, in either the bridged or fused series, is required to produce fragmented Golgi membranes in the pericentriolar region that is characteristic of macfarlandin E.

  12. New acylated clionasterol glycosides from Valeriana officinalis.

    PubMed

    Pullela, Srinivas V; Choi, Young Whan; Khan, Shabana I; Khan, Ikhlas A

    2005-10-01

    The chloroform extract of Valeriana officinalis led to the isolation of clionasterol-3-O-beta-D-glucopyranoside and a mixture of 6'-O-acyl-beta-D-glucosyl-clionasterols. The acyl moieties were identified as hexadecanoyl, 8 E,11 E-octadecadienoyl and 14-methylpentadecanoyl by alkaline hydrolysis followed by GC-MS analysis. The isolated compounds did not exhibit any anti-inflammatory, anticancer or cytotoxic activity when tested in a variety of in vitro cell-based assays.

  13. Triacetic acid lactone production in industrial Saccharomyces yeast strains

    USDA-ARS?s Scientific Manuscript database

    Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into thirteen industrial yeast strains of varied genetic background. TAL produ...

  14. Suzuki-miyaura cross-coupling in acylation reactions, scope and recent developments.

    PubMed

    Blangetti, Marco; Rosso, Heléna; Prandi, Cristina; Deagostino, Annamaria; Venturello, Paolo

    2013-01-17

    Since the first report and due to its handiness and wide scope, the Suzuki-Miyaura (SM) cross coupling reaction has become a routine methodology in many laboratories worldwide. With respect to other common transition metal catalyzed cross couplings, the SM reaction has been so far less exploited as a tool to introduce an acyl function into a specific substrate. In this review, the various approaches found in the literature will be considered, starting from the direct SM acylative coupling to the recent developments of cross coupling between boronates and acyl chlorides or anhydrides. Special attention will be dedicated to the use of masked acyl boronates, alkoxy styryl and alkoxy dienyl boronates as coupling partners. A final section will be then focused on the acyl SM reaction as key synthetic step in the framework of natural products synthesis.

  15. Quantitative analysis of sesquiterpene lactones in extract of Arnica montana L. by 1H NMR spectroscopy.

    PubMed

    Staneva, Jordanka; Denkova, Pavletta; Todorova, Milka; Evstatieva, Ljuba

    2011-01-05

    (1)H NMR spectroscopy was used as a method for quantitative analysis of sesquiterpene lactones present in a crude lactone fraction isolated from Arnica montana. Eight main components - tigloyl-, methacryloyl-, isobutyryl- and 2-methylbutyryl-esters of helenalin (H) and 11α,13-dihydrohelenalin (DH) were identified in the studied sample. The method allows the determination of the total amount of sesquiterpene lactones and the quantity of both type helenalin and 11α,13-dihydrohelenalin esters separately. Furthermore, 6-O-tigloylhelenalin (HT, 1), 6-O-methacryloylhelenalin (HM, 2), 6-O-tigloyl-11α,13-dihydrohelenalin (DHT, 5), and 6-O-methacryloyl-11α,13-dihydrohelenalin (DHM, 6) were quantified as individual components. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. [Preparation of hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata and study on its in vitro release mechanism].

    PubMed

    Xu, Fang-Fang; Shi, Wei; Zhang, Hui; Guo, Qing-Ming; Wang Zhen-Zhong; Bi, Yu-An; Wang, Zhi-Min; Xiao, Wei

    2015-01-01

    In this study, hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata were prepared and the in vitro release behavior were also evaluated. The optimal prescription was achieved by studying the main factor of the type and amount of hydroxypropyl methylcellulose (HPMC) using single factor test and evaluating through cumulative release of three lactones. No burst drug release from the obtained matrix tablets was observed. Drug release sustained to 14 h. The release mechanism of three lactones from A. paniculata was accessed by zero-order, first-order, Higuchi and Peppas equation. The release behavior of total lactones from A. paniculata was better agreed with Higuchi model and the drug release from the tablets was controlled by degradation of the matrix. The preparation of hydrophilic matrix sustained release tablets of total lactones from A. paniculata with good performance of drug release was simple.

  17. Two lactones in the androconial scent of the lycaenid butterfly Celastrina argiolus ladonides

    NASA Astrophysics Data System (ADS)

    Ômura, Hisashi; Yakumaru, Kazuhisa; Honda, Keiichi; Itoh, Takao

    2013-04-01

    Male adult butterflies of many species have characteristic odors originating from the disseminating organs known as androconia. Despite the fact that androconia exist in several species, there have been few investigations on adult scents from the lycaenid species. Celastrina argiolus ladonides (Lycaenidae) is a common species in Eurasia. We have reported that male adults of this species emit a faint odor, and the major components causing this odor have been newly found in the Insecta. By using field-caught individuals, we determined the chemical nature and location of this odor in the butterfly. Gas chromatography-mass spectrometry (GC-MS) analyses revealed that two lactone compounds, lavender lactone and δ-decalactone, are present in the extracts of males but absent in those of the females. On an average, approximately 50 ng of each compound was found per male. Chiral GC analyses performed using enantiomerically pure standards revealed that the natural lavender lactone was a mixture of two enantiomers with an R/ S ratio of 32:68, whereas the natural δ-decalactone contained only the R-enantiomer. When the analyses were conducted using different parts—forewings, hindwings, and body—of three males, the lactones were more abundantly found on the forewings and hindwings than on the body. Microscopic observation of the wings demonstrated that battledore scales known as androconia are scattered on the upper surface of both the wings of C. argiolus ladonides males. These results indicate that the specialized scales on the wings of males serve as scent-disseminating organs.

  18. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  19. Fatty acyl-CoA reductases of birds

    PubMed Central

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  20. The activity of Rhizomuchor miehei lipase as a biocatalyst in enzymatic acylation of cyclic alcohol

    NASA Astrophysics Data System (ADS)

    Iftitah, Elvina Dhiaul; Srihardyastuti, Arie; Ariefin, Mokhamat

    2017-03-01

    We report the activity of Rhizomuchor miehei lipase (RML) as a biocatalyst, in particular the investigations concerning the effort of substrate-structure reactivity on the enzymatic acylation. The acylation was studied using acetic anhydride as an acyl donor and performed in n-hexane as a solvent. The selectivity of the enzymatic acylation was revealed by Gas Chromatography-Mass Spectra. We observed that, RML has shown different behavior when catalyzing the acylation of isopulegol and mixture of isopulegol and citronellal (ratio 1:1). The chemoselectivity for the O-acylation was improved when the acyl acceptor included mixture of isopulegol and citronellal

  1. AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

    PubMed

    González, Alex; Bellenberg, Sören; Mamani, Sigde; Ruiz, Lina; Echeverría, Alex; Soulère, Laurent; Doutheau, Alain; Demergasso, Cecilia; Sand, Wolfgang; Queneau, Yves; Vera, Mario; Guiliani, Nicolas

    2013-04-01

    Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

  2. Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43.

    PubMed

    Tomatis, Vanesa M; Trenchi, Alejandra; Gomez, Guillermo A; Daniotti, Jose L

    2010-11-30

    An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

  3. Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

    PubMed

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

    2016-08-01

    The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

  4. Acyl Meldrum's acid derivatives: application in organic synthesis

    NASA Astrophysics Data System (ADS)

    Janikowska, K.; Rachoń, J.; Makowiec, S.

    2014-07-01

    This review is focused on an important class of Meldrum's acid derivatives commonly known as acyl Meldrum's acids. The preparation methods of these compounds are considered including the recently proposed and rather rarely used ones. The chemical properties of acyl Meldrum's acids are described in detail, including thermal stability and reactions with various nucleophiles. The possible mechanisms of these transformations are analyzed. The bibliography includes 134 references.

  5. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  6. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans.

    PubMed

    Tuck, Laura R; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D; Campopiano, Dominic J; Clarke, David J; Marles-Wright, Jon

    2016-02-22

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes.

  7. Maintenance of Paraoxonase 2 Activity as a Strategy to Attenuate P. Aeruginosa Virulence

    DTIC Science & Technology

    2015-12-01

    oxododecanoyl)-L-homoserine lactone (3OC12) is a key Pa QS signal that has been shown to be necessary for biofilm maturation and full expression of...virulence in Pa animal infection models (5- 8). Concentrations up to 600 µM 3OC12 have been measured in Pa biofilms in vitro (9). Concentrations of over...3OC12 from 1-10 µM have been measured in planktonic cultures, concentrations of ~ 600 µM have been measured in Pa biofilms grown in vitro (9

  8. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    DOE PAGES

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

  9. Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

    Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

  10. Method to produce water-soluble sugars from biomass using solvents containing lactones

    DOEpatents

    Dumesic, James A.; Luterbacher, Jeremy S.

    2015-06-02

    A process to produce an aqueous solution of carbohydrates that contains C6-sugar-containing oligomers, C6 sugar monomers, C5-sugar-containing oligomers, C5 sugar monomers, or any combination thereof is presented. The process includes the steps of reacting biomass or a biomass-derived reactant with a solvent system including a lactone and water, and an acid catalyst. The reaction yields a product mixture containing water-soluble C6-sugar-containing oligomers, C6-sugar monomers, C5-sugar-containing oligomers, C5-sugar monomers, or any combination thereof. A solute is added to the product mixture to cause partitioning of the product mixture into an aqueous layer containing the carbohydrates and a substantially immiscible organic layer containing the lactone.

  11. Method to produce water-soluble sugars from biomass using solvents containing lactones

    DOEpatents

    Dumesic, James A.; Luterbacher, Jeremy S.

    2017-08-08

    A process to produce an aqueous solution of carbohydrates that contains C6-sugar-containing oligomers, C6 sugar monomers, C5-sugar-containing oligomers, C5 sugar monomers, or any combination thereof is presented. The process includes the steps of reacting biomass or a biomass-derived reactant with a solvent system including a lactone and water, and an acid catalyst. The reaction yields a product mixture containing water-soluble C6-sugar-containing oligomers, C6-sugar monomers, C5-sugar-containing oligomers, C5-sugar monomers, or any combination thereof. A solute is added to the product mixture to cause partitioning of the product mixture into an aqueous layer containing the carbohydrates and a substantially immiscible organic layer containing the lactone.

  12. A validated Fourier transform infrared spectroscopy method for quantification of total lactones in Inula racemosa and Andrographis paniculata.

    PubMed

    Shivali, Garg; Praful, Lahorkar; Vijay, Gadgil

    2012-01-01

    Fourier transform infrared (FT-IR) spectroscopy is a technique widely used for detection and quantification of various chemical moieties. This paper describes the use of the FT-IR spectroscopy technique for the quantification of total lactones present in Inula racemosa and Andrographis paniculata. To validate the FT-IR spectroscopy method for quantification of total lactones in I. racemosa and A. paniculata. Dried and powdered I. racemosa roots and A. paniculata plant were extracted with ethanol and dried to remove ethanol completely. The ethanol extract was analysed in a KBr pellet by FT-IR spectroscopy. The FT-IR spectroscopy method was validated and compared with a known spectrophotometric method for quantification of lactones in A. paniculata. By FT-IR spectroscopy, the amount of total lactones was found to be 2.12 ± 0.47% (n = 3) in I. racemosa and 8.65 ± 0.51% (n = 3) in A. paniculata. The method showed comparable results with a known spectrophotometric method used for quantification of such lactones: 8.42 ± 0.36% (n = 3) in A. paniculata. Limits of detection and quantification for isoallantolactone were 1 µg and 10 µg respectively; for andrographolide they were 1.5 µg and 15 µg respectively. Recoveries were over 98%, with good intra- and interday repeatability: RSD ≤ 2%. The FT-IR spectroscopy method proved linear, accurate, precise and specific, with low limits of detection and quantification, for estimation of total lactones, and is less tedious than the UV spectrophotometric method for the compounds tested. This validated FT-IR spectroscopy method is readily applicable for the quality control of I. racemosa and A. paniculata. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

    PubMed

    Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

    2013-01-01

    Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

  14. Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

    PubMed Central

    Tizzano, Marco; Gulbransen, Brian D.; Vandenbeuch, Aurelie; Clapp, Tod R.; Herman, Jake P.; Sibhatu, Hiruy M.; Churchill, Mair E. A.; Silver, Wayne L.; Kinnamon, Sue C.; Finger, Thomas E.

    2010-01-01

    The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms. PMID:20133764

  15. Encapsulation of Autoinducer Sensing Reporter Bacteria in Reinforced Alginate-Based Microbeads.

    PubMed

    Li, Ping; Müller, Mareike; Chang, Matthew Wook; Frettlöh, Martin; Schönherr, Holger

    2017-07-12

    Quorum sensing, in which bacteria communities use signaling molecules for inter- and intracellular communication, has been intensively studied in recent decades. In order to fabricate highly sensitive easy-to-handle point of care biosensors that detect quorum sensing molecules, we have developed, as is reported here, reporter bacteria loaded alginate-methacrylate (alginate-MA) hydrogel beads. The alginate-MA beads, which were obtained by electrostatic extrusion, were reinforced by photo-cross-linking to increase stability and thereby to reduce bacteria leaching. In these beads the genetically engineered fluorescent reporter bacterium Escherichia coli pTetR-LasR-pLuxR-GFP (E. coli pLuxR-GFP) was encapsulated, which responds to the autoinducer N-(3-oxododecanoyl)homoserine lactone secreted by Pseudomonas aeruginosa. After encapsulation in alginate-MA hydrogel beads with diameters in the range of 100-300 μm that were produced by an electrostatic extrusion method and rapid photo-cross-linking, the E. coli pLuxR-GFP were found to possess a high degree of viability and sensing activity. The encapsulated bacteria could proliferate inside the hydrogel beads, when exposed to bacteria culture medium. In media containing the autoinducer N-(3-oxododecanoyl)homoserine lactone, the encapsulated reporter bacteria responded with a strong fluorescence signal due to an increased green fluorescent protein (GFP) expression. A prototype dipstick type sensor developed here underlines the potential of encapsulation of viable and functional reporter bacteria inside reinforced alginate-methacrylate hydrogel beads for whole cell sensors for bacteria detection.

  16. Sesquiterpene lactones from Gynoxys verrucosa and their anti-MRSA activity.

    PubMed

    Ordóñez, Paola E; Quave, Cassandra L; Reynolds, William F; Varughese, Kottayil I; Berry, Brian; Breen, Philip J; Malagón, Omar; Smeltzer, Mark S; Compadre, Cesar M

    2011-09-02

    Because of its virulence and antibiotic resistance, Staphylococcus aureus is a more formidable pathogen now than at any time since the pre-antibiotic era. In an effort to identify and develop novel antimicrobial agents with activity against this pathogen, we have examined Gynoxys verrucosa Wedd (Asteraceae), an herb used in traditional medicine in southern Ecuador for the treatment and healing of wounds. The sesquiterpene lactones leucodine (1) and dehydroleucodine (2) were extracted and purified from the aerial parts of Gynoxys verrucosa, and their structure was elucidated by spectroscopic methods and single-crystal X-ray analysis. The in vitro anti-microbial activity of Gynoxys verrucosa extracts and its purified constituents was determined against six clinical isolates including Staphylococcus aureus and Staphylococcus epidermidis strains with different drug-resistance profiles, using the microtiter broth method. Compound 1 has very low activity, while compound 2 has moderate activity with MIC(50)s between 49 and 195 μg/mL. The extract of Gynoxys verrucosa has weak activity with MIC(50)s between 908 and 3290 μg/mL. We are reporting the full assignment of the (1)H NMR and (13)C NMR of both compounds, and the crystal structure of compound 2, for the first time. Moreover, the fact that compound 2 has antimicrobial activity and compound 1 does not, demonstrates that the exocyclic conjugated methylene in the lactone ring is essential for the antimicrobial activity of these sesquiterpene lactones. However, the weak activity observed for the plant extracts, does not explain the use of Gynoxys verrucosa in traditional medicine for the treatment of wounds and skin infections. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. Sesquiterpene Lactones from Gynoxys verrucosa and their Anti-MRSA Activity

    PubMed Central

    Ordóñez, Paola E.; Quave, Cassandra L.; Reynolds, William F.; Varughese, Kottayil I.; Berry, Brian; Breen, Philip J.; Malagón, Omar; Smeltzer, Mark S.; Compadre, Cesar M.

    2011-01-01

    Ethnopharmacological relevance Because of its virulence and antibiotic resistance, Staphylococcus aureus is a more formidable pathogen now than at any time since the pre-antibiotic era. In an effort to identify and develop novel antimicrobial agents with activity against this pathogen, we have examined Gynoxys verrucosa Wedd (Asteraceae), an herb used in traditional medicine in southern Ecuador for the treatment and healing of wounds. Materials and Methods The sesquiterpene lactones leucodine (1) and dehydroleucodine (2) were extracted and purified from the aerial parts of G. verrucosa, and their structure was elucidated by spectroscopic methods and single-crystal X-ray analysis. The in vitro anti-microbial activity of G. verrucosa extracts and its purified constituents was determined against six clinical isolates including S. aureus and Staphylococcus epidermidis strains with different drug-resistance profiles, using the microtiter broth method. Results Compound 1 has very low activity, while compound 2 has moderate activity with MIC50s between 49 and195 μg/mL. The extract of G. verrucosa has weak activity with MIC50s between 908 and 3290 μg/mL. Conclusions We are reporting the full assignment of the 1H-NMR and 13C-NMR of both compounds, and the crystal structure of compound 2, for the first time. Moreover, the fact that compound 2 has antimicrobial activity and compound 1 does not, demonstrates that the exocyclic conjugated methylene in the lactone ring is essential for the antimicrobial activity of these sesquiterpene lactones. However, the weak activity observed for the plants extracts, does not explain the use of G. verrucosa in traditional medicine for the treatment of wounds and skin infections. PMID:21782013

  18. Head-group acylation of monogalactosyldiacylglycerol is a common stress response, but the acyl-galactose acyl composition varies with the plant species and applied stress

    USDA-ARS?s Scientific Manuscript database

    Head group acylation of monogalactosyldiacylglycerol is a plant lipid modification occurring during bacterial infection. Little is known about the range of stresses that induce this lipid modification, the molecular species induced, and the function of the modification. Lipidomic analysis using trip...

  19. Acyl transfer from membrane lipids to peptides is a generic process.

    PubMed

    Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

    2013-11-15

    The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

  20. Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

    PubMed

    Fujiwara, Yuko; Hama, Kotaro; Tsukahara, Makoto; Izumi-Tsuzuki, Ryosuke; Nagai, Toru; Ohe-Yamada, Mihoko; Inoue, Keizo; Yokoyama, Kazuaki

    2018-01-01

    Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [ 14 C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [ 14 C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [ 14 C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

  1. Structure determination of 3-O-caffeoyl-epi-gamma-quinide, an orphan bitter lactone in roasted coffee.

    PubMed

    Frank, Oliver; Blumberg, Simone; Krümpel, Gudrun; Hofmann, Thomas

    2008-10-22

    Recent investigations on the bitterness of coffee as well as 5- O-caffeoyl quinic acid roasting mixtures indicated the existence of another, yet unknown, bitter lactone besides the previously identified bitter compounds 5- O-caffeoyl- muco-gamma-quinide, 3- O-caffeoyl-gamma-quinide, 4- O-caffeoyl- muco-gamma-quinide, 5- O-caffeoyl- epi-delta-quinide, and 4- O-caffeoyl-gamma-quinide. In the present study, this orphan bitter lactone was isolated from the reaction products generated by dry heating of 5- O-caffeoylquinic acid model, and its structure was determined as the previously unreported 3- O-caffeoyl- epi-gamma-quinide by means of liquid chromatography-mass spectrometry (LC-MS) and one-/two-dimensional NMR experiments. The occurrence of this bitter lactone, exhibiting a low bitter recognition threshold of 58 micromol/L, in coffee beverages could be confirmed by LC-MS/MS (negative electrospray ionization) operating in the multiple reaction monitoring mode.

  2. Structural Basis for Substrate Fatty Acyl Chain Specificity

    PubMed Central

    McAndrew, Ryan P.; Wang, Yudong; Mohsen, Al-Walid; He, Miao; Vockley, Jerry; Kim, Jung-Ja P.

    2008-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ∼400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an α-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial β-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. PMID:18227065

  3. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

    PubMed

    Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

    2011-08-10

    Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

  4. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  5. Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

    PubMed

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

    2017-04-01

    Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

  6. The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

    PubMed

    Marcella, Aaron M; Barb, Adam W

    2017-12-01

    The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

  7. Total and acylated ghrelin in liver cirrhosis: correlation with clinical and nutritional status.

    PubMed

    El-Shehaby, Amal M; Obaia, Eman M; Alwakil, Sahar S; Hiekal, Ahmed A

    2010-07-01

    The pathogenesis of anorexia in cirrhotic patients is complex and the appetite-modulating hormone ghrelin could be involved. Acylated ghrelin is the biologically active form that modifies insulin sensitivity and body composition. The aim of the present study was to compare acylated and total ghrelin concentration in patients with liver cirrhosis and to investigate the possible relationship between ghrelin and clinical and nutritional parameters. Sixty patients with viral liver cirrhosis who did not have hepatocellular carcinoma or acute infections were studied. Twenty healthy volunteers were recruited after matching for age, gender, and body mass index with the patients and served as controls. Fasting levels of total, acylated ghrelin, leptin, TNF-alpha and insulin were measured in all subjects, in addition, clinical and nutrition parameters were assessed. In cirrhotic patients, plasma levels of both acylated and total ghrelin were significantly higher than those in the controls. The mean plasma acylated ghrelin levels were significantly higher in Child C cirrhosis compared to Child A and B. Ghrelin (total and acylated) were negatively correlated with leptin in cirrhotic patients confirming the fact that leptin acts as a physiological counterpart of ghrelin. Nutritional and metabolic abnormalities in cirrhotic patients may be dependent on the changes in the ghrelin/leptin systems, mainly the acylated form of ghrelin.

  8. Chlorogenic acids and the acyl-quinic acids: discovery, biosynthesis, bioavailability and bioactivity.

    PubMed

    Clifford, Michael N; Jaganath, Indu B; Ludwig, Iziar A; Crozier, Alan

    2017-12-13

    Covering: 2000 up to late 2017This review is focussed upon the acyl-quinic acids, the most studied group within the ca. 400 chlorogenic acids so far reported. The acyl-quinic acids, the first of which was characterised in 1846, are a diverse group of plant-derived compounds produced principally through esterification of an hydroxycinnamic acid and 1l-(-)-quinic acid. Topics addressed in this review include the confusing nomenclature, quantification and characterisation by NMR and MS, biosynthesis and role in planta, and the occurrence of acyl-quinic acids in coffee, their transformation during roasting and delivery to the beverage. Coffee is the major human dietary source world-wide of acyl-quinic acids and consideration is given to their absorption and metabolism in the upper gastrointestinal tract, and the colon where the microbiota play a key role in the formation of catabolites. Evidence on the potential of the in vivo metabolites and catabolites of acyl-quinic acids to promote the consumer's health is evaluated.

  9. Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives.

    PubMed

    Bi, Huiping; Bai, Yanfen; Cai, Tao; Zhuang, Yibin; Liang, Xiaomei; Zhang, Xueli; Liu, Tao; Ma, Yanhe

    2013-12-01

    Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96 ± 0.06 mg/L chloramphenicol-3-isobutyrate and 3.94 ± 0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77 ± 3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.

  10. Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides.

    PubMed

    Ziaullah; Rupasinghe, H P Vasantha

    2016-04-01

    This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-2'-glucoside, using Candida antarctica lipase B (Novozyme 435(®)), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4-5 folds. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans

    PubMed Central

    Sidani, Reem M.; Garcia, Anna E.; Antoun, Joseph; Isbell, James M.; Abumrad, Naji N.

    2016-01-01

    Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (iv) infusions of glucose. These “isoglycemic clamps” enabled us to determine the contribution of the GI tract and postabsorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared with gastric glucose administration (P < 0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P ≤ 0.01). However, gastric and isoglycemic iv glucose infusions resulted in similar degrees of acyl ghrelin suppression (P > 0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion. PMID:27279247

  12. SELECTIVE HYDROGENATION OF ANHYDRIDES TO LACTONES UNDER SUPERCRITICAL CARBON DIOXIDE MEDIUM

    EPA Science Inventory

    Selective Hydrogenation of Anhydrides to Lactones Under Supercritical Carbon Dioxide Medium

    Endalkachew Sahle-Demessie Unnikrishnan R Pillai
    U.S. EPA , 26 W. Martin Luther King Dr. Cincinnati, OH 45268 Phone: 513-569-7739
    Fax: 513-569-7677
    Abstract:
    Hydrogenat...

  13. Antiquorum sensing, antibiofilm formation and cytotoxicity activity of commonly used medicinal plants by inhabitants of Borabu sub-county, Nyamira County, Kenya

    PubMed Central

    Omwenga, Eric Omori; Hensel, Andreas; Pereira, Susana; Shitandi, Alfred Anakalo

    2017-01-01

    Productions of various bacterial traits like production of virulence factors (e.g. toxins, enzymes), biofilm formation, luminescence among others, have been known to be controlled by quorum sensing (QS), a process that is dependent on chemical signals or autoinducers (AIs). Bacteria known to rely on such AIs are known to be virulent and tend to be resistant against various antimicrobial agents. Therefore, strategies aimed at the inhibition of QS pathways, are regarded as potential novel therapies in managing bacterial virulence hence reducing their ability to induce infections in humans. In the present study, a portfolio of 25 medicinal plant extracts (ethanol 50% v/v) used in southwestern Kenya were assayed against a transformed E. coli Top 10 reporter QS strain. This biosensor responds to the exogenous addition of 3-oxo-N-hexanoyl homoserine lactone (3OC6HSL) expressing green fluorescent protein (GFP). The large majority of the screened medicinal plants seemed to exhibit toxic effects and almost none of them induced antiquorum sensing (AQS) activity. This could be the consequence of the presence of mixed compounds in the extracts. Elaeodendron buchananii Loes and Acacia gerrardii Benth extracts that seemed to show AQS activity were further proved found to possess mild AQS but with defined antimicrobial activities, and no antibiofilm formation inhibition. As a control, an E. coli pBCA9145_jtk2828::sfGFP strain that produces constitutively GFP was used and confirmed that none of the two extracts quenched the fluorescence of sfGFP. Cytotoxicity assays with mammalian MDCK cells also did indicate that the selected extracts with putative AQS activity, also reduced the cell viability. Therefore, further studies will be needed to separate and re-test the individual compounds especially from the selected two promising plants. PMID:29091715

  14. Quorum Sensing in a Methane-Oxidizing Bacterium.

    PubMed

    Puri, Aaron W; Schaefer, Amy L; Fu, Yanfen; Beck, David A C; Greenberg, E Peter; Lidstrom, Mary E

    2017-03-01

    Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum , a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N -3-hydroxydecanoyl-l-homoserine lactone (3-OH-C 10 -HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level. Copyright © 2017 American Society for

  15. Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

    NASA Astrophysics Data System (ADS)

    Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

    2017-06-01

    Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

  16. Two new diterpenoid lactones isolated from Andrographis paniculata.

    PubMed

    Wang, Gui-Yang; Wen, Ting; Liu, Fei-Fei; Tian, Hai-Yan; Chun-Lin, Fan; Huang, Xiao-Jun; Ye, Wen-Cai; Wang, Ying

    2017-06-01

    In the present study, two new diterpenoid lactones, 3-deoxy-andrographoside (1) and 14-deoxy-15-methoxy-andrographolide (2), were isolated from the aerial parts of Andrographis paniculata. Their structures were elucidated by combination of NMR, MS, and chemical methods. The configurations of 1 and 2 were established based on the analysis of ROESY data and single crystal X-ray diffraction experiment. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  17. Acyl hydrazides as acyl donors for the synthesis of diaryl and aryl alkyl ketones.

    PubMed

    Akhbar, Ahmed R; Chudasama, Vijay; Fitzmaurice, Richard J; Powell, Lyn; Caddick, Stephen

    2014-01-21

    In this communication we describe a novel strategy for the formation of valuable diaryl and aryl alkyl ketones from acyl hydrazides. A wide variety of ketones are prepared and the mild reaction conditions allow for the use of a range of functionalities, especially in the synthesis of diaryl ketones.

  18. [Determination of terpene lactones in Ginkgo biloba leaves in different ages by UPLC-TQ-MS].

    PubMed

    Yao, Xin; Zhou, Gui-Sheng; Tang, Yu-Ping; Qian, Ye-Fei; Shang, Er-Xin; Su, Shu-Lan; Qian, Da-Wei; Duan, Jin-Ao

    2013-02-01

    To establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass (UPLC-TQ-MS) for determination of four terpene lactones. Chromatographic separation was carried out on a ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with isocratic elution of 70% methanol at a flow rate of 0.4 mL x min(-1), the column temperature was set at 30 degrees C; Waters Xevo TQ worked in multiple reaction monitoring mode. All calibration curves were linear (r > 0.990 3) over the tested ranges. The average recoveries ranged from 98.83% to 103.9% with RSD value below 3.0%. The contents of total terpene lactones in Ginkgo biloba leaves were significantly different in different ages. The contents in the leaves of young ginkgo tree were higher than that in old tree. The method was simple and fast with high precision, sensitivity and repeatability, which can be used for qualitative and quantitative analysis of terpene lactones in G. biloba leaves.

  19. Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate.

    PubMed

    Karlen, Steven D; Free, Heather C A; Padmakshan, Dharshana; Smith, Bronwen G; Ralph, John; Harris, Philip J

    2018-06-01

    Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p -coumarate. The Poaceae, or grass family, is a member of this group, and most of the p -coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p -coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. © 2018 American Society of Plant Biologists. All rights reserved.

  20. SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

    PubMed Central

    Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

    2017-01-01

    Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

  1. Identification of N-acyl-fumonisin B1 as new cytotoxic metabolites of fumonisin mycotoxins.

    PubMed

    Harrer, Henning; Laviad, Elad L; Humpf, Hans Ulrich; Futerman, Anthony H

    2013-03-01

    Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. LOCATION OF ACYL GROUPS ON TWO PARTLY ACYLATED GLYCOLIPIDS FROM STRAINS OF USTILAGO (SMUT FUNGI),

    DTIC Science & Technology

    erythritol from Ustilago sp. (probably U. nuda (Jens.) Rostr. = U. tritici (Pers.) Rostr.) PRL-627 were acetalated with methyl vinyl ether, deacylated...Partly acylated ustilagic acids 8 (from Ustilago maydis (DC) Corda (= U. zeae Unger) PRL-119), consisting of partially esterified beta-cellobiosyl

  3. Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition in Bufo arenarum oocytes.

    PubMed

    Sánchez Toranzo, G; López, L A; Martínez, J Zapata; Bühler, M C Gramajo; Bühler, M I

    2010-02-01

    Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring. It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown. The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL). The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL. Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed. Our

  4. Acylation-dependent protein export in Leishmania.

    PubMed

    Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

    2000-04-14

    The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

  5. Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

    PubMed Central

    Austin, Caitlin M.; Stoy, William; Su, Peter; Harber, Marie C.; Bardill, J. Patrick; Hammer, Brian K.; Forest, Craig R.

    2014-01-01

    Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices. PMID:25379076

  6. Label-free molecular imaging of bacterial communities of the opportunistic pathogen Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Baig, Nameera; Polisetti, Sneha; Morales-Soto, Nydia; Dunham, Sage J. B.; Sweedler, Jonathan V.; Shrout, Joshua D.; Bohn, Paul W.

    2016-09-01

    Biofilms, such as those formed by the opportunistic human pathogen Pseudomonas aeruginosa are complex, matrix enclosed, and surface-associated communities of cells. Bacteria that are part of a biofilm community are much more resistant to antibiotics and the host immune response than their free-floating counterparts. P. aeruginosa biofilms are associated with persistent and chronic infections in diseases such as cystic fibrosis and HIV-AIDS. P. aeruginosa synthesizes and secretes signaling molecules such as the Pseudomonas quinolone signal (PQS) which are implicated in quorum sensing (QS), where bacteria regulate gene expression based on population density. Processes such as biofilms formation and virulence are regulated by QS. This manuscript describes the powerful molecular imaging capabilities of confocal Raman microscopy (CRM) and surface enhanced Raman spectroscopy (SERS) in conjunction with multivariate statistical tools such as principal component analysis (PCA) for studying the spatiotemporal distribution of signaling molecules, secondary metabolites and virulence factors in biofilm communities of P. aeruginosa. Our observations reveal that the laboratory strain PAO1C synthesizes and secretes 2-alkyl-4-hydroxyquinoline N-oxides and 2-alkyl-4-hydroxyquinolones in high abundance, while the isogenic acyl homoserine lactone QS-deficient mutant (ΔlasIΔrhlI) strain produces predominantly 2-alkyl-quinolones during biofilm formation. This study underscores the use of CRM, along with traditional biological tools such as genetics, for studying the behavior of microbial communities at the molecular level.

  7. Effect of Quorum Quenching Lactonase in Clinical Isolates of Pseudomonas aeruginosa and Comparison with Quorum Sensing Inhibitors

    PubMed Central

    Guendouze, Assia; Plener, Laure; Bzdrenga, Janek; Jacquet, Pauline; Rémy, Benjamin; Elias, Mikael; Lavigne, Jean-Philippe; Daudé, David; Chabrière, Eric

    2017-01-01

    Pseudomonas aeruginosa is a Gram negative pathogenic bacterium involved in many human infections including otitis, keratitis, pneumonia, and diabetic foot ulcers. P. aeruginosa uses a communication system, referred to as quorum sensing (QS), to adopt a group behavior by synchronizing the expression of certain genes. Among the regulated traits, secretion of proteases or siderophores, motility and biofilm formation are mainly involved in the pathogenicity. Many efforts have been dedicated to the development of quorum sensing inhibitors (QSI) and quorum quenching (QQ) agents to disrupt QS. QQ enzymes have been particularly considered as they may act in a catalytic way without entering the cell. Here we focus on the lactonase SsoPox which was previously investigated for its ability to degrade the signaling molecules, acyl-homoserine lactones, in particular on the engineered variant SsoPox-W263I. We highlight the potential of SsoPox-W263I to inhibit the virulence of 51 clinical P. aeruginosa isolates from diabetic foot ulcers by decreasing the secretion of two virulence factors, proteases and pyocyanin, as well as biofilm formation. We further compared the effect of SsoPox-W263I to the comprehensively described QSI, 5-fluorouracil and C-30. We found the lactonase SsoPox-W263I to be significantly more effective than the tested QSI at their respective concentration optimum and to retain its activity after immobilization steps, paving the way for future therapeutic applications. PMID:28261183

  8. Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference

    PubMed Central

    Dong, Yi-Hu; Zhang, Xi-Fen; Xu, Jin-Ling; Zhang, Lian-Hui

    2004-01-01

    It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections. PMID:14766576

  9. Quorum Sensing Signaling and Quenching in the Multidrug-Resistant Pathogen Stenotrophomonas maltophilia

    PubMed Central

    Huedo, Pol; Coves, Xavier; Daura, Xavier; Gibert, Isidre; Yero, Daniel

    2018-01-01

    Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf-1 and rpf-2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies. PMID:29740543

  10. Review on Molecular Mechanisms of Antifouling Compounds: An Update since 2012.

    PubMed

    Chen, Lianguo; Qian, Pei-Yuan

    2017-08-28

    Better understanding of the mechanisms of antifouling compounds is recognized to be of high value in establishing sensitive biomarkers, allowing the targeted optimization of antifouling compounds and guaranteeing environmental safety. Despite vigorous efforts to find new antifouling compounds, information about the mechanisms of antifouling is still scarce. This review summarizes the progress into understanding the molecular mechanisms underlying antifouling activity since 2012. Non-toxic mechanisms aimed at specific targets, including inhibitors of transmembrane transport, quorum sensing inhibitors, neurotransmission blockers, adhesive production/release inhibitors and enzyme/protein inhibitors, are put forward for natural antifouling products or shelf-stable chemicals. Several molecular targets show good potential for use as biomarkers in future mechanistic screening, such as acetylcholine esterase for neurotransmission, phenoloxidase/tyrosinase for the formation of adhesive plaques, N -acyl homoserine lactone for quorum sensing and intracellular Ca 2+ levels as second messenger. The studies on overall responses to challenges by antifoulants can be categorized as general targets, including protein expression/metabolic activity regulators, oxidative stress inducers, neurotransmission blockers, surface modifiers, biofilm inhibitors, adhesive production/release inhibitors and toxic killing. Given the current situation and the knowledge gaps regarding the development of alternative antifoulants, a basic workflow is proposed that covers the indispensable steps, including preliminary mechanism- or bioassay-guided screening, evaluation of environmental risks, field antifouling performance, clarification of antifouling mechanisms and the establishment of sensitive biomarkers, which are combined to construct a positive feedback loop.

  11. Indole production provides limited benefit to Escherichia coli during co-culture with Enterococcus faecalis.

    PubMed

    Pringle, Shelly L; Palmer, Kelli L; McLean, Robert J C

    2017-01-01

    Escherichia coli lives in the gastrointestinal tract and elsewhere, where it coexists within a mixed population. Indole production enables E. coli to grow with other gram-negative bacteria as indole inhibits N-acyl-homoserine lactone (AHL) quorum regulation. We investigated whether E. coli indole production enhanced competition with gram-positive Enterococcus faecalis, wherein quorum signaling is mediated by small peptides. During planktonic co-culture with E. faecalis, the fitness and population density of E. coli tnaA mutants (unable to produce indole) equaled or surpassed that of E. coli wt. During biofilm growth, the fitness of both populations of E. coli stabilized around 100 %, whereas the fitness of E. faecalis declined over time to 85-90 %, suggesting that biofilm and planktonic populations have different competition strategies. Media supplementation with indole removed the competitive advantage of E. coli tnaA in planktonic populations but enhanced it in biofilm populations. E. coli wt and tnaA showed similar growth in Luria-Bertani (LB) broth. However, E. coli growth was inhibited in the presence of filter-sterilized spent LB from E. faecalis, with inhibition being enhanced by indole. Similarly, there was also an inhibition of E. faecalis growth by proteinaceous components (likely bacteriocins) from spent culture media from both E. coli strains. We conclude that E. coli indole production is not a universal competition strategy, but rather works against gram-negative, AHL-producing bacteria.

  12. Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1.

    PubMed

    Rajesh, P S; Ravishankar Rai, V

    2014-01-01

    Quorum sensing mechanism allows the microorganisms to resist the antibiotic treatment by forming biofilms. Quorum quenching is one of the mechanisms to control the development of drug resistance in microbes. Endophyte bacteria are beneficial to plant growth as they support the immune system against the pathogen attack. The endophytic bacteria present in Pterocarpus santalinus were screened for the presence of N-acyl homoserine lactones (AHLs) degrading bacteria using biosensor strains and further confirmed by quantifying the violacein production. Cell-free lysate of endophytic bacteria, Bacillus firmus PT18 and Enterobacter asburiae PT39 exhibited potent AHL degrading ability by inhibiting about 80% violacein production in biosensor strain. Furthermore, when the cell-free lysate was applied to Pseudomonas aeruginosa PAO1 and PAO1-JP2 biofilm it resulted in significant (p<0.01) inhibition of biofilm formation. The biofilm inhibition was confirmed by visualization of biofilm slides under fluorescence microscopy, which showed decrease in total biomass formation in treated slides. Isolation and amplification of the gene (aiiA) indicated that the presence of AHL lactonase in cell-free lysate and sequence alignment indicated that AiiA contains a "HXHXDH" zinc-binding motif that is being conserved in several groups of metallohydrolases. Therefore, the study shows the potential of AHLs degradation by AHL lactonase present in cell-free lysate of isolated endophytic bacteria and inhibition of quorum sensing regulated biofilm formation in P. aeruginosa PAO1. Copyright © 2013 Elsevier GmbH. All rights reserved.

  13. Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Norton, Jeanette M.; Klotz, Martin G; Stein, Lisa Y

    2008-01-01

    The complete genome of the ammonia-oxidizing bacterium, Nitrosospira multiformis (ATCC 25196T), consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2827 putative proteins. Of these, 2026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and N. eutropha were the best match for 42% of the predicted genes in N. multiformis. The genome contains three nearly identical copies of amo and hao gene clusters as large repeats. Distinguishing features compared to N. europaea include: the presencemore » of gene clusters encoding urease and hydrogenase, a RuBisCO-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced AOB genomes. Gene clusters encoding proteins associated with outer membrane and cell envelope functions including transporters, porins, exopolysaccharide synthesis, capsule formation and protein sorting/export were abundant. Numerous sensory transduction and response regulator gene systems directed towards sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate and cyanophycin storage and utilization were identified providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.« less

  14. Review on Molecular Mechanisms of Antifouling Compounds: An Update since 2012

    PubMed Central

    Chen, Lianguo; Qian, Pei-Yuan

    2017-01-01

    Better understanding of the mechanisms of antifouling compounds is recognized to be of high value in establishing sensitive biomarkers, allowing the targeted optimization of antifouling compounds and guaranteeing environmental safety. Despite vigorous efforts to find new antifouling compounds, information about the mechanisms of antifouling is still scarce. This review summarizes the progress into understanding the molecular mechanisms underlying antifouling activity since 2012. Non-toxic mechanisms aimed at specific targets, including inhibitors of transmembrane transport, quorum sensing inhibitors, neurotransmission blockers, adhesive production/release inhibitors and enzyme/protein inhibitors, are put forward for natural antifouling products or shelf-stable chemicals. Several molecular targets show good potential for use as biomarkers in future mechanistic screening, such as acetylcholine esterase for neurotransmission, phenoloxidase/tyrosinase for the formation of adhesive plaques, N-acyl homoserine lactone for quorum sensing and intracellular Ca2+ levels as second messenger. The studies on overall responses to challenges by antifoulants can be categorized as general targets, including protein expression/metabolic activity regulators, oxidative stress inducers, neurotransmission blockers, surface modifiers, biofilm inhibitors, adhesive production/release inhibitors and toxic killing. Given the current situation and the knowledge gaps regarding the development of alternative antifoulants, a basic workflow is proposed that covers the indispensable steps, including preliminary mechanism- or bioassay-guided screening, evaluation of environmental risks, field antifouling performance, clarification of antifouling mechanisms and the establishment of sensitive biomarkers, which are combined to construct a positive feedback loop. PMID:28846624

  15. Effects of an inducible aiiA gene on disease resistance in Eucalyptus urophylla × Eucalyptus grandis.

    PubMed

    Ouyang, L J; Li, L M

    2016-08-01

    N-acyl-homoserine lactones (AHLs) are metabolites of mostly gram-negative bacteria and are critical signaling molecules in bacterial quorum-sensing systems. At threshold concentrations, AHLs can activate the expression of pathogenic genes and induce diseases. Therefore, reducing AHL concentrations is a key point of disease control in plants. AHL-lactonase, which is expressed by aiiA, is widespread in Bacillus sp and can hydrolyze AHLs. In the present study, we cloned aiiA from Bacillus subtilis by PCR. A plant expression vector of aiiA was constructed and name Pcam-PPP3-aiiA, in which expression of aiiA was controlled by the pathogen-inducible plant promoter PPP3. The recombinant plasmid was transferred into Eucalyptus × urophylla × E. grandis by an Agrobacterium-mediated transformation. PCR and Southern blotting showed that aiiA was successfully integrated into the E. urophylla × E. grandis genome and its expression was induced by Ralstonia solanacearum 12 h after inoculation, as shown by reverse transcription-PCR. The transcription efficacy of aiiA increased 43.88-, 30.65-, and 18.95-fold after inoculation with R. solanacearum, Erwinia carotovora ssp. zeae (Sabet) and Cylindrocladium quinqueseptatum, respectively as shown by RT-real-time PCR. Transgenic E.urophylla × E.grandis expressing the AIIA protein exhibited significantly enhanced disease resistance compared to non-transgenic plants by delaying the onset of wilting and reducing the disease index.

  16. Isolation and Genomic Characterization of ‘Desulfuromonas soudanensis WTL’, a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine

    PubMed Central

    Badalamenti, Jonathan P.; Summers, Zarath M.; Chan, Chi Ho; Gralnick, Jeffrey A.; Bond, Daniel R.

    2016-01-01

    Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, ‘Desulfuromonas soudanensis’ strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that ‘D. soudanensis’ releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. ‘D. soudanensis’ contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of ‘D. soudanensis’ underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats. PMID:27445996

  17. Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).

    PubMed

    Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C

    2011-04-01

    The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

  18. Evolution of the acyl-CoA binding protein (ACBP)

    PubMed Central

    Burton, Mark; Rose, Timothy M.; Færgeman, Nils J.; Knudsen, Jens

    2005-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein that binds C12–C22 acyl-CoA esters with high affinity. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified in all four eukaryotic kingdoms, Animalia, Plantae, Fungi and Protista, and eleven eubacterial species. ACBP homologues were not detected in any other known bacterial species, or in archaea. Nearly all of the ACBP-containing bacteria are pathogenic to plants or animals, suggesting that an ACBP gene could have been acquired from a eukaryotic host by horizontal gene transfer. Many bacterial, fungal and higher eukaryotic species only harbour a single ACBP homologue. However, a number of species, ranging from protozoa to vertebrates, have evolved two to six lineage-specific paralogues through gene duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage-specific paralogues have evolved altered functions. The appearance of ACBP very early on in evolution points towards a fundamental role of ACBP in acyl-CoA metabolism, including ceramide synthesis and in signalling. PMID:16018771

  19. Acylated and unacylated ghrelin confer neuroprotection to mesencephalic neurons.

    PubMed

    Wagner, Johanna; Vulinović, Franca; Grünewald, Anne; Unger, Marcus M; Möller, Jens C; Klein, Christine; Michel, Patrick P; Ries, Vincent; Oertel, Wolfgang H; Alvarez-Fischer, Daniel

    2017-12-04

    The polypeptide ghrelin is an endogenous ligand at the growth hormone secretagogue receptor 1a. To ghrelin multiple functions have been ascribed including promotion of gastrointestinal motility. Postprandial ghrelin levels have been reported to be reduced in patients suffering from Parkinson disease (PD). Experimental studies revealed neuroprotective effects of ghrelin in different PD models. The purpose of the present study was (i) to further elucidate the mechanism underlying the neuroprotective action of ghrelin and (ii) to determine whether these effects occur with both the acylated and the unacylated form. The study was conducted in primary mesencephalic cultures treated with mitochondrial complex I and complex II inhibitors. We show that protective effects of ghrelin against complex I inhibition with MPP + were independent of the acylation status of ghrelin, although acylated ghrelin appeared to be more potent. Protection by both forms was also observed when neurons were exposed to the complex II inhibitor 3-NP. Both forms led to higher oxygen consumption rates upon electron transport chain uncoupling, indicating that the two peptides may exert uncoupling effects themselves. We demonstrate that the rescue provided by ghrelin required calcium influx through L-type voltage-gated calcium channels. Whereas the protective effects of acylated ghrelin required receptor binding, effects of the unacylated form remained unaffected by treatment with a ghrelin receptor antagonist. Importantly, inhibition of ghrelin O-acyltransferase failed to reduce the activity of unacylated ghrelin. Overall, our data suggest that both acylated and unacylated ghrelin afford protection to dopamine neurons but through mechanisms that only partially overlap. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. Determination of Lactones in Wines by Headspace Solid-Phase Microextraction and Gas Chromatography Coupled with Mass Spectrometry

    PubMed Central

    Pérez-Olivero, S. J.; Pérez-Pont, M. L.; Conde, J. E.; Pérez-Trujillo, J. P.

    2014-01-01

    Application of headspace solid-phase microextraction (HS-SPME) coupled with high-resolution gas chromatographic (HRGC) analysis was studied for determining lactones in wines. Six different SPME fibers were tested, and the influence of different factors such as temperature and time of desorption, ionic strength, time of extraction, content of sugar, ethanol, tannins and anthocyanins, and pH and influence of SO2 were studied. The proposed HS-SPME-GC method is an appropriate technique for the quantitative analysis of γ-butyrolactone, γ-hexalactone, trans-whiskey lactone, γ-octalactone, cis-whiskey lactone, γ-nonalactone, γ-decalactone, δ-decalactone, and γ-undecalactone in wines. Method reproducibility and repeatability ranged between 0.6 and 5.2% for all compounds. Detection limit for γ-butyrolactone was 0.17 mg/L and a few μg/L for the rest of the compounds. The optimized method has been applied to several wine samples. PMID:24782943

  1. Free radical-scavenging delta-lactones from Boletus calopus.

    PubMed

    Kim, Jin-Woo; Yoo, Ick-Dong; Kim, Won-Gon

    2006-12-01

    The methanol extracts from the fruiting body of the mushroom Boletus calopus showed free radical-scavenging activity. Bioactivity-guided fractionation of the methanol extracts led to a new hydroxylated calopin named calopin B, along with the known delta-lactones calopin and cyclocalopin A. The structure of the new calopin analogue was elucidated by spectroscopic methods. All compounds showed potent free radical-scavenging activity against superoxide, DPPH, and ABTS radicals with IC (50) values of 1.2 - 5.4 microg/mL.

  2. A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine.

    PubMed

    Ilc, Tina; Halter, David; Miesch, Laurence; Lauvoisard, Florian; Kriegshauser, Lucie; Ilg, Andrea; Baltenweck, Raymonde; Hugueney, Philippe; Werck-Reichhart, Danièle; Duchêne, Eric; Navrot, Nicolas

    2017-01-01

    Monoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound. Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool. Our analysis of (E)-8-carboxylinalool in Riesling × Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries. Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  3. A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

    PubMed Central

    Lohans, Christopher T.; van Groesen, Emma; Kumar, Kiran; Tooke, Catherine L.; Spencer, James; Paton, Robert S.; Brem, Jürgen

    2018-01-01

    Abstract β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required. PMID:29236332

  4. Acyl spermidines in inflorescence extracts of elder (Sambucus nigra L., Adoxaceae) and elderflower drinks.

    PubMed

    Kite, Geoffrey C; Larsson, Sonny; Veitch, Nigel C; Porter, Elaine A; Ding, Ning; Simmonds, Monique S J

    2013-04-10

    LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

  5. Diversion of a thioglycoligase for the synthesis of 1-O-acyl arabinofuranoses.

    PubMed

    Pavic, Quentin; Tranchimand, Sylvain; Lemiègre, Loïc; Legentil, Laurent

    2018-05-15

    An arabinofuranosylhydrolase from the GH51 family was transformed into an acyl transferase by mutation of the catalytic acid/base amino acid. The resulting enzyme was able to transfer carboxylic acid onto the anomeric position of arabinose with complete chemo- and stereoselectivity. A wide range of acyl α-l-arabinofuranoses was obtained with yields ranging from 25 to 83%. Using this method, ibuprofen and N-Boc phenylalanine were successfully transformed into their corresponding acyl conjugates, expanding the scope of the reaction to drugs and amino acids.

  6. Exogenous Supply of Pantoyl Lactone to Excised Leaves Increases their Pantothenate Levels

    PubMed Central

    RATHINASABAPATHI, BALA; RAMAN, SURESH BABU

    2005-01-01

    • Background and Aims All plants synthesize pantothenate but its synthesis and regulation are not well understood. The aim of this work is to study the effect of exogenous supply of precursor compounds on pantothenate levels in leaves. • Methods Precursor compounds were supplied in solution to excised leaves and the pantothenate content was measured using a microbial method. • Key Results Pantothenate levels in excised leaves of Limonium latifolium, tomato (Lycopersicon esculentum), bean (Phaseolus vulgaris) and grapefruit (Citrus × paradisi) were examined following an exogenous supply of the precursor compounds pantoyl lactone or β-alanine. Significantly higher levels of extractable pantothenate were found when pantoyl lactone was supplied, but not when β-alanine was supplied despite a measurable uptake of β-alanine into the leaf. • Conclusions The results suggested that the pantoate supply may be rate-limiting or regulating pantothenate synthesis in leaves. PMID:15767268

  7. Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana.

    PubMed

    Joel, Daniel M; Chaudhuri, Swapan K; Plakhine, Dina; Ziadna, Hammam; Steffens, John C

    2011-05-01

    The germination of the obligate root parasites of the Orobanchaceae depends on the perception of chemical stimuli from host roots. Several compounds, collectively termed strigolactones, stimulate the germination of the various Orobanche species, but do not significantly elicit germination of Orobanche cumana, a specific parasite of sunflower. Phosphate starvation markedly decreased the stimulatory activity of sunflower root exudates toward O. cumana, and fluridone - an inhibitor of the carotenoid biosynthesis pathway - did not inhibit the production of the germination stimulant in both shoots and roots of young sunflower plants, indicating that the stimulant is not a strigolactone. We identified the natural germination stimulant from sunflower root exudates by bioassay-driven purification. Its chemical structure was elucidated as the guaianolide sesquiterpene lactone dehydrocostus lactone (DCL). Low DCL concentrations effectively stimulate the germination of O. cumana seeds but not of Phelipanche aegyptiaca (syn. Orobanche aegyptiaca). DCL and other sesquiterpene lactones were found in various plant organs, but were previously not known to be exuded to the rhizosphere where they can interact with other organisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Shifting Native Chemical Ligation into Reverse through N→S Acyl Transfer

    PubMed Central

    Macmillan, Derek; Adams, Anna; Premdjee, Bhavesh

    2011-01-01

    Peptide thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for further development and discovery. PMID:22347724

  9. Exploring Cooperative Effects in Oxidative NHC Catalysis: Regioselective Acylation of Carbohydrates.

    PubMed

    Cramer, David L; Bera, Srikrishna; Studer, Armido

    2016-05-23

    The utility of oxidative NHC catalysis for both the regioselective and chemoselective functionalization of carbohydrates is explored. Chiral NHCs allow for the highly regioselective oxidative esterification of various carbohydrates using aldehydes as acylation precursors. The transformation was also shown to be amenable to both cis/trans diol isomers, free amino groups, and selective for specific sugar epimers in competition experiments. Efficiency and regioselectivity of the acylation can be improved upon using two different NHC catalysts that act cooperatively. The potential of the method is documented by the regioselective acylation of an amino-linked neodisaccharide. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Acute aerobic exercise differentially alters acylated ghrelin and perceived fullness in normal-weight and obese individuals.

    PubMed

    Heden, Timothy D; Liu, Ying; Park, Youngmin; Dellsperger, Kevin C; Kanaley, Jill A

    2013-09-01

    Adiposity alters acylated ghrelin concentrations, but it is unknown whether adiposity alters the effect of exercise and feeding on acylated ghrelin responses. Therefore, the purpose of this study was to determine whether adiposity [normal-weight (NW) vs. obese (Ob)] influences the effect of exercise and feeding on acylated ghrelin, hunger, and fullness. Fourteen NW and 14 Ob individuals completed two trials in a randomized counterbalanced fashion, including a prior exercise trial (EX) and a no exercise trial (NoEX). During the EX trial, the participants performed 1 h of treadmill walking (55-60% peak O2 uptake) during the evening, 12 h before a 4-h standardized mixed meal test. Frequent blood samples were taken and analyzed for acylated ghrelin, and a visual analog scale was used to assess perceived hunger and fullness. In NW individuals, EX, compared with NoEX, reduced fasting acylated ghrelin concentrations by 18% (P = 0.03), and, in response to feeding, the change in acylated ghrelin (P = 0.02) was attenuated by 39%, but perceived hunger and fullness were unaltered. In Ob individuals, despite no changes in fasting or postprandial acylated ghrelin concentrations with EX, postprandial fullness was attenuated by 46% compared with NoEX (P = 0.05). In summary, exercise performed the night before a meal suppresses acylated ghrelin concentrations in NW individuals without altering perceived hunger or fullness. In Ob individuals, despite no changes in acylated ghrelin concentrations, EX reduced the fullness response to the test meal. Acylated ghrelin and perceived fullness responses are differently altered by acute aerobic exercise in NW and Ob individuals.

  11. Quantum chemical study of penicillin: Reactions after acylation

    NASA Astrophysics Data System (ADS)

    Li, Rui; Feng, Dacheng; Zhu, Feng

    The density functional theory methods were used on the model molecules of penicillin to determine the possible reactions after their acylation on ?-lactamase, and the results were compared with sulbactam we have studied. The results show that, the acylated-enzyme tetrahedral intermediate can evolves with opening of ?-lactam ring as well as the thiazole ring; the thiazole ring-open products may be formed via ?-lactam ring-open product or from tetrahedral intermediate directly. Those products, in imine or enamine form, can tautomerize via hydrogen migration. In virtue of the water-assisted, their energy barriers are obviously reduced.

  12. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.

    PubMed Central

    Faergeman, N J; Knudsen, J

    1997-01-01

    The intracellular concentration of free unbound acyl-CoA esters is tightly controlled by feedback inhibition of the acyl-CoA synthetase and is buffered by specific acyl-CoA binding proteins. Excessive increases in the concentration are expected to be prevented by conversion into acylcarnitines or by hydrolysis by acyl-CoA hydrolases. Under normal physiological conditions the free cytosolic concentration of acyl-CoA esters will be in the low nanomolar range, and it is unlikely to exceed 200 nM under the most extreme conditions. The fact that acetyl-CoA carboxylase is active during fatty acid synthesis (Ki for acyl-CoA is 5 nM) indicates strongly that the free cytosolic acyl-CoA concentration is below 5 nM under these conditions. Only a limited number of the reported experiments on the effects of acyl-CoA on cellular functions and enzymes have been carried out at low physiological concentrations in the presence of the appropriate acyl-CoA-buffering binding proteins. Re-evaluation of many of the reported effects is therefore urgently required. However, the observations that the ryanodine-senstitive Ca2+-release channel is regulated by long-chain acyl-CoA esters in the presence of a molar excess of acyl-CoA binding protein and that acetyl-CoA carboxylase, the AMP kinase kinase and the Escherichia coli transcription factor FadR are affected by low nanomolar concentrations of acyl-CoA indicate that long-chain acyl-CoA esters can act as regulatory molecules in vivo. This view is further supported by the observation that fatty acids do not repress expression of acetyl-CoA carboxylase or Delta9-desaturase in yeast deficient in acyl-CoA synthetase. PMID:9173866

  13. Metabolite profiling of sesquiterpene lactones from Lactuca species. Major latex components are novel oxalate and sulfate conjugates of lactucin and its derivatives.

    PubMed

    Sessa, R A; Bennett, M H; Lewis, M J; Mansfield, J W; Beale, M H

    2000-09-01

    Wounding leaves or stems of Lactuca species releases a milky latex onto the plant surface. We have examined the constituents of latex from Lactuca sativa (lettuce) cv. Diana. The major components were shown to be novel 15-oxalyl and 8-sulfate conjugates of the guaianolide sesquiterpene lactones, lactucin, deoxylactucin, and lactucopicrin. The oxalates were unstable, reverting to the parent sesquiterpene lactone on hydrolysis. Oxalyl derivatives have been reported rarely from natural sources. The sulfates were stable and are the first reported sesquiterpene sulfates from plants. Unusual tannins based on 4-hydroxyphenylacetyl conjugates of glucose were also identified. Significant qualitative and quantitative variation was found in sesquiterpene lactone profiles in different lettuce varieties and in other Lactuca spp. The proportions of each conjugate in latex also changed depending on the stage of plant development. A similar profile was found in chicory, in which oxalyl conjugates were identified, but the 8-sulfate conjugates were notably absent. The presence of the constitutive sesquiterpene lactones was not correlated with resistance to pathogens but may have a significant bearing on the molecular basis of the bitter taste of lettuce and related species. The induced sesquiterpene lactone phytoalexin, lettucenin A, was found in the Lactuca spp. but not in chicory.

  14. Quorum sensing is a key regulator for the antifungal and biocontrol activity of chitinase-producing Chromobacterium sp. C61.

    PubMed

    Kim, In Seon; Yang, Si Young; Park, Seur Kee; Kim, Young Cheol

    2017-01-01

    Chromobacterium sp. strain C61 has strong biocontrol activity; however, the genetic and biochemical determinants of its plant disease suppression activity are not well understood. Here, we report the identification and characterization of two new determinants of its biocontrol activity. Transposon mutagenesis was used to identify mutants that were deficient in fungal suppression. One of these mutants had an insertion in a homologue of depD, a structural gene in the dep operon, that encodes a protein involved in non-ribosomal peptide synthesis. In the second mutant, the insertion was in a homologue of the luxI gene, which encodes a homoserine lactone synthase. The luxI - and depD - mutants had no antifungal activity in vitro and a dramatically reduced capacity to suppress various plant diseases in planta. Antifungal production and biocontrol were restored by complementation of the luxI - mutant. Other phenotypes associated with effective biological control, including motility and lytic enzyme secretion, were also affected by the luxI mutation. Biochemical analysis of ethyl acetate extracts of culture filtrates of the mutant and wild-type strains showed that a key antifungal compound, chromobactomycin, was produced by wild-type C61 and the complemented luxI - mutant, but not by the luxI - or depD - mutant. These data suggest that multiple biocontrol-related phenotypes are regulated by homoserine lactones in C61. Thus, quorum sensing plays an essential role in the biological control potential of diverse bacterial lineages. © 2016 BSPP and John Wiley & Sons Ltd.

  15. Dendocarbin A: a sesquiterpene lactone from Drimys winteri.

    PubMed

    Paz Robles, Cristian; Burgos, Viviana; Suarez, Sebastián; Baggio, Ricardo

    2014-11-01

    The natural compound dendocarbin A, C15H22O3, is a sesquiterpene lactone isolated for the first time from Drimys winteri for var chilensis. The compound crystallizes in the orthorhombic space group P2₁2₁2₁ and its X-ray crystal structure confirmed the S/R character of the chiral centres at C-5/C-10 and C-9/C-11, respectively. The α-OH group at C-11 was found to be involved in intermolecular hydrogen bonding, defining chains along the <100> 2₁ screw axis.

  16. Anorexia in hemodialysis patients: the possible role of des-acyl ghrelin.

    PubMed

    Muscaritoli, Maurizio; Molfino, Alessio; Chiappini, Maria Grazia; Laviano, Alessandro; Ammann, Thomas; Spinsanti, Paola; Melchiorri, Daniela; Inui, Akio; Alegiani, Filippo; Rossi Fanelli, Filippo

    2007-01-01

    Anorexia is frequently found in end-stage renal disease and is a reliable predictor of morbidity and mortality in hemodialysis (HD) patients. The pathogenesis of anorexia is complex and the appetite-modulating hormone ghrelin could be involved. Two forms of circulating ghrelin have been described: acylated ghrelin (<10% of circulating ghrelin) which promotes food intake, and des-acyl ghrelin which induces a negative energy balance. The aim of this cross-sectional study is to clarify whether anorexia and body weight change in HD patients relate to plasma des-acyl ghrelin levels. 34 HD patients and 15 healthy controls were studied. The presence of anorexia was assessed by a questionnaire. Serum des-acyl ghrelin was measured in HD patients and in 15 body mass index-, sex- and age-matched controls by ELISA. Energy intake was assessed by a 3-day dietary diary, and fat-free mass (FFM) was evaluated by body impedance analysis. Data have been statistically analyzed and are presented as mean +/- SD. 14 patients (41%) were found to be anorexic, and 20 patients (59%) non-anorexic. Energy intake (kcal/day) was significantly lower in anorexic than in non-anorexic patients (1,682 +/- 241 vs. 1,972.50 +/- 490; p < 0.05). FFM (%) was lower in anorexic than in non-anorexic patients (65.8 +/- 4.4 vs. 70.9 +/- 8.7; p = 0.05). Plasma des-acyl ghrelin levels (fmol/ml) were significantly higher in HD patients than in controls (214.88 +/- 154.24 vs. 128.93 +/- 51.07; p < 0.05), and in anorexic HD patients than in non-anorexic (301.7 +/- 162.4 vs. 159.1 +/- 115.5; p < 0.01). Anorexia is highly prevalent among HD patients and des-acyl ghrelin could be involved in its pathogenesis. Copyright 2007 S. Karger AG, Basel.

  17. Heliolactone, a non-sesquiterpene lactone germination stimulant for root parasitic weeds from sunflower.

    PubMed

    Ueno, Kotomi; Furumoto, Toshio; Umeda, Shuhei; Mizutani, Masaharu; Takikawa, Hirosato; Batchvarova, Rossitza; Sugimoto, Yukihiro

    2014-12-01

    Root exudates of sunflower (Helianthus annuus L.) line 2607A induced germination of seeds of root parasitic weeds Striga hermonthica, Orobanche cumana, Orobanche minor, Orobanche crenata, and Phelipanche aegyptiaca. Bioassay-guided purification led to the isolation of a germination stimulant designated as heliolactone. FT-MS analysis indicated a molecular formula of C20H24O6. Detailed NMR spectroscopic studies established a methylfuranone group, a common structural component of strigolactones connected to a methyl ester of a C14 carboxylic acid via an enol ether bridge. The cyclohexenone ring is identical to that of 3-oxo-α-ionol and the other part of the molecule corresponds to an oxidized carlactone at C-19. It is a carlactone-type molecule and functions as a germination stimulant for seeds of root parasitic weeds. Heliolactone induced seed germination of the above mentioned root parasitic weeds, while dehydrocostus lactone and costunolide, sesquiterpene lactones isolated from sunflower root exudates, were effective only on O. cumana and O. minor. Heliolactone production in aquacultures increased when sunflower seedlings were grown hydroponically in tap water and decreased on supplementation of the culture with either phosphorus or nitrogen. Costunolide, on the other hand, was detected at a higher concentration in well-nourished medium as opposed to nutrient-deficient media, thus suggesting a contrasting contribution of heliolactone and the sesquiterpene lactone to the germination of O. cumana under different soil fertility levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Cascade Synthesis of Five-Membered Lactones using Biomass-Derived Sugars as Carbon Nucleophiles.

    PubMed

    Yamaguchi, Sho; Matsuo, Takeaki; Motokura, Ken; Miyaji, Akimitsu; Baba, Toshihide

    2016-06-06

    We report the cascade synthesis of five-membered lactones from a biomass-derived triose sugar, 1,3-dihydroxyacetone, and various aldehydes. This achievement provides a new synthetic strategy to generate a wide range of valuable compounds from a single biomass-derived sugar. Among several examined Lewis acid catalysts, homogeneous tin chloride catalysts exhibited the best performance to form carbon-carbon bonds. The scope and limitations of the synthesis of five-membered lactones using aldehyde compounds are investigated. The cascade reaction led to high product selectivity as well as diastereoselectivity, and the mechanism leading to the diastereoselectivity was discussed based on isomerization experiments and density functional theory (DFT) calculations. The present results are expected to support new approaches for the efficient utilization of biomass-derived sugars. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

    PubMed Central

    Shipston, Michael J.

    2014-01-01

    Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

  20. Variecolactol: A New Sesterterpene Lactone from the Sclerotia of Aspergillus auricomus (Guegen) Saito

    USDA-ARS?s Scientific Manuscript database

    Variecolactol (1), a new sesterterpene lactone related to variecolin (2), has been isolated from the organic extracts of Aspergillus auricomus. Structure determination of this compound was achieved primarily through HMQC, HMBC, and NOESY experiments. The known compounds dihydropenicillic acid (3) ...

  1. Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sato, Miho; Nakahara, Keiko; Goto, Shintaro

    Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [{sup 125}I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmedmore » that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.« less

  2. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

    PubMed

    Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-10-04

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

  3. Toward Green Acylation of (Hetero)arenes: Palladium-Catalyzed Carbonylation of Olefins to Ketones

    PubMed Central

    2017-01-01

    Green Friedel–Crafts acylation reactions belong to the most desired transformations in organic chemistry. The resulting ketones constitute important intermediates, building blocks, and functional molecules in organic synthesis as well as for the chemical industry. Over the past 60 years, advances in this topic have focused on how to make this reaction more economically and environmentally friendly by using green acylating conditions, such as stoichiometric acylations and catalytic homogeneous and heterogeneous acylations. However, currently well-established methodologies for their synthesis either produce significant amounts of waste or proceed under harsh conditions, limiting applications. Here, we present a new protocol for the straightforward and selective introduction of acyl groups into (hetero)arenes without directing groups by using available olefins with inexpensive CO. In the presence of commercial palladium catalysts, inter- and intramolecular carbonylative C–H functionalizations take place with good regio- and chemoselectivity. Compared to classical Friedel–Crafts chemistry, this novel methodology proceeds under mild reaction conditions. The general applicability of this methodology is demonstrated by the direct carbonylation of industrial feedstocks (ethylene and diisobutene) as well as of natural products (eugenol and safrole). Furthermore, synthetic applications to drug molecules are showcased. PMID:29392174

  4. The influence of quorum sensing in compartment II of the MELiSSA loop

    NASA Astrophysics Data System (ADS)

    Condori, Sandra; Mastroleo, Felice; Wattiez, Ruddy; Leys, Natalie

    MELiSSA (Micro-Ecological Life Support System Alternative) has been conceived as a 5 compartments microorganisms and higher plants recycling system for long haul space flights. Rhodospirillum rubrum S1H colonizes compartment II. Previous work reported that continuous culture of the bacterium in a photobioreactor could lead to thick biofilm formation, leading to bioreactor arrest. Our aim is to investigate the unknown quorum sensing (QS) system of R. rubrum S1H, specifically under MELiSSA relevant culture conditions meaning light anaerobic (LAN) and using acetate as carbon source. In that purpose an autoinducer synthase gene (Rru_A3396) knockout mutant was constructed by allelic exchange generating strain M68. In addition phenotypic comparison between wild type (WT) and M68 was performed. Results of thin layer chromatography assay where Agrobacterium tumefaciens NT1 have been used as reporter strain showed that WT produces acyl-homoserine lactones (AHLs) from C4 to C12 acyl carbon chain length; however, in M68 no AHLs were detected confirming that gene Rru_A3396 (named rruI) encodes an autoinducer synthase. Interestingly under a low shear or static environment M68 showed cell aggregation similar as reported in a closely related bacterium Rhodobacter sphaeroides (cerI mutant). In contrast to WT, M68 did not form biofilm and exhibited a decreased motility and pigment content. M68 vs wild type transcriptomics results showed that 326 genes were statistically significant differentially expressed. Downregulation of genes related to photosynthesis e.g., reaction center subunits, light harvesting complex and photosynthetic assembly proteins was observed. Similar results were obtained for preliminary proteomic analysis. Results obtained showed that in R. rubrum S1H the AHL-based QS system regulates almost 8% of the genome which is linked to biofilm formation among other biological processes described above. Since strain M68 could not be used in compartment II due to its less

  5. Des-acyl ghrelin prevents heatstroke-like symptoms in rats exposed to high temperature and high humidity.

    PubMed

    Inoue, Yoshiyuki; Hayashi, Yujiro; Kangawa, Kenji; Suzuki, Yoshihiro; Murakami, Noboru; Nakahara, Keiko

    2016-02-26

    We have shown previously that des-acyl ghrelin decreases body temperature in rats through activation of the parasympathetic nervous system. Here we investigated whether des-acyl ghrelin ameliorates heatstroke in rats exposed to high temperature. Peripheral administration of des-acyl ghrelin significantly attenuated hyperthermia induced by exposure to high-temperature (35°C) together with high humidity (70-80%). Although biochemical analysis revealed that exposure to high temperature significantly increased hematocrit and the serum levels of aspartate amino transferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine and electrolytes (Na(+), K(+), Cl(-)), most of these heatstroke-associated reactions were significantly reduced by treatment with des-acyl ghrelin. The level of des-acyl ghrelin in plasma was also found to be significantly increased under high-temperature conditions. These results suggest that des-acyl ghrelin could be useful for preventing heatstroke under high temperature condition. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Binding modes of phosphotriesterase-like lactonase complexed with δ-nonanoic lactone and paraoxon using molecular dynamics simulations.

    PubMed

    Guan, Shanshan; Zhao, Li; Jin, Hanyong; Shan, Ning; Han, Weiwei; Wang, Song; Shan, Yaming

    2017-02-01

    Phosphotriesterase-like lactonases (PLLs) have received much attention because of their physical and chemical properties. They may have widespread applications in various fields. For example, they show potential for quorum-sensing signaling pathways and organophosphorus (OP) detoxification in agricultural science. However, the mechanism by which PLLs hydrolyze, which involves OP compounds and lactones and a variety of distinct catalytic efficiencies, has only rarely been explored. In the present study, molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of DrPLL, a member of the PLL superfamily in Deinococcus radiodurans, bound to two substrates, δ-nonanoic lactone and paraoxon. It has been observed that there is a 16-fold increase in the catalytic efficiency of the two mutant strains of DrPLL (F26G/C72I) vs. the wild-type enzyme toward the hydrolysis of paraoxon, but an explanation for this behavior is currently lacking. The analysis of the molecular trajectories of DrPLL bound to δ-nonanoic lactone indicated that lactone-induced conformational changes take place in loop 8, which is near the active site. Binding to paraoxon may lead to conformational displacement of loop 1 residues, which could lead to the deformation of the active site and so trigger the entry of the paraoxon into the active site. The efficiency of the F26G/C72I mutant was increased by decreasing the displacement of loop 1 residues and increasing the flexibility of loop 8 residues. These results provide a molecular-level explanation for the experimental behavior.

  7. Novel anticancer alkene lactone from Persea americana.

    PubMed

    Falodun, Abiodun; Engel, Nadja; Kragl, Udo; Nebe, Barbara; Langer, Peter

    2013-06-01

    Persea americana Mill (Lauraceae) root bark is used in ethnomedicine for a variety of diseases including cancer. To isolate and characterize the chemical constituent in P. americana, and also to determine the anticancer property of a new alkene lactone from the root bark of P. americana. The MCF-7 cells were treated with different concentrations of the pure compound for 48 h. The percentage of cells in the various phases, online monitoring of metabolic changes and integrin receptor expression determined by flow cytometry. One novel alkene lactone (4-hydroxy-5-methylene-3-undecyclidenedihydrofuran-2 (3H)-one) (1) was isolated and characterized using 1D-NMR, 2D-NMR, infrared, UV and MS. At a concentration of 10 µg/mL, significant reduction of proliferation of MCF-7 was induced while MCF-12 A cell was significantly stimulated by 10 µg/mL. The IC50 value for MCF-7 cells is 20.48 µg/mL. Lower concentration of 1 harbor no significant effect on either MCF-7 or MCF-12A. The apoptotic rates of MCF-7 cells were increased significantly. At the final concentration 10 µg/mL, up to 80% of all breast cancer cells were dead. On the non-tumorigenic cell line MCF-12A, the same concentrations (1 and 10 µg/mL) of compound 1 caused significant enhanced apoptotic rates. A total of 1 µg/mL of 1 caused a decrease of α4-, α6-, β1- and β3-integrin expression. The compound caused a stimulatory effect on non-tumorigenic MCF-12A cells with respect to cell adhesion while tumorigenic MCF-7 cells detached continuously. This is the first report on the anticancer effects of this class of compound.

  8. Commelinid Monocotyledon Lignins Are Acylated by p-Coumarate1[OPEN

    PubMed Central

    Free, Heather C.A.; Smith, Bronwen G.

    2018-01-01

    Commelinid monocotyledons are a monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and p-coumarate. The Poaceae, or grass family, is a member of this group, and most of the p-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked p-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases. PMID:29724771

  9. New macrocyclic lactones with acaricidal and nematocidal activities from a genetically engineered strain Streptomyces bingchenggensis BCJ60.

    PubMed

    Li, Jian-Song; Zhang, Hui; Zhang, Shao-Yong; Wang, Hai-Yan; Zhang, Ji; Chen, An-Liang; Wang, Ji-Dong; Xiang, Wen-Sheng

    2017-04-01

    Two new macrocyclic lactones, 4,25-diethyl-4,25-demethyl-milbemycin β 3 (1) and 27-formaldehyde-milbemycin β 14 (2), were isolated from a genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as ESI-MS and comparison with data from the literature. The acaricidal and nematocidal capacities of compounds 1 and 2 were evaluated against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, respectively. The results showed that the two new macrocyclic lactones 1 and 2 possessed potent acaricidal and nematocidal activities.

  10. New sesquiterpene lactones from Ambrosia cumanensis Kunth.

    PubMed

    Jimenez-Usuga, Nora Del Socorro; Malafronte, Nicola; Cotugno, Roberta; De Leo, Marinella; Osorio, Edison; De Tommasi, Nunziatina

    2016-09-01

    Eleven sesquiterpene lactones, including three new natural products (1-3), were isolated from the n-butanolic extract of Ambrosia cumanensis Kunth. aerial parts. The structure of all isolated compounds was elucidated by 1D- and 2D-NMR, and MS analyses. All compounds were tested for their antiproliferative activity on HeLa, Jurkat, and U937 cell lines. Compound 3, 2,3-dehydropsilostachyn C, showed cytotoxic activity with different potency in all cell lines. By means of flow cytometric studies, compound 3 was demonstrated to induce in Jurkat cells a G2/M cell cycle block, while in U937 elicited both cytostatic and cytotoxic responses. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. N-Acyl derivatives of Asn, new bacterial N-acyl D-amino acids with surfactant activity.

    PubMed

    Peypoux, F; Laprévote, O; Pagadoy, M; Wallach, J

    2004-03-01

    New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.

  12. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

  13. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

  14. 40 CFR 721.10056 - Benzenemethanaminium, N-(3-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)-N,N-dimethyl-, N-soya acyl derivs., chlorides. 721.10056 Section 721.10056 Protection of Environment...-aminopropyl)-N,N-dimethyl-, N-soya acyl derivs., chlorides. (a) Chemical substance and significant new uses...-dimethyl-, N-soya acyl derivs., chlorides (PMN P-03-47; CAS No. 90194-13-1) is subject to reporting under...

  15. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  16. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

    1998-01-06

    Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  17. Development of Acid Functional Groups and Lactones During the Thermal Degradation of Wood and Wood Components

    USGS Publications Warehouse

    Rutherford, David W.; Wershaw, Robert L.; Reeves, James B.

    2008-01-01

    Black carbon (pyrogenic materials including chars) in soils has been recognized as a substantial portion of soil organic matter, and has been shown to play a vital role in nutrient cycling; however, little is known concerning the properties of this material. Previous studies have largely been concerned with the creation of high-surface-area materials for use as sorbents. These materials have been manufactured at high temperature and have often been activated. Chars occurring in the environment can be formed over a wide range of temperature. Because it is extremely difficult to isolate black carbon once it has been incorporated in soils, chars produced in the laboratory under controlled conditions can be used to investigate the range of properties possible for natural chars. This report shows that charring conditions (temperature and time) have substantial impact on the acid functional group and lactone content of chars. Low temperatures (250?C) and long charring times (greater than 72 hours) produce chars with the highest acid functional group and lactone content. The charring of cellulose appears to be responsible for the creation of the acid functional group and lactones. The significance of this study is that low-temperature chars can have acid functional group contents comparable to humic materials (as high as 8.8 milliequivalents per gram). Acid functional group and lactone content decreases as charring temperature increases. The variation in formation conditions expected under natural fire conditions will result in a wide range of sorption properties for natural chars which are an important component of soil organic matter. By controlling the temperature and duration of charring, it is possible to tailor the sorption properties of chars, which may be used as soil amendments.

  18. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcescens.

    PubMed

    Srobel, G; Li, J Y; Sugawara, F; Koshino, H; Harper, J; Hess, W M

    1999-12-01

    A unique chlorinated macrocyclic lactone, termed oocydin A, was isolated from a strain of Serratia marcescens growing as an epiphyte on Rhyncholacis pedicillata, an aquatic plant native to the Carrao river of the Venezuelan-Guyanan region of South America. The lactone has a molecular mass of 470 Da, and contains one atom of chlorine, a carboxyl group and a tetrahydrofuran ring internal to a larger macrocyclic ring. MICs of approximately 0.03 microg ml(-1) were noted for oocydin A against such phytopathogenic oomycetes as Pythium ultimum, Phytophthora parasitica, Phytophthora cinnamomi and Phytophthora citrophora. With regard to the true fungi, oocydin A had either minimal or no effect against certain Fungi Imperfecti (including several pathogens of humans), two ascomycetes and a basidiomycete. Oocydin A may have potential as an antimycotic in agricultural applications and especially for crop protection.

  19. The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

    PubMed Central

    Stockbridge, Randy B.; Wolfenden, Richard

    2009-01-01

    To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16 m for N-acetylgalactosamine kinase, 7.4 × 10−17 m for homoserine kinase, and 6.4 × 10−18 m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. PMID:19531469

  20. Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

    PubMed

    Hiramine, Yasushi; Tanabe, Toshizumi

    2011-06-01

    Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

  1. Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

    PubMed

    Garg, Hari G; Mrabat, Hicham; Yu, Lunyin; Hales, Charles A; Li, Boyangzi; Moore, Casey N; Zhang, Fuming; Linhardt, Robert J

    2011-08-01

    Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

  2. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

    PubMed Central

    Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-01-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  3. Association of acylated ghrelin profiles with chronic inflammatory markers in overweight and obese postmenopausal women: a MONET study.

    PubMed

    St-Pierre, David H; Bastard, Jean-Philippe; Coderre, Lise; Brochu, Martin; Karelis, Antony D; Lavoie, Marie-Eve; Malita, Florin; Fontaine, Jonathan; Mignault, Diane; Cianflone, Katherine; Imbeault, Pascal; Doucet, Eric; Rabasa-Lhoret, Rémi

    2007-10-01

    Recent reports have suggested that the existence of associations between hormonal dysregulation and chronic upregulation of inflammatory markers, which may cause obesity-related disturbances. Thus, we examined whether acylated ghrelin (AcylG) and total ghrelin (TotG) levels could be associated with the following inflammatory markers: C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), and soluble TNF receptor 1 (sTNF-R1). Cross-sectional study consisting of 50 overweight and obese postmenopausal women. AcylG and TotG levels were assessed at 0, 60, 160, 170, and 180 min of the euglycemic/hyperinsulinemic clamp (EHC). We evaluated insulin sensitivity, body composition, and blood lipid profiles as well as fasting concentrations of CRP, TNF-alpha, and sTNF-R1. In fasting conditions, sTNF-R1 was negatively correlated with AcylG (r = -0.48, P < 0.001) levels. In addition, AcylG/TotG was associated negatively with sTNF-R1 (r = -0.44, P = 0.002) and positively with TNF-alpha (r = 0.38, P = 0.009) values. During the EHC, TotG (at all time points) and AcylG (at 60 and 160 min) values were significantly decreased from fasting concentrations. AcylG maximal reduction and area under the curve (AUC) values were correlated to sTNF-R1 (r = -0.35, P = 0.02 and r = -0.34, P = 0.02, respectively). Meanwhile, the AcylG/TotG AUC ratio was associated negatively with sTNF-R1 (r = -0.29, P < 0.05) and positively with TNF-alpha (r = 0.36, P = 0.02). Following adjustments for total adiposity, sTNF-R1 remained correlated with fasting and maximal reduction AcylG values. Similarly, AcylG/TotG ratios remained significantly correlated with sTNF-R1 and TNF-alpha. Importantly, 23% of the variation in sTNF-R1 was independently predicted by fasting AcylG. These results are the first to suggest that both fasting and EHC-induced AcylG profiles are correlated with fasting values of sTNF-R1, a component of the TNF-alpha system. Thus, AcylG may act, at least in part, as one mediator of

  4. Hepatocurative potential of sesquiterpene lactones of Taraxacum officinale on carbon tetrachloride induced liver toxicity in mice.

    PubMed

    Mahesh, A; Jeyachandran, R; Cindrella, L; Thangadurai, D; Veerapur, V P; Muralidhara Rao, D

    2010-06-01

    The hepatocurative potential of ethanolic extract (ETO) and sesquiterpene lactones enriched fraction (SL) of Taraxacum officinale roots was evaluated against carbon tetrachloride (CCl 4 ) induced hepatotoxicity in mice. The diagnostic markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin contents were significantly elevated, whereas significant reduction in the level of reduced glutathione (GSH) and enhanced hepatic lipid peroxidation, liver weight and liver protein were observed in CCl 4 induced hepatotoxicity in mice. Post-treatment with ETO and SL significantly protected the hepatotoxicity as evident from the lower levels of hepatic enzyme markers, such as serum transaminase (ALT, AST), ALP and total bilirubin. Further, significant reduction in the liver weight and liver protein in drug-treated hepatotoxic mice and also reduced oxidative stress by increasing reduced glutathione content and decreasing lipid peroxidation level has been noticed. The histopathological evaluation of the liver also revealed that ETO and SL reduced the incidence of liver lesions induced by CCl 4 . The results indicate that sesquiterpene lactones have a protective effect against acute hepatotoxicity induced by the administration of CCl 4 in mice. Furthermore, observed activity of SL may be due to the synergistic action of two sesquiterpene lactones identified from enriched ethyl acetate fraction by HPLC method.

  5. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

  6. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

  7. 40 CFR 721.7270 - 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs., chloride.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-trimethyl-N-soya acyl derivs., chloride. 721.7270 Section 721.7270 Protection of Environment ENVIRONMENTAL...-soya acyl derivs., chloride. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as 1-propanaminium, 3-amino-, N,N,N-trimethyl-N-soya acyl derivs...

  8. Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11.

    PubMed

    Lagrange, Brice; Benaoudia, Sacha; Wallet, Pierre; Magnotti, Flora; Provost, Angelina; Michal, Fanny; Martin, Amandine; Di Lorenzo, Flaviana; Py, Bénédicte F; Molinaro, Antonio; Henry, Thomas

    2018-01-16

    Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

  9. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

    1998-01-06

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

  10. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

  11. Production of Cell-Cell Signaling Molecules by Bacteria Isolated From Human Chronic Wounds

    PubMed Central

    Rickard, Alexander H.; Colacino, Katelyn R.; Manton, Katelynn M.; Morton, Robert I.; Pulcini, Elinor; Pfeil, Joanne; Rhoads, Daniel; Wolcott, Randall D.; James, Garth

    2009-01-01

    AIM To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cell-cell signaling molecules and (ii) determine if chronic wound debridement samples might contain these molecules. METHODS AND RESULTS Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains as belonging to nine genera. Using bio-reporter assays, 69.6% of the chronic wound strains were inferred to produce AI-2 while 19.6% were inferred to produced AHL molecules. At-least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch-cultures was growth-phase-dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signaling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. CONCLUSION Chronic wound bacteria produce cell-cell signaling molecules. Resident species generally produce AI-2 molecules and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cell-cell signals are present in human chronic wounds. SIGNIFICANCE AND IMPACT OF STUDY Inter-bacterial cell-cell signaling may be an important factor influencing wound development and the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cell −cell signaling may provide a novel strategy for improving wound healing. PMID:19840177

  12. A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium

    PubMed Central

    Ramsay, Joshua P.; Williamson, Neil R.; Spring, David R.; Salmond, George P. C.

    2011-01-01

    Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air–liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen. PMID:21873216

  13. A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium.

    PubMed

    Ramsay, Joshua P; Williamson, Neil R; Spring, David R; Salmond, George P C

    2011-09-06

    Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.

  14. Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios

    PubMed Central

    Gromek, Samantha M.; Suria, Andrea M.; Fullmer, Matthew S.; Garcia, Jillian L.; Gogarten, Johann Peter; Nyholm, Spencer V.; Balunas, Marcy J.

    2016-01-01

    Female members of many cephalopod species house a bacterial consortium in the accessory nidamental gland (ANG), part of the reproductive system. These bacteria are deposited into eggs that are then laid in the environment where they must develop unprotected from predation, pathogens, and fouling. In this study, we characterized the genome and secondary metabolite production of Leisingera sp. JC1, a member of the roseobacter clade (Rhodobacteraceae) of Alphaproteobacteria isolated from the jelly coat of eggs from the Hawaiian bobtail squid, Euprymna scolopes. Whole genome sequencing and MLSA analysis revealed that Leisingera sp. JC1 falls within a group of roseobacters associated with squid ANGs. Genome and biochemical analyses revealed the potential for and production of a number of secondary metabolites, including siderophores and acyl-homoserine lactones involved with quorum sensing. The complete biosynthetic gene cluster for the pigment indigoidine was detected in the genome and mass spectrometry confirmed the production of this compound. Furthermore, we investigated the production of indigoidine under co-culture conditions with Vibrio fischeri, the light organ symbiont of E. scolopes, and with other vibrios. Finally, both Leisingera sp. JC1 and secondary metabolite extracts of this strain had differential antimicrobial activity against a number of marine vibrios, suggesting that Leisingera sp. JC1 may play a role in host defense against other marine bacteria either in the eggs and/or ANG. These data also suggest that indigoidine may be partially, but not wholly, responsible for the antimicrobial activity of this squid-associated bacterium. PMID:27660622

  15. Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.

    PubMed

    Williamson, Lynn L; Borlee, Bradley R; Schloss, Patrick D; Guan, Changhui; Allen, Heather K; Handelsman, Jo

    2005-10-01

    The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.

  16. Spatial organization of Pseudomonas aeruginosa biofilms probed by combined matrix-assisted laser desorption ionization mass spectrometry and confocal Raman microscopy.

    PubMed

    Masyuko, Rachel N; Lanni, Eric J; Driscoll, Callan M; Shrout, Joshua D; Sweedler, Jonathan V; Bohn, Paul W

    2014-11-21

    Bacteria growing as surface attached biofilms differ significantly from planktonic cells in several important traits that are reflected in the spatiotemporal organization of the cells and the extracellular polymeric substances they secrete. The structural and chemical features that define these biofilms are explored here using a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and confocal Raman microspectroscopies (CRM) to characterize and compare the composition and distribution of biomolecules found in biofilms and planktonic cells of the bacterium Pseudomonas aeruginosa. Three-day old P. aeruginosa biofilms show dramatic differences in molecular composition compared to planktonic cultures. CRM reveals that wild-type planktonic cell Raman spectra are characterized by bands linked to cellular constituents and are dominated by contributions from DNA- and RNA-related bands. In contrast, biofilm spectra are dominated by bands characteristic of glycolipids - rhamnolipids - polysaccharides and by secreted proteins. LDI MS was applied in turn to identify the rhamnolipids present in the biofilm. Experiments were also conducted using an acyl homoserine lactone quorum sensing-deficient mutant (ΔlasIΔrhlI), which is incapable of producing rhamnolipids. CRM and LDI MS analyses revealed that while molecular composition of the planktonic quorum sensing-deficient cells is similar to that of the wild-type planktonic cells, several compositional differences are observed in the mutant after biofilm growth, including complete absence of detectable rhamnolipids. CRM vibrational spectra of the mutant cells are very similar for planktonic and biofilm growth conditions, indicating that biofilm formation is greatly hindered in the absence of functioning quorum sensing machinery.

  17. Signal Integration in Quorum Sensing Enables Cross-Species Induction of Virulence in Pectobacterium wasabiae

    PubMed Central

    Valente, Rita S.; Nadal-Jimenez, Pol; Carvalho, André F. P.; Vieira, Filipe J. D.

    2017-01-01

    ABSTRACT Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora), two signaling networks—the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway—control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. PMID:28536283

  18. A novel l-isoleucine-4′-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis

    PubMed Central

    Smirnov, Sergey V; Sokolov, Pavel M; Kotlyarova, Veronika A; Samsonova, Natalya N; Kodera, Tomohiro; Sugiyama, Masakazu; Torii, Takayoshi; Hibi, Makoto; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-01-01

    A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone. PMID:23554367

  19. Expression of putative pathogenicity-related genes in Xylella fastidiosa grown at low and high cell density conditions in vitro.

    PubMed

    Scarpari, Leandra M; Lambais, Marcio R; Silva, Denise S; Carraro, Dirce M; Carrer, Helaine

    2003-05-16

    Xylella fastidiosa is the causal agent of economically important plant diseases, including citrus variegated chlorosis and Pierce's disease. Hitherto, there has been no information on the molecular mechanisms controlling X. fastidiosa-plant interactions. To determine whether predicted open reading frames (ORFs) encoding putative pathogenicity-related factors were expressed by X. fastidiosa 9a5c cells grown at low (LCD) and high cell density (HCD) conditions in liquid modified PW medium, reverse Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed. Our results indicated that ORFs XF2344, XF2369, XF1851 and XF0125, encoding putative Fur, GumC, a serine-protease and RsmA, respectively, were significantly suppressed at HCD conditions. In contrast, ORF XF1115, encoding putative RpfF, was significantly induced at HCD conditions. Expressions of ORFs XF2367, XF2362 and XF0290, encoding putative GumD, GumJ and RpfA, respectively, were detected only at HCD conditions, whereas expression of ORF XF0287, encoding putative RpfB was detected only at LCD conditions. Bioassays with an Agrobacterium traG::lacZ reporter system indicated that X. fastidiosa does not synthesize N-acyl-homoserine lactones, whereas bioassays with a diffusible signal factor (DSF)-responsive Xanthomonas campestris pv. campestris mutant indicate that X. fastidiosa synthesizes a molecule similar to DSF in modified PW medium. Our data also suggest that the synthesis of the DSF-like molecule and fastidian gum by X. fastidiosa is affected by cell density in vitro.

  20. Non-ribosomal Peptide Synthases from Pseudomonas aeruginosa Play a Role in Cyclodipeptide Biosynthesis, Quorum-Sensing Regulation, and Root Development in a Plant Host.

    PubMed

    González, Omar; Ortíz-Castro, Randy; Díaz-Pérez, César; Díaz-Pérez, Alma L; Magaña-Dueñas, Viridiana; López-Bucio, José; Campos-García, Jesús

    2017-04-01

    Diverse molecules mediate cross-kingdom communication between bacteria and their eukaryotic partners and determine pathogenic or symbiotic relationships. N-acyl-L-homoserine lactone-dependent quorum-sensing signaling represses the biosynthesis of bacterial cyclodipeptides (CDPs) that act as auxin signal mimics in the host plant Arabidopsis thaliana. In this work, we performed bioinformatics, biochemical, and plant growth analyses to identify non-ribosomal peptide synthase (NRPS) proteins of Pseudomonas aeruginosa, which are involved in CDP synthesis. A reverse genetics strategy allowed the identification of the genes encoding putative multi-modular-NRPS (MM-NRPS). Mutations in these genes affected the synthesis of the CDPs cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Leu), and cyclo(L-Pro-L-Tyr), while showing wild-type-like levels of virulence factors, such as violacein, elastase, and pyocyanin. When analyzing the bioactivity of purified, naturally produced CDPs, it was found that cyclo(L-Pro-L-Tyr) and cyclo(L-Pro-L-Val) were capable of antagonizing quorum-sensing-LasR (QS-LasR)-dependent signaling in a contrasting manner in the cell-free supernatants of the selected NRPS mutants, which showed QS induction. Using a bacteria-plant interaction system, we further show that the pvdJ, ambB, and pchE P. aeruginosa mutants failed to repress primary root growth, but improved root branching in A. thaliana seedlings. These results indicated that the CDP production in P. aeruginosa depended on the functional MM-NRPS, which influences quorum-sensing of bacteria and plays a role in root architecture remodeling.

  1. Contribution of Stress Responses to Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Franklin, Michael J.; Williamson, Kerry S.; Folsom, James P.; Boegli, Laura; James, Garth A.

    2015-01-01

    Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin. PMID:25870065

  2. The role of microbial signals in plant growth and development

    PubMed Central

    Ortíz-Castro, Randy; Contreras-Cornejo, Hexon Angel; Macías-Rodríguez, Lourdes

    2009-01-01

    Plant growth and development involves a tight coordination of the spatial and temporal organization of cell division, cell expansion and cell differentiation. Orchestration of these events requires the exchange of signaling molecules between the root and shoot, which can be affected by both biotic and abiotic factors. The interactions that occur between plants and their associated microorganisms have long been of interest, as knowledge of these processes could lead to the development of novel agricultural applications. Plants produce a wide range of organic compounds including sugars, organic acids and vitamins, which can be used as nutrients or signals by microbial populations. On the other hand, microorganisms release phytohormones, small molecules or volatile compounds, which may act directly or indirectly to activate plant immunity or regulate plant growth and morphogenesis. In this review, we focus on recent developments in the identification of signals from free-living bacteria and fungi that interact with plants in a beneficial way. Evidence has accumulated indicating that classic plant signals such as auxins and cytokinins can be produced by microorganisms to efficiently colonize the root and modulate root system architecture. Other classes of signals, including N-acyl-L-homoserine lactones, which are used by bacteria for cell-to-cell communication, can be perceived by plants to modulate gene expression, metabolism and growth. Finally, we discuss the role played by volatile organic compounds released by certain plant growth-promoting rhizobacteria in plant immunity and developmental processes. The picture that emerges is one in which plants and microbes communicate themselves through transkingdom signaling systems involving classic and novel signals. PMID:19820333

  3. Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii.

    PubMed

    Koutsoudis, Maria D; Tsaltas, Dimitrios; Minogue, Timothy D; von Bodman, Susanne B

    2006-04-11

    The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo/capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI/EsaR quorum-sensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI/EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.

  4. Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii

    PubMed Central

    Koutsoudis, Maria D.; Tsaltas, Dimitrios; Minogue, Timothy D.; von Bodman, Susanne B.

    2006-01-01

    The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo/capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI/EsaR quorum-sensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI/EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control. PMID:16585516

  5. Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism*

    PubMed Central

    Ikezawa, Nobuhiro; Göpfert, Jens Christian; Nguyen, Don Trinh; Kim, Soo-Un; O'Maille, Paul E.; Spring, Otmar; Ro, Dae-Kyun

    2011-01-01

    Sesquiterpene lactones (STLs) are terpenoid natural products possessing the γ-lactone, well known for their diverse biological and medicinal activities. The occurrence of STLs is sporadic in nature, but most STLs have been isolated from plants in the Asteraceae family. Despite the implication of the γ-lactone group in many reported bioactivities of STLs, the biosynthetic origins of the γ-lactone ring remains elusive. Germacrene A acid (GAA) has been suggested as a central precursor of diverse STLs. The regioselective (C6 or C8) and stereoselective (α or β) hydroxylation on a carbon of GAA adjacent to its carboxylic acid at C12 is responsible for the γ-lactone formation. Here, we report two cytochrome P450 monooxygenases (P450s) capable of catalyzing 6α- and 8β-hydroxylation of GAA from lettuce and sunflower, respectively. To identify these P450s, sunflower trichomes were isolated to generate a trichome-specific transcript library, from which 10 P450 clones were retrieved. Expression of these clones in a yeast strain metabolically engineered to synthesize substrate GAA identified a P450 catalyzing 8β-hydroxylation of GAA, but the STL was not formed by spontaneous lactonization. Subsequently, we identified the closest homolog of the GAA 8β-hydroxylase from lettuce and discovered 6α-hydroxylation of GAA by the recombinant enzyme. The resulting 6α-hydroxy-GAA spontaneously undergoes a lactonization to yield the simplest form of STL, costunolide. Furthermore, we demonstrate the milligram per liter scale de novo synthesis of costunolide using the lettuce P450 in an engineered yeast strain, an important advance that will enable exploitation of STLs. Evolution and homology models of these two P450s are discussed. PMID:21515683

  6. Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton.

    PubMed

    Bruns, Alke; Nübel, Ulrich; Cypionka, Heribert; Overmann, Jörg

    2003-04-01

    The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 micro M) of the signal compounds N-(oxohexanoyl)-DL-homoserine lactone, N-(butyryl)-DL-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-(3)H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low K(m) value of

  7. Physical characterisation of high amylose maize starch and acylated high amylose maize starches.

    PubMed

    Lim, Ya-Mei; Hoobin, Pamela; Ying, DanYang; Burgar, Iko; Gooley, Paul R; Augustin, Mary Ann

    2015-03-06

    The particle size, water sorption properties and molecular mobility of high amylose maize starch (HAMS) and high amylose maize starch acylated with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) were investigated. Acylation increased the mean particle size (D(4,3)) and lowered the specific gravity (G) of the starch granules with an inverse relationship between the length of the fatty acid chain and particle size. Acylation of HAMS with fatty acids lowered the monolayer moisture content with the trend being HAMSBacylated starches and that drying and storage of the starch granules further reduced T2 long. Analysis of the Free Induction Decay (FID) focussing on the short components of T2 (correlated to the solid matrix), indicated that drying and subsequent storage resulted in alterations of starch at 0.33a(w) and that these changes were reduced with acylation. In vitro enzymatic digestibility of heated starch dispersions by bacterial α-amylase was increased by acylation (HAMS

  8. Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat.

    PubMed

    Grillo, M P; Benet, L Z

    2001-08-01

    Clofibric acid (CA) is metabolized to chemically reactive acylating products that can transacylate glutathione to form clofibryl-S-acyl-glutathione (CA-SG) in vitro and in vivo. We investigated the first step in the degradation of CA-SG to the mercapturic acid conjugate, clofibryl-S-acyl-N-acetylcysteine (CA-SNAC), which is catalyzed by gamma-glutamyltranspeptidase (gamma-GT). After gamma-GT mediated cleavage of glutamate from CA-SG, the product clofibryl-S-acyl-cysteinylglycine (CA-S-CG) should undergo an intramolecular rearrangement reaction [Tate, S. S. (1975) FEBS Lett. 54, 319-322] to form clofibryl-N-acyl-cysteinylglycine (CA-N-CG). We performed in vitro studies incubating CA-SG with gamma-GT to determine the products formed, and in vivo studies examining the products excreted in urine after dosing rats with CA-SG or CA. Thus, CA-SG (0.1 mM) was incubated with gamma-GT (0.1 unit/mL) in buffer (pH 7.4, 25 degrees C) and analyzed for products formed by reversed-phase HPLC and electrospray mass spectrometry (ESI/MS). Results showed that CA-SG is degraded completely after 6 h of incubation leading to the formation of two products, CA-N-CG and its disulfide, with no detection of CA-S-CG thioester. After 36 h of incubation, only the disulfide remained in the incubation. Treatment of the disulfide with dithiothreitol led to the reappearance of CA-N-CG. ESI/LC/MS analysis of urine (16 h) extracts of CA-SG-dosed rats (200 mg/kg, iv) showed that CA-SG is degraded to CA-N-CG, CA-N-acyl-cysteine (CA-N-C) and their respective S-methylated products. The mercapturic acid conjugate (CA-SNAC) was found as a minor product. Analysis of urine extracts from CA-dosed rats (200 mg/kg, ip) resulted in the detection of clofibryl-N-acyl-cysteine (CA-N-C), but no evidence for the formation of CA-SNAC was obtained. These in vitro and in vivo experiments indicate that gamma-GT mediated degradation of clofibryl-S-acyl-glutathione leads primarily to the formation and excretion of clofibryl-N-acyl

  9. Lanthanum Tricyanide-Catalyzed Acyl Silane-Ketone Benzoin Additions and Kinetic Resolution of Resultant α-Silyloxyketones

    PubMed Central

    Tarr, James C.

    2010-01-01

    We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic α-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant α-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction. PMID:20392127

  10. Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions and kinetic resolution of resultant alpha-silyloxyketones.

    PubMed

    Tarr, James C; Johnson, Jeffrey S

    2010-05-21

    We report the full account of our efforts on the lanthanum tricyanide-catalyzed acyl silane-ketone benzoin reaction. The reaction exhibits a wide scope in both acyl silane (aryl, alkyl) and ketone (aryl-alkyl, alkyl-alkyl, aryl-aryl, alkenyl-alkyl, alkynyl-alkyl) coupling partners. The diastereoselectivity of the reaction has been examined in both cyclic and acyclic systems. Cyclohexanones give products arising from equatorial attack by the acyl silane. The diastereoselectivity of acyl silane addition to acyclic alpha-hydroxy ketones can be controlled by varying the protecting group to obtain either Felkin-Ahn or chelation control. The resultant alpha-silyloxyketone products can be resolved with selectivity factors from 10 to 15 by subjecting racemic ketone benzoin products to CBS reduction.

  11. Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

    PubMed Central

    Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

    2013-01-01

    Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

  12. The absence of the N-acyl-homoserine-lactone autoinducer synthase genes tral and ngrl increases the copy number of the symbiotic plasmid in Sinorhizobium fredii NGR234

    USDA-ARS?s Scientific Manuscript database

    Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be...

  13. Lanthanum tricyanide-catalyzed acyl silane-ketone benzoin additions.

    PubMed

    Tarr, James C; Johnson, Jeffrey S

    2009-09-03

    Lanthanum tricyanide efficiently catalyzes a benzoin-type coupling between acyl silanes and ketones. Yields range from moderate to excellent over a broad substrate scope encompassing aryl, alkyl, electron-rich, and sterically hindered ketones.

  14. Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

    PubMed

    Wang, Nan; Rudolf, Jeffrey D; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2018-06-04

    Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

  15. Acylated ghrelin concentrations are markedly decreased during pregnancy in mothers with and without gestational diabetes: relationship with cholinesterase.

    PubMed

    Tham, Elaine; Liu, Jianhua; Innis, Sheila; Thompson, David; Gaylinn, Bruce D; Bogarin, Roberto; Haim, Alon; Thorner, Michael O; Chanoine, Jean-Pierre

    2009-05-01

    Acylated (octanoylated) ghrelin stimulates food intake and growth hormone secretion and is deacylated into desacyl ghrelin by butyrylcholinesterase. Acylated and desacyl ghrelin both promote adipogenesis. Ghrelin concentrations decrease with hyperglycemia and hyperinsulinism. We hypothesized that 1) acylated ghrelin increases during pregnancy, contributing positively to energy balance, but is lower in women with gestational diabetes and 2) butyrylcholinesterase activity is inversely correlated with acylated ghrelin concentrations. In a first group of subjects, using two-site sandwich ghrelin assays that specifically detect full-length forms, we investigated women with and without gestational diabetes (n = 14/group) during pregnancy and after delivery. We examined whether changes in ghrelin during a test meal were correlated with changes in pituitary growth hormone [assessed through calculation of the area under the curve (AUC) during the test meal]. In postpartum subjects, the percent of total ghrelin that is acylated was four to five times higher than previously observed using single antibody assays. During pregnancy, acylated ghrelin concentrations (mean +/- SE) were lower compared with the postpartum period throughout the meal (AUC 1.2 +/- 0.2 vs. 10.2 +/- 1.9 ng.ml(-1).90 min(-1), P < 0.001). In the postpartum, acylated ghrelin and growth hormone were positively correlated (r = 0.50, P = 0.007). Desacyl (but not acylated) ghrelin was increased in subjects with gestational diabetes during and after pregnancy (AUC 15.4 +/- 1.9 vs. 8.6 +/- 1.2 ng.ml(-1).90 min(-1), P = 0.005). In a second group of subjects (n = 13), acylated ghrelin was similarly suppressed during pregnancy. Circulating octanoate concentrations (3.1 +/- 0.5 vs. 4.5 +/- 0.6 microg/ml, P = 0.029) and cholinesterase activity (705 +/- 33 vs. 1,013 +/- 56 U/ml, P < 0.001) were lower during pregnancy compared with the postpartum period. In conclusion, acylated ghrelin markedly decreases during pregnancy

  16. Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.

    PubMed

    Vergnolle, Olivia; Xu, Hua; Blanchard, John S

    2013-09-27

    Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

  17. Quorum Sensing Disruption in Vibrio harveyi Bacteria by Clay Materials.

    PubMed

    Naik, Sajo P; Scholin, Jonathon; Ching, San; Chi, Fang; Herpfer, Marc

    2018-01-10

    This work describes the use of clay minerals as catalysts for the degradation of quorum sensing molecule N-(3-oxooctanoyl)-dl-homoserine lactone. Certain clay minerals as a result of their surface properties and porosity can catalytically degrade the quorum sensing molecule into smaller fragments. The disruption of quorum sensing by clay in a growing Gram-negative Vibrio harveyi bacteria culture was also studied by monitoring luminescence and population density of the bacteria, wherein quenching of bacterial quorum sensing activity was observed by means of luminescence reduction. The results of this study show that food-grade clays can be used as biocatalysts in disrupting bacterial activity in various media.

  18. Conformationally Constrained Analogues of Diacylglycerol. 29. Cells Sort Diacylglycerol-Lactone Chemical Zip Codes to Produce Diverse and Selective Biological Activities

    PubMed Central

    Duan, Dehui; Sigano, Dina M.; Kelley, James A.; Lai, Christopher C.; Lewin, Nancy E.; Kedei, Noemi; Peach, Megan L.; Lee, Jeewoo; Abeyweera, Thushara P.; Rotenberg, Susan A.; Kim, Hee; Kim, Young Ho; Kazzouli, Saïd El; Chung, Jae-Uk; Young, Howard A.; Young, Matthew R.; Baker, Alyson; Colburn, Nancy H.; Haimovitz-Friedman, Adriana; Truman, Jean-Philip; Parrish, Damon A.; Deschamps, Jeffrey R.; Perry, Nicholas A.; Surawski, Robert J.; Blumberg, Peter M.; Marquez, Victor E.

    2008-01-01

    Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have termed “chemical zip codes”, are able to transform a relatively small chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these “chemical zip codes”. It is postulated that after binding to protein kinase C (PKC) isozymes or other non-kinase target proteins that contain diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCα to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses. PMID:18698758

  19. Inverse association of des-acyl ghrelin with worksite blood pressure in overweight/obese male workers.

    PubMed

    Narisada, Akihiko; Hasegawa, Tomomi; Nakahigashi, Maki; Hirobe, Takaaki; Ikemoto, Tatsunori; Ushida, Takahiro; Kobayashi, Fumio

    2015-05-01

    Job strain, defined as a combination of high job demands and low job control, has been reported to elevate blood pressure (BP) during work. Meanwhile, a recent experimental study showed that ghrelin blunted the BP response to such mental stress. In the present study, we examined the hypothesis that des-acyl ghrelin may have some beneficial effects on worksite BP through modulating the BP response to work-related mental stress, i.e., job strain. Subjects were 34 overweight/obese male day-shift workers (mean age 41.7 ± 6.7 years). No subjects had received any anti-hypertensive medication. A 24-h ambulatory BP monitoring was recorded every 30 min on a regular working day. The average BP was calculated for Work BP, Morning BP, and Home BP. Job strain was assessed using the short version of the Japanese Job Content Questionnaire. Des-acyl ghrelin showed significant inverse correlations with almost all BPs except Morning SBP, Morning DBP, and Home DBP. In multiple regression analysis, des-acyl ghrelin inversely correlated with Work SBP after adjusting for confounding factors. Des-acyl ghrelin was also negatively associated with BP changes from Sleep to Morning, Sleep to Work, and Sleep to Home. Des-acyl ghrelin was inversely associated with Worksite BP, suggesting a unique beneficial effect of des-acyl ghrelin on Worksite BP in overweight/obese male day-shift workers.

  20. Kinetics of acyl transfer reactions in organic media catalysed by Candida antarctica lipase B.

    PubMed

    Martinelle, M; Hult, K

    1995-09-06

    The acyl transfer reactions catalysed by Candida antartica lipase B in organic media followed a bi-bi ping-pong mechanism, with competitive substrate inhibition by the alcohols used as acyl acceptors. The effect of organic solvents on Vm and Km was investigated. The Vm values in acetonitrile was 40-50% of those in heptane. High Km values in acetonitrile compared to those in heptane could partly be explained by an increased solvation of the substrates in acetonitrile. Substrate solvation caused a 10-fold change in substrate specificity, defined as (Vm/Km)ethyl octanoate/(Vm/Km)octanoic acid, going from heptane to acetonitrile. Deacylation was the rate determining step for the acyl transfer in heptane with vinyl- and ethyl octanoate as acyl donors and (R)-2-octanol as acyl acceptor. With 1-octanol, a rate determining deacylation step in heptane was indicated using the same acyl donors. Using 1-octanol as acceptor in heptane, S-ethyl thiooctanoate had a 25- to 30-fold lower Vm/Km value and vinyl octanoate a 4-fold higher Vm/Km value than that for ethyl octanoate. The difference showed to be a Km effect for vinyl octanoate and mainly a Km effect for S-ethyl thiooctanoate. The Vm values of the esterification of octanoic acid with different alcohols was 10-30-times lower than those for the corresponding transesterification of ethyl octanoate. The low activity could be explained by a low pH around the enzyme caused by the acid or a withdrawing of active enzyme by nonproductive binding by the acid.