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Sample records for aeruginosa extracellular products

  1. Phenazine production enhances extracellular DNA release via hydrogen peroxide generation in Pseudomonas aeruginosa

    PubMed Central

    Das, Theerthankar; Manefield, Mike

    2013-01-01

    In Pseudomonas aeruginosa eDNA is a crucial component essential for biofilm formation and stability. In this study we report that release of eDNA is influenced by the production of phenazine in P. aeruginosa. A ∆phzA-G mutant of P. aeruginosa PA14 deficient in phenazine production generated significantly less eDNA in comparison with the phenazine producing strains. The relationship between eDNA release and phenazine production is bridged via hydrogen peroxide (H2O2) generation and subsequent H2O2 mediated cell lysis and ultimately release of chromosomal DNA into the extracellular environment as eDNA. PMID:23710274

  2. Abiotic factors in colony formation: effects of nutrition and light on extracellular polysaccharide production and cell aggregates of Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Yang, Zhen; Kong, Fanxiang

    2013-07-01

    Colony morphology is important for Microcystis to sustain a competitive advantage in eutrophic lakes. The mechanism of colony formation in Microcystis is currently unclear. Extracellular polysaccharide (EPS) has been reported to play an important role in cell aggregate formation of some phytoplankton. Microcystis aeruginosa was cultivated under varied abiotic conditions, including different nutrient, light, and temperature conditions, to investigate their effects on EPS production and morphological change. The results show that nutrient concentration and light intensity have great effects on EPS productionin M. aeruginosa. There was a considerable increase in EPS production after M. aeruginosa was cultivated in adjusted culture conditions similar to those present in the field (28.9 mg C/L, 1.98 mg N/L, 0.65 mg P/L, light intensity: 100 μmol/(m2 · s)). These results indicate that abiotic factors might be one of the triggers for colony formation in Microcystis.

  3. Effects of iron on growth, antioxidant enzyme activity, bound extracellular polymeric substances and microcystin production of Microcystis aeruginosa FACHB-905.

    PubMed

    Wang, Chao; Wang, Xun; Wang, Peifang; Chen, Bin; Hou, Jun; Qian, Jin; Yang, Yangyang

    2016-10-01

    Toxic cyanobacterial blooms have occurred in various water bodies during recent decades and made serious health hazards to plants, animals and humans. Iron is an important micronutrient for algal growth and recently, the concentration of which has increased remarkably in freshwaters. In this paper, the cyanobacterium Microcystis aeruginosa FACHB-905 was cultivated under non-iron (0μM), iron-limited (10μM) and iron-replete (100μM) conditions to investigate the effects of iron on growth, antioxidant enzyme activity, EPS and microcystin production. The results showed that algal cell density and chlorophyll-a content were maximal at the highest iron concentration. Antioxidant enzymes activity increased notably under all three conditions in the early stage of experiment, of which the SOD activity recovered soon from oxidative stress in 10μM group. The productions of some protein-like substances and humic acid-like substances of bound EPS were inhibited in iron-containing groups in the early stage of experiment while promoted after the adaptation period of Microcystis aeruginosa. Iron addition is a factor affecting the formation of cyanobacterial blooms through its impact on the content of LB-EPS and the composition of TB-EPS. The intracellular MC-LR concentration and the productivity potential of MC-LR were the lowest in 0μM group and highest in 10μM group. No obvious extracellular release of MC-LR was observed during the cultivation time. Therefore, iron addition can promote the physiological activities of M. aeruginosa, but a greater harm could be brought into environment under iron-limited (10μM) condition than under iron-replete (100μM) condition. PMID:27337497

  4. Effects of iron on growth, antioxidant enzyme activity, bound extracellular polymeric substances and microcystin production of Microcystis aeruginosa FACHB-905.

    PubMed

    Wang, Chao; Wang, Xun; Wang, Peifang; Chen, Bin; Hou, Jun; Qian, Jin; Yang, Yangyang

    2016-10-01

    Toxic cyanobacterial blooms have occurred in various water bodies during recent decades and made serious health hazards to plants, animals and humans. Iron is an important micronutrient for algal growth and recently, the concentration of which has increased remarkably in freshwaters. In this paper, the cyanobacterium Microcystis aeruginosa FACHB-905 was cultivated under non-iron (0μM), iron-limited (10μM) and iron-replete (100μM) conditions to investigate the effects of iron on growth, antioxidant enzyme activity, EPS and microcystin production. The results showed that algal cell density and chlorophyll-a content were maximal at the highest iron concentration. Antioxidant enzymes activity increased notably under all three conditions in the early stage of experiment, of which the SOD activity recovered soon from oxidative stress in 10μM group. The productions of some protein-like substances and humic acid-like substances of bound EPS were inhibited in iron-containing groups in the early stage of experiment while promoted after the adaptation period of Microcystis aeruginosa. Iron addition is a factor affecting the formation of cyanobacterial blooms through its impact on the content of LB-EPS and the composition of TB-EPS. The intracellular MC-LR concentration and the productivity potential of MC-LR were the lowest in 0μM group and highest in 10μM group. No obvious extracellular release of MC-LR was observed during the cultivation time. Therefore, iron addition can promote the physiological activities of M. aeruginosa, but a greater harm could be brought into environment under iron-limited (10μM) condition than under iron-replete (100μM) condition.

  5. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Das, Theerthankar; Kutty, Samuel K.; Tavallaie, Roya; Ibugo, Amaye I.; Panchompoo, Janjira; Sehar, Shama; Aldous, Leigh; Yeung, Amanda W. S.; Thomas, Shane R.; Kumar, Naresh; Gooding, J. Justin; Manefield, Mike

    2015-01-01

    Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation. PMID:25669133

  6. The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

    PubMed

    Qaisar, Uzma; Kruczek, Cassandra J; Azeem, Muhammed; Javaid, Nasir; Colmer-Hamood, Jane A; Hamood, Abdul N

    2016-08-01

    Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope. PMID:27480638

  7. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

    PubMed Central

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard

    2015-01-01

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid l-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype. PMID:26552982

  8. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard; Lewenza, Shawn

    2016-01-01

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype. PMID:26552982

  9. Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Charron-Mazenod, Laetitia; Moore, Richard; Lewenza, Shawn

    2015-11-09

    Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

  10. Extracellular biogenic nanomaterials inhibit pyoverdine production in Pseudomonas aeruginosa: a novel insight into impacts of metal(loid)s on environmental bacteria.

    PubMed

    Mohanty, Anee; Liu, Yang; Yang, Liang; Cao, Bin

    2015-02-01

    Anthropogenic activities such as mining, smelting, and industrial use have caused serious problems of metal(loid) pollution in nearly every country in the world. A wide range of environmental microorganisms are capable of transforming metal(loid)s into nanomaterials, i.e., biogenic nanomaterials (bio-NMs), in the environment. Although the impacts of various metal(loid)s on the ecosystems have been extensively studied, the potential influence of the bio-NMs generated in the environment to environmental organisms is largely unexplored. Using tellurium nanomaterials transformed from tellurite by a metal-reducing bacterium as model bio-NMs, we demonstrated that the bio-NMs significantly decreased siderophore production in an environmental bacterium Pseudomonas aeruginosa in both planktonic cultures and biofilms. Transcriptomic analysis revealed that the bio-NMs inhibited the expression of genes involved in biosynthesis and transport of siderophores. Siderophores secreted by certain bacteria in microbial communities can be considered as public goods that can be exploited by local communities, playing an important role in shaping microbial communities. The inhibition of siderophore production by the bio-NMs implies that bio-NMs may have an important influence on the ecosystems through altering specific functions of environmental bacteria. Taken together, this study provides a novel insight into the environmental impacts of metal(loid)s. PMID:25273177

  11. Biodegradation and extracellular enzymatic activities of Pseudomonas aeruginosa strain GF31 on β-cypermethrin.

    PubMed

    Tang, Aixing; Wang, Bowen; Liu, Youyan; Li, Qingyun; Tong, Zhangfa; Wei, Yingjun

    2015-09-01

    Pseudomonas aeruginosa strain GF31, isolated from a contaminated soil, can effectively degrade β-cypermethrin (β-CP), as well as fenpropathrin, fenvalerate, and cyhalothrin. The highest level of degradation (81.2 %) was achieved with the addition of peptone. Surprisingly, the enzyme responsible for degradation was mainly localized to the extracellular areas of the bacteria, in contrast to the other known pyrethroid-degrading enzymes, which are intracellular. Although intact bacterial cells function at about 30 °C for biodegradation, similar to other degrading strains, the crude extracellular extract of strain GF31 remained biologically active at 60 °C. Moreover, the extract fraction showed good storage stability, maintaining >50 % of its initial activity following storage at 25 °C for at least 20 days. Significant differences in the characteristics of the crude GF31 extracellular extract compared with the known pyrethroid-degrading enzymes indicate the presence of a novel pyrethroid-degrading enzyme. Furthermore, the identification of 3-phenoxybenzoic acid and 2,2-dimethylcyclopropanecarboxylate from the degradation products suggests the possibility that β-CP degradation by both the strain and the crude extracellular fraction is achieved through a hydrolysis pathway. Further degradation of these two metabolites may lead to the development of an efficient method for the mineralization of these types of pollutants.

  12. Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    Background As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa. Results Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain. Conclusion We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed

  13. Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Mulcahy, Heidi; Charron-Mazenod, Laetitia; Lewenza, Shawn

    2008-01-01

    Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation. PMID:19023416

  14. Effects of sulfate on microcystin production, photosynthesis, and oxidative stress in Microcystis aeruginosa.

    PubMed

    Chen, Lei; Gin, Karina Y H; He, Yiliang

    2016-02-01

    Increasing sulfate in freshwater systems, caused by human activities and climate change, may have negative effects on aquatic organisms. Microcystis aeruginosa (M. aeruginosa) is both a major primary producer and a common toxic cyanobacterium, playing an important role in the aquatic environment. This study first investigated the effects of sulfate on M. aeruginosa. The experiment presented here aims at analyzing the effects of sulfate on physiological indices, molecular levels, and its influencing mechanism. The results of our experiment showed that sulfate (at 40, 80, and 300 mg L(-1)) inhibited M. aeruginosa growth, increased both intracellular and extracellular toxin contents, and enhanced the mcyD transcript level. Sulfate inhibited the photosynthesis of M. aeruginosa, based on the decrease in pigment content and the down-regulation of photosynthesis-related genes after sulfate exposure. Furthermore, sulfate decreased the maximum electron transport rate, causing the cell to accumulate surplus electrons and form reactive oxygen species (ROS). Sulfate also increased the malondialdehyde (MDA) content, which showed that sulfate damaged the cytomembrane. This damage contributed to the release of intracellular toxin to the culture medium. Although sulfate increased superoxide dismutase (SOD) activities, expression of sod, and total antioxidant capacity in M. aeruginosa, it still overwhelmed the antioxidant system since the ROS level simultaneously increased, and finally caused oxidative stress. Our results indicate that sulfate has direct effects on M. aeruginosa, inhibits photosynthesis, causes oxidative stress, increases toxin production, and affects the related genes expression in M. aeruginosa. PMID:26490939

  15. Effects of sulfate on microcystin production, photosynthesis, and oxidative stress in Microcystis aeruginosa.

    PubMed

    Chen, Lei; Gin, Karina Y H; He, Yiliang

    2016-02-01

    Increasing sulfate in freshwater systems, caused by human activities and climate change, may have negative effects on aquatic organisms. Microcystis aeruginosa (M. aeruginosa) is both a major primary producer and a common toxic cyanobacterium, playing an important role in the aquatic environment. This study first investigated the effects of sulfate on M. aeruginosa. The experiment presented here aims at analyzing the effects of sulfate on physiological indices, molecular levels, and its influencing mechanism. The results of our experiment showed that sulfate (at 40, 80, and 300 mg L(-1)) inhibited M. aeruginosa growth, increased both intracellular and extracellular toxin contents, and enhanced the mcyD transcript level. Sulfate inhibited the photosynthesis of M. aeruginosa, based on the decrease in pigment content and the down-regulation of photosynthesis-related genes after sulfate exposure. Furthermore, sulfate decreased the maximum electron transport rate, causing the cell to accumulate surplus electrons and form reactive oxygen species (ROS). Sulfate also increased the malondialdehyde (MDA) content, which showed that sulfate damaged the cytomembrane. This damage contributed to the release of intracellular toxin to the culture medium. Although sulfate increased superoxide dismutase (SOD) activities, expression of sod, and total antioxidant capacity in M. aeruginosa, it still overwhelmed the antioxidant system since the ROS level simultaneously increased, and finally caused oxidative stress. Our results indicate that sulfate has direct effects on M. aeruginosa, inhibits photosynthesis, causes oxidative stress, increases toxin production, and affects the related genes expression in M. aeruginosa.

  16. Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms.

    PubMed

    Pezzoni, Magdalena; Pizarro, Ramón A; Costa, Cristina S

    2014-02-01

    One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA(+) strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment. PMID:24491420

  17. Variation in assimilable organic carbon formation during chlorination of Microcystis aeruginosa extracellular organic matter solutions.

    PubMed

    Sun, Xingbin; Yuan, Ting; Ni, Huishan; Li, Yanpeng; Hu, Yang

    2016-07-01

    This study investigated the chlorination of Microcystis aeruginosa extracellular organic matter (EOM) solutions under different conditions, to determine how the metabolites produced by these organisms affect water safety and the formation of assimilable organic carbon (AOC). The effects of chlorine dosages, coagulant dosage, reaction time and temperature on the formation of AOC were investigated during the disinfection of M.aeruginosa metabolite solutions. The concentration of AOC followed a decreasing and then increasing pattern with increasing temperature and reaction time. The concentration of AOC decreased and then increased with increasing chlorination dosage, followed by a slight decrease at the highest level of chlorination. However, the concentration of AOC decreased continuously with increasing coagulant dosage. The formation of AOC can be suppressed under appropriate conditions. In this study, chlorination at 4mg/L, combined with a coagulant dose of 40mg/L at 20°C over a reaction time of 12hr, produced the minimum AOC. PMID:27372113

  18. Imaging Pseudomonas aeruginosa Biofilm Extracellular Polymer Scaffolds with Amphiphilic Carbon Dots.

    PubMed

    Ritenberg, Margarita; Nandi, Sukhendu; Kolusheva, Sofiya; Dandela, Rambabu; Meijler, Michael M; Jelinek, Raz

    2016-05-20

    Biofilm formation is a critical facet of pathogenesis and resilience of human, animal, and plant bacteria. Extracellular polymeric substances (EPS) constitute the physical scaffolding for bacterial biofilms and thus play central roles in their development and virulence. We show that newly synthesized amphiphilic fluorescent carbon dots (C-dots) readily bind to the EPS scaffold of Pseudomonas aeruginosa, a major biofilm-forming pathogen, resulting in unprecedented microscopic visualization of the EPS structural features. Fluorescence microscopy analysis utilizing the C-dots reveals that the P. aeruginosa EPS matrix exhibits a remarkable dendritic morphology. The experiments further illuminate the growth kinetics of the EPS and the effect of external factors such as temperature. We also show that the amphiphilic C-dot platform enabled screening of substances disrupting biofilm development, specifically quorum sensing inhibitors. PMID:26882175

  19. Optimization of process parameters influencing the submerged fermentation of extracellular lipases from Pseudomonas aeruginosa, candida albicans and Aspergillus flavus.

    PubMed

    Padhiar, Jigita; Das, Arijit; Bhattacharya, Sourav

    2011-11-15

    The present study was aimed at optimization, production and partial purification of lipases from Pseudomonas aeruginosa, Candida albicans and Aspergillus flavus. Various nutritional and physical parameters affecting lipase production such as carbon and nitrogen supplements, pH, temperature, agitation speed and incubation time were studied. Refined sunflower oil (1% v/v) and tryptone at a pH of 6.2 favored maximum lipase production in Pseudomonas at 30 degrees C and 150 rpm, when incubated for 5 days. In C. albicans refined sunflower oil (3% v/v) and peptone resulted in maximum lipase production at pH 5.2, 30 degrees C and 150 rpm, when incubated for 5 days. In A. flavus coconut oil (3% v/v) and peptone yielded maximum lipase at pH 6.2, 37 degrees C, 200 rpm after an incubation period of 5 days. The lipases were partially purified by ammonium sulphate precipitation and dialysis. In P. aeruginosa enzyme activity of the dialyzed fraction was found to be 400 U mL-' and for C. albicans 410 U mL(-1). The dialysed lipase fraction from A. flavus demonstrated an activity of 460 U mL(-1). The apparent molecular weights of the dialyzed lipases were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dialyzed lipase fraction obtained from P. aeruginosa revealed molecular weights of 47, 49 and 51 kDa, whereas, lipases from C. albicans and A. flavus demonstrated 3 bands (16.5, 27 and 51 kDa) and one band (47 kDa), respectively. These extracellular lipases may find wide industrial applications.

  20. Optimization of process parameters influencing the submerged fermentation of extracellular lipases from Pseudomonas aeruginosa, candida albicans and Aspergillus flavus.

    PubMed

    Padhiar, Jigita; Das, Arijit; Bhattacharya, Sourav

    2011-11-15

    The present study was aimed at optimization, production and partial purification of lipases from Pseudomonas aeruginosa, Candida albicans and Aspergillus flavus. Various nutritional and physical parameters affecting lipase production such as carbon and nitrogen supplements, pH, temperature, agitation speed and incubation time were studied. Refined sunflower oil (1% v/v) and tryptone at a pH of 6.2 favored maximum lipase production in Pseudomonas at 30 degrees C and 150 rpm, when incubated for 5 days. In C. albicans refined sunflower oil (3% v/v) and peptone resulted in maximum lipase production at pH 5.2, 30 degrees C and 150 rpm, when incubated for 5 days. In A. flavus coconut oil (3% v/v) and peptone yielded maximum lipase at pH 6.2, 37 degrees C, 200 rpm after an incubation period of 5 days. The lipases were partially purified by ammonium sulphate precipitation and dialysis. In P. aeruginosa enzyme activity of the dialyzed fraction was found to be 400 U mL-' and for C. albicans 410 U mL(-1). The dialysed lipase fraction from A. flavus demonstrated an activity of 460 U mL(-1). The apparent molecular weights of the dialyzed lipases were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dialyzed lipase fraction obtained from P. aeruginosa revealed molecular weights of 47, 49 and 51 kDa, whereas, lipases from C. albicans and A. flavus demonstrated 3 bands (16.5, 27 and 51 kDa) and one band (47 kDa), respectively. These extracellular lipases may find wide industrial applications. PMID:22514878

  1. Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation.

    PubMed

    Das, Theerthankar; Kutty, Samuel K; Kumar, Naresh; Manefield, Mike

    2013-01-01

    Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions. PMID:23505483

  2. Extracellular polymeric substances facilitate the biosorption of phenanthrene on cyanobacteria Microcystis aeruginosa.

    PubMed

    Bai, Leilei; Xu, Huacheng; Wang, Changhui; Deng, Jiancai; Jiang, Helong

    2016-11-01

    Phytoplankton-derived extracellular polymeric substances (EPS) are of vital importance for the biogeochemical cycles of hydrophobic organic pollutants in lake ecosystems. In this study, roles of loosely-bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) in biosorption of phenanthrene (PHE) on a typical cyanobacteria Microcystis aeruginosa were investigated. The results showed that the biosorption of PHE on M. aeruginosa cell varied lasted 24 h, while the binding of PHE to LB-EPS and TB-EPS reached equilibrium within less than 2 h. The equilibrium biosorption capacities of M. aeruginosa cell, LB-EPS and TB-EPS were 6.78, 12.31, and 9.47 μg mg(-1), respectively, indicating that the binding of PHE to EPS was a considerable process involved in biosorption. Fluorescence quenching titration revealed that increasing temperature induced more binding sites in EPS for PHE and the binding process was driven by electrostatic force and hydrophobic interactions. Interestingly, dynamic and static quenching processes occurred simultaneously for the binding of PHE to protein-like substances in EPS, whereas the binding of PHE to humic-like substances belonged to static quenching. The relatively higher contents of proteins in LB-EPS produced a stronger binding capacity of PHE. Overall, the interactions between hydrophobic organic pollutants and cyanobacterial EPS are favorable to the bioaccumulation of hydrophobic organic pollutants in cyanobacteria and facilitate the regulatory function of cyanobacterial biomass as a biological pump. PMID:27497347

  3. Development of potent inhibitors of pyocyanin production in Pseudomonas aeruginosa

    PubMed Central

    Miller, Laura C.; O’Loughlin, Colleen T.; Zhang, Zinan; Siryaporn, Albert; Silpe, Justin E.; Bassler, Bonnie L.; Semmelhack, Martin F.

    2015-01-01

    The development of new approaches for the treatment of antimicrobial-resistant infections is an urgent public health priority. The Pseudomonas aeruginosa pathogen, in particular, is a leading source of infection in hospital settings, with few available treatment options. In the context of an effort to develop antivirulence strategies to combat bacterial infection, we identified a series of highly effective small molecules that inhibit the production of pyocyanin, a redox-active virulence factor produced by P. aeruginosa. Interestingly, these new antagonists appear to suppress P. aeruginosa virulence factor production through a pathway that is independent of LasR and RhlR. PMID:25597392

  4. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

  5. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  6. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

    PubMed Central

    Hoyne, P A; Haas, R; Meyer, T F; Davies, J K; Elleman, T C

    1992-01-01

    Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell. Images PMID:1358873

  7. Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

    PubMed Central

    Wiens, Jacinta R.; Vasil, Adriana I.; Schurr, Michael J.; Vasil, Michael L.

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. PMID:24496793

  8. Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa.

    PubMed

    Penterman, Jon; Singh, Pradeep K; Walker, Graham C

    2014-09-01

    LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.

  9. Biological Cost of Pyocin Production during the SOS Response in Pseudomonas aeruginosa

    PubMed Central

    Singh, Pradeep K.

    2014-01-01

    LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs. PMID:25022851

  10. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  11. Iron Depletion Enhances Production of Antimicrobials by Pseudomonas aeruginosa

    PubMed Central

    Nguyen, Angela T.; Jones, Jace W.; Ruge, Max A.; Kane, Maureen A.

    2015-01-01

    ABSTRACT Cystic fibrosis (CF) is a heritable disease characterized by chronic, polymicrobial lung infections. While Staphylococcus aureus is the dominant lung pathogen in young CF patients, Pseudomonas aeruginosa becomes predominant by adulthood. P. aeruginosa produces a variety of antimicrobials that likely contribute to this shift in microbial populations. In particular, secretion of 2-alkyl-4(1H)-quinolones (AQs) contributes to lysis of S. aureus in coculture, providing an iron source to P. aeruginosa both in vitro and in vivo. We previously showed that production of one such AQ, the Pseudomonas quinolone signal (PQS), is enhanced by iron depletion and that this induction is dependent upon the iron-responsive PrrF small RNAs (sRNAs). Here, we demonstrate that antimicrobial activity against S. aureus during coculture is also enhanced by iron depletion, and we provide evidence that multiple AQs contribute to this activity. Strikingly, a P. aeruginosa ΔprrF mutant, which produces very little PQS in monoculture, was capable of mediating iron-regulated growth suppression of S. aureus. We show that the presence of S. aureus suppresses the ΔprrF1,2 mutant's defect in iron-regulated PQS production, indicating that a PrrF-independent iron regulatory pathway mediates AQ production in coculture. We further demonstrate that iron-regulated antimicrobial production is conserved in multiple P. aeruginosa strains, including clinical isolates from CF patients. These results demonstrate that iron plays a central role in modulating interactions of P. aeruginosa with S. aureus. Moreover, our studies suggest that established iron regulatory pathways of these pathogens are significantly altered during polymicrobial infections. IMPORTANCE Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are a significant cause of morbidity and mortality, as the interplay between these two organisms exacerbates infection. This is in part due to enhanced

  12. Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance.

    PubMed

    Braud, Armelle; Geoffroy, Valérie; Hoegy, Françoise; Mislin, Gaëtan L A; Schalk, Isabelle J

    2010-06-01

    In order to get access to iron, Pseudomonas aeruginosa strain PAO1 produces two major siderophores pyoverdine (PVD) and pyochelin (PCH). Both siderophores are able to chelate many other metals in addition to iron. However, despite this property, only iron is transported efficiently into the bacteria by the PVD and PCH uptake pathways. Growth studies with P. aeruginosa strains showed a lower sensitivity to toxic metals for the siderophore-producing strain than for the mutants unable to produce siderophores. Moreover, addition of PVD or PCH to the growth medium of a siderophore-deficient strain considerably reduced the toxicity of toxic metals present at concentrations of 100 µM in iron-limited and iron-supplemented growth conditions. Measurement by Inductively Coupled Plasma-Atomic Emission Spectrometry of the concentration of metals present in bacteria incubated with metals in the presence or absence of PVD or PCH indicated that both siderophores were able to sequester metals from the extracellular medium of the bacteria, decreasing metal diffusion into the bacteria. Pyoverdine was able to sequester Al(3+) , Co(2+) , Cu(2+) , Eu(3+) , Ni(2+) , Pb(2+) , Tb(3+) and Zn(2+) from the extracellular medium, and PCH, Al(3+) , Co(2+) , Cu(2+) , Ni(2+) , Pb(2+) and Zn(2+) . Moreover, the presence of 100 µM Cu(2+) and Ni(2+) increased PVD production by 290% and 380%, respectively, in a medium supplemented with iron. All these data suggest that PVD and PCH may contribute to P. aeruginosa resistance to heavy metals. PMID:23766115

  13. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa

    PubMed Central

    Wang, Shoubing; Xu, Ziran

    2016-01-01

    Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice. PMID:27755566

  14. Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants

    SciTech Connect

    Koch, A.K.; Fiechter, A.; Reiser, J. ); Kaeppeli, O. )

    1991-07-01

    The authors isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up {sup 14}C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C{sub 12} to C{sub 19}. However, growth on these alkanes and uptake of ({sup 14}C)hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up ({sup 14}C)hexadecane uptake. The addition of small amounts of rhamnolipids restored on alkanes and ({sup 14}C)hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P.aeruginosa.

  15. Antimicrobial targets localize to the extracellular vesicle-associated proteome of Pseudomonas aeruginosa grown in a biofilm.

    PubMed

    Park, Amber J; Surette, Matthew D; Khursigara, Cezar M

    2014-01-01

    Microbial biofilms are particularly resistant to antimicrobial therapies. These surface-attached communities are protected against host defenses and pharmacotherapy by a self-produced matrix that surrounds and fortifies them. Recent proteomic evidence also suggests that some bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, undergo modifications within a biofilm that make them uniquely resistant compared to their planktonic (free-living) counterparts. This study examines 50 proteins in the resistance subproteome of both surface-associated and free-living P. aeruginosa PAO1 over three time points. Proteins were grouped into categories based on their roles in antimicrobial: (i) binding, (ii) efflux, (iii) resistance, and (iv) susceptibility. In addition, the extracellular outer membrane vesicle-associated proteome is examined and compared between the two growth modes. We show that in whole cells between 12-24% of the proteins are present at significantly different abundance levels over time, with some proteins being unique to a specific growth mode; however, the total abundance levels in the four categories remain consistent. In contrast, marked differences are seen in the protein content of the outer membrane vesicles, which contain a greater number of drug-binding proteins in vesicles purified from late-stage biofilms. These results show how the method of analysis can impact the interpretation of proteomic data (i.e., individual proteins vs. systems), and highlight the advantage of using protein-based methods to identify potential antimicrobial resistance mechanisms in extracellular sample components. Furthermore, this information has the potential to inform the development of specific antipseudomonal therapies that quench possible drug-sequestering vesicle proteins. This strategy could serve as a novel approach for combating the high-level of antimicrobial resistance in P. aeruginosa biofilms.

  16. Elastase and alkaline protease production by Pseudomonas aeruginosa strains: comparison of two procedures.

    PubMed

    Yagci, A; Tuc, Y; Soyletir, G

    2002-04-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can cause fatal infections in immunocompromised hosts. The virulence of P. aeruginosa is associated with the presence of various extracellular factors like elastase and alkaline protease. These enzymes are suggested to contribute to tissue destruction and assist bacterial invasion during infection. Therefore it seems likely that determination of these virulence factors will be an important prognostic marker in the near future especially for follow up of cystic fibrosis patients, to start antimicrobial agents that are directly or indirectly inhibit microbial growth or virulence factor production. Herein, we suggest a simple test procedure to be used in routine laboratories for estimation of elastase and alkaline protease levels and compare them with quantitative methods in the literature. We detected the amount of elastase and alkaline protease in 49 clinical P. aeruginosa isolates by comparing agar plate method and colorimetric assay. The resulting values were in the range reported in the literature and differed from one strain to another(elastase: 0-1390 mg/ml, alkaline protease: 0- 770 mg/ml). Linear relationships were found when assays compared in pairs and significant correlation coefficients were obtained(r>0.788 for alkaline protease, p<0.0001- r>0.926 for elastase, p<0.0001). Our method can be applied in laboratories regardless of the availability of technical equipment.

  17. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1

    PubMed Central

    Pereira Jr, Nei; Freire, Denise M.G.

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L−1–10.9 g L−1). These results offer promising pathways for the optimization of processes for the production of rhamnolipids. PMID:27257553

  18. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1.

    PubMed

    Soares Dos Santos, Alexandre; Pereira, Nei; Freire, Denise M G

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L(-1)-10.9 g L(-1)). These results offer promising pathways for the optimization of processes for the production of rhamnolipids.

  19. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1.

    PubMed

    Soares Dos Santos, Alexandre; Pereira, Nei; Freire, Denise M G

    2016-01-01

    Rhamnolipids are biosurfactants with potential for diversified industrial and environmental uses. The present study evaluated three strategies for increasing the production of rhamnolipid-type biosurfactants produced by Pseudomonas aeruginosa strain PA1. The influence of pH, the addition of P. aeruginosa spent culture medium and the use of a fed-batch process were examined. The culture medium adjusted to pH 7.0 was the most productive. Furthermore, the pH of the culture medium had a measurable effect on the ratio of synthesized mono- and dirhamnolipids. At pH values below 7.3, the proportion of monorhamnolipids decreased from 45 to 24%. The recycling of 20% of the spent culture medium in where P. aeruginosa was grown up to the later stationary phase was responsible for a 100% increase in rhamnolipid volumetric productivity in the new culture medium. Finally, the use of fed-batch operation under conditions of limited nitrogen resulted in a 3.8-fold increase in the amount of rhamnolipids produced (2.9 g L(-1)-10.9 g L(-1)). These results offer promising pathways for the optimization of processes for the production of rhamnolipids. PMID:27257553

  20. Azithromycin increases in vitro fibronectin production through interactions between macrophages and fibroblasts stimulated with Pseudomonas aeruginosa

    PubMed Central

    Cory, Theodore J.; Birket, Susan E.; Murphy, Brian S.; Mattingly, Cynthia; Breslow-Deckman, Jessica M.; Feola, David J.

    2013-01-01

    Objectives Chronic azithromycin therapy has been associated with improved clinical outcomes in patients with cystic fibrosis (CF) who are chronically infected with Pseudomonas aeruginosa. We have previously demonstrated that azithromycin polarizes macrophages towards an alternatively activated phenotype, thereby blunting inflammation associated with infection. Because this phenotype is pro-fibrotic, it is important to evaluate azithromycin's consequential effects upon fibroblast function and extracellular matrix (ECM) protein production. Methods We co-cultured macrophages and fibroblasts together and stimulated them by adding P. aeruginosa or lipopolysaccharide to assess the ability of azithromycin to alter the macrophage phenotype, along with the impact exerted upon the production of fibronectin and other effectors that govern tissue remodelling, including transforming growth factor β (TGFβ), matrix metalloproteinase-9 (MMP-9) and arginase. We supported these studies by evaluating the impact of azithromycin treatment on these proteins in a mouse model of P. aeruginosa infection. Results Azithromycin increased arginase expression in vitro, as well as the activation of latent TGFβ, consistent with polarization to the alternative macrophage phenotype. While the drug increased fibronectin concentrations after stimulation in vitro, secretion of the ECM-degrading enzyme MMP-9 was also increased. Neutralization of active TGFβ resulted in the ablation of azithromycin's ability to increase fibronectin concentrations, but did not alter its ability to increase MMP-9 expression. In P. aeruginosa-infected mice, azithromycin significantly decreased MMP-9 and fibronectin concentrations in the alveolar space compared with non-treated, infected controls. Conclusions Our results suggest that azithromycin's effect on MMP-9 is regulated independently of TGFβ activity. Additionally, the beneficial effects of azithromycin may be partially due to effects on homeostasis in which ECM

  1. Gallium induces the production of virulence factors in Pseudomonas aeruginosa.

    PubMed

    García-Contreras, Rodolfo; Pérez-Eretza, Berenice; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Coria-Jiménez, Rafael; Rangel-Vega, Adrián; Maeda, Toshinari; Wood, Thomas K

    2014-02-01

    The novel antimicrobial gallium is a nonredox iron III analogue with bacteriostatic and bactericidal properties, effective for the treatment of Pseudomonas aeruginosa in vitro and in vivo in mouse and rabbit infection models. It interferes with iron metabolism, transport, and presumably its homeostasis. As gallium exerts its antimicrobial effects by competing with iron, we hypothesized that it ultimately will lead cells to an iron deficiency status. As iron deficiency promotes the expression of virulence factors in vitro and promotes the pathogenicity of P. aeruginosa in animal models, it is anticipated that treatment with gallium will also promote the production of virulence factors. To test this hypothesis, the reference strain PA14 and two clinical isolates from patients with cystic fibrosis were exposed to gallium, and their production of pyocyanin, rhamnolipids, elastase, alkaline protease, alginate, pyoverdine, and biofilm was determined. Gallium treatment induced the production of all the virulence factors tested in the three strains except for pyoverdine. In addition, as the Ga-induced virulence factors are quorum sensing controlled, co-administration of Ga and the quorum quencher brominated furanone C-30 was assayed, and it was found that C-30 alleviated growth inhibition from gallium. Hence, adding both C-30 and gallium may be more effective in the treatment of P. aeruginosa infections.

  2. Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms.

    PubMed

    Couto, Narciso; Schooling, Sarah R; Dutcher, John R; Barber, Jill

    2015-10-01

    In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations. PMID:26303878

  3. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix

    PubMed Central

    Jennings, Laura K.; Storek, Kelly M.; Ledvina, Hannah E.; Coulon, Charlène; Marmont, Lindsey S.; Sadovskaya, Irina; Secor, Patrick R.; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J.; Howell, P. Lynne; Parsek, Matthew R.

    2015-01-01

    Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components. PMID:26311845

  4. Extracellular polysaccharide production by thraustochytrid protists.

    PubMed

    Jain, Ruchi; Raghukumar, Seshagiri; Tharanathan, R; Bhosle, N B

    2005-01-01

    Four strains of marine stramenopilan protists, the thraustochytrids, were studied for their ability to produce extracellular polysaccharides (EPSs). Observations by light and scanning electron microscopy revealed the production of a matrix of EPS around groups of cells in stationary cultures. EPS in shake culture filtrates ranged from 0.3 to 1.1 g/L. EPS production, which was studied in greater detail in 2 isolates, SC-1 and CW1, increased with age of cultures, reaching a peak in the stationary phase. Anion exchange chromatography yielded a single fraction of the EPS of both species. The EPS contained 39% to 53% sugars, besides proteins, lipids, uronic acids, and sulfates. Molecular weight of the EPS produced by SC-1 was approximately 94 kDa, and that of CW1, 320 kDa. Glucose formed the major component in the EPS of both isolates-galactose, mannose, and arabinose being the other components. Cultures of both isolates survived air-drying up to a period of 96 hours, suggesting a role for EPS in preventing desiccation of cells.

  5. Extracellular synthesis of silver nanoparticles using culture supernatant of Pseudomonas aeruginosa.

    PubMed

    Kumar, C Ganesh; Mamidyala, Suman Kumar

    2011-06-01

    Bio-directed synthesis of metal nanoparticles is gaining importance due to their biocompatibility, low toxicity and eco-friendly nature. We used culture supernatant of Pseudomonas aeruginosa strain BS-161R for the simple and cost effective green synthesis of silver nanoparticles. The reduction of silver ions occurred when silver nitrate solution was treated with the Pseudomonas aeruginosa culture supernatant at room temperature. The nanoparticles were characterized by UV-visible, TEM, EDAX, FT-IR and XRD spectroscopy. The nanoparticles exhibited an absorption peak around 430 nm, a characteristic surface plasmon resonance band of silver nanoparticles. They were mono-dispersed and spherical in shape with an average particle size of 13 nm. The EDAX analysis showed the presence of elemental silver signal in the synthesized nanoparticles. The FT-IR analysis revealed that the protein component in the form of enzyme nitrate reductase and the rhamnolipids produced by the isolate in the culture supernatant may be responsible for reduction and as a capping material. The XRD spectrum showed the characteristic Bragg peaks of 111, 200, 220 and 311 facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature. The prepared silver nanoparticles exhibited strong antimicrobial activity against gram-positive, gram-negative and different Candida species at concentrations ranging between 4 and 32 μg ml(-1).

  6. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene.

    PubMed Central

    Totten, P A; Lara, J C; Lory, S

    1990-01-01

    The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism. Images FIG. 1 FIG. 2 PMID:2152909

  7. High-level expression of pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, in Escherichia coli and its purification.

    PubMed

    Odunuga, Odutayo O; Adekoya, Olayiwola A; Sylte, Ingebrigt

    2015-09-01

    Pseudolysin is the extracellular elastase of Pseudomonas aeruginosa and belongs to the thermolysin-like family of metallopeptidases. Pseudolysin has been identified as a robust drug target and a biotechnologically important enzyme in the tanning industry. Previous attempts to purify active pseudolysin from P. aeruginosa or by expression in Escherichia coli yielded low quantities. Considerable expression and purification of secreted pseudolysin from Pichia pastoris has been reported but it is time-consuming and not cost-effective. We report the successful large-scale expression of pseudolysin in E. coli and purification of the correctly folded and active protein. The lasB gene that codes for the enzymatically active mature 33-kilodalton pseudolysin was expressed with a histidine tag under the control of the T7 promoter. Pseudolysin expressed highly in E. coli and was solubilized and purified in 8M urea by metal affinity chromatography. The protein was simultaneously further purified, refolded and buffer-exchanged on a preparative Superdex 200 column by a modified urea reverse-gradient size exclusion chromatography. Using this technique, precipitation of pseudolysin was completely eliminated. Refolded pseudolysin was found to be active as assessed by its ability to hydrolyze N-succinyl-ala-ala-ala-p-nitroanilide. The purification scheme yielded approximately 40 mg of pseudolysin per liter of expression culture and specific activity of 3.2U/mg of protein using N-succinyl-ala-ala-ala-p-nitroanilide as substrate. This approach provides a reproducible strategy for high-level expression and purification of active metallopeptidases and perhaps other inclusion body-forming and precipitation-prone proteins.

  8. Light availability may control extracellular phosphatase production in turbid environments.

    PubMed

    Rychtecký, Pavel; Řeháková, Klára; Kozlíková, Eliška; Vrba, Jaroslav

    2015-01-01

    Extracellular phosphatase production by phytoplankton was investigated in the moderately eutrophic Lipno reservoir, Czech Republic during 2009 and 2010. We hypothesized that production of extracellular phosphatases is an additional mechanism of phosphorus acquisition enabling producers to survive rather than to dominate the phytoplankton. Hence, we examined the relationship between light availability and phosphatase production, as light plays an important role in polymictic environments. Bulk phosphatase activity was measured using a common fluorometric assay, and the production of phosphatases was studied using the Fluorescently Labelled Enzyme Activity technique, which enabled direct microscopic detection of phosphatase-positive cells. In total, 29 taxa of phytoplankton were identified during both years. Only 17 taxa from the total number of 29 showed production of extracellular phosphatases. Species dominating the phytoplankton rarely produced extracellular phosphatases. In contrast, taxa exhibiting phosphatase activity were present in low biomass in the phytoplankton assemblage. Moreover, there was a significant relationship between the proportion of phosphatase positive species in samples and the Z(eu):Z(mix) ratio (a proxy of light availability). A laboratory experiment with different light intensities confirmed the influence of light on production of phosphatases. Our seasonal study confirmed that extracellular phosphatase production is common in low-abundance populations but not in dominant taxa of the phytoplankton. It also suggested the importance of sufficient light conditions for the production of extracellular phosphatases.

  9. Influence of hydrophobic/hydrophilic fractions of extracellular organic matters of Microcystis aeruginosa on ultrafiltration membrane fouling.

    PubMed

    Zhou, Shiqing; Shao, Yisheng; Gao, Naiyun; Li, Lei; Deng, Jing; Tan, Chaoqun; Zhu, Mingqiu

    2014-02-01

    Fouling is a major obstacle to maintain the efficiency of ultrafiltration-based drinking water treatment process. Algal extracellular organic matters (EOMs) are currently considered as one of the major sources of membrane fouling. The objective of this study was to investigate the influence of different hydrophobic/hydrophilic fractions of EOM extracted from Microcystis aeruginosa on ultrafiltration membrane fouling at lab scale. The experimental data indicated that EOM exhibited similar flux decline trends on polyethersulfone (PES) and regenerated cellulose (RC) membranes but caused greater irreversible fouling on PES membrane than RC membrane due to its hydrophobic property. It was also observed that charged hydrophilic (CHPI) and neutral hydrophilic (NHPI) fractions caused greater flux decline over hydrophobic (HPO) and transphilic (TPI) fractions. For PES membrane, the order of the irreversible fouling potentials for the four fractions was HPO>TPI>CHPI>NHPI, while the irreversible fouling potentials of RC membrane were tiny and could be ignored. Fluorescence excitation-emission matrix (EEM) spectra and Fourier transform infrared (FTIR) spectra suggested that protein-like, polysaccharide-like and humic-like substances were the major components responsible for membrane fouling. The results also indicated that the irreversible fouling increased as the pH decreased. The addition of calcium to feed solutions led to more severe flux decline and irreversible fouling.

  10. Pyocyanin Production by Pseudomonas aeruginosa Confers Resistance to Ionic Silver

    PubMed Central

    Merrett, Neil D.

    2014-01-01

    Silver in its ionic form (Ag+), but not the bulk metal (Ag0), is toxic to microbial life forms and has been used for many years in the treatment of wound infections. The prevalence of bacterial resistance to silver is considered low due to the nonspecific nature of its toxicity. However, the recent increased use of silver as an antimicrobial agent for medical, consumer, and industrial products has raised concern that widespread silver resistance may emerge. Pseudomonas aeruginosa is a common pathogen that produces pyocyanin, a redox toxin and a reductant for molecular oxygen and ferric (Fe3+) ions. The objective of this study was to determine whether pyocyanin reduces Ag+ to Ag0, which may contribute to silver resistance due to lower bioavailability of the cation. Using surface plasmon resonance spectroscopy and scanning electron microscopy, pyocyanin was confirmed to be a reductant for Ag+, forming Ag0 nanoparticles and reducing the bioavailability of free Ag+ by >95% within minutes. Similarly, a pyocyanin-producing strain of P. aeruginosa (PA14) reduced Ag+ but not a pyocyanin-deficient (ΔphzM) strain of the bacterium. Challenge of each strain with Ag+ (as AgNO3) gave MICs of 20 and 5 μg/ml for the PA14 and ΔphzM strains, respectively. Removal of pyocyanin from the medium strain PA14 was grown in or its addition to the medium that ΔphzM mutant was grown in gave MICs of 5 and 20 μg/ml, respectively. Clinical isolates demonstrated similar pyocyanin-dependent resistance to Ag+. We conclude that pseudomonal silver resistance exists independently of previously recognized intracellular mechanisms and may be more prevalent than previously considered. PMID:25001302

  11. Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa.

    PubMed

    Daniels, Jonathan B; Scoffield, Jessica; Woolnough, Jessica L; Silo-Suh, Laura

    2014-12-01

    Pseudomonas aeruginosa establishes life-long chronic infections in the cystic fibrosis (CF) lung by utilizing various adaptation strategies. Some of these strategies include altering metabolic pathways to utilize readily available nutrients present in the host environment. The airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphatidylcholine, a major component of lung surfactant. Pseudomonas aeruginosa can degrade phosphatidylcholine to glycerol and fatty acids to increase the availability of usable carbon sources in the CF lung. In this study, we show that some CF-adapted P. aeruginosa isolates utilize glycerol more efficiently as a carbon source than nonadapted isolates. Furthermore, a mutation in a gene required for glycerol utilization impacts the production of several virulence factors in both acute and chronic isolates of P. aeruginosa. Taken together, the results suggest that interference with this metabolic pathway may have potential therapeutic benefits. PMID:25409940

  12. Production of extracellular fatty acid using engineered Escherichia coli

    PubMed Central

    2012-01-01

    Background As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics. Results In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL) produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired product. The strain p

  13. Enzymatic Production of Extracellular Reactive Oxygen Species by Marine Microorganisms

    NASA Astrophysics Data System (ADS)

    Diaz, J. M.; Andeer, P. F.; Hansel, C. M.

    2014-12-01

    Reactive oxygen species (ROS) serve as intermediates in a myriad of biogeochemically important processes, including cell signaling pathways, cellular oxidative stress responses, and the transformation of both nutrient and toxic metals such as iron and mercury. Abiotic reactions involving the photo-oxidation of organic matter were once considered the only important sources of ROS in the environment. However, the recent discovery of substantial biological ROS production in marine systems has fundamentally shifted this paradigm. Within the last few decades, marine phytoplankton, including diatoms of the genus Thalassiosira, were discovered to produce ample extracellular quantities of the ROS superoxide. Even more recently, we discovered widespread production of extracellular superoxide by phylogenetically and ecologically diverse heterotrophic bacteria at environmentally significant levels (up to 20 amol cell-1 hr-1), which has introduced the revolutionary potential for substantial "dark" cycling of ROS. Despite the profound biogeochemical importance of extracellular biogenic ROS, the cellular mechanisms underlying the production of this ROS have remained elusive. Through the development of a gel-based assay to identify extracellular ROS-producing proteins, we have recently found that enzymes typically involved in antioxidant activity also produce superoxide when molecular oxygen is the only available electron acceptor. For example, large (~3600 amino acids) heme peroxidases are involved in extracellular superoxide production by a bacterium within the widespread Roseobacter clade. In Thalassiosira spp., extracellular superoxide is produced by flavoproteins such as glutathione reductase and ferredoxin NADP+ reductase. Thus, extracellular ROS production may occur via secreted and/or cell surface enzymes that modulate between producing and degrading ROS depending on prevailing geochemical and/or ecological conditions.

  14. The macrophage chemotactic activity of Edwardsiella tarda extracellular products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from ...

  15. Antimicrobial effects of Pseudomonas aeruginosa on survivability and recovery of Campylobacter jejuni on poultry products.

    PubMed

    Davis, M A; Conner, D E

    2007-04-01

    Three types of poultry products representing differences in skin coverage were tested to determine the ability of Pseudomonas aeruginosa to inhibit growth of Campylobacter jejuni. Processed ready-to-cook poultry carcasses were obtained from the Poultry Research Unit at Auburn University and were not subjected to any treatment to reduce or eliminate the native microflora on the carcasses. Carcasses were cut into wing sections (drumette, flat, tip), split breast pieces (with and without bone), and boneless, skinless breast pieces. Equal numbers of the 3 product types were subjected to 1 of 6 treatments: 1) uninoculated, 2) C. jejuni only, 3) P. aeruginosa type 1 only, 4) P. aeruginosa type 2 only, 5) C. jejuni + P. aeruginosa type 1, or 6) C. jejuni + P. aeruginosa type 2. Products were inoculated at 10(4) to 10(5) cfu. Postinoculation, equal numbers of product type were also subjected to the following: 1) aerobic or vacuum packaging, 2) storage temperature of 4 or 10 degrees C, and 3) storage of 0, 1, 2, 3, or 4 d. Products were sampled after storage duration to determine the population of C. jejuni and P. aeruginosa. Individual pieces were rinsed with 50 mL of buffered peptone water. The recovered rinse was used to make appropriate dilutions and spiral plated onto Campy-Cefex and Pseudomonas P agars. Campy-Cefex plates were incubated microaerophilically at 42 degrees C for 48 h, whereas Pseudomonas P plates were incubated aerobically at 37 degrees C for 24 to 48 h. Random suspect colonies on Campy-Cefex plates were confirmed by cell morphology when viewed under microscopic examination. Suspect colonies on Pseudomonas P plates produced a blue color in the medium indicative of glycerol reduction. At both 4 and 10 degrees C, neither type of P. aeruginosa inhibited the growth or survival of C. jejuni compared to plates that were inoculated with C. jejuni only. PMID:17369550

  16. Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice.

    PubMed

    Rocha, Maria V P; Souza, Maria C M; Benedicto, Sofia C L; Bezerra, Márcio S; Macedo, Gorete R; Pinto, Gustavo A Saavedra; Gonçalves, Luciana R B

    2007-04-01

    In this work, the ability of biosurfactant production by Pseudomonas aeruginosa in batch cultivation using cashew apple juice (CAJ) and mineral media was evaluated. P. aeruginosa was cultivated in CAJ, which was supplemented with peptone (5.0 g/L) and nutritive broth. All fermentation assays were performed in Erlenmeyer flasks containing 300 mL, incubated at 30 degrees C and 150 rpm. Cell growth (biomass and cell density), pH, and superficial tension were monitored vs time. Surface tension was reduced by 10.58 and 41% when P. aeruginosa was cultivated in nutrient broth and CAJ supplemented with peptone, respectively. These results indicated that CAJ is an adequate medium for growth and biosurfactant production. Best results of biosurfactant production were obtained when CAJ was supplemented with peptone.

  17. Production of Biosurfactant by Pseudomonas aeruginosa Grown on Cashew Apple Juice

    NASA Astrophysics Data System (ADS)

    Rocha, Maria V. P.; Souza, Maria C. M.; Benedicto, Sofia C. L.; Bezerra, Márcio S.; Macedo, Gorete R.; Saavedra Pinto, Gustavo A.; Gonçalves, Luciana R. B.

    In this work, the ability of biosurfactant production by Pseudomonas aeruginosa in batch cultivation using cashew apple juice (CAJ) and mineral media was evaluated. P. aeruginosa was cultivated in CAJ, which was supplemented with peptone (5.0 g/L) and nutritive broth. All fermentation assays were performed in Erlenmeyer flasks containing 300 mL, incubated at 30°C and 150 rpm. Cell growth (biomass and cell density), pH, and superficial tension were monitored vs time. Surface tension was reduced by 10.58 and 41% when P. aeruginosa was cultivated in nutrient broth and CAJ supplemented with peptone, respectively. These results indicated that CAJ is an adequate medium for growth and biosurfactant production. Best results of biosurfactant production were obtained when CAJ was supplemented with peptone.

  18. Metabolite transfer with the fermentation product 2,3-butanediol enhances virulence by Pseudomonas aeruginosa

    PubMed Central

    Venkataraman, Arvind; Rosenbaum, Miriam A; Werner, Jeffrey J; Winans, Stephen C; Angenent, Largus T

    2014-01-01

    The respiratory tract of cystic fibrosis (CF) patients harbor persistent microbial communities (CF airway microbiome) with Pseudomonas aeruginosa emerging as a dominant pathogen. Within a polymicrobial infection, interactions between co-habitant microbes can be important for pathogenesis, but even when considered, these interactions are not well understood. Here, we show with in vitro experiments that, compared with glucose, common fermentation products from co-habitant bacteria significantly increase virulence factor production, antimicrobial activity and biofilm formation of P. aeruginosa. The maximum stimulating effect was produced with the fermentation product 2,3-butanediol, which is a substrate for P. aeruginosa, resulting in a metabolic relationship between fermenters and this pathogen. The global transcription regulator LasI LasR, which controls quorum sensing, was upregulated threefold with 2,3-butanediol, resulting in higher phenazine and exotoxin concentrations and improved biofilm formation. This indicates that the success of P. aeruginosa in CF airway microbiomes could be governed by the location within the food web with fermenting bacteria. Our findings suggest that interbacterial metabolite transfer in polymicrobial infections stimulates virulence of P. aeruginosa and could have a considerable impact on disease progression. PMID:24401856

  19. PRODUCTION OF EXTRACELLULAR GUANOSINE-5'-MONOPHOSPHATE BY BACILLUS SUBTILIS

    PubMed Central

    Demain, A. L.; Miller, I. M.; Hendlin, D.

    1964-01-01

    Demain, A. L. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), I. M. Miller, and D. Hendlin. Production of extracellular guanosine-5'-monophosphate by Bacillus subtilis. J. Bacteriol. 88:991–995. 1964.—Wild-type Bacillus subtilis colonies were found to feed purineless mutants. A strain with high feeding capacity was selected for study, with a guanineless mutant of B. subtilis used as the assay organism. The factor was excreted during its growth phase in a complex medium containing starch and soybean meal extract. Nutritional studies led to the development of a defined medium to be used for biochemical studies and to aid in the isolation of the factor. Starch was replaced by maltose and the soybean meal extract by Mn++. Production of the factor was sensitive to the pH of the medium during growth. Practically its entire extracellular accumulation occurred before visible lysis. The factor was identified as guanosine-5'-monophosphate derived by extracellular enzymatic hydrolysis of excreted ribonucleic acid. PMID:14219064

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

    PubMed

    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

    1993-06-01

    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.

  1. 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

  2. Characterization of adhesive exopolysaccharide (EPS) produced by Pseudomonas aeruginosa under starvation conditions.

    PubMed

    Myszka, Kamila; Czaczyk, Katarzyna

    2009-06-01

    Pseudomonas aeruginosa synthesizes large quantities of exopolysaccharide (EPS), making it an excellent model organism for the study of EPS-mediated adhesion. The purpose of this investigation was to evaluate the influence of limited nutrients availability in the culture medium on the composition of EPS produced by P. aeruginosa. The relationship between the EPS production and the adhesion process of the P. aeruginosa cells to stainless steel surface (type 316 L) under starvation conditions were also examined. In all experimental variants P. aeruginosa produced more EPS with an increase of incubation period upon starvation conditions. Under limited nutrients condition, glucose dominated in the EPS materials. After 6 days of the process, only glucosyl units were detected in the extracellular matrix produced by nutrient-deprived P. aeruginosa cells. These extracellular molecules promoted more advanced stages of P. aeruginosa biofilm formation on the surface of stainless steel.

  3. Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

    PubMed

    Smith, Karen; Rajendran, Ranjith; Kerr, Stephen; Lappin, David F; Mackay, William G; Williams, Craig; Ramage, Gordon

    2015-09-01

    In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis. PMID:26162475

  4. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Cho, Moo Hwan; Lee, Jintae

    2014-12-01

    The opportunistic pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilms of this bacterium are much more resistant to antibiotics than planktonic cells. Thirty-six metal ions have been investigated to identify antivirulence and antibiofilm metal ions. Zinc ions and ZnO nanoparticles were found to markedly inhibit biofilm formation and the production of pyocyanin, Pseudomonas quinolone signal (PQS), pyochelin, and hemolytic activity of P. aeruginosa without affecting the growth of planktonic cells. Transcriptome analyses showed that ZnO nanoparticles induce the zinc cation efflux pump czc operon and several important transcriptional regulators (porin gene opdT and type III repressor ptrA), but repress the pyocyanin-related phz operon, which explains observed phenotypic changes. A mutant study showed that the effects of ZnO nanoparticles on the control of pyocyanin production and biofilm formation require the czc regulator CzcR. In addition, ZnO nanoparticles markedly increased the cellular hydrophilicity of P. aeruginosa cells. Our results support that ZnO nanoparticles are potential antivirulence materials against recalcitrant P. aeruginosa infections and possibly other important pathogens. PMID:24958247

  5. The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

    PubMed

    Schultz, M J; Speelman, P; Zaat, S A; Hack, C E; van Deventer, S J; van der Poll, T

    2000-11-01

    To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

  6. Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice

    PubMed Central

    Zhou, Qing; Xu, Yudi; Vanogtrop, Floris; Guo, Qijin; Liu, Guofeng; Yan, Shaohua

    2015-01-01

    This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg−1, which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn’s method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD50) of acidolysis product in mice was >9.09 g kg−1 body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient. PMID:25649189

  7. Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice.

    PubMed

    Han, Shiqun; Zhou, Qing; Xu, Yudi; Vanogtrop, Floris; Guo, Qijin; Liu, Guofeng; Yan, Shaohua

    2015-10-01

    This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg(-1), which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn's method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD50) of acidolysis product in mice was >9.09 g kg(-1) body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient.

  8. Optimization of extracellular fungal peroxidase production by 2 Coprinus species.

    PubMed

    Ikehata, Keisuke; Pickard, Michael A; Buchanan, Ian D; Smith, Daniel W

    2004-12-01

    Optimum culture conditions for the batch production of extracellular peroxidase by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were explored using 2 statistical experimental designs, including 2-level, 7-factor fractional factorial design and 2-factor central composite design. Of the 7 factors examined in the screening study, the concentrations of carbon (glucose) and nitrogen (peptone or casitone) sources showed significant effects on the peroxidase production by Coprinus sp. UAMH 10067. The optimum glucose and peptone concentrations were determined as 2.7% and 0.8% for Coprinus sp. UAMH 10067, and 2.9% and 1.4% for C. cinereus UAMH 4103, respectively. Under the optimized culture condition the maximum peroxidase activity achieved in this study was 34.5 U x mL(-1) for Coprinus sp. UAMH 10067 and 68.0 U x mL(-1) for C. cinereus UAMH 4103, more than 2-fold higher than the results of previous studies.

  9. Extracellular zinc induces phosphoethanolamine addition to Pseudomonas aeruginosa lipid A via the ColRS two-component system

    PubMed Central

    Nowicki, Emily M.; O'Brien, John P.; Brodbelt, Jennifer S.; Trent, M. Stephen

    2015-01-01

    Summary Gram-negative bacteria survive harmful environmental stressors by modifying their outer membrane. Much of this protection is afforded upon remodeling of the lipid A region of the major surface molecule lipopolysaccharide (LPS). For example, the addition of cationic substituents, such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoehthanolamine (pEtN) at the lipid A phosphate groups is often induced in response to specific environmental flux stabilizing the outer membrane. The work herein represents the first report of pEtN addition to P. aeruginosa lipid A. We have identified the key pEtN transferase which we named EptAPa and characterized its strict activity on only one position of lipid A, contrasting from previously studied EptA enzymes. We further show that transcription of eptAPa is regulated by zinc via the ColRS two-component system instead of the PmrAB system responsible for eptA regulation in E. coli and S. enterica. Further, although L-Ara4N is readily added to the same position of lipid A as pEtN under certain environmental conditions, ColR specifically induces pEtN addition to lipid A in lieu of L-Ara4N when Zn2+ is present. The unique, specific regulation of eptAPa transcription and enzymatic activity described in this work demonstrates the tight yet inducible control over LPS modification in P. aeruginosa. PMID:25846400

  10. The role of two Pseudomonas aeruginosa anthranilate synthases in tryptophan and quorum signal production

    PubMed Central

    Palmer, Gregory C.; Jorth, Peter A.

    2013-01-01

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes infections in the lungs of individuals with the genetic disease cystic fibrosis. Density-dependent production of toxic factors regulated by the Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone; PQS) have been proposed to be involved in P. aeruginosa virulence. PQS biosynthesis requires conversion of the central metabolite chorismate to anthranilate by anthranilate synthase. This reaction is also the first step in tryptophan biosynthesis. P. aeruginosa possesses two functional anthranilate synthases, TrpEG and PhnAB, and these enzymes are not functionally redundant, as trpEG mutants are tryptophan auxotrophs but produce PQS while mutants in phnAB are tryptophan prototrophs but do not produce PQS in minimal media. The goal of the work described in this paper was to determine the mechanism for this lack of functional complementation of TrpEG and PhnAB. Our results reveal that overexpression of either enzyme compensates for tryptophan auxotrophy and PQS production in the trpEG and phnAB mutants respectively, leading to the hypothesis that differential regulation of these genes is responsible for the lack of functional complementation. In support of this hypothesis, trpEG was shown to be expressed primarily during low-density growth while phnAB was expressed primarily at high density. Furthermore, dysregulation of phnAB expression eliminated tryptophan auxotrophy in the P. aeruginosa trpEG mutant. Based on these data, we propose a model for anthranilate sequestration by differential transcriptional regulation of the two P. aeruginosa anthranilate synthase enzymes. PMID:23449919

  11. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate.

    PubMed Central

    Goldberg, J B; Ohman, D E

    1984-01-01

    Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation. PMID:6427188

  12. Bacteria associated with obstructive pulmonary disease elaborate extracellular products that stimulate mucin secretion by explants of guinea pig airways.

    PubMed Central

    Adler, K. B.; Hendley, D. D.; Davis, G. S.

    1986-01-01

    Certain cell-free filtrates from broth cultures of Pseudomonas aeruginosa, Hemophilus influenzae and Streptococcus pneumoniae stimulate secretion of glycoconjugates by explants of guinea pig trachea. The stimulatory effect is not related to toxicity or damage to the respiratory mucosa, as well as could be determined by ultrastructural examination of the explants after exposure. Bacteria isolated from patients with a history of chronic obstructive lung disease (P aeruginosa from cystic fibrosis, H influenzae, and S pneumoniae from chronic bronchitis) do not demonstrate increased frequency of positive strains or greater stimulation of secretion than organisms isolated from other individuals. At least three stimulatory substances are found in cell-free filtrates of P aeruginosa. They appear to be proteins of molecular weight 60,000-100,000 as determined by gel filtration. Within the crude filtrate, they are relatively stable to heat, proteolysis, and storage at 4 C and in liquid nitrogen. The stimulatory activity is not lost upon subculture of the bacteria. When isolated from the filtrate by column chromatography, they become labile to heat and trypsin. Isolated active fractions show proteolytic activity coinciding with mucin-stimulating capacity, suggesting a relationship with Pseudomonas proteases. Stimulatory substances released by S pneumoniae and H influenzae appear to be different from those elaborated by Pseudomonas. They are extremely labile to heat and storage, and the capacity to stimulate secretion is lost on subculture. Preliminary gel filtration indicates the S pneumoniae stimulatory substance(s) is in a molecular weight range of 100,000-300,000 daltons, while that of H influenzae is between 50,000 and 200,000. The results suggest bacteria which chronically infect or colonize respiratory airways of individuals suffering from obstructive lung disease can elaborate extracellular product(s) capable of stimulating secretion of mucin. Thus, the bacteria

  13. Production and characterization of an extracellular lipase from Candida guilliermondii

    PubMed Central

    Oliveira, Anne Caroline Defranceschi; Fernandes, Maria Luiza; Mariano, André Bellin

    2014-01-01

    Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL−1) in the presence of 5 mmol L−1 NaCl at 30 °C and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL−1. The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone) and different acid:alcohol molar ratios. Higher ester conversion rate (81%) was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium. PMID:25763060

  14. Production of a polyester degrading extracellular hydrolase from Thermomonospora fusca.

    PubMed

    Gouda, Mona K; Kleeberg, Ilona; van den Heuvel, Joop; Müller, Rolf-Joachim; Deckwer, Wolf-Dieter

    2002-01-01

    The production of a polyester-degrading hydrolase from the thermophilic actinomycete Thermomonospora fusca was investigated with regard to its potential technical application. Only in the presence of a polyester (random aliphatic-aromatic copolyester from 1,4-butanediol, terephthalic acid, and adipic acid with around 40-50 mol % terephthalic acid in the acid component), the excretion of the extracellular enzyme could be achieved with an optimized synthetic medium using pectin and NH(4)Cl as nitrogen source. Compared to complex media, a significantly higher specific activity at comparable volumetric yields could be obtained, thus reducing the expenditure for purification. The activity profile in the medium is controlled by a complex process involving (1) induction of enzyme excretion, (2) enzyme adsorption on the hydrophobic polyester surface, (3) inhibition of enzyme generation by monomers produced by polyester cleavage, and (4) enzyme denaturation. Diafiltration with cellulose acetate membranes as the sole downstream processing step led to a product of high purity and with sufficient yield (60% of total activity). Scaling-up from shaking flasks to a fermentor scale of 100 L revealed no specific problems. However, the excretion of the hydrolase by the actinomycete turned out to be inhibited by the degradation products (monomers) of the aliphatic-aromatic copolyester used as inductor for the enzyme production. The crude enzyme exhibited generally similar properties (temperature and pH optimum) as the highly purified hydrolase described previously; however, the storage capability and thermal stability is improved when the crude enzyme solution is diafiltrated.

  15. Evaluation of disinfection by-product formation potential (DBPFP) during chlorination of two algae species--Blue-green Microcystis aeruginosa and diatom Cyclotella meneghiniana.

    PubMed

    Liao, Xiaobin; Liu, Jinjin; Yang, Mingli; Ma, Hongfang; Yuan, Baoling; Huang, Ching-Hua

    2015-11-01

    Microcystis aeruginosa (blue-green alga) commonly blooms in summer and Cyclotella meneghiniana (diatom) outbreaks in fall in the reservoirs that serve as drinking water sources in Southeast China. Herein, an evaluation of disinfection by-product formation potential (DBPFP) from them during chlorination should be conducted. Five DBPs including trichloromethane (TCM), trichloronitromethane (TCNM), dichloroacetonitrile (DCAN), 1,1-dichloropropanone (1,1-DCP) and 1,1,1-trichloropropanone (1,1,1-TCP) were monitored. The formation potential of TCM and TCNM was enhanced with the increase of reaction time and chlorine dosage, whereas that of DCAN, 1,1-DCP and 1,1,1-TCP increased first and then fell with continuing reaction time. M. aeruginosa showed higher DBPFP than C. meneghiniana, the yield of DBPs varied with components of algal cells. The DBPFP order from components of M. aeruginosa was cell suspension (CS) ≈ intracellular organic matter (IOM) > extracellular organic matter (EOM) > cell debris (CD), which indicated that IOM was the main DBP precursors for M. aeruginosa. The yields of DBPs from components of C. meneghiniana were in the order of CS>IOM≈ CD ≈ EOM, suggesting that three components made similar contributions to the total DBP formation. The amount of IOM with higher DBPFP leaked from both algae species increased with the chlorine dosage, indicating that chlorine dosage should be considered carefully in the treatment of eutrophic water for less destroying of the cell integrity. Though fluorescence substances contained in both algae species varied significantly, the soluble microbial products (SMPs) and aromatic protein-like substances were the main cellular components that contributed to DBP formation for both algae.

  16. Effect of linoleic acid sustained-release microspheres on Microcystis aeruginosa antioxidant enzymes activity and microcystins production and release.

    PubMed

    Ni, Lixiao; Jie, Xiaoting; Wang, Peifang; Li, Shiyin; Wang, Guoxiang; Li, Yiping; Li, Yong; Acharya, Kumud

    2015-02-01

    The objective of this work was to identify the optimal dose range for good anti-algal effect of linoleic acid (LA) sustained-release microspheres and investigate their impact on the antioxidant enzymes (super oxide dismutase, Catalase and Peroxidase) activity changes of Microcystis aeruginosa, as well as the production and release of microcystins (MCs). Based on measured changes in algal cell density and inhibitory ratio (IR), the optimal dose of LA microspheres was 0.3 g L(-1) with over 90% of IR in this study. The Chlorophyll a content and antioxidant enzymes activity in the LA microspheres group decreased markedly until beyond the minimal detection limit after 16 d and 9 d, respectively. In addition, LA microspheres demonstrated no significant impact on the extracellular release of MCs during the culturing period. The amount of intracellular microcystin-LR (MC-LR) per 10(6) algal cells in LA microspheres group was highest among all groups during the whole experimental process. Under the sustained stress of LA released from LA microspheres, the LA microspheres could decrease the production and release of algal toxins. There was no increase in the total amount of MC-LR in the algal cell culture medium. These indicated that LA sustained-release microspheres represent a high degree of ecological safety and their practical applications for the treatment of water undergoing algal blooms need further study.

  17. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    PubMed

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples.

  18. Ultrafiltration membrane fouling by extracellular organic matters (EOM) of Microcystis aeruginosa in stationary phase: influences of interfacial characteristics of foulants and fouling mechanisms.

    PubMed

    Qu, Fangshu; Liang, Heng; Wang, Zhaozhi; Wang, Hui; Yu, Huarong; Li, Guibai

    2012-04-01

    This paper focused on the membrane fouling caused by extracellular organic matters (EOM) which was extracted from lab-cultured Microcystis aeruginosa in stationary phase. The characteristics of EOM such as molecular weight distribution, hydrophobicity and fluorescence were measured. It was found that high molecular weight (MW) and hydrophilic organics accounted for the major parts of algal EOM which was comprised of protein-like, polysaccharide-like and humic-like substances. Ultrafiltration (UF) experiments were carried out in a stirring cell and hydrophobic polyethersulfone (PES) membranes which carried negative charge were used. Prefiltration, calcium addition and XAD fractionation were employed to change the interfacial characteristics of EOM. Then the effects of these interfacial characteristics on flux decline, reversibility and mass balance of organics were compared. Algal EOM proved to cause serious membrane fouling during UF. The fraction of algal EOM between 0.45 μm and 100 kDa contributed a significant portion of the fouling. Hydrophobic organics in EOM tended to adhere to membrane surface causing irreversible fouling, while the cake layer formed by hydrophilic organics caused greater resistance to water flow due to hydrophilic interaction such as hydrogen bond and led to faster flux decline during UF. The results also indicated that the algal EOM was negatively charged and the electrostatic repulsion could prevent organics from adhering to membrane surface. In term of fouling mechanisms, cake layer formation, hydrophobic adhesion and pore plugging were the main mechanisms for membrane fouling caused by algal EOM. PMID:22178303

  19. Efficient production and enhanced tumor delivery of engineered extracellular vesicles.

    PubMed

    Watson, Dionysios C; Bayik, Defne; Srivatsan, Avinash; Bergamaschi, Cristina; Valentin, Antonio; Niu, Gang; Bear, Jenifer; Monninger, Mitchell; Sun, Mei; Morales-Kastresana, Aizea; Jones, Jennifer C; Felber, Barbara K; Chen, Xiaoyuan; Gursel, Ihsan; Pavlakis, George N

    2016-10-01

    Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded ∼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods. PMID:27522254

  20. Production, purification, and characterization of an extracellular chitosanase from Streptomyces.

    PubMed Central

    Price, J S; Storck, R

    1975-01-01

    The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall. Images PMID:371

  1. Quercetin Attenuates Lactate Production and Extracellular Matrix Secretion in Keratoconus

    PubMed Central

    McKay, T. B.; Lyon, D.; Sarker-Nag, A.; Priyadarsini, S.; Asara, J. M.; Karamichos, D.

    2015-01-01

    Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC. PMID:25758533

  2. Pseudomonas aeruginosa inducing rice resistance against Rhizoctonia solani: production of salicylic acid and peroxidases.

    PubMed

    Saikia, R; Kumar, R; Arora, D K; Gogoi, D K; Azad, P

    2006-01-01

    Three isolates of Pseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice against Rhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) by P. aeruginosa both in vitro and in vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King's B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0-200 g/L showed no inhibition of mycelial growth of R. solani in vitro, while at > or =300 g/L it inhibited it. P. aeruginosa-pretreated rice plants challenged inoculation with R. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungi R. solani, Pyricularia oryzae and Helminthosporium oryzae. PMID:17176755

  3. Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume

    PubMed Central

    Pottier, Laurent; Huet, Joelle; Rabemanantsoa, Christian; Kiendrebeogo, Martin; Andriantsimahavandy, Abel; Rasamindrakotroka, Andry; Stévigny, Caroline; Duez, Pierre; El Jaziri, Mondher

    2015-01-01

    Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms. PMID:26186595

  4. Effects of UV-B radiation on microcystin production of a toxic strain of Microcystis aeruginosa and its competitiveness against a non-toxic strain.

    PubMed

    Yang, Zhen; Kong, Fanxiang; Shi, Xiaoli; Yu, Yang; Zhang, Min

    2015-01-01

    Microcystins (MCs) produced by toxic cyanobacteria pose a health hazard to humans and animals. Some environmental factors can alter the MC concentrations by affecting the abundance of toxin-producing strains in a cyanobacteria population and/or their toxin production. In this study, we designed a monoculture and competition experiment to investigate the impacts of UV-B radiation on MC production and the competition between toxin and non-toxin producing strains of Microcystis aeruginosa. UV-B radiation resulted in higher inhibition of the growth and photosynthetic activity of the non-toxin producing strain relative to that observed for the toxin-producing strain. Both intracellular and extracellular MC contents decreased markedly when the toxin-producing strain was exposed to UV-B radiation. In addition, a quantitative real-time PCR assay revealed that the ratio of toxin-producing M. aeruginosa under UV-B exposure was higher than that under PAR alone at an early stage of the experiment. However, its abundance under UV-B exposure was lower compared with the PAR alone treatment after day 12. Our study demonstrated that UV-B radiation has a great impact on the abundance of the toxin-producing strain in the Microcystis population and their toxin production, which suggests that the fluctuation of UV-B radiation affects the MC level of cyanobacteria blooms. PMID:25464282

  5. Effects of UV-B radiation on microcystin production of a toxic strain of Microcystis aeruginosa and its competitiveness against a non-toxic strain.

    PubMed

    Yang, Zhen; Kong, Fanxiang; Shi, Xiaoli; Yu, Yang; Zhang, Min

    2015-01-01

    Microcystins (MCs) produced by toxic cyanobacteria pose a health hazard to humans and animals. Some environmental factors can alter the MC concentrations by affecting the abundance of toxin-producing strains in a cyanobacteria population and/or their toxin production. In this study, we designed a monoculture and competition experiment to investigate the impacts of UV-B radiation on MC production and the competition between toxin and non-toxin producing strains of Microcystis aeruginosa. UV-B radiation resulted in higher inhibition of the growth and photosynthetic activity of the non-toxin producing strain relative to that observed for the toxin-producing strain. Both intracellular and extracellular MC contents decreased markedly when the toxin-producing strain was exposed to UV-B radiation. In addition, a quantitative real-time PCR assay revealed that the ratio of toxin-producing M. aeruginosa under UV-B exposure was higher than that under PAR alone at an early stage of the experiment. However, its abundance under UV-B exposure was lower compared with the PAR alone treatment after day 12. Our study demonstrated that UV-B radiation has a great impact on the abundance of the toxin-producing strain in the Microcystis population and their toxin production, which suggests that the fluctuation of UV-B radiation affects the MC level of cyanobacteria blooms.

  6. Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

    PubMed

    Hollands, Andrew; Corriden, Ross; Gysler, Gabriela; Dahesh, Samira; Olson, Joshua; Raza Ali, Syed; Kunkel, Maya T; Lin, Ann E; Forli, Stefano; Newton, Alexandra C; Kumar, Geetha B; Nair, Bipin G; Perry, J Jefferson P; Nizet, Victor

    2016-07-01

    Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound. PMID:27226531

  7. Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

    PubMed

    Hollands, Andrew; Corriden, Ross; Gysler, Gabriela; Dahesh, Samira; Olson, Joshua; Raza Ali, Syed; Kunkel, Maya T; Lin, Ann E; Forli, Stefano; Newton, Alexandra C; Kumar, Geetha B; Nair, Bipin G; Perry, J Jefferson P; Nizet, Victor

    2016-07-01

    Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

  8. Comparative effects of inorganic and organic nitrogen on the growth and microcystin production of Microcystis aeruginosa.

    PubMed

    Yan, YangWei; Dai, RuiHua; Liu, Yan; Gao, JiaYi; Wu, XuanHao

    2015-05-01

    Nitrogen causes the frequent occurrence of harmful algal blooms and possible microcystin production. The effects of ammonia and alanine (Ala) on the growth and microcystin production of Microcystis aeruginosa were investigated using an isotope tracer ((15)N). The results indicated that Ala was directly used by M. aeruginosa and contributed to biomass formation amounting to 2.1 × 10(7) cells mL(-1) on day 48, compared with only 6.2 × 10(6) cells mL(-1) from ammonia alone. Microcystin-LR production with Ala was less than that of ammonia, which peaked at 50.2 fg cell(-1) on day 6. Liquid chromatographic analysis with tandem mass spectrometry of (15)N-microcystin-LR suggested that (15)N from ammonia was probably synthesized into the arginine residue. By contrast, (15)N from Ala was assimilated into the Ala, leucine, the iso-linked (2R,3S)-3-methylaspartic acid, arginine, and certain unusual C20 amino acid residues. The results represent the forward steps in the determination of the nitrogen forms that fuel toxin production and blooms. PMID:25726035

  9. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-05-11

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.

  10. Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors

    PubMed Central

    Lee, Moon Geon; Seo, Hyo Jin; Shin, Jin Hyuk; Shin, Tai Sun; Kim, Min Yong; Choi, Jong Il

    2016-01-01

    Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media) and additional ingredients such as aminolevulinic acid (2 mM), lysine (2.28 mM), alanine (1 mM), and Naphthalene acetic acid (1 mM) as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 μmolm−2s−1 intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL) as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL). The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL) was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production. PMID:27556034

  11. Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors.

    PubMed

    Khan, Muhammad Imran; Lee, Moon Geon; Seo, Hyo Jin; Shin, Jin Hyuk; Shin, Tai Sun; Yoon, Yang Ho; Kim, Min Yong; Choi, Jong Il; Kim, Jong Deog

    2016-01-01

    Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media) and additional ingredients such as aminolevulinic acid (2 mM), lysine (2.28 mM), alanine (1 mM), and Naphthalene acetic acid (1 mM) as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 μmolm(-2)s(-1) intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL) as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL). The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL) was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production. PMID:27556034

  12. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    PubMed Central

    Morkunas, Bernardas; Gal, Balint; Galloway, Warren R J D; Hodgkinson, James T; Ibbeson, Brett M; Sing Tan, Yaw; Welch, Martin

    2016-01-01

    Summary Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable. PMID:27559393

  13. Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread.

    PubMed

    Picão, Renata Cristina; Carrara-Marroni, Floristher Elaine; Gales, Ana Cristina; Venâncio, Emerson José; Xavier, Danilo Elias; Tognim, Maria Cristina Bronharo; Pelayo, Jacinta Sanchez

    2012-09-01

    The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes. PMID:22990963

  14. Improved production of biosurfactant by a Pseudomonas aeruginosa mutant using vegetable oil refinery wastes.

    PubMed

    Raza, Zulfiqar Ali; Rehman, Asma; Khan, Muhammad Saleem; Khalid, Zafar M

    2007-02-01

    Biosurfactant production by Pseudomonas aeruginosa EBN-8 mutant was studied in shake flasks on separate wastes from canola, soybean and corn oil refineries. Of the substrates tested, canola oil refinery waste (COD=20 g l(-1)) supplemented with sodium nitrate (at COD/N=20) showed the best microbial growth (4.50 g l(-1)) and rhamnolipid production (8.50 g l(-1)), at 10 d of incubation with the specific growth rate of 0.316 h(-1) and specific product yield of 0.597 g g(-1) h. Its cell-free supernatant showed the critical micelle dilution (CMD) of 150 and surface tension (ST) of 28.5 mN m(-1).

  15. Extracellular polymeric substances buffer against the biocidal effect of H2O2 on the bloom-forming cyanobacterium Microcystis aeruginosa.

    PubMed

    Gao, Lei; Pan, Xiangliang; Zhang, Daoyong; Mu, Shuyong; Lee, Duu-Jong; Halik, Umut

    2015-02-01

    H2O2 is an emerging biocide for bloom-forming cyanobacteria. It is important to investigate the H2O2 scavenging ability of extracellular polymeric substances (EPS) of cyanobacteria because EPS with strong antioxidant activity may "waste" considerable amounts of H2O2 before it kills the cells. In this study, the buffering capacity against H2O2 of EPS from the bloom-forming cyanobacterium Microcystis aeruginosa was investigated. IC50 values for the ability of EPS and vitamin C (VC) to scavenge 50% of the initial H2O2 concentration were 0.097 and 0.28 mg mL(-1), respectively, indicating the higher H2O2 scavenging activity of EPS than VC. Both proteins and polysaccharides are significantly decomposed by H2O2 and the polysaccharides were more readily decomposed than proteins. H2O2 consumed by the EPS accounted for 50% of the total amount of H2O2 consumed by the cells. Cell growth and photosynthesis were reduced more for EPS-free cells than EPS coated cells when the cells were treated with 0.1 or 0.2 mg mL(-1) H2O2, and the maximum photochemical efficiency Fv/Fm of EPS coated cells recovered to higher values than EPS-free cells. Concentrations of H2O2 above 0.3 mg mL(-1) completely inhibited photosynthesis and no recovery was observed for both EPS-free and EPS coated cells. This shows that EPS has some buffering capacity against the killing effect of H2O2 on cyanobacterial cells. Such a strong H2O2 scavenging ability of EPS is not favorable for killing bloom-forming cyanobacteria. The high H2O2 scavenging capacity means considerable amounts of H2O2 have to be used to break through the EPS barrier before H2O2 exerts any killing effects on the cells. It is therefore necessary to determine the H2O2 scavenging capacity of the EPS of various bloom-forming cyanobacteria so that the cost-effective amount of H2O2 needed to be used for killing the cyanobacteria can be estimated.

  16. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space.

    PubMed

    Yang, Zhen; Kong, Fanxiang

    2015-10-01

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m² not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space. PMID:26492272

  17. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space.

    PubMed

    Yang, Zhen; Kong, Fanxiang

    2015-10-20

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m² not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space.

  18. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space

    PubMed Central

    Yang, Zhen; Kong, Fanxiang

    2015-01-01

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m2 not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space. PMID:26492272

  19. Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production.

    PubMed

    Heyd, M; Weigold, P; Franzreb, M; Berensmeier, S

    2009-01-01

    During the last decades, whole-cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high-gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis-à-vis pure alginate beads was achieved with alginate beads with embedded Bayoxide magnetite or MagPrep silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.

  20. Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production.

    PubMed

    Heyd, M; Weigold, P; Franzreb, M; Berensmeier, S

    2009-01-01

    During the last decades, whole-cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high-gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis-à-vis pure alginate beads was achieved with alginate beads with embedded Bayoxide magnetite or MagPrep silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task. PMID:19691121

  1. Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa

    PubMed Central

    Eraqi, Walaa A.; Yassin, Aymen S.; Ali, Amal E.; Amin, Magdy A.

    2016-01-01

    Biosurfactants are produced by bacteria or yeast utilizing different substrates as sugars, glycerol, or oils. They have important applications in the detergent, oil, and pharmaceutical industries. Glycerol is the product of biodiesel industry and the existing glycerol market cannot accommodate the excess amounts generated; consequently, new markets for refined glycerol need to be developed. The aim of present work is to optimize the production of microbial rhamnolipid using waste glycerol. We have developed a process for the production of rhamnolipid biosurfactants using glycerol as the sole carbon source by a local Pseudomonas aeruginosa isolate that was obtained from an extensive screening program. A factorial design was applied with the goal of optimizing the rhamnolipid production. The highest production yield was obtained after 2 days when cells were grown in minimal salt media at pH 6, containing 1% (v/v) glycerol and 2% (w/v) sodium nitrate as nitrogen source, at 37°C and at 180 rpm, and reached 2.164 g/L after 54 hours (0.04 g/L h). Analysis of the produced rhamnolipids by TLC, HPLC, and FTIR confirmed the nature of the biosurfactant as monorhamnolipid. Glycerol can serve as a source for the production of rhamnolipid from microbial isolates providing a cheap and reliable substrate. PMID:26942014

  2. Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

    PubMed

    Niazy, Abdurahman; Hughes, Lee E

    2015-08-01

    The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 μM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate. PMID:25917504

  3. Inhibition of Biofilm Formation, Quorum Sensing and Infection in Pseudomonas aeruginosa by Natural Products-Inspired Organosulfur Compounds

    PubMed Central

    Cady, Nathaniel C.; McKean, Kurt A.; Behnke, Jason; Kubec, Roman; Mosier, Aaron P.; Kasper, Stephen H.; Burz, David S.; Musah, Rabi A.

    2012-01-01

    Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed. PMID:22715388

  4. Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models.

    PubMed

    Madsen, Jonas S; Lin, Yu-Cheng; Squyres, Georgia R; Price-Whelan, Alexa; de Santiago Torio, Ana; Song, Angela; Cornell, William C; Sørensen, Søren J; Xavier, Joao B; Dietrich, Lars E P

    2015-12-01

    As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.

  5. Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa.

    PubMed

    Costa, Siddhartha G V A O; Lépine, François; Milot, Sylvain; Déziel, Eric; Nitschke, Marcia; Contiero, Jonas

    2009-08-01

    Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography-mass spectrophotometry (MS) and by high-performance liquid chromatography-MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l(-1). Under these conditions, the surface tension of the culture decreased to 30 mN m(-1), and the critical micelle concentration was 26.5 mg l(-1). It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.

  6. Production of a specific extracellular inhibitor of TRPM3 channels

    PubMed Central

    Naylor, J; Milligan, C J; Zeng, F; Jones, C; Beech, D J

    2008-01-01

    Background and purpose: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. Experimental approach: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca2+ measurement. Key results: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca2+ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. Conclusions and implications: The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers. PMID:18604232

  7. A new extracellular von Willebrand A domain-containing protein is involved in silver uptake in Microcystis aeruginosa exposed to silver nanoparticles.

    PubMed

    Chen, Si; Jin, Yujian; Lavoie, Michel; Lu, Haiping; Zhu, Kun; Fu, Zhengwei; Qian, Haifeng

    2016-10-01

    Silver nanoparticles (AgNPs) can be toxic for cyanobacteria when present at low nanomolar concentrations, but the molecular mechanisms whereby AgNPs (or free Ag(+) released from AgNPs) interact with these prokaryotic algal cells remain elusive. Here, we studied Ag uptake mechanisms in the prokaryotic cyanobacterium Microcystis aeruginosa exposed to AgNPs by measuring growth inhibition in the absence or presence of high-affinity Ag-binding ligands and by genetic transformation of E. coli with a protein predicted to be involved in Ag uptake. We discovered a new von Willebrand A (vWA) domain-containing protein in M. aeruginosa that mediates Ag uptake from AgNPs when expressed in E. coli. This new Ag transport protein, which is absent in eukaryotic algae, is a potential candidate explaining the higher AgNPs toxicity in cyanobacteria such as M. aeruginosa than that in eukaryotic algae. The present study provides new insights on Ag uptake mechanisms in the prokaryotic algae M. aeruginosa. PMID:27412462

  8. Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora

    PubMed Central

    Rüttimann-Johnson, Carmen; Salas, Loreto; Vicuña, Rafael; Kirk, T. Kent

    1993-01-01

    The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize 14C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly. PMID:16348955

  9. [Extracellular slime production and adhesion of Morganella morganii strains to polystyrene].

    PubMed

    Michalska, Anna; Zalas-Wiecek, Patrycja; Sielska, Barbara; Gospodarek, Eugenia

    2011-01-01

    The aim of this study was the evaluation of the ability of extracellular slime production and adhesive properties of M. morganii strains. This study included 50 of M. morganii strains isolated from clinical samples. All of these strains were isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital in 2008-2009. Five (10.0%) out of 50. M. morganii strains demonstrated extracellular slime production. Adherence to polystyrene revealed 36 (72.0%) of M. morganii strains in it 6 strains (12.0%) adhered strongly, medium - 12 (24.0%) and weakly - 18 (36.0%).

  10. Enhancement of Rhamnolipid Production in Residual Soybean Oil by an Isolated Strain of Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    de Lima, C. J. B.; França, F. P.; Sérvulo, E. F. C.; Resende, M. M.; Cardoso, V. L.

    In the present work, the production of rhamnolipid from residual soybean oil (RSO) from food frying facilities was studied using a strain of Pseudomonas aeruginosa of contaminated lagoon, isolated from a hydrocarbon contaminated soil. The optimization of RSO, amonium nitrate, and brewery residual yeast concentrations was accomplished by a central composite experimental design and surface response analysis. The experiments were performed in 500-mL Erlenmeyer flasks containing 50mL of mineral medium, at 170 rpm and 30±1°C, for a 48-h fermentation period. Rhamnolipid production has been monitored by measurements of surface tension, rhamnose concentration, and emulsifying activity. The best-planned results, located on the central point, have corresponded to 22g/L of RSO, 5.625 g/ L of NH4NO3' and 11.5 g/L of brewery yeast. At the maximum point the values for rhamnose and emulsifying index were 2.2g/L and 100%, respectively.

  11. Extracellular enzyme activities during cassava fermentation for 'fufu' production.

    PubMed

    Oyewole, O B; Odunfa, S A

    1992-01-01

    Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.

  12. Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa.

    PubMed

    Buzid, Alyah; Muimhneacháin, Eoin Ó; Reen, F Jerry; Hayes, Phyllis E; Pardo, Leticia M; Shang, Fengjun; O'Gara, Fergal; Sperry, Jonathan; Luong, John H T; Glennon, Jeremy D; McGlacken, Gerard P

    2016-09-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1-10 μM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 μM, respectively. Graphical abstract Electrochemical detection of barakacin. PMID:27473426

  13. Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds.

    PubMed

    Roach, Thomas; Beckett, Richard P; Minibayeva, Farida V; Colville, Louise; Whitaker, Claire; Chen, Hongying; Bailly, Christophe; Kranner, Ilse

    2010-01-01

    Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O(2)(.-)) production upon desiccation in Castanea sativa seeds, mechanisms of O(2)(.-) production and the effect of exogenously supplied ROS. A transient increase in extracellular O(2)(.-) production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H(2)O(2))-dependent O(2)(.-) production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of approximately 50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O(2)(.-) production. H(2)O(2), applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.

  14. Glycerol as substrate for the production of biosurfactant by Pseudomonas aeruginosa UCP0992.

    PubMed

    Silva, S N R L; Farias, C B B; Rufino, R D; Luna, J M; Sarubbo, L A

    2010-08-01

    In this work the influence of the carbon source concentration, of the type and concentration of the nitrogen source and of the cultivation conditions (temperature, aeration and agitation speed) in mineral medium formulated with glycerol was studied for biosurfactant production by Pseudomonas aeruginosa UCP0992. The kinetics of microorganism growth and biosurfactant production have been described for the medium supplemented with 3% glycerol and 0.6% NaNO(3), at 28 degrees C during 120 h under 200 rpm, showing a growth-associated production. The isolated biosurfactant corresponded to a concentration of 8.0 g/l after 96 h. The medium surface tension was reduced to 27.4 mN/m and the emulsification index of hexadecane reached 75-80% after 72 h. A CMC of 700 mg/l and an interfacial tension against hexadecane of 2 mN/m were obtained. The biosurfactant showed stability when submitted at 90 degrees C during 2 h, and thermal (4-120 degrees C) and pH (4-12) stability regarding the surface tension reduction and the emulsification capacity of vegetable oils and hydrocarbons, and tolerance under high salt concentrations (2-10%). The biosurfactant was characterized as a group of rhamnolipids with anionic nature. The crude biosurfactant did not show toxicity against the microcrustacean Artemia salina and the cabbage (Brassica oleracea), while the isolated biosurfactant showed toxicity against the microcrustacean at the CMC. The application of the biosurfactant in diesel recovery from sand was demonstrated by removal percentiles above 85%. The results obtained in this work are noteworthy for possible biosurfactant production from glycerol with potential of application in the environment.

  15. Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

    PubMed Central

    Madsen, Jonas S.; Lin, Yu-Cheng; Squyres, Georgia R.; Price-Whelan, Alexa; de Santiago Torio, Ana; Song, Angela; Cornell, William C.; Sørensen, Søren J.

    2015-01-01

    As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities. PMID:26431965

  16. Extracellular ligninolytic enzymes production by Pleurotus eryngii on agroindustrial wastes.

    PubMed

    Akpinar, Merve; Urek, Raziye Ozturk

    2014-01-01

    Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ligninolytic ability to produce laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) enzymes through solid-state fermentation using apricot and pomegranate agroindustrial wastes. The reducing sugar, protein, lignin, and cellulose levels in these were studied. Also, the production of these ligninolytic enzymes was researched over the growth of the microorganism throughout 20 days, and the reducing sugar, protein, and nitrogen levels were recorded during the stationary cultivation at 28 ± 0.5°C. The highest Lac activity was obtained as 1618.5 ± 25 U/L on day 12 of cultivation using apricot. The highest MnP activity was attained as 570.82 ± 15 U/L on day 17 in pomegranate culture and about the same as apricot culture. There were low LiP activities in both cultures. The maximum LiP value detected was 16.13 ± 0.8 U/L in apricot cultures. In addition, AAO activities in both cultures showed similar trends up to day 17 of cultivation, with the highest AAO activity determined as 105.99 ± 6.3 U/L on day 10 in apricot cultures. Decolorization of the azo dye methyl orange was also achieved with produced ligninolytic enzymes by P. eryngii using apricot and pomegranate wastes. PMID:24279903

  17. A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa.

    PubMed

    Ben Mohamed, Fatima; Mohamed, Fatima Ben; Garcia-Verdugo, Ignacio; Medina, Mathieu; Balloy, Viviane; Chignard, Michel; Ramphal, Reuben; Touqui, Lhousseine

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis. PMID:22768318

  18. Hfq regulates antibacterial antibiotic biosynthesis and extracellular lytic-enzyme production in Lysobacter enzymogenes OH11

    PubMed Central

    Xu, Gaoge; Zhao, Yuxin; Du, Liangcheng; Qian, Guoliang; Liu, Fengquan

    2015-01-01

    Lysobacter enzymogenes is an important biocontrol agent with the ability to produce a variety of lytic enzymes and novel antibiotics. Little is known about their regulatory mechanisms. Understanding these will be helpful for improving biocontrol of crop diseases and potential medical application. In the present study, we generated an hfq (encoding a putative ribonucleic acid chaperone) deletion mutant, and then utilized a new genomic marker-free method to construct an hfq-complemented strain. We showed for the first time that Hfq played a pleiotropic role in regulating the antibacterial antibiotic biosynthesis and extracellular lytic enzyme activity in L. enzymogenes. Mutation of hfq significantly increased the yield of WAP-8294A2 (an antibacterial antibiotic) as well as the transcription of its key biosynthetic gene, waps1. However, inactivation of hfq almost abolished the extracellular chitinase activity and remarkably decreased the activity of both extracellular protease and cellulase in L. enzymogenes. We further showed that the regulation of hfq in extracellular chitinase production was in part through the impairment of the secretion of chitinase A. Collectively, our results reveal the regulatory roles of hfq in antibiotic metabolite and extracellular lytic enzymes in the underexplored genus of Lysobacter. PMID:25683974

  19. Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.

    PubMed

    Mookerjee, Shona A; Brand, Martin D

    2015-12-12

    Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis(1). Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate(2), the same is not true for extracellular acid production and its relationship to glycolytic rate (3-6). Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate(-) + H(+) and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3(-) + H(+) is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification (6). Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

  20. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  1. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

  2. Growing Menace of Antibacterial Resistance in Clinical Isolates of Pseudomonas aeruginosa in Nepal: An Insight of Beta-Lactamase Production

    PubMed Central

    Dhital, Rabindra; Puri, Ram; Chaudhary, Niraj; Khatiwada, Suresh

    2016-01-01

    Introduction. Pseudomonas aeruginosa is the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate in Pseudomonas aeruginosa isolated from different clinical specimens. Methods. Pseudomonas aeruginosa recovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC. Results. Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively. Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance.

  3. Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

    PubMed Central

    Prince, R W; Cox, C D; Vasil, M L

    1993-01-01

    A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants. Images PMID:8478325

  4. Growth, microcystin-production and proteomic responses of Microcystis aeruginosa under long-term exposure to amoxicillin.

    PubMed

    Liu, Ying; Chen, Shi; Zhang, Jian; Gao, Baoyu

    2016-04-15

    Ecological risk of antibiotics due to the induction of antibiotic-resistant bacteria has been widely investigated, while studies on the hazard of antibiotic contaminants via the regulation of cyanobacteria were still limited. This study focused on the long-term action effect and mechanism of amoxicillin (a broadly used antibiotic) in Microcystis aeruginosa at environmentally relevant concentrations through 30 days of semi-continuous culture. Amoxicillin stimulated the photosynthesis activity and the production of microcystins, and interaction of differential proteins under amoxicillin exposure further manifested the close correlation between the two processes. D1 protein, ATP synthase subunits alpha and beta, enolase, triosephosphate isomerase and phosphoglycerate kinase were candidate target positions of amoxicillin in M. aeruginosa under long-term exposure. Amoxicillin affected the cellular biosynthesis process and the metabolism of carbohydrate and nucleoside phosphate according to the proteomic responses. Under exposure to amoxicillin, stimulated growth rate at the beginning phase and increased production and release of microcystins during the whole exposure period would lead to a higher contamination of M. aeruginosa cells and microcystins, indicating that amoxicillin was harmful to aquatic environments through the promotion of cyanobacterial bloom. PMID:26900975

  5. Growing Menace of Antibacterial Resistance in Clinical Isolates of Pseudomonas aeruginosa in Nepal: An Insight of Beta-Lactamase Production.

    PubMed

    Ansari, Shamshul; Dhital, Rabindra; Shrestha, Sony; Thapa, Sangita; Puri, Ram; Chaudhary, Niraj; Khatiwada, Suresh; Gautam, Rajendra

    2016-01-01

    Introduction. Pseudomonas aeruginosa is the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate in Pseudomonas aeruginosa isolated from different clinical specimens. Methods. Pseudomonas aeruginosa recovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC. Results. Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively. Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance. PMID:27642599

  6. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

    PubMed

    Whitney, John C; Whitfield, Gregory B; Marmont, Lindsey S; Yip, Patrick; Neculai, A Mirela; Lobsanov, Yuri D; Robinson, Howard; Ohman, Dennis E; Howell, P Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  7. Growing Menace of Antibacterial Resistance in Clinical Isolates of Pseudomonas aeruginosa in Nepal: An Insight of Beta-Lactamase Production

    PubMed Central

    Dhital, Rabindra; Puri, Ram; Chaudhary, Niraj; Khatiwada, Suresh

    2016-01-01

    Introduction. Pseudomonas aeruginosa is the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate in Pseudomonas aeruginosa isolated from different clinical specimens. Methods. Pseudomonas aeruginosa recovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC. Results. Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively. Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance. PMID:27642599

  8. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

  9. Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa.

    PubMed

    Ross-Gillespie, A; Dumas, Z; Kümmerli, R

    2015-01-01

    Public goods cooperation is common in microbes, and there is much interest in understanding how such traits evolve. Research in recent years has identified several important factors that shape the evolutionary dynamics of such systems, yet few studies have investigated scenarios involving interactions between multiple public goods. Here, we offer general predictions about the evolutionary trajectories of two public goods traits having positive, negative or neutral regulatory influence on one another's expression, and we report on a test of some of our predictions in the context of Pseudomonas aeruginosa's production of two interlinked iron-scavenging siderophores. First, we confirmed that both pyoverdine and pyochelin siderophores do operate as public goods under appropriate environmental conditions. We then tracked their production in lines experimentally evolved under different iron-limitation regimes known to favour different siderophore expression profiles. Under strong iron limitation, where pyoverdine represses pyochelin, we saw a decline in pyoverdine and a concomitant increase in pyochelin - consistent with expansion of pyoverdine-defective cheats derepressed for pyochelin. Under moderate iron limitation, pyochelin declined - again consistent with an expected cheat invasion scenario - but there was no concomitant shift in pyoverdine because cross-suppression between the traits is unidirectional only. Alternating exposure to strong and moderate iron limitation caused qualitatively similar though lesser shifts compared to the constant-environment regimes. Our results confirm that the regulatory interconnections between public goods traits can significantly modulate the course of evolution, yet also suggest how we can start to predict the impacts such complexities will have on phenotypic divergence and community stability.

  10. Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa.

    PubMed

    Ross-Gillespie, A; Dumas, Z; Kümmerli, R

    2015-01-01

    Public goods cooperation is common in microbes, and there is much interest in understanding how such traits evolve. Research in recent years has identified several important factors that shape the evolutionary dynamics of such systems, yet few studies have investigated scenarios involving interactions between multiple public goods. Here, we offer general predictions about the evolutionary trajectories of two public goods traits having positive, negative or neutral regulatory influence on one another's expression, and we report on a test of some of our predictions in the context of Pseudomonas aeruginosa's production of two interlinked iron-scavenging siderophores. First, we confirmed that both pyoverdine and pyochelin siderophores do operate as public goods under appropriate environmental conditions. We then tracked their production in lines experimentally evolved under different iron-limitation regimes known to favour different siderophore expression profiles. Under strong iron limitation, where pyoverdine represses pyochelin, we saw a decline in pyoverdine and a concomitant increase in pyochelin - consistent with expansion of pyoverdine-defective cheats derepressed for pyochelin. Under moderate iron limitation, pyochelin declined - again consistent with an expected cheat invasion scenario - but there was no concomitant shift in pyoverdine because cross-suppression between the traits is unidirectional only. Alternating exposure to strong and moderate iron limitation caused qualitatively similar though lesser shifts compared to the constant-environment regimes. Our results confirm that the regulatory interconnections between public goods traits can significantly modulate the course of evolution, yet also suggest how we can start to predict the impacts such complexities will have on phenotypic divergence and community stability. PMID:25421271

  11. Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27.

    PubMed

    Deive, Francisco J; Carvalho, Elisabete; Pastrana, Lorenzo; Rúa, Maria L; Longo, Maria A; Sanroman, M Angeles

    2009-07-01

    In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

  12. Tasco®: A Product of Ascophyllum nodosum Enhances Immune Response of Caenorhabditis elegans Against Pseudomonas aeruginosa Infection

    PubMed Central

    Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin; Critchley, Alan T.; Prithiviraj, Balakrishnan

    2012-01-01

    The effects of Tasco®, a product made from the brown seaweed (Ascophyllum nodosum) were tested for the ability to protect Caenorhabditis elegans against Pseudomonas aeruginosa infection. A water extract of Tasco® (TWE) reduced P. aeruginosa inflicted mortality in the nematode. The TWE, at a concentration of 300 µg/mL, offered the maximum protection and induced the expression of innate immune response genes viz.; zk6.7 (Lypases), lys-1 (Lysozyme), spp-1 (Saponin like protein), f28d1.3 (Thaumatin like protein), t20g5.7 (Matridin SK domain protein), abf-1 (Antibacterial protein) and f38a1.5 (Lectin family protein). Further, TWE treatment also affected a number of virulence components of the P. aeuroginosa and reduced its secreted virulence factors such as lipase, proteases and toxic metabolites; hydrogen cyanide and pyocyanin. Decreased virulence factors were associated with a significant reduction in expression of regulatory genes involved in quorum sensing, lasI, lasR, rhlI and rhlR. In conclusion, the TWE-treatment protected the C. elegans against P. aeruginosa infection by a combination of effects on the innate immunity of the worms and direct effects on the bacterial quorum sensing and virulence factors. PMID:22363222

  13. Tasco®: a product of Ascophyllum nodosum enhances immune response of Caenorhabditis elegans against Pseudomonas aeruginosa infection.

    PubMed

    Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin; Critchley, Alan T; Prithiviraj, Balakrishnan

    2012-01-01

    The effects of Tasco®, a product made from the brown seaweed (Ascophyllum nodosum) were tested for the ability to protect Caenorhabditis elegans against Pseudomonas aeruginosa infection. A water extract of Tasco® (TWE) reduced P. aeruginosa inflicted mortality in the nematode. The TWE, at a concentration of 300 µg/mL, offered the maximum protection and induced the expression of innate immune response genes viz.; zk6.7 (Lypases), lys-1 (Lysozyme), spp-1 (Saponin like protein), f28d1.3 (Thaumatin like protein), t20g5.7 (Matridin SK domain protein), abf-1 (Antibacterial protein) and f38a1.5 (Lectin family protein). Further, TWE treatment also affected a number of virulence components of the P. aeuroginosa and reduced its secreted virulence factors such as lipase, proteases and toxic metabolites; hydrogen cyanide and pyocyanin. Decreased virulence factors were associated with a significant reduction in expression of regulatory genes involved in quorum sensing, lasI, lasR, rhlI and rhlR. In conclusion, the TWE-treatment protected the C. elegans against P. aeruginosa infection by a combination of effects on the innate immunity of the worms and direct effects on the bacterial quorum sensing and virulence factors. PMID:22363222

  14. Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

    PubMed

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  15. Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

    PubMed

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. PMID:25705212

  16. Effects of cyanobacterial extracellular products and gibberellic acid on salinity tolerance in Oryza sativa L

    PubMed Central

    Rodríguez, AA; Stella, AM; Storni, MM; Zulpa, G; Zaccaro, MC

    2006-01-01

    Salt stress is one of the most serious factors limiting the productivity of rice, the staple diet in many countries. Gibberellic acid has been reported to reduce NaCl-induced growth inhibition in some plants including rice. Most paddy soils have a natural population of Cyanobacteria, prokaryotic photosynthethic microorganisms, which synthesize and liberate plant growth regulators such as gibberellins that could exert a natural beneficial effect on salt stressed rice plants. The aim of this work was to evaluate the effect of the cyanobacterium Scytonema hofmanni extracellular products on the growth of rice seedlings inhibited by NaCl and to compare it with the effect of the gibberellic acid in the same stress condition. Growth (length and weight of the seedlings) and biochemical parameters (5-aminolevulinate dehydratase activity, total free porphyrin and pigments content) were evaluated. Salt exposure negatively affected all parameters measured, with the exception of chlorophyll. Chlrorophyll concentrations nearly doubled upon exposure to high salt. Gibberellic acid counteracted the effect of salt on the length and dry weight of the shoot, and on carotenoid and chlorophyll b contents. Extracellular products nullified the salt effect on shoot dry weight and carotenoid content; partially counteracted the effect on shoot length (from 54% to 38% decrease), root dry weight (from 59% to 41% decrease) and total free porphyrin (from 31 to 13% decrease); reduced by 35% the salt increase of chlorophyll a; had no effect on root length and chlorophyll b. Gibberellic acid and extracellular products increased 5-aminolevulinate dehydratase activity over the control without salt. When coincident with high salinity, exposure to either EP or GA3, resulted in a reversal of shoot-related responses to salt stress. We propose that Scytonema hofmanni extracellular products may counteract altered hormone homeostasis of rice seedlings under salt stress by producing gibberellin-like plant

  17. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    PubMed Central

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. PMID:25705212

  18. The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis.

    PubMed

    Gałczyńska, Katarzyna; Kurdziel, Krystyna; Adamus-Białek, Wioletta; Wąsik, Sławomir; Szary, Karol; Drabik, Marcin; Węgierek-Ciuk, Aneta; Lankoff, Anna; Arabski, Michał

    2015-01-01

    Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals. PMID:26645324

  19. Influence of cultivation parameters on growth and microcystin production of Microcystis aeruginosa (Cyanophyceae) isolated from Lake Chao (China).

    PubMed

    Krüger, Thomas; Hölzel, Nadine; Luckas, Bernd

    2012-01-01

    Microcystis aeruginosa isolated in 2005 from the shallow eutrophic Lake Chao (Anhui, China) was investigated in terms of growth parameters and microcystin production under varying nutrient concentrations (P, N) and pH values (abiotic factors) as well as under the influence of spent medium of the non-toxic cyanobacterium Synechocystis sp. (biotic factors). Stimulating effects on growth were observed at the alkaline pH value (10.5), whereas toxin production was significantly increased under phosphate-P limitation (0.6 mg L(-1) medium). Within a broad range of nitrate-N concentrations (41.2-247.2 mg L(-1) medium), no significant influence on cell growth and microcystin production was observed; however, N-starvation resulted in a typical decrease of growth and toxicity. In addition, cryopreservation of M. aeruginosa evidenced the decrease of toxin production by time-dependent exposure with the cryoprotectant dimethyl sulfoxide under thawing conditions without affecting the growth of the cyanobacterial cells.

  20. Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source.

    PubMed

    Thaniyavarn, Jiraporn; Chongchin, Aree; Wanitsuksombut, Nopparat; Thaniyavarn, Suthep; Pinphanichakarn, Pairoh; Leepipatpiboon, Natthanant; Morikawa, Masaaki; Kanaya, Shigenori

    2006-08-01

    Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively. PMID:17116970

  1. Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans.

    PubMed

    Katz, M E; Flynn, P K; vanKuyk, P A; Cheetham, B F

    1996-04-10

    The extracellular proteases of Aspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designated xprE, located on chromosome VI. The xprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations, xprF1, xprF2 and xprG1, which suppress xprE1, were characterised. Both xprF and xprG map to chromosome VII but the two genes are unlinked. The xprF1, xprF2 and xprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that the xprE1 and xprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.

  2. Sodium houttuyfonate affects production of N-acyl homoserine lactone and quorum sensing-regulated genes expression in Pseudomonas aeruginosa

    PubMed Central

    Wu, Daqiang; Huang, Weifeng; Duan, Qiangjun; Li, Fang; Cheng, Huijuan

    2014-01-01

    Quorum sensing (QS) is a means of cell-to-cell communication that uses diffusible signaling molecules that are sensed by the population to determine population density, thus allowing co-ordinate gene regulation in response to population density. In Pseudomonas aeruginosa, production of the QS signaling molecule, N-acyl homoserine lactone (AHL), co-ordinates expression of key factors of pathogenesis, including biofilm formation and toxin secretion. It is predicted that the inhibition of AHL sensing would provide an effective clinical treatment to reduce the expression of virulence factors and increase the effectiveness of antimicrobial agents. We previously demonstrated that sodium houttuyfonate (SH), commonly used in traditional Chinese medicine to treat infectious diseases, can effectively inhibit QS-regulated processes, including biofilm formation. Here, using a model system, we demonstrate that SH causes the dose-dependent inhibition of AHL production, through down-regulation of the AHL biosynthesis gene, lasI. Addition of SH also resulted in down-regulation of expression of the AHL sensor and transcriptional regulator, LasR, and inhibited the production of the QS-regulated virulence factors, pyocyanin and LasA. These results suggest that the antimicrobial activity of SH may be due to its ability to disrupt QS in P. aeruginosa. PMID:25505457

  3. Lead-enhanced siderophore production and alteration in cell morphology in a Pb-resistant Pseudomonas aeruginosa strain 4EA.

    PubMed

    Naik, Milind Mohan; Dubey, Santosh Kumar

    2011-02-01

    A lead-resistant bacterial strain 4EA from soil contaminated with car battery waste from Goa, India was isolated and identified as Pseudomonas aeruginosa. This lead-resistant bacterial isolate interestingly revealed lead-enhanced siderophore (pyochelin and pyoverdine) production up to 0.5 mM lead nitrate whereas cells exhibit a significant decline in siderophore production above 0.5 mM lead nitrate. The bacterial cells also revealed significant alteration in cell morphology as size reduction when exposed to 0.8 mM lead nitrate. Enhanced production of siderophore was evidently detected by chrome azurol S agar diffusion (CASAD) assay as increase in diameter of orange halo, and reduction in bacterial size along with significant biosorption of lead was recorded by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDX). Pseudomonas aeruginosa strain 4EA also exhibits cross tolerance to other toxic metals viz. cadmium, mercury, and zinc besides resistance to multiple antibiotics such as ampicillin, erythromycin, amikacin, cephalexin, co-trimoxazole, mecillinam, lincomycin, ciphaloridine, oleondamycin, and nalidixic acid. PMID:20661573

  4. Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps.

    PubMed

    Yamaguchi, Rui; Kawata, Jin; Yamamoto, Toshitaka; Ishimaru, Yasuji; Sakamoto, Arisa; Ono, Tomomichi; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-08-01

    Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

  5. Pseudomonas aeruginosa LBI production as an integrated process using the wastes from sunflower-oil refining as a substrate.

    PubMed

    Benincasa, Maria; Accorsini, Fábio Raphael

    2008-06-01

    Pseudomonas aeruginosa LBI produced surface active rhamnolipids when cultivated on waste from the sunflower-oil process under different conditions. These biosurfactants, which reduce the superficial and interfacial tensions between fluids, offer advantages over their chemical counterparts, especially because of their ecological acceptability. These molecules can be used in fields as diverse as chemical, pharmaceutical and petrochemical industries. In this work, we present the effect of C/N ratio on growth and production yield. The best production yields (Y P/S) were achieved for C/N ratios (in g/g) of 8/1 (0.22) and 6.4/1 (0.23). The product concentration was very satisfactory (7.3g/L) at C/N ratio of 8/1, especially when considering that the substrate was basically composed of wastes that would otherwise constitute an environmental disposal problem.

  6. The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

    PubMed Central

    Darzins, A

    1993-01-01

    The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili. Images PMID:8104179

  7. Inhibitors of Serine Proteases in Regulating the Production and Function of Neutrophil Extracellular Traps

    PubMed Central

    Majewski, Pawel; Majchrzak-Gorecka, Monika; Grygier, Beata; Skrzeczynska-Moncznik, Joanna; Osiecka, Oktawia; Cichy, Joanna

    2016-01-01

    Neutrophil extracellular traps (NETs), DNA webs released into the extracellular environment by activated neutrophils, are thought to play a key role in the entrapment and eradication of microbes. However, NETs are highly cytotoxic and a likely source of autoantigens, suggesting that NET release is tightly regulated. NET formation involves the activity of neutrophil elastase (NE), which cleaves histones, leading to chromatin decondensation. We and others have recently demonstrated that inhibitors of NE, such as secretory leukocyte protease inhibitor (SLPI) and SerpinB1, restrict NET production in vitro and in vivo. SLPI was also identified as a NET component in the lesional skin of patients suffering from the autoinflammatory skin disease psoriasis. SLPI-competent NET-like structures (a mixture of SLPI with neutrophil DNA and NE) stimulated the synthesis of interferon type I (IFNI) in plasmacytoid dendritic cells (pDCs) in vitro. pDCs uniquely respond to viral or microbial DNA/RNA but also to nucleic acids of “self” origin with the production of IFNI. Although IFNIs are critical in activating the antiviral/antimicrobial functions of many cells, IFNIs also play a role in inducing autoimmunity. Thus, NETs decorated by SLPI may regulate skin immunity through enhancing IFNI production in pDCs. Here, we review key aspects of how SLPI and SerpinB1 can control NET production and immunogenic function. PMID:27446090

  8. Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro.

    PubMed

    Helman, Yael; Natale, Frank; Sherrell, Robert M; Lavigne, Michèle; Starovoytov, Valentin; Gorbunov, Maxim Y; Falkowski, Paul G

    2008-01-01

    The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell-cell and cell-substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of (45)Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization. PMID:18162537

  9. Antimicrobial activity of neutralized extracellular culture filtrates of lactic acid bacteria isolated from a cultured Indian milk product ('dahi').

    PubMed

    Varadaraj, M C; Devi, N; Keshava, N; Manjrekar, S P

    1993-12-01

    Neutralized extracellular culture filtrate obtained from isolates of Lactobacillus acidophilus, Lactobacillus delbruecki ssp. bulgaricus, Lactobacillus salivarius and Lactococcus lactis ssp. lactis from 'dahi' showed weak to moderate inhibition of Staphylococcus aureus, Bacillus cereus, Escherichia coli, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus laterosporus, Bacillus subtilis and Pseudomonas aeruginosa when tested by the diffusion agar well assay method. The effective minimum quantity of lactic culture filtrates required to obtain complete inhibition of an inoculum of 10(3) cfu/ml of the bacteria tested was between 20 and 26% (vol/vol), as determined by the agar incorporation method. Neutralized extracellular culture filtrate of these lactic cultures added at a level of 10% in sterile, 10% reconstituted non-fat dry milk was able to either suppress or retard growth of selected bacterial cultures when incubated at 37 degrees C for 24 h. This study indicated the antimicrobial activity of dahi and the potential of using neutralized extracellular culture filtrate of lactic acid bacteria in the biopreservation of foods.

  10. Extracellular enzyme production by Rhizopus and Mucor species on solid media.

    PubMed

    Thompson, D P; Eribo, B E

    1984-01-01

    Solid media were employed to determine the presence and absence of extracellular enzyme production by two genera of fruit-rot fungi, Rhizopus and Mucor. The results of this investigation revealed that phosphatase was released into the cultural medium by all the fungi examined; however, only R. oryzae, R. tritici, M. mucedo, and M. piriformis showed the possibility of being high producers of the enzyme. Protease, urease, ribonuclease, pectate lyase, and polygalacturonase, at varying levels of activity, were detected, in the majority of the fungi, in the cultural medium.

  11. Extracellular production of a sphingomyelinase from Streptomyces griseocarneus using Streptomyces lividans.

    PubMed

    Sugimori, Daisuke; Tomita, Yu; Matsumoto, Yusaku; Ogino, Chiaki

    2011-04-01

    The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl(2) which was about 50-fold more than that with 10 mM MgCl(2).

  12. Production and composition of extracellular polysaccharide synthesized by a Rhizobium isolate of Vigna mungo (L.) Hepper.

    PubMed

    Mandal, Santi Mohan; Ray, Bimalendu; Dey, Satyahari; Pati, Bikas Ranjan

    2007-08-01

    An extracellular polysaccharide (EPS) was produced by a Rhizobium sp. isolated from the root nodules of Vigna mungo (L.) Hepper. Maximum EPS production (346 mg l(-1)) was when the yeast extract basal medium was supplemented with mannitol (1%), biotin (1.5 mg l(-1)) and asparagine (0.3%). Ribose (53%) and mannose (47%) were the principle monomers of the EPS. Chemical, chromatographic and spectroscopic analysis showed that this polymer, which has Man(4)Rib(1) as an oligomeric subunit, has an apparent molecular mass of 750 kDa.

  13. Chlorination of Microcystis aeruginosa: toxin release and oxidation, cellular chlorine demand and disinfection by-products formation.

    PubMed

    Zamyadi, Arash; Fan, Yan; Daly, Rob I; Prévost, Michèle

    2013-03-01

    Direct chlorination of toxic cyanobacteria cells can occur at various stages of treatment. The objectives of this work are to determine and model the extent of Microcystis aeruginosa cells lysis, toxins and organic compounds release and oxidation, and quantify the subsequent disinfection by-products formation. Chlorine exposure (CT) values of 296 and 100 mg min/L were required to obtain 76% cell lysis and oxidation of released cell-bound toxins at levels below the provisional World Health Organisation guideline value (1 μg/L MC-LR). Toxin oxidation rates were similar or faster than cell lysis rates in ultrapure water. This work presents much needed unit M. aeruginosa cellular chlorine demand (5.6 ± 0.2 pgCl(2)/cell) which could be used to adjust the chlorination capacity to satisfy the total chlorine demand associated with the presence of cells. Furthermore, a novel successive reaction kinetics model is developed using the kinetics of the chlorine reaction with cyanobacterial cells and cell-bound toxins. Chlorination of dense cell suspensions (500,000 cells/mL) in ultrapure water at CT up to 3051 mg min/L resulted in modest concentrations of trihalomethanes (13 μg/L) and haloacetic acids (below detection limit).

  14. Evidence of the Cost of the Production of Microcystins by Microcystis aeruginosa under Differing Light and Nitrate Environmental Conditions

    PubMed Central

    Briand, Enora; Bormans, Myriam; Quiblier, Catherine; Salençon, Marie-José; Humbert, Jean-François

    2012-01-01

    The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains. PMID:22276137

  15. Extracellular xylanase production by Pleurotus species on lignocellulosic wastes under in vivo condition using novel pretreatment.

    PubMed

    Singh, M P; Pandey, A K; Vishwakarma, S K; Srivastava, A K; Pandey, V K

    2012-01-01

    The production of extracellular xylanase by three species of Pleurotus species i.e. P. florida, P. flabellatus and P. sajor caju was studied under in vivo condition during their cultivation on pretreated lignocellulosic wastes. Neem (Azadirachta indica) oil and ashoka (Saraca indica) leaves extract were used for pretreatment of paddy straw and wheat straw. Between these two wastes, paddy straw pretreated with neem oil, supported better xylanase production than wheat straw. Initially, xylanase production was low but it increased in subsequent days and reached at peak on 25th day of cultivation of Pleurotus species. Thereafter, there was decrease in the activity of the enzyme. On 25th day of incubation P. florida produced maximum xylanase on neem oil pretreated paddy straw i.e. 10.59 Uh—1ml—1. Among the three species, P. florida showed maximum enzyme activity followed by P. flabellatus and P. sajor caju. PMID:23273208

  16. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    PubMed

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production.

  17. Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale.

    PubMed

    Jasim, B; Anisha, C; Rohini, Sabu; Kurian, Jacob Manoj; Jyothis, Mathew; Radhakrishnan, E K

    2014-05-01

    Ginger (Zingiber officinale) is cultivated commercially in most parts of the world especially in India for its culinary and medicinal applications. One of the major challenges that limit the yield of ginger is rhizome rot disease caused by organisms including Pythium myriotylum. A feasible ecofriendly method is yet to be devised to prevent the plant from this threatening disease. Recent studies on plant microbiome show the possibility of having endophytic organisms with plant protective characteristics associated with the plants. Because of the uniquely evolved underground nature of the ginger rhizome and its peculiar survival in soil for a long time, many interesting endophytic microbes with plant protective characters can be well expected from it. In the current study, previously isolated endophytic Pseudomonas aeruginosa from ginger was investigated in detail for its effect on Pythium myriotylum. The rhizome protective effect of the organism was also studied by co-inoculation studies, which confirmed that Pseudomonas aeruginosa has very potent inhibitory effect on Pythium myriotylum. On further studies, the active antifungal compound was identified as phenazine 1-carboxylic acid.

  18. 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.

  19. Extracellular electron transfer from cathode to microbes: application for biofuel production.

    PubMed

    Choi, Okkyoung; Sang, Byoung-In

    2016-01-01

    Extracellular electron transfer in microorganisms has been applied for bioelectrochemical synthesis utilizing microbes to catalyze anodic and/or cathodic biochemical reactions. Anodic reactions (electron transfer from microbe to anode) are used for current production and cathodic reactions (electron transfer from cathode to microbe) have recently been applied for current consumption for valuable biochemical production. The extensively studied exoelectrogenic bacteria Shewanella and Geobacter showed that both directions for electron transfer would be possible. It was proposed that gram-positive bacteria, in the absence of cytochrome C, would accept electrons using a cascade of membrane-bound complexes such as membrane-bound Fe-S proteins, oxidoreductase, and periplasmic enzymes. Modification of the cathode with the addition of positive charged species such as chitosan or with an increase of the interfacial area using a porous three-dimensional scaffold electrode led to increased current consumption. The extracellular electron transfer from the cathode to the microbe could catalyze various bioelectrochemical reductions. Electrofermentation used electrons from the cathode as reducing power to produce more reduced compounds such as alcohols than acids, shifting the metabolic pathway. Electrofuel could be generated through artificial photosynthesis using electrical energy instead of solar energy in the process of carbon fixation. PMID:26788124

  20. Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

    PubMed

    Chandra, Vikash; Karamitri, Angeliki; Richards, Paul; Cormier, Françoise; Ramond, Cyrille; Jockers, Ralf; Armanet, Mathieu; Albagli-Curiel, Olivier; Scharfmann, Raphael

    2016-01-01

    Acute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation. PMID:27166427

  1. 7-fluoroindole as an antivirulence compound against Pseudomonas aeruginosa.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Cho, Moo Hwan; Kim, Jung-Ae; Lee, Jintae

    2012-04-01

    The emergence of antibiotic resistance has necessitated new therapeutic approaches for combating persistent bacterial infection. An alternative approach is regulation of bacterial virulence instead of growth suppression, which can readily lead to drug resistance. The virulence of the opportunistic human pathogen Pseudomonas aeruginosa depends on a large number of extracellular factors and biofilm formation. Thirty-one natural and synthetic indole derivatives were screened. 7-fluoroindole (7FI) was identified as a compound that inhibits biofilm formation and blood hemolysis without inhibiting the growth of planktonic P. aeruginosa cells. Moreover, 7FI markedly reduced the production of quorum-sensing (QS)-regulated virulence factors 2-heptyl-3-hydroxy-4(1H)-quinolone, pyocyanin, rhamnolipid, two siderophores, pyoverdine and pyochelin. 7FI clearly suppressed swarming motility, protease activity and the production of a polymeric matrix in P. aeruginosa. However, unlike natural indole compounds, synthetic 7FI did not increase antibiotic resistance. Therefore, 7FI is a potential candidate for use in an antivirulence approach against persistent P. aeruginosa infection. PMID:22251040

  2. Influences of environmental factors on bacterial extracellular polymeric substances production in porous media

    NASA Astrophysics Data System (ADS)

    Xia, Lu; Zheng, Xilai; Shao, Haibing; Xin, Jia; Peng, Tao

    2014-11-01

    Bioclogging of natural porous media occurs frequently under a wide range of conditions. It may influence the performance of permeable reactive barrier and constructed wetland. It is also one of the factors that determine the effect of artificial groundwater recharge and in situ bioremediation process. In this study, a series of percolation column experiments were conducted to simulate bioclogging process in porous media. The predominant bacteria in porous media which induced clogging were identified to be Methylobacterium, Janthinobacterium, Yersinia, Staphylococcus and Acidovorax, most of which had been shown to effectively produce viscous extracellular polymeric substances (EPS). The column in which EPS production was maximized also coincided with the largest reduction in saturated hydraulic conductivity of porous media. In addition, carbon concentration was the most significant factor to affect polysaccharide, protein and EPS secretion, followed by phosphorus concentration and temperature. The coupled effect of carbon and phosphorus concentration was also very important to stimulate polysaccharide and EPS production.

  3. Extracellular peroxidase production by Coprinus species from urea-treated soil.

    PubMed

    Ikehata, Keisuke; Buchanan, Ian D; Smith, Daniel W

    2004-01-01

    Thirteen strains of inky-cap mushroom Coprinus species were evaluated for the production of extracellular peroxidase. The liquid fermentation was carried out in shake flasks containing 1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract broth at 25 degrees C. Peroxidase activity was detected in the liquid culture of several Coprinus species, including C. echinosporus NBRC 30630; C. macrocephalus NBRC 30117; Coprinus spp. UAMH 10065, UAMH 10066, UAMH 10067, and 074, after 10 days of growth. Peroxidase production by Coprinus sp. UAMH 10067, a Coprinus species isolated from urea-treated soil, was comparable to that of C. cinereus and reached 15 U.mL(-1) after 10 days. In addition, the peroxidase from Coprinus sp. UAMH 10067 was apparently more thermally stable than the enzyme produced by C. cinereus.

  4. Quorum sensing systems differentially regulate the production of phenazine-1-carboxylic acid in the rhizobacterium Pseudomonas aeruginosa PA1201

    PubMed Central

    Sun, Shuang; Zhou, Lian; Jin, Kaiming; Jiang, Haixia; He, Ya-Wen

    2016-01-01

    Pseudomonas aeruginosa strain PA1201 is a newly identified rhizobacterium that produces high levels of the secondary metabolite phenazine-1-carboxylic acid (PCA), the newly registered biopesticide Shenqinmycin. PCA production in liquid batch cultures utilizing a specialized PCA-promoting medium (PPM) typically occurs after the period of most rapid growth, and production is regulated in a quorum sensing (QS)-dependent manner. PA1201 contains two PCA biosynthetic gene clusters phz1 and phz2; both clusters contribute to PCA production, with phz2 making a greater contribution. PA1201 also contains a complete set of genes for four QS systems (LasI/LasR, RhlI/RhlR, PQS/MvfR, and IQS). By using several methods including gene deletion, the construction of promoter-lacZ fusion reporter strains, and RNA-Seq analysis, this study investigated the effects of the four QS systems on bacterial growth, QS signal production, the expression of phz1 and phz2, and PCA production. The possible mechanisms for the strain- and condition-dependent expression of phz1 and phz2 were discussed, and a schematic model was proposed. These findings provide a basis for further genetic engineering of the QS systems to improve PCA production. PMID:27456813

  5. Species-level variability in extracellular production rates of reactive oxygen species by diatoms

    NASA Astrophysics Data System (ADS)

    Schneider, Robin; Roe, Kelly; Hansel, Colleen; Voelker, Bettina

    2016-03-01

    Biological production and decay of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O2-) likely have significant effects on the cycling of trace metals and carbon in marine systems. In this study, extracellular production rates of H2O2 and O2- were determined for five species of marine diatoms in the presence and absence of light. Production of both ROS was measured in parallel by suspending cells on filters and measuring the ROS downstream using chemiluminescence probes. In addition, the ability of these organisms to break down O2- and H2O2 was examined by measuring recovery of O2- and H2O2 added to the influent medium. O2- production rates ranged from undetectable to 7.3 x 10-16 mol cell-1 hr-1, while H2O2 production rates ranged from undetectable to 3.4 x 10-16 mol cell-1 hr-1. Results suggest that extracellular ROS production occurs through a variety of pathways even amongst organisms of the same genus. Thalassiosira spp. produced more O2- in light than dark, even when the organisms were killed, indicating that O2- is produced via a passive photochemical process on the cell surface. The ratio of H2O¬2 to O2- production rates was consistent with production of H2O2 solely through dismutation of O2- for T. oceanica, while T. pseudonana made much more H2O2 than O2 . T. weissflogii only produced H2O2 when stressed or killed. P. tricornutum cells did not make cell-associated ROS, but did secrete H2O2-producing substances into the growth medium. In all organisms, recovery rates for killed cultures (94-100% H2O2; 10-80% O2-) were consistently higher than those for live cultures (65-95% H2O2; 10-50% O2-). While recovery rates for killed cultures in H2O2 indicate that nearly all H2O2 was degraded by active cell processes, O2- decay appeared to occur via a combination of active and passive processes. Overall, this study shows that the rates and pathways for ROS production and decay vary greatly among diatom species, even between those that are

  6. Species-Level Variability in Extracellular Production Rates of Reactive Oxygen Species by Diatoms.

    PubMed

    Schneider, Robin J; Roe, Kelly L; Hansel, Colleen M; Voelker, Bettina M

    2016-01-01

    Biological production and decay of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O[Formula: see text]) likely have significant effects on the cycling of trace metals and carbon in marine systems. In this study, extracellular production rates of H2O2 and O[Formula: see text] were determined for five species of marine diatoms in the presence and absence of light. Production of both ROS was measured in parallel by suspending cells on filters and measuring the ROS downstream using chemiluminescence probes. In addition, the ability of these organisms to break down O[Formula: see text] and H2O2 was examined by measuring recovery of O[Formula: see text] and H2O2 added to the influent medium. O[Formula: see text] production rates ranged from undetectable to 7.3 × 10(-16) mol cell(-1) h(-1), while H2O2 production rates ranged from undetectable to 3.4 × 10(-16) mol cell(-1) h(-1). Results suggest that extracellular ROS production occurs through a variety of pathways even amongst organisms of the same genus. Thalassiosira spp. produced more O[Formula: see text] in light than dark, even when the organisms were killed, indicating that O[Formula: see text] is produced via a passive photochemical process on the cell surface. The ratio of H2O2 to O[Formula: see text] production rates was consistent with production of H2O2 solely through dismutation of O[Formula: see text] for T. oceanica, while T. pseudonana made much more H2O2 than O[Formula: see text]. T. weissflogii only produced H2O2 when stressed or killed. P. tricornutum cells did not make cell-associated ROS, but did secrete H2O2-producing substances into the growth medium. In all organisms, recovery rates for killed cultures (94-100% H2O2; 10-80% O[Formula: see text]) were consistently higher than those for live cultures (65-95% H2O2; 10-50% O[Formula: see text]). While recovery rates for killed cultures in H2O2 indicate that nearly all H2O2 was degraded by active cell processes, O

  7. Species-Level Variability in Extracellular Production Rates of Reactive Oxygen Species by Diatoms

    PubMed Central

    Schneider, Robin J.; Roe, Kelly L.; Hansel, Colleen M.; Voelker, Bettina M.

    2016-01-01

    Biological production and decay of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O2-) likely have significant effects on the cycling of trace metals and carbon in marine systems. In this study, extracellular production rates of H2O2 and O2- were determined for five species of marine diatoms in the presence and absence of light. Production of both ROS was measured in parallel by suspending cells on filters and measuring the ROS downstream using chemiluminescence probes. In addition, the ability of these organisms to break down O2- and H2O2 was examined by measuring recovery of O2- and H2O2 added to the influent medium. O2- production rates ranged from undetectable to 7.3 × 10−16 mol cell−1 h−1, while H2O2 production rates ranged from undetectable to 3.4 × 10−16 mol cell−1 h−1. Results suggest that extracellular ROS production occurs through a variety of pathways even amongst organisms of the same genus. Thalassiosira spp. produced more O2- in light than dark, even when the organisms were killed, indicating that O2- is produced via a passive photochemical process on the cell surface. The ratio of H2O2 to O2- production rates was consistent with production of H2O2 solely through dismutation of O2- for T. oceanica, while T. pseudonana made much more H2O2 than O2-. T. weissflogii only produced H2O2 when stressed or killed. P. tricornutum cells did not make cell-associated ROS, but did secrete H2O2-producing substances into the growth medium. In all organisms, recovery rates for killed cultures (94–100% H2O2; 10–80% O2-) were consistently higher than those for live cultures (65–95% H2O2; 10–50% O2-). While recovery rates for killed cultures in H2O2 indicate that nearly all H2O2 was degraded by active cell processes, O2- decay appeared to occur via a combination of active and passive processes. Overall, this study shows that the rates and pathways for ROS production and decay vary greatly among diatom species, even

  8. Growth, toxin production, active oxygen species and catalase activity of Microcystis aeruginosa (Cyanophyceae) exposed to temperature stress.

    PubMed

    Giannuzzi, Leda; Krock, Bernd; Minaglia, Melina Celeste Crettaz; Rosso, Lorena; Houghton, Christian; Sedan, Daniela; Malanga, Gabriela; Espinosa, Mariela; Andrinolo, Darío; Hernando, Marcelo

    2016-11-01

    Microcystis are known for their potential ability to synthesize toxins, mainly microcystins (MCs). In order to evaluate the effects of temperature on chlorophyll a (Chl a), growth, physiological responses and toxin production of a native Microcystis aeruginosa, we exposed the cells to low (23°C) and high (29°C) temperature in addition to a 26°C control treatment. Exponential growth rate was significantly higher at 29°C compared to 23°C and control, reaching 0.43, 0.32 and 0.33day(-)(1) respectively. In addition, there was a delay of the start of exponential growth at 23°C. However, the intracellular concentration of Chl a decreased significantly due to temperature change. A significant increase in intracellular ROS was observed in coincidence with the activation of enzymatic antioxidant catalase (CAT) during the first two days of exposure to 23° and 29°C in comparison to the control experiment, decreasing thereafter to nearly initial values. Five MCs were determined by LC-MS/MS analysis. In the experiments, the highest MC concentration, 205fg [Leu(1)] MC-LR.cell(-1) expressed as MC-LR equivalent was measured in the beginning of the experiment and subsequently declined to 160fg.cell(-1) on day 2 and 70fg.cell(-1) on day 4 in cells exposed to 29°C. The same trend was observed for all other MCs except for the least abundant MC-LR which showed a continuous increase during exposure time. Our results suggest a high ability of M. aeruginosa to perceive ROS and to rapidly initiate antioxidant defenses with a differential response on MC production.

  9. Extracellular α-Galactosidase from Trichoderma sp. (WF-3): Optimization of Enzyme Production and Biochemical Characterization

    PubMed Central

    Chauhan, Aishwarya Singh; Kumar, Arunesh; Siddiqi, Nikhat J.; Sharma, B.

    2015-01-01

    Trichoderma spp. have been reported earlier for their excellent capacity of secreting extracellular α-galactosidase. This communication focuses on the optimization of culture conditions for optimal production of enzyme and its characterization. The evaluation of the effects of different enzyme assay parameters such as stability, pH, temperature, substrate concentrations, and incubation time on enzyme activity has been made. The most suitable buffer for enzyme assay was found to be citrate phosphate buffer (50 mM, pH 6.0) for optimal enzyme activity. This enzyme was fairly stable at higher temperature as it exhibited 72% activity at 60°C. The enzyme when incubated at room temperature up to two hours did not show any significant loss in activity. It followed Michaelis-Menten curve and showed direct relationship with varying substrate concentrations. Higher substrate concentration was not inhibitory to enzyme activity. The apparent Michaelis-Menten constant (Km), maximum rate of reaction (Vmax), Kcat, and catalytic efficiency values for this enzyme were calculated from the Lineweaver-Burk double reciprocal plot and were found to be 0.5 mM, 10 mM/s, 1.30 U mg−1, and 2.33 U mg−1 mM−1, respectively. This information would be helpful in understanding the biophysical and biochemical characteristics of extracellular α-galactosidase from other microbial sources. PMID:26609435

  10. Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli.

    PubMed

    Ujiie, Ayana; Nakano, Hideo; Iwasaki, Yugo

    2016-03-01

    An Escherichia coli expression system was established to produce recombinant extracellular Pseudozyma (Candida) antarctica lipase B (CALB). With the aim of producing the genuine CALB without additional amino acid residues, the mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3) using the pET system. Inducing gene expression at low temperature (20°C) was crucial for the production of active CALB; higher temperatures caused inclusion body formation. Prolonged induction for 48 h at 20°C allowed for the enzyme to be released into the culture medium, with more than half of the activity detected in the culture supernatant. A catalytically inactive CALB mutant (S105A) protein was similarly released, suggesting that the lipid-hydrolyzing activity of the enzyme was not the reason for the release. The CALB production level was further improved by optimizing the culture medium. Under the optimized conditions, the CALB in the culture supernatant amounted to 550 mg/L. The recombinant CALB was purified from the culture supernatant, yielding 5.67 mg of purified CALB from 50 mL of culture. N-terminal sequencing and ESI-MS analyses showed proper removal of the pelB signal sequence and the correct molecular weight of the protein, respectively, confirming the structural integrity of the recombinant CALB. The kinetic parameters towards p-nitrophenylbutyrate and the enantiomeric selectivity on rac-1-phenylethylacetate of the recombinant CALB were consistent with those of the authentic CALB. This is the first example of E. coli-based extracellular production of a CALB enzyme without extra amino acid residues.

  11. Medium factors on anaerobic production of rhamnolipids by Pseudomonas aeruginosa SG and a simplifying medium for in situ microbial enhanced oil recovery applications.

    PubMed

    Zhao, Feng; Zhou, Jidong; Han, Siqin; Ma, Fang; Zhang, Ying; Zhang, Jie

    2016-04-01

    Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO4(2-) and Mg(2+) are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO3, 5.49 g/l KH2PO4, 6.9 g/l K2HPO4·3H2O and 0.40 g/l MgSO4 was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI24 = 82.5 %. The simplifying medium was promising for in situ MEOR applications.

  12. Medium factors on anaerobic production of rhamnolipids by Pseudomonas aeruginosa SG and a simplifying medium for in situ microbial enhanced oil recovery applications.

    PubMed

    Zhao, Feng; Zhou, Jidong; Han, Siqin; Ma, Fang; Zhang, Ying; Zhang, Jie

    2016-04-01

    Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO4(2-) and Mg(2+) are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO3, 5.49 g/l KH2PO4, 6.9 g/l K2HPO4·3H2O and 0.40 g/l MgSO4 was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI24 = 82.5 %. The simplifying medium was promising for in situ MEOR applications. PMID:26925616

  13. Influence of O polysaccharides on biofilm development and outer membrane vesicle biogenesis in Pseudomonas aeruginosa PAO1.

    PubMed

    Murphy, Kathleen; Park, Amber J; Hao, Youai; Brewer, Dyanne; Lam, Joseph S; Khursigara, Cezar M

    2014-04-01

    Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA(-) and OSA(-) cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA(-) cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development. PMID:24464462

  14. The Gac/Rsm and cyclic-di-GMP signalling networks coordinately regulate iron uptake in Pseudomonas aeruginosa.

    PubMed

    Frangipani, Emanuela; Visaggio, Daniela; Heeb, Stephan; Kaever, Volkhard; Cámara, Miguel; Visca, Paolo; Imperi, Francesco

    2014-03-01

    Pseudomonas aeruginosa is a versatile bacterial pathogen capable of occupying diverse ecological niches. To cope with iron limitation, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, whose ability to deliver iron to the cell is crucial for biofilm formation and pathogenicity. In this study, we describe a link between iron uptake and the Gac/Rsm system, a conserved signal transducing pathway of P. aeruginosa that controls the production of extracellular products and virulence factors, as well as the switch from planktonic to biofilm lifestyle. We have observed that pyoverdine and pyochelin production in P. aeruginosa is strongly dependent on the activation state of the Gac/Rsm pathway, which controls siderophore regulatory and biosynthetic genes at the transcriptional level, in a manner that does not involve regulation of ferric uptake regulator (Fur) expression. Gac/Rsm-mediated regulation of iron uptake genes appears to be conserved in different P. aeruginosa strains. Further experiments led to propose that the Gac/Rsm system regulates siderophore production through modulation of the intracellular levels of the second messenger c-di-GMP, indicating that the c-di-GMP and the Gac/Rsm regulatory networks essential for biofilm formation can also coordinately control iron uptake in P. aeruginosa. PMID:23796404

  15. NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

    SciTech Connect

    Xiong Jing; Wang Yang; Zhu, Zhonghua; Liu Jianshe; Wang Yumei; Zhang Chun; Hammes, Hans-Peter; Lang, Florian; Feng Yuxi

    2007-10-05

    As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM.

  16. Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

    PubMed Central

    Fleuri, Luciana F.; Kawaguti, Haroldo Y.; Sato, Hélia H.

    2009-01-01

    This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25°C and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25°C and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts. PMID:24031407

  17. Inhibition and Dispersal of Pseudomonas aeruginosa Biofilms by Combination Treatment with Escapin Intermediate Products and Hydrogen Peroxide.

    PubMed

    Santiago, Ariel J; Ahmed, Marwa N A; Wang, Shu-Lin; Damera, Krishna; Wang, Binghe; Tai, Phang C; Gilbert, Eric S; Derby, Charles D

    2016-09-01

    Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix. PMID:27401562

  18. Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

    PubMed

    Rocha, Juliana D B; Nascimento, Michelle T C; Decote-Ricardo, Debora; Côrte-Real, Suzana; Morrot, Alexandre; Heise, Norton; Nunes, Marise P; Previato, José Osvaldo; Mendonça-Previato, Lucia; DosReis, George A; Saraiva, Elvira M; Freire-de-Lima, Célio G

    2015-01-01

    In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. PMID:25620354

  19. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology.

    PubMed

    Jia, Jia; Yang, Xiaofeng; Wu, Zhiliang; Zhang, Qian; Lin, Zhi; Guo, Hongtao; Lin, Carol Sze Ki; Wang, Jianying; Wang, Yunshan

    2015-01-01

    Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N) ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production. PMID:26366414

  20. Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates.

    PubMed

    Allison, Steven D; Lu, Lucy; Kent, Alyssa G; Martiny, Adam C

    2014-01-01

    Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis. We observed a similar decline in cultures inoculated with producers only, suggesting that cheater mutants arose de novo and swept the population. DNA sequencing showed that genetic changes most likely occurred outside the protease operon. In one experimental replicate, the population regained the ability to produce protease, likely due to further genetic changes or population dynamics. Under spatially structured conditions on agar plates, cheaters did not sweep the population. Instead, we observed a significant increase in the variation of enzyme activity levels expressed by clones isolated from the population. Together these results suggest that restricted diffusion favors a diversity of enzyme production strategies. In contrast, well-mixed conditions favor population sweeps by cheater strains, consistent with theoretical predictions. Cheater and producer strategies likely coexist in natural environments with the frequency of cheating increasing with diffusion rate.

  1. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology

    PubMed Central

    Jia, Jia; Yang, Xiaofeng; Wu, Zhiliang; Zhang, Qian; Lin, Zhi; Guo, Hongtao; Lin, Carol Sze Ki; Wang, Jianying; Wang, Yunshan

    2015-01-01

    Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N) ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production. PMID:26366414

  2. Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates

    PubMed Central

    Allison, Steven D.; Lu, Lucy; Kent, Alyssa G.; Martiny, Adam C.

    2014-01-01

    Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis. We observed a similar decline in cultures inoculated with producers only, suggesting that cheater mutants arose de novo and swept the population. DNA sequencing showed that genetic changes most likely occurred outside the protease operon. In one experimental replicate, the population regained the ability to produce protease, likely due to further genetic changes or population dynamics. Under spatially structured conditions on agar plates, cheaters did not sweep the population. Instead, we observed a significant increase in the variation of enzyme activity levels expressed by clones isolated from the population. Together these results suggest that restricted diffusion favors a diversity of enzyme production strategies. In contrast, well-mixed conditions favor population sweeps by cheater strains, consistent with theoretical predictions. Cheater and producer strategies likely coexist in natural environments with the frequency of cheating increasing with diffusion rate. PMID:24782855

  3. Direct Regulation of Extracellular Chitinase Production by the Transcription Factor LeClp in Lysobacter enzymogenes OH11.

    PubMed

    Xu, Huiyong; Chen, Hongfu; Shen, Yuemao; Du, Liangcheng; Chou, Shan-Ho; Liu, Hongxia; Qian, Guoliang; Liu, Fengquan

    2016-09-01

    Lysobacter enzymogenes is a gram-negative bacterial biological control agent that produces abundant extracellular enzymes capable of degrading the cell walls of fungal pathogens. In strain OH11, an isolate from China, the global regulator LeClp controls the production of extracellular chitinase by regulating the transcription of the chitinase-encoding gene chiA. Using a combination of bioinformatic, genetic, and biochemical methods, we show that LeClp regulates chiA transcription by directly binding to the chiA promoter region. Although LeClp appears to be important in this role, it is not the sole regulator of chiA transcription. Furthermore, the sequence analysis of putative LeClp binding sites indicated that the LeClp homolog could be involved in the regulation of extracellular chitinase production in diverse Lysobacter spp. by a mechanism similar to that in L. enzymogenes. Our findings present new insights into the molecular mechanism of LeClp in controlling extracellular chitinase activity, providing a fundamental road to elucidate how LeClp regulates the production of other extracellular lytic enzymes in L. enzymogenes. PMID:27385597

  4. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed Central

    Higerd, T B; Vesole, D H; Goust, J M

    1978-01-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. Images PMID:689736

  5. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed

    Higerd, T B; Vesole, D H; Goust, J M

    1978-08-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response. PMID:689736

  6. Production and characterization of an extracellular polysaccharide from Streptomyces violaceus MM72.

    PubMed

    Manivasagan, Panchanathan; Sivasankar, Palaniappan; Venkatesan, Jayachandran; Senthilkumar, Kalimuthu; Sivakumar, Kannan; Kim, Se-Kwon

    2013-08-01

    The isolation, optimization, purification and characterization of an extracellular polysaccharide (EPS) from a marine actinobacterium, Streptomyces violaceus MM72 were investigated. Medium composition and culture conditions for the EPS production by S. violaceus MM72 were optimized using two statistical methods: Plackett-Burman design applied to find the key ingredients and conditions for the best yield of EPS production and central composite design used to optimize the concentration of the three significant variables: glucose, tryptone and NaCl. The preferable culture conditions for EPS production were pH 7.0, temperature 35°C and NaCl concentration 2.0% for 120h with fructose and yeast extract as best carbon and nitrogen sources, respectively. The results showed that S. violaceus MM72 produced a kind of EPS having molecular weight of 8.96×10(5)Da. In addition, the EPS showed strong DPPH radical-scavenging activity, superoxide scavenging and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities determined in this study. These results showed the great potential of EPS produced by S. violaceus MM72 could be used in industry in place of synthetic compounds. The EPS from S. violaceus MM72 may be a new source of natural antioxidants with potential value for health, food and therapeutics. PMID:23597709

  7. Production of extracellular polysaccharide by Bacillus megaterium RB-05 using jute as substrate.

    PubMed

    Chowdhury, Sougata Roy; Basak, Ratan Kumar; Sen, Ramkrishna; Adhikari, Basudam

    2011-06-01

    Bacillus megaterium RB-05 was grown on glucose and on "tossa-daisee" (Corchorus olitorius)-derived jute, and production and composition of extracellular polysaccharide (EPS) were monitored. An EPS yield of 0.065 ± 0.013 and of 0.297 g ± 0.054 g(-1) substrate after 72 h was obtained for glucose and jute, respectively. EPS production in the presence of jute paralleled bacterial cellulase activity. High performance liquid chromatography (HPLC), matrix assisted LASER desorption/ionization-time of flight (MALDI-ToF) mass spectroscopy, and fourier transform infrared (FT-IR) spectroscopy demonstrated that the EPS synthesized in jute culture (JC) differed from that synthesized in glucose mineral salts medium (GMSM). While fucose was only a minor constituent (4.9 wt.%) of EPS from GMSM, it a major component (41.9 wt.%) of EPS synthesized in JC. This study establishes jute as an effective fermentation substrate for EPS production by a cellulase-producing bacterium.

  8. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    PubMed

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production. PMID:25277968

  9. The role of 2,4-dihydroxyquinoline (DHQ) in Pseudomonas aeruginosa pathogenicity

    PubMed Central

    Gruber, Jordon D.; Chen, Wei; Parnham, Stuart; Beauchesne, Kevin; Moeller, Peter; Flume, Patrick A.

    2016-01-01

    Bacteria synchronize group behaviors using quorum sensing, which is advantageous during an infection to thwart immune cell attack and resist deleterious changes in the environment. In Pseudomonas aeruginosa, the Pseudomonas quinolone signal (Pqs) quorum-sensing system is an important component of an interconnected intercellular communication network. Two alkylquinolones, 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), activate transcriptional regulator PqsR to promote the production of quinolone signals and virulence factors. Our work focused on the most abundant quinolone produced from the Pqs system, 2,4-dihydroxyquinoline (DHQ), which was shown previously to sustain pyocyanin production and antifungal activity of P. aeruginosa. However, little is known about how DHQ affects P. aeruginosa pathogenicity. Using C. elegans as a model for P. aeruginosa infection, we found pqs mutants only able to produce DHQ maintained virulence towards the nematodes similar to wild-type. In addition, DHQ-only producing mutants displayed increased colonization of C. elegans and virulence factor production compared to a quinolone-null strain. DHQ also bound to PqsR and activated the transcription of pqs operon. More importantly, high extracellular concentration of DHQ was maintained in both aerobic and anaerobic growth. High levels of DHQ were also detected in the sputum samples of cystic fibrosis patients. Taken together, our findings suggest DHQ may play an important role in sustaining P. aeruginosa pathogenicity under oxygen-limiting conditions. PMID:26788419

  10. Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils.

    PubMed

    Haba, E; Espuny, M J; Busquets, M; Manresa, A

    2000-03-01

    World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.

  11. Solid state fermentation for extracellular polysaccharide production by Lactobacillus confusus with coconut water and sugar cane juice as renewable wastes.

    PubMed

    Seesuriyachan, Phisit; Techapun, Charin; Shinkawa, Hidenori; Sasaki, Ken

    2010-01-01

    Extracellular polysaccharide (EPS) production by Lactobacillus confusus in liquid and solid state fermentation was carried out using coconut water and sugarcane juice as renewable wastes. High concentrations of EPS of 62 (sugarcane juice) and 18 g/l of coconut water were produced in solid state fermentation when nitrogen sources were reduced 5-fold from the original medium.

  12. Vaccination of channel catfish with extracellular products of Aeromonas hydrophila provides protection against infection by the pathogen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aeromonas hydrophila, a Gram-negative bacterium, is one of the economically-important pathogens in modern aquaculture. Among various traits, extracellular products (ECP) secreted by the bacterium are considered to be essential factors for virulence. Whether vaccination with the ECP could produce imm...

  13. Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa.

    PubMed

    Salvador, Daniel; Churro, Catarina; Valério, Elisabete

    2016-04-01

    Cyanobacteria are phytoplanktonic organisms widely occurring in freshwaters, being frequently associated with the production of toxins, namely microcystins (MCs). MCs are produced non-ribosomally by a multienzyme complex (mcy genes). It has been reported that environmental factors, such as light intensity, can influence toxin production. The aim of this study was to assess the influence of light intensity in the transcription of the mcyA gene and corresponding production of microcystins in toxic isolates of Planktothrix agardhii, where little is known, and compare them to Microcystis aeruginosa. For that purpose, cultures were exposed to three different light intensities (4, 20 and 30 μmol photons m(-2) s(-1)) for 18 days at 20 ± 1 °C. The growth was followed daily using absorbance readings. Samples were collected at each growth stage for cell counting, microcystins quantification and RNA extraction. The level of transcripts was quantified by RT-qPCR and the relative expression determined using 16S rDNA, gltA and rpoC1 as reference genes. The most stable reference genes in M. aeruginosa were rpoC1 and gltA, whereas in P. agardhii were 16S rDNA and gltA. There was a correspondence between the growth rate and light intensity in M. aeruginosa and P. agardhii. The growth rates for both species were lower at 4 and higher at 30 μmol photons m(-2) s(-1). Microcystin concentration per cell was similar between light intensities in M. aeruginosa and over time, while in P. agardhii it was higher in the stationary phase at 4 μmol photons m(-2) s(-1). There were differences in the expression of mcyA between the two species. In M. aeruginosa, the highest levels of expression occurred at 4 μmol photons m(-2) s(-1) in the adaptation phase, whereas for P. agardhii it was at 4μmol photons m(-2) s(-1) in the exponential growth phase. Our results indicate that the light intensities tested had distinct influences on the growth, microcystin production and mcyA expression levels

  14. Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa.

    PubMed

    Salvador, Daniel; Churro, Catarina; Valério, Elisabete

    2016-04-01

    Cyanobacteria are phytoplanktonic organisms widely occurring in freshwaters, being frequently associated with the production of toxins, namely microcystins (MCs). MCs are produced non-ribosomally by a multienzyme complex (mcy genes). It has been reported that environmental factors, such as light intensity, can influence toxin production. The aim of this study was to assess the influence of light intensity in the transcription of the mcyA gene and corresponding production of microcystins in toxic isolates of Planktothrix agardhii, where little is known, and compare them to Microcystis aeruginosa. For that purpose, cultures were exposed to three different light intensities (4, 20 and 30 μmol photons m(-2) s(-1)) for 18 days at 20 ± 1 °C. The growth was followed daily using absorbance readings. Samples were collected at each growth stage for cell counting, microcystins quantification and RNA extraction. The level of transcripts was quantified by RT-qPCR and the relative expression determined using 16S rDNA, gltA and rpoC1 as reference genes. The most stable reference genes in M. aeruginosa were rpoC1 and gltA, whereas in P. agardhii were 16S rDNA and gltA. There was a correspondence between the growth rate and light intensity in M. aeruginosa and P. agardhii. The growth rates for both species were lower at 4 and higher at 30 μmol photons m(-2) s(-1). Microcystin concentration per cell was similar between light intensities in M. aeruginosa and over time, while in P. agardhii it was higher in the stationary phase at 4 μmol photons m(-2) s(-1). There were differences in the expression of mcyA between the two species. In M. aeruginosa, the highest levels of expression occurred at 4 μmol photons m(-2) s(-1) in the adaptation phase, whereas for P. agardhii it was at 4μmol photons m(-2) s(-1) in the exponential growth phase. Our results indicate that the light intensities tested had distinct influences on the growth, microcystin production and mcyA expression levels

  15. Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

    PubMed Central

    Tanaka, E; Kawamoto, S; Fukushima, J; Hamajima, K; Onishi, H; Miyagi, Y; Inami, S; Morihara, K; Okuda, K

    1991-01-01

    The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain. Images PMID

  16. Evaluation of Mannosidase and Trypsin Enzymes Effects on Biofilm Production of Pseudomonas aeruginosa Isolated from Burn Wound Infections

    PubMed Central

    Banar, Maryam; Emaneini, Mohammad; Satarzadeh, Mhboubeh; Abdellahi, Nafiseh; Beigverdi, Reza; van Leeuwen, Willem B.; Jabalameli, Fereshteh

    2016-01-01

    Biofilm is an important virulence factor in Pseudomonas aeruginosa and has a substantial role in antibiotic resistance and chronic burn wound infections. New therapeutic agents against P. aeruginosa, degrading biofilms in burn wounds and improving the efficacy of current antimicrobial agents, are required. In this study, the effects of α-mannosidase, β-mannosidase and trypsin enzymes on the degradation of P. aeruginosa biofilms and on the reduction of ceftazidime minimum biofilm eliminating concentrations (MBEC) were evaluated. All tested enzymes, destroyed the biofilms and reduced the ceftazidime MBECs. However, only trypsin had no cytotoxic effect on A-431 human epidermoid carcinoma cell lines. In conclusion, since trypsin had better features than mannosidase enzymes, it can be a promising agent in combatting P. aeruginosa burn wound infections. PMID:27736961

  17. Anthranilate deteriorates the structure of Pseudomonas aeruginosa biofilms and antagonizes the biofilm-enhancing indole effect.

    PubMed

    Kim, Soo-Kyoung; Park, Ha-Young; Lee, Joon-Hee

    2015-04-01

    Anthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation of Pseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product of P. aeruginosa, had an opposite effect on P. aeruginosa biofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment of P. aeruginosa at the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole on P. aeruginosa biofilm formation.

  18. Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

    PubMed Central

    Kim, Soo-Kyoung; Park, Ha-Young

    2015-01-01

    Anthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation of Pseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product of P. aeruginosa, had an opposite effect on P. aeruginosa biofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment of P. aeruginosa at the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole on P. aeruginosa biofilm formation. PMID:25616795

  19. Rhamnolipids Modulate Swarming Motility Patterns of Pseudomonas aeruginosa

    PubMed Central

    Caiazza, Nicky C.; Shanks, Robert M. Q.; O'Toole, G. A.

    2005-01-01

    Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed. PMID:16237018

  20. Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity.

    PubMed

    Nisole, Audrey; Lussier, François-Xavier; Morley, Krista L; Shareck, François; Kazlauskas, Romas J; Dupont, Claude; Pelletier, Joelle N

    2006-04-01

    Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.

  1. The combined effects of Dolichospermum flos-aquae, light, and temperature on microcystin production by Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Chen, Ruoqi; Li, Fangfang; Liu, Jiadong; Zheng, Hongye; Shen, Fei; Xue, Yarong; Liu, Changhong

    2016-11-01

    The effects of light, temperature, and coculture on the intracellular microcystin-LR (MC-LR) quota of Microcystis aeruginosa were evaluated based on coculture experiments with nontoxic Dolichospermum ( Anabaena) flos-aquae. The MC-LR quota and transcription of mcyB and mcyD genes encoding MC synthetases in M. aeruginosa were evaluated on the basis of cell counts, high-performance liquid chromatography, and reverse-transcription quantitative real-time PCR. The MC-LR quotas of M. aeruginosa in coculture with a 1/1 ratio of inoculum of the two species were significantly lower relative to monocultures 6-d after inoculation. Decreased MC-LR quotas under coculture conditions were enhanced by increasing the D. flos-aquae to M. aeruginosa ratio in the inoculum and by environmental factors, such as temperature and light intensity. Moreover, the transcriptional concentrations of mcyB and mcyD genes in M. aeruginosa were significantly inhibited by D. flos-aquae competition in coculture ( P <0.01), lowered to 20% of initial concentrations within 8 days. These data suggested that coculture eff ects by D. flos-aquae not only reduced M. aeruginosa's intracellular MC-LR quota via inhibition of genes encoding MC synthetases, but also that this eff ect was regulated by environmental factors, including temperature and light intensities.

  2. Constitutive Extracellular Polysaccharide (EPS) Production by Specific Isolates of Crocosphaera watsonii

    PubMed Central

    Sohm, Jill A.; Edwards, Bethanie R.; Wilson, Brian G.; Webb, Eric A.

    2011-01-01

    Unicellular dinitrogen (N2) fixing cyanobacteria have only recently been identified in the ocean and recognized as important contributors to global N2 fixation. The only cultivated representatives of the open ocean unicellular diazotrophs are multiple isolates of Crocosphaera watsonii. Although constituents of the genus are nearly genetically identical, isolates have been described in two size classes, large ∼5 μm and small ∼3 μm cell diameters. We show here that the large size class constitutively produces substantial amounts of extracellular polysaccharides (EPS) during exponential growth, up to 10 times more than is seen in the small size class, and does so under both N2 fixing and non-N2 fixing conditions. The EPS production exceeds the amount produced by larger phytoplankton such as diatoms and coccolithophores by one to two orders of magnitude, is ∼22% of the total particulate organic C in the culture, and is depleted in N compared to cellular material. The large difference in observed EPS production may be accounted for by consistently higher photochemical efficiency of photosystem II in the large (0.5) vs. small (∼0.35) strains. While it is known that Crocosphaera plays an important role in driving the biological carbon (C) pump through the input of new nitrogen (N) to the open ocean, we hypothesize that this species may also contribute directly to the C cycle through the constitutive production of EPS. Indeed, at two stations in the North Pacific Subtropical Gyre, ∼70% of large Crocosphaera cells observed were embedded in EPS. The evolutionary advantage of releasing such large amounts of fixed C is still unknown, but in regions where Crocosphaera can be abundant (i.e., the warm oligotrophic ocean) this material will likely have important biogeochemical consequences. PMID:22110469

  3. Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere.

    PubMed

    Mallikharjuna Rao, K L N; Siva Raju, K; Ravisankar, H

    2016-01-01

    Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30°C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

  4. Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

    PubMed Central

    Mallikharjuna Rao, K.L.N.; Siva Raju, K.; Ravisankar, H.

    2016-01-01

    Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco. PMID:26887223

  5. High-yield production of extracellular type-I cellulose by the cyanobacterium Synechococcus sp. PCC 7002.

    PubMed

    Zhao, Chi; Li, Zhongkui; Li, Tao; Zhang, Yingjiao; Bryant, Donald A; Zhao, Jindong

    2015-01-01

    Cellulose synthase, encoded by the cesA gene, is responsible for the synthesis of cellulose in nature. We show that the cell wall of the cyanobacterium Synechococcus sp. PCC 7002 naturally contains cellulose. Cellulose occurs as a possibly laminated layer between the inner and outer membrane, as well as being an important component of the extracellular glycocalyx in this cyanobacterium. Overexpression of six genes, cmc-ccp-cesAB-cesC-cesD-bgl, from Gluconacetobacter xylinus in Synechococcus sp. PCC 7002 resulted in very high-yield production of extracellular type-I cellulose. High-level cellulose production only occurred when the native cesA gene was inactivated and when cells were grown at low salinity. This system provides a method for the production of lignin-free cellulose from sunlight and CO2 for biofuel production and other biotechnological applications. PMID:27462405

  6. High-yield production of extracellular type-I cellulose by the cyanobacterium Synechococcus sp. PCC 7002.

    PubMed

    Zhao, Chi; Li, Zhongkui; Li, Tao; Zhang, Yingjiao; Bryant, Donald A; Zhao, Jindong

    2015-01-01

    Cellulose synthase, encoded by the cesA gene, is responsible for the synthesis of cellulose in nature. We show that the cell wall of the cyanobacterium Synechococcus sp. PCC 7002 naturally contains cellulose. Cellulose occurs as a possibly laminated layer between the inner and outer membrane, as well as being an important component of the extracellular glycocalyx in this cyanobacterium. Overexpression of six genes, cmc-ccp-cesAB-cesC-cesD-bgl, from Gluconacetobacter xylinus in Synechococcus sp. PCC 7002 resulted in very high-yield production of extracellular type-I cellulose. High-level cellulose production only occurred when the native cesA gene was inactivated and when cells were grown at low salinity. This system provides a method for the production of lignin-free cellulose from sunlight and CO2 for biofuel production and other biotechnological applications.

  7. High-yield production of extracellular type-I cellulose by the cyanobacterium Synechococcus sp. PCC 7002

    PubMed Central

    Zhao, Chi; Li, Zhongkui; Li, Tao; Zhang, Yingjiao; Bryant, Donald A; Zhao, Jindong

    2015-01-01

    Cellulose synthase, encoded by the cesA gene, is responsible for the synthesis of cellulose in nature. We show that the cell wall of the cyanobacterium Synechococcus sp. PCC 7002 naturally contains cellulose. Cellulose occurs as a possibly laminated layer between the inner and outer membrane, as well as being an important component of the extracellular glycocalyx in this cyanobacterium. Overexpression of six genes, cmc–ccp–cesAB–cesC–cesD–bgl, from Gluconacetobacter xylinus in Synechococcus sp. PCC 7002 resulted in very high-yield production of extracellular type-I cellulose. High-level cellulose production only occurred when the native cesA gene was inactivated and when cells were grown at low salinity. This system provides a method for the production of lignin-free cellulose from sunlight and CO2 for biofuel production and other biotechnological applications. PMID:27462405

  8. Characterization of wetland quorum quenching Pseudomonas aeruginosa strain 2SW8 and its 2-heptyl-3-hydroxy-4-quinolone production.

    PubMed

    Wong, Cheng-Siang; Yin, Wai-Fong; Sam, Choon-Kook; Koh, Chong-Lek; Chan, Kok-Gan

    2012-01-01

    Most Proteobacteria produce N-acylhomoserine lactones for bacterial cell-to-cell communication, a process called quorum sensing. Interference of quorum sensing, commonly known as quorum quenching, represents an important way to control quorum sensing. This work reports the isolation of quorum quenching bacterium strain 2WS8 from Malaysia tropical wetland water (2°11'8"N, 102°15'2"E, in 2007) by using a modified version of a previously reported KG medium. Strain 2WS8 was isolated based on its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) as the sole source of energy. This bacterium clustered closely to Pseudomonas aeruginosa PAO1. Strain 2SW8 possesses both quiP and pvdQ homologue acylase genes. Rapid Resolution Liquid Chromatography analysis confirmed that strain 2SW8 preferentially degraded N-acylhomoserine lactones with 3-oxo group substitution but not those with unsubstituted groups at C3 position in the acyl side chain. Strain 2SW8 also showed 2-heptyl-3-hydroxy-4-quinolone production.

  9. Evaluation of extracellular products and mutagenicity in cyanobacteria cultures separated from a eutrophic reservoir.

    PubMed

    Huang, Winn-Jung; Lai, Chun-Hsi; Cheng, Yung-Ling

    2007-05-15

    The algal extracellular products (ECPs) in three cultures of cyanobacteria species (Anabaena, Microcystis, and Oscillatoria) dominating the eutrophic reservoir populations and their toxins have been investigated in the present work. Using gas chromatography coupled with high-resolution electron-impact mass spectrometry (GC/EI-MS) and high performance anion-exchange chromatography (HPAEC) techniques, more than 20 compounds were found in the algal culture (including cells and filtrates) extracts. The main identified ECPs were classified to polysaccharides, hydrocarbons, and aldehydes. Odor causing substances such as trans-1,10-dimethyl-trans-9-decalol (geosmin) and 2-methylisoborneol (2-MIB)were also found in the algal cultures. The potential mutagenicity of the algal suspensions was also studied with the Ames test. The organic extracts of the algal suspension from the axenic cultures were mutagenicity in TA98 without S9 mix and in TA100 with and without S9 mix. The results indicate that the ECPs of three algae species dominating the eutrophic reservoir were mutagenic clearly in the bacterial test. PMID:17391736

  10. Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

    PubMed Central

    Arala-Chaves, M P; Porto, M T; Arnaud, P; Saraiva, M J; Geada, H; Patrick, C C; Fudenberg, H H

    1981-01-01

    The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si. Images PMID:6454698

  11. Bioremediation of Direct Blue 14 and Extracellular Ligninolytic Enzyme Production by White Rot Fungi: Pleurotus Spp.

    PubMed Central

    Singh, M. P.; Vishwakarma, S. K.; Srivastava, A. K.

    2013-01-01

    In the present investigation, four species of white rot fungi (Pleurotus), that is, P. flabellatus, P. florida, P. ostreatus and P. sajor-caju were used for decolorization of direct blue 14 (DB14). Among all four species of Pleurotus, P. flabellatus showed the fastest decolorization in petri plates on different concentration, that is, 200 mg/L, 400 mg/L, and 600 mg/L. All these four species were also evaluated for extracellular ligninolytic enzymes (laccase and manganese peroxidase) production and it was observed that the twelve days old culture of P. flabellatus showed the maximum enzymatic activity, that is, 915.7 U/mL and 769.2 U/mL of laccase and manganese peroxidase, respectively. Other three Pleurotus species took more time for dye decolorization and exhibited less enzymatic activities. The rate of decolorization of DB14 dye solution (20 mg/L) by crude enzymes isolated from P. flabellatus was very fast, and it was observed that up to 90.39% dye solution was decolorized in 6 hrs of incubation. PMID:23841054

  12. Lipolytic Potential of Aspergillus japonicus LAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

    PubMed Central

    Souza, Lívia Tereza Andrade; Oliveira, Jamil S.; dos Santos, Vera L.; Regis, Wiliam C. B.; Santoro, Marcelo M.; Resende, Rodrigo R.

    2014-01-01

    Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+ showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and Vmax values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications. PMID:25530954

  13. Lipolytic potential of Aspergillus japonicus LAB01: production, partial purification, and characterisation of an extracellular lipase.

    PubMed

    Souza, Lívia Tereza Andrade; Oliveira, Jamil S; dos Santos, Vera L; Regis, Wiliam C B; Santoro, Marcelo M; Resende, Rodrigo R

    2014-01-01

    Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu(2+), Ni(2+), and Al(3+) showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

  14. Virulence determinants and production of extracellular enzymes in Enterococcus spp. from surface water sources.

    PubMed

    Molale, Lesego Gertrude; Bezuidenhout, Cornelius Carlos

    2016-01-01

    Virulence factors in Enterococcus may be indicative of potential pathogenicity. The aim of this study was to determine the relationship between the presence of clinically relevant virulence genes, in Enterococcus spp. from environmental water, and their in vitro expression. One hundred and twenty-four Enterococcus isolates (seven species), from five surface water systems in the North West Province, South Africa, were screened for the presence of asa1, cylA, esp, gelE and hyl using polymerase chain reaction. The expression of cylA, hyl and gelE was determined by phenotypic assessments. Sixty-five percent of the isolates were positive for one virulence gene and 13% for two or more. Most frequently detected genes were gelE (32%) and cylA (28%). Enterococcal surface protein was absent in all isolates screened. The presence of virulence genes was correlated with their extracellular enzyme production. The results show that a large percentage of these environmental Enterococcus spp. possess virulence factors that could be expressed in vitro. This is a cause for concern and could have implications for individuals using this water for recreational and cultural purposes. Further investigation is required into the sources of these potential pathogenic Enterococcus isolates and measures to minimize their presence in water sources.

  15. A process for extracellular thermostable lipase production by a novel Bacillus thermoamylovorans strain.

    PubMed

    Deive, Francisco J; Álvarez, María S; Morán, Paloma; Sanromán, M Angeles; Longo, María A

    2012-08-01

    A lipolytic enzyme-producing thermophilic microorganism, recently isolated from a hot spring in Galicia (North Western Spain), has been investigated. First, the strain was genetically identified and tentatively named Bacillus thermoamylovorans CH6B. It produced significant levels (around 450 U/L) of extracellular lipolytic activity in shake flask cultures, and the most suitable conditions for this biological process were found at temperatures between 50 and 55 °C, and an initial pH value around 7.0. Next, a preliminary scaling up of the process was carried out in a 5-L stirred tank bioreactor, and it was concluded that operation at agitation and aeration rates of 300 rpm and 0.33 vvm, respectively, were advisable. In both type of cultures, the results were successfully fitted to logistic equations, and the relationship between lipase production and cell growth was investigated. Furthermore, some relevant properties of the crude lipolytic enzyme extracts were assessed. The crude biocatalyst preferentially hydrolysed p-nitrophenyl esters of medium and long-chain fatty acids. Thermal stability in aqueous solution of the produced enzyme was also promising, and the deactivation profiles were fitted to a series-type deactivation model. PMID:22237683

  16. Role of extracellular polymeric substances (EPS) production in bioaggregation: application to wastewater treatment.

    PubMed

    Ding, Zhiji; Bourven, Isabelle; Guibaud, Gilles; van Hullebusch, Eric D; Panico, Antonio; Pirozzi, Francesco; Esposito, Giovanni

    2015-12-01

    This paper reviews the formation, structure, and stability of bioaggregates with an emphasis on the composition and distribution of extracellular polymeric substances (EPS) and their role in bioaggregation. Bioaggregation is ubiquitous in natural environment and is of great importance in biological wastewater treatment processes. It greatly influences the flocculability, settleability, and dewaterability for flocs and sludge retention and shear resistance for biofilms. The physico-chemical and microbial structures of bioaggregates are dependent on operational conditions as well as microbial diversity and spatial distribution. The formation of bioaggregates is mediated by the physico-chemical interactions as well as the microbial interactions such as EPS production and quorum sensing. EPS are composed of a mixture of macromolecules including proteins, polysaccharides, humic-like substances, and nucleic acids, which entrap the microbial cells in a three-dimensional matrix. The composition and physico-chemical characteristics of EPS have significant influence on the maintenance of the bioaggregate structure and the process performance of the wastewater treatment. However, the mechanisms of bioaggregation are still unclear and the conclusions on the role of EPS were mostly drawn from the established correlations and hypotheses. This paper expects to provide up-to-date knowledge on bioaggregation and insights for further studies and applications. PMID:26381665

  17. Production and properties of an extracellular bacteriocin from Streptococcus mutans bacteriocidal for group A and other streptococci.

    PubMed Central

    Paul, D; Slade, H D

    1975-01-01

    An extracellular bacteriocidal substance is produced by a serotype c strain of Streptococcus mutans in liquid meduim during the stationary phase of growth. The lethal effect of the substance was demonstrated by the decrease in viable counts of a standardized suspension of group A streptococci in broth. No lysis of affected cells was observed and no changes in appearance of these cells was seen in electron micrographs. The material was effective against certain strains of immmunological groups A, C, D, G, H, L, and O streptococci. It was inactive against strains of S. mutans belonging to the a, b, c, and d serotypes, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The factor was purified 273-fold from the culture fluid by column chromatography. It was sensitive to trysin and Pronase and resistant to catalase. It possessed a molecular weight of more than 20,000 and was not dialyzable. The properties of this substance indicate that it is a bacteriocin. Group A streptococci, which had been treated with antiserum specific for the cell wall group and type antigens, were susceptible to the bacteriocin. Streptococcal strains resistant to the lethal action of the bacteriocin adsorbed the bacteriocin from the solutions, as did the sensitive cells. The bacteriocin was not adsorbed at 0 C. Images PMID:1107225

  18. Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production

    PubMed Central

    Kusunoki, Yoshihiro; Nakazawa, Daigo; Shida, Haruki; Hattanda, Fumihiko; Miyoshi, Arina; Masuda, Sakiko; Nishio, Saori; Tomaru, Utano; Atsumi, Tatsuya; Ishizu, Akihiro

    2016-01-01

    Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 106/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared

  19. Production of polyol oils from soybean oil by Pseudomonas aeruginosa E03-12.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy-polyols are important starting materials for the manufacture of polymers such as polyurethane. We have been trying to develop a bioprocess for the production of polyol oils directly from soybean oil. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus ...

  20. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    PubMed Central

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

  1. Inquisition of Microcystis aeruginosa and Synechocystis nanowires: characterization and modelling.

    PubMed

    Sure, Sandeep; Torriero, Angel A J; Gaur, Aditya; Li, Lu Hua; Chen, Ying; Tripathi, Chandrakant; Adholeya, Alok; Ackland, M Leigh; Kochar, Mandira

    2015-11-01

    Identification of extracellular conductive pilus-like structures (PLS) i.e. microbial nanowires has spurred great interest among scientists due to their potential applications in the fields of biogeochemistry, bioelectronics, bioremediation etc. Using conductive atomic force microscopy, we identified microbial nanowires in Microcystis aeruginosa PCC 7806 which is an aerobic, photosynthetic microorganism. We also confirmed the earlier finding that Synechocystis sp. PCC 6803 produces microbial nanowires. In contrast to the use of highly instrumented continuous flow reactors for Synechocystis reported earlier, we identified simple and optimum culture conditions which allow increased production of nanowires in both test cyanobacteria. Production of these nanowires in Synechocystis and Microcystis were found to be sensitive to the availability of carbon source and light intensity. These structures seem to be proteinaceous in nature and their diameter was found to be 4.5-7 and 8.5-11 nm in Synechocystis and M. aeruginosa, respectively. Characterization of Synechocystis nanowires by transmission electron microscopy and biochemical techniques confirmed that they are type IV pili (TFP) while nanowires in M. aeruginosa were found to be similar to an unnamed protein (GenBank : CAO90693.1). Modelling studies of the Synechocystis TFP subunit i.e. PilA1 indicated that strategically placed aromatic amino acids may be involved in electron transfer through these nanowires. This study identifies PLS from Microcystis which can act as nanowires and supports the earlier hypothesis that microbial nanowires are widespread in nature and play diverse roles.

  2. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation.

    PubMed

    Laverty, Garry; Gorman, Sean P; Gilmore, Brendan F

    2014-07-18

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

  3. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You

    2016-01-01

    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa. PMID:27175902

  4. The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

    PubMed

    Yang, Nana; Ding, Shuting; Chen, Feifei; Zhang, Xue; Xia, Yongjie; Di, Hongxia; Cao, Qiao; Deng, Xin; Wu, Min; Wong, Catherine C L; Tian, Xiao-Xu; Yang, Cai-Guang; Zhao, Jing; Lan, Lefu

    2015-05-01

    Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhl QS signal C4-HSL. Crc is unable to bind lon mRNA in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhl QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhl QS in P. aeruginosa.

  5. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1.

    PubMed

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You

    2016-01-01

    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa.

  6. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1.

    PubMed

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You

    2016-01-01

    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa. PMID:27175902

  7. Sequestration and Distribution Characteristics of Cd(II) by Microcystis aeruginosa and Its Role in Colony Formation

    PubMed Central

    Bi, Xiangdong; Yan, Ran; Li, Fenxiang; Dai, Wei; Jiao, Kewei; Liu, Qi

    2016-01-01

    To investigate the sequestration and distribution characteristics of Cd(II) by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II) concentrations for 10 days. Cd(II) exposure caused hormesis in the growth of M. aeruginosa. Low concentrations of Cd(II) significantly induced formation of small Microcystis colonies (P < 0.05) and increased the intracellular polysaccharide (IPS) and bound extracellular polysaccharide (bEPS) contents of M. aeruginosa significantly (P < 0.05). There was a linear relationship between the amount of Cd(II) sequestrated by algal cells and the amount added to cultures in the rapid adsorption process that occurred during the first 5 min of exposure. After 10 d, M. aeruginosa sequestrated nearly 80% of 0.2 mg L−1 added Cd(II), while >93% of Cd(II) was sequestrated in the groups with lower added concentrations of Cd(II). More than 80% of the sequestrated Cd(II) was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II) could stimulate the production of IPS and bEPS via increasing Cd(II) bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II) and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies. PMID:27777956

  8. Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

    PubMed Central

    Goldberg, J B; Ohman, D E

    1987-01-01

    The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome. Images PMID:3031015

  9. Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production.

    PubMed Central

    Xu, P L; Iwata, M; Leong, S; Sequeira, L

    1990-01-01

    Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum. Images PMID:2163393

  10. Experimental investigation and optimization of process variables affecting the production of extracellular lipase by Kluyveromyces marxianus IFO 0288.

    PubMed

    Stergiou, Panagiota-Yiolanda; Foukis, Athanasios; Sklivaniti, Helen; Zacharaki, Paraskevi; Papagianni, Maria; Papamichael, Emmanuel M

    2012-10-01

    In this study, the production and optimization of extracellular lipase from Kluyveromyces marxianus IFO 0288 was investigated by using optimized nutritional and cultural conditions in a yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150 rpm). The influence of four fermentation parameters (type of lipidic source, initial culture pH, temperature, and length of fermentation) on growth and lipase production was investigated and evaluated using the conventional "one variable at a time" approach and response surface methodology. An 18-fold increase in lipase production during 65 h of fermentation was obtained with optimized nutritional (0.5 % olive oil) and cultivation (pH 6.5, 35 °C) conditions by employing the conventional optimization method. By applying the response surface methodology technique the initial pH value of 6.4 and temperature of 32.5 °C were identified as optimal and led to further improvements (up to 18-fold) of extracellular lipase production. The results provide, for the first time, evidence that K. marxianus has the potential to be used as an efficient producer of extracellular lipase with prospective application in a variety of industrial and biotechnological areas.

  11. Extracellular heat shock protein 90 binding to TGFβ receptor I participates in TGFβ-mediated collagen production in myocardial fibroblasts.

    PubMed

    García, Raquel; Merino, David; Gómez, Jenny M; Nistal, J Francisco; Hurlé, María A; Cortajarena, Aitziber L; Villar, Ana V

    2016-10-01

    The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor β (TGFβ) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling by stabilizing the TGFβ signaling cascade. We detected extracellular Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. This was supported by in silico studies predicting Hsp90 dimers and TGFβRI extracellular domain interaction. Both, Hsp90aa1 and Hsp90ab1 isoforms participate in TGFβRI complex. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFβ signaling cascade, and collagen protein synthesis was drastically reduced in Hsp90aa1 KO mice. These observations together with the significant increase in activity of Hsp90 at the plasma membrane pointed to a functional cooperative partnership between Hsp90 and TGFβRI in the fibrotic process. We propose that a surface population of Hsp90 extracellularly binds TGFβRI and this complex behaves as an active participant in collagen production in TGFβ-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 and its isoforms during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice.

  12. Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo

    PubMed Central

    Westman, Johannes; Papareddy, Praveen; Dahlgren, Madelene W.; Chakrakodi, Bhavya; Norrby-Teglund, Anna; Smeds, Emanuel; Linder, Adam; Mörgelin, Matthias; Johansson-Lindbom, Bengt; Egesten, Arne; Herwald, Heiko

    2015-01-01

    The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection. PMID:26646682

  13. High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae

    PubMed Central

    Hu, Yanmei; Palmer, Stephanie O.; Munoz, Hector; Bullard, James M.

    2015-01-01

    Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the Km’s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNAPhe were determined to be 48, 200, and 1.2 μM, respectively, while the Km’s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNAPhe were 21, 225 and 0.94 μM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50’s of 2.3 and 4.9 μM, respectively. The compound had a Ki of 0.83 and 0.98 μM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 μg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with AT P. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed. PMID:25601215

  14. Motility activity, slime production, biofilm formation and genetic typing by ERIC-PCR for Pseudomonas aeruginosa strains isolated from bovine and other sources (human and environment).

    PubMed

    Wolska, K; Szweda, P; Lada, K; Rytel, E; Gucwa, K; Kot, B; Piechota, M

    2014-01-01

    The molecular-typing strategy, ERIC-PCR was used in an attempt to determine the genomic relationship of 28 P. aeruginosa strains isolated from faeces of healthy bovine, bovine mastitis and from faeces of hospital patients as well as from environment. ERIC-PCR fingerprinting revealed large molecular differentiation within this group of isolates. Twenty two out of 28 strains tested generated unique patterns of DNA bands and only three genotypes consisted of two isolates each were identified. We also tested the P. aeruginosa isolates for their ability to form a biofilm on abiotic surfaces including polyvinylchloride and polystyrene. Different biofilm-forming abilities were demonstrated among strains; however, most of them (64.3%) showed moderate-biofilm forming ability. The strains with increased swimming and twitching motility displayed elevated biofilm formation. However, a negative correlation was found between slime and initial biofilm production. On the basis of the results obtained, we suggest that there are no major differences in phenotypic properties between P. aeruginosa strains isolated from different sources.

  15. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

    PubMed Central

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

    2015-01-01

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

  16. Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

    PubMed

    Brown, A; Horobin, A; Blount, D G; Hill, P J; English, J; Rich, A; Williams, P M; Pritchard, D I

    2012-12-01

    In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

  17. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  18. In vitro antibiofilm activity of Murraya koenigii essential oil extracted using supercritical fluid CO₂ method against Pseudomonas aeruginosa PAO1.

    PubMed

    Ganesh, P Sankar; Vittal, Ravishankar Rai

    2015-01-01

    The antibiofilm activity of Murraya koenigii essential oil (EO) against Pseudomonas aeruginosa PAO1 was investigated in this study. A decrease in the production of rhamnolipid, extracellular polymeric substance and swarming motility was observed by the EO treatment (0.3% v/v). The static microtitre plate assay revealed 80% reduction in biofilm formation by P. aeruginosa PAO1 on M. koenigii EO treatment. Fluorescence microscopy and scanning electron microscopy analyses confirmed the reduction of biofilm formation in P. aeruginosa PAO1 when treated with M. koenigii EO. Gas chromatography-mass spectrometry analysis of the EO revealed the presence of well-known antibiofilm agents such as spathulenol (5.85%), cinnamaldehyde (0.37%) and linalool (0.04%). Cinnamaldehyde has not been previously reported in M. koenigii EO. The potent antibiofilm properties of M. koenigii EO may be effectively exploited in food and pharmaceutical industries as well as in controlling Pseudomonas biofilms on indwelling medical devices. PMID:25635569

  19. Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

    PubMed Central

    Lee, Jintae; Attila, Can; Cirillo, Suat L. G.; Cirillo, Jeffrey D.; Wood, Thomas K.

    2009-01-01

    Summary Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  20. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence.

    PubMed

    Lee, Jintae; Attila, Can; Cirillo, Suat L G; Cirillo, Jeffrey D; Wood, Thomas K

    2009-01-01

    Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  1. Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum.

    PubMed

    Pereira, Patrícia; Pedro, Augusto Q; Tomás, Joana; Maia, Cláudio J; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-04-01

    The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics. PMID:26860940

  2. Environmental conditions associating microcystins production to Microcystis aeruginosa in a reservoir of Thailand.

    PubMed

    Wang, Xiaofeng; Parkpian, Preeda; Fujimoto, Naoshi; Ruchirawat, Khunying Mathuros; DeLaune, R D; Jugsujinda, A

    2002-08-01

    Three heptapeptide toxins, microcystin-RR, microcystin-RY and microcystin-LR, which can cause health problems in animals and humans were monitored in Bang Phra Reservoir, Thailand using reversed-phase high performance liquid chromatography. The concentrations of the three toxins in the reservoir varied greatly depending on location and time water samples were collected. Water quality parameters such as light intensity, temperature, pH, dissolved oxygen, suspended solid, chemical oxygen demand, dissolved organic carbon, total nitrogen, total phosphorus, ammonia, nitrate, phosphate, total dissolved nitrogen, total dissolved phosphorus and chlorophyll-a were also measured in parallel with microcystin determinations. Relationships among water quality parameters, toxins and chlorophyll-a were established. Toxin concentration increased in proportion to increases in total phosphorus, fraction of dissolved phosphorus, but was inversely correlated with water pH and total suspended solids. The other measured parameters in the study showed no correlations to toxin level in reservoir water. Significant correlations between chlorophyll-a and suspended solids, phosphate, nitrate and ammonia were observed suggesting that nitrogen and phosphorus are the two major nutrients governing growth of algae in the reservoir. This relationship suggests that algal production as well as toxin concentration are dependant on nutrient levels in the water body, since both measured light intensity and temperature level was favorable for algal growth. A small algal bloom observed in the rainy season of each year (lasting for only a couple of months) paralleled measured increases in toxin concentration, chlorophyll-a, TP and TN in the water column. Toxin level in the water column remain detectable for 3-4 months period following the initiation of algal bloom. Results indicate that major blooms are likely to occur following the raining season which usually occurs near the end of October when runoff

  3. Environmental conditions associating microcystins production to Microcystis aeruginosa in a reservoir of Thailand.

    PubMed

    Wang, Xiaofeng; Parkpian, Preeda; Fujimoto, Naoshi; Ruchirawat, Khunying Mathuros; DeLaune, R D; Jugsujinda, A

    2002-08-01

    Three heptapeptide toxins, microcystin-RR, microcystin-RY and microcystin-LR, which can cause health problems in animals and humans were monitored in Bang Phra Reservoir, Thailand using reversed-phase high performance liquid chromatography. The concentrations of the three toxins in the reservoir varied greatly depending on location and time water samples were collected. Water quality parameters such as light intensity, temperature, pH, dissolved oxygen, suspended solid, chemical oxygen demand, dissolved organic carbon, total nitrogen, total phosphorus, ammonia, nitrate, phosphate, total dissolved nitrogen, total dissolved phosphorus and chlorophyll-a were also measured in parallel with microcystin determinations. Relationships among water quality parameters, toxins and chlorophyll-a were established. Toxin concentration increased in proportion to increases in total phosphorus, fraction of dissolved phosphorus, but was inversely correlated with water pH and total suspended solids. The other measured parameters in the study showed no correlations to toxin level in reservoir water. Significant correlations between chlorophyll-a and suspended solids, phosphate, nitrate and ammonia were observed suggesting that nitrogen and phosphorus are the two major nutrients governing growth of algae in the reservoir. This relationship suggests that algal production as well as toxin concentration are dependant on nutrient levels in the water body, since both measured light intensity and temperature level was favorable for algal growth. A small algal bloom observed in the rainy season of each year (lasting for only a couple of months) paralleled measured increases in toxin concentration, chlorophyll-a, TP and TN in the water column. Toxin level in the water column remain detectable for 3-4 months period following the initiation of algal bloom. Results indicate that major blooms are likely to occur following the raining season which usually occurs near the end of October when runoff

  4. Effect of light on growth, intracellular and extracellular pigment production by five pigment-producing filamentous fungi in synthetic medium.

    PubMed

    Velmurugan, Palanivel; Lee, Yong Hoon; Venil, Chidambaram Kulandaisamy; Lakshmanaperumalsamy, Perumalsamy; Chae, Jong-Chan; Oh, Byung-Taek

    2010-04-01

    The competence of the living creatures to sense and respond to light is well known. The effect of darkness and different color light quality on biomass, extracellular and intracellular pigment yield of five potent pigment producers Monascus purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides and Penicillium purpurogenum, with different color shades such as red, pink, reddish brown and yellow, were investigated. Incubation in total darkness increased the biomass, extracellular and intracellular pigment production in all the fungi. Extracellular red pigment produced by M. purpureus resulted maximum in darkness 36.75 + or - 2.1 OD and minimum in white unscreened light 5.90 + or - 1.1 OD. Similarly, intracellular red pigment produced by M. purpureus resulted maximum in darkness 18.27 + or - 0.9 OD/g and minimum in yellow light 8.03 + or - 0.6 OD/g of substrate. The maximum biomass production was also noticed in darkness 2.51 g/L and minimum in yellow light 0.5 g/L of dry weight. In contrast, growth of fungi in green and yellow wavelengths resulted in low biomass and pigment yield. It was found that darkness, (red 780-622 nm, blue 492-455 nm) and white light influenced pigment and biomass yield. PMID:20226375

  5. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

    PubMed

    Kim, Sun-Ki; Min, Won-Ki; Park, Yong-Cheol; Seo, Jin-Ho

    2015-11-01

    Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.

  6. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

    PubMed

    Kim, Sun-Ki; Min, Won-Ki; Park, Yong-Cheol; Seo, Jin-Ho

    2015-11-01

    Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins. PMID:26320714

  7. Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

    PubMed Central

    Britigan, B E; Roeder, T L; Rasmussen, G T; Shasby, D M; McCormick, M L; Cox, C D

    1992-01-01

    Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection. PMID:1469082

  8. Extracellular Polysaccharide Production in a Scytonemin-Deficient Mutant of Nostoc punctiforme Under UVA and Oxidative Stress.

    PubMed

    Soule, Tanya; Shipe, Dexter; Lothamer, Justin

    2016-10-01

    Some cyanobacteria can protect themselves from ultraviolet radiation by producing sunscreen pigments. In particular, the sheath pigment scytonemin protects cells against long-wavelength UVA radiation and is only found in cyanobacteria which are capable of extracellular polysaccharide (EPS) production. The presence of a putative glycosyltransferase encoded within the scytonemin gene cluster, along with the localization of scytonemin and EPS to the extracellular sheath, prompted us to investigate the relationship between scytonemin and EPS production under UVA stress. In this study, it was hypothesized that there would be a relationship between the biosynthesis of scytonemin and EPS under both UVA and oxidative stress, since the latter is a by-product of UVA radiation. EPS production was measured following exposure of wild-type Nostoc punctiforme and the non-scytonemin-producing strain SCY59 to UVA and oxidative stress. Under UVA, SCY59 produced significantly more EPS than the unstressed controls and the wild type, while both strains produced more EPS under oxidative stress compared to the controls. The results suggest that EPS secretion occurs in response to the oxidative stress by-product of UVA rather than as a direct response to UVA radiation. PMID:27301251

  9. An Assay to Quantify Chemotactic Properties of Degradation Products from Extracellular Matrix

    PubMed Central

    Sicari, Brian M.; Zhang, Li; Londono, Ricardo; Badylak, Stephen F.

    2015-01-01

    The endogenous chemotaxis of cells toward sites of tissue injury and/or biomaterial implantation is an important component of the host response. Implanted biomaterials capable of recruiting host stem/progenitor cells to a site of interest may obviate challenges associated with cell transplantation. An assay for the identification and quantification of chemotaxis induced by surgically placed biologic scaffolds composed of extracellular matrix is described herein. PMID:24155230

  10. Proteolytic activity of extracellular products from Arthrobotrys musiformis and their effect in vitro against Haemonchus contortus infective larvae

    PubMed Central

    Acevedo-Ramírez, Perla María del Carmen; Figueroa-Castillo, Juan Antonio; Ulloa-Arvizú, Raúl; Martínez-García, Luz Gisela; Guevara-Flores, Alberto; Rendón, Juan Luis; Valero-Coss, Rosa Ofelia; Mendoza-de Gives, Pedro; Quiroz-Romero, Héctor

    2015-01-01

    Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of Amusiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs. PMID:26392902

  11. A study on large scale cultivation of Microcystis aeruginosa under open raceway pond at semi-continuous mode for biodiesel production.

    PubMed

    Ashokkumar, Veeramuthu; Agila, Elango; Salam, Zainal; Ponraj, Mohanadoss; Din, Mohd Fadhil Md; Ani, Farid Nasir

    2014-11-01

    The study explores on upstream and downstream process in Microcystis aeruginosa for biodiesel production. The alga was isolated from temple tank, acclimatized and successfully mass cultivated in open raceway pond at semi-continuous mode. A two step combined process was designed and harvested 99.3% of biomass, the daily dry biomass productivity was recorded up to 28gm(-2)day(-1). The lipid extraction was optimized and achieved 21.3%; physicochemical properties were characterized and found 11.7% of FFA, iodine value 72% and 99.2% of ester content. The lipid was transesterified by a two step simultaneous process and produced 90.1% of biodiesel; the calorific value of the biodiesel was 38.8MJ/kg. Further, the physicochemical properties of biodiesel was characterized and found to be within the limits of American ASTM D6751. Based on the areal and volumetric biomass productivity estimation, M. aeruginosa can yield 84.1 tons of dry biomass ha(-1)year(-1).

  12. A study on large scale cultivation of Microcystis aeruginosa under open raceway pond at semi-continuous mode for biodiesel production.

    PubMed

    Ashokkumar, Veeramuthu; Agila, Elango; Salam, Zainal; Ponraj, Mohanadoss; Din, Mohd Fadhil Md; Ani, Farid Nasir

    2014-11-01

    The study explores on upstream and downstream process in Microcystis aeruginosa for biodiesel production. The alga was isolated from temple tank, acclimatized and successfully mass cultivated in open raceway pond at semi-continuous mode. A two step combined process was designed and harvested 99.3% of biomass, the daily dry biomass productivity was recorded up to 28gm(-2)day(-1). The lipid extraction was optimized and achieved 21.3%; physicochemical properties were characterized and found 11.7% of FFA, iodine value 72% and 99.2% of ester content. The lipid was transesterified by a two step simultaneous process and produced 90.1% of biodiesel; the calorific value of the biodiesel was 38.8MJ/kg. Further, the physicochemical properties of biodiesel was characterized and found to be within the limits of American ASTM D6751. Based on the areal and volumetric biomass productivity estimation, M. aeruginosa can yield 84.1 tons of dry biomass ha(-1)year(-1). PMID:25262427

  13. Genetics of O-Antigen Biosynthesis in Pseudomonas aeruginosa

    PubMed Central

    Rocchetta, H. L.; Burrows, L. L.; Lam, J. S.

    1999-01-01

    Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of d-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants. PMID:10477307

  14. Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients.

    PubMed

    Jabalameli, Fereshteh; Mirsalehian, Akbar; Khoramian, Babak; Aligholi, Marzieh; Khoramrooz, Seyed Sajjad; Asadollahi, Parisa; Taherikalani, Morovat; Emaneini, Mohammad

    2012-12-01

    Pseudomonas aeruginosa is one of the common pathogenic causes of serious infections in burn patients throughout the world. Type III secretion toxins are thought to promote the dissemination of P. aeruginosa from the site of infection, the bacterial evasion of the host immune response and inhibition of DNA synthesis leading to host cell death. A total of 96 isolates of P. aeruginosa were collected from wound infections of burn patients, from April to July 2010. Antimicrobial susceptibility of the isolates were determined by disk agar diffusion method. Polymerase chain reaction (PCR)-based method was used for targeting the genes encoding the type III secretion toxins. The quantitative determination of biofilm-forming capacity was determined by a colorimetric microtiter plate assay. All the isolates were resistant to cefixime and ceftriaxone. More than 90% of the isolates were resistant to amikacin, carbenicillin, cefepime, cefotaxime, cefpodoxime, gatifloxacin, gentamicin, piperacillin/tazobactam, ticarcillin and tobramycin. All the isolates carried the exoT gene, 95% carried exoY, 64.5% carried exoU and 29% carried the exoS gene. Most of the isolates (58%) carried both exoY and exoU genes while 24% showed the concomitant presence of exoS and exoY and 1% carried both exoS and exoU. Coexistence of exoS, exoY and exoU was seen in 4% of the isolates. Biofilm formation was seen in more than 96% of the isolates among which 47% were strong biofilm producers, 26% were moderate and 22.9% were weak biofilm formers. In conclusion, the findings of this study show that the genes, particularly the exoU gene, encoding the type III secretion toxins, are commonly disseminated among the P. aeruginosa strains isolated from burn patients.

  15. Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures.

    PubMed

    Hamilton, G E; Luechau, F; Burton, S C; Lyddiatt, A

    2000-04-28

    Direct product sequestration of extracellular proteins from microbial batch cultures can be achieved by continuous or intermittent broth recycle through an external extractive loop. Here, we describe the development of a fluidisable, mixed mode adsorbent, designed to tolerate increasing ionic strength (synonymous with extended productive batch cultures). This facilitated operations for the integrated recovery of an extracellular acid protease from cultures of Yarrowia lipolytica. Mixed mode adsorbents were prepared using chemistries containing hydrophobic and ionic groups. Matrix hydrophobicity and titration ranges were matched to the requirements of integrated protease adsorption. A single expanded bed was able to service the productive phase of growth without recourse to the pH adjustment of the broth previously required for ion exchange adsorption. This resulted in increased yields of product, accompanied by further increases in enzyme specific activity. A step change from pH 4.5 to 2.6, across the isoelectric point of the protease, enabled high resolution fixed bed elution induced by electrostatic repulsion. The generic application of mixed mode chemistries, which combine the physical robustness of ion-exchange ligands in sanitisation and sterilisation procedures with a selectivity, which approaches that of affinity interactions, is discussed.

  16. Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system.

    PubMed

    Ram, Senthoor K; Raval, Keyur; JagadeeshBabu, P E

    2015-01-01

    Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

  17. Temperature and Light Effects on Extracellular Superoxide Production by Algal and Bacterial Symbionts in Corals: Implications for Coral Bleaching

    NASA Astrophysics Data System (ADS)

    Brighi, C.; Diaz, J. M.; Apprill, A.; Hansel, C. M.

    2014-12-01

    Increased surface seawater temperature due to global warming is one of the main causes of coral bleaching, a phenomenon in which corals lose their photosynthetic algae. Light and temperature induced production of superoxide and other reactive oxygen species (ROS) by these symbiotic algae has been implicated in the breakdown of their symbiotic association with the coral host and subsequent coral bleaching. Nevertheless, a direct link between Symbiodinium ROS production and coral bleaching has not been demonstrated. In fact, given the abundance and diversity of microorganisms within the coral holobiont, the concentration and fluxes of ROS within corals may involve several microbial sources and sinks. Here, we explore the role of increased light and temperature on superoxide production by coral-derived cultures of Symbiodinium algae and Oceanospirillales bacteria of the genus Endozoicomonas, which are globally common and abundant associates of corals. Using a high sensitivity chemiluminescent technique, we find that heat stress (exposure to 34°C vs. 23°C for 2hr or 24hr) has no significant effect on extracellular superoxide production by Symbiodinium isolates within clades B and C, regardless of the level of light exposure. Exposure to high light, however, increased superoxide production by these organisms at both 34°C and 23°C. On the other hand, extracellular superoxide production by Endozoicomonas bacteria tested under the same conditions was stimulated by the combined effects of thermal and light stress. The results of this research suggest that the sources and physical triggers for biological superoxide production within corals are more complex than currently assumed. Thus, further investigations into the biological processes controlling ROS dynamics within corals are required to improve our understanding of the mechanisms underpinning coral bleaching and to aid in the development of mitigation strategies.

  18. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    PubMed

    Willsey, Graham G; Wargo, Matthew J

    2015-01-01

    Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes) involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment.

  19. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    PubMed

    Willsey, Graham G; Wargo, Matthew J

    2015-01-01

    Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes) involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment. PMID:26599415

  20. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members

    PubMed Central

    Willsey, Graham G.; Wargo, Matthew J.

    2015-01-01

    Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes) involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment. PMID:26599415

  1. Adaptation of Iron Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine Mutant in the Cystic Fibrosis Lung

    PubMed Central

    Nguyen, Angela T.; O'Neill, Maura J.; Watts, Annabelle M.; Robson, Cynthia L.; Lamont, Iain L.; Wilks, Angela

    2014-01-01

    Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections, most notably by Pseudomonas aeruginosa, which persists for decades in the lungs and undergoes extensive evolution. P. aeruginosa requires iron for virulence and uses the fluorescent siderophore pyoverdine to scavenge and solubilize ferric iron during acute infections. Pyoverdine mutants accumulate in the lungs of some CF patients, however, suggesting that the heme and ferrous iron acquisition pathways of P. aeruginosa are more important in this environment. Here, we sought to determine how evolution of P. aeruginosa in the CF lung affects iron acquisition and regulatory pathways through the use of longitudinal CF isolates. These analyses demonstrated a significant reduction of siderophore production during the course of CF lung infection in nearly all strains tested. Mass spectrometry analysis of one of these strains showed that the later CF isolate has streamlined the metabolic flux of extracellular heme through the HemO heme oxygenase, resulting in more-efficient heme utilization. Moreover, gene expression analysis shows that iron regulation via the PrrF small RNAs (sRNAs) is enhanced in the later CF isolate. Finally, analysis of P. aeruginosa gene expression in the lungs of various CF patients demonstrates that both PrrF and HemO are consistently expressed in the CF lung environment. Combined, these results suggest that heme is a critical source of iron during prolonged infection of the CF lung and that changes in iron and heme regulatory pathways play a crucial role in adaptation of P. aeruginosa to this ever-changing host environment. PMID:24727222

  2. Extracellular enzyme production and phylogenetic distribution of yeasts in wastewater treatment systems.

    PubMed

    Yang, Qingxiang; Zhang, Hao; Li, Xueling; Wang, Zhe; Xu, Ying; Ren, Siwei; Chen, Xuanyu; Xu, Yuanyuan; Hao, Hongxin; Wang, Hailei

    2013-02-01

    The abilities of yeasts to produce different extracellular enzymes and their distribution characteristics were studied in municipal, inosine fermentation, papermaking, antibiotic fermentation, and printing and dyeing wastewater treatment systems. The results indicated that of the 257 yeasts, 16, 14, 55, and 11 produced lipase, protease, manganese dependant peroxidase (MnP), and lignin peroxidase (LiP), respectively. They were distributed in 12 identified and four unidentified genera, in which Candida rugosa (AA-M17) and an unidentified Saccharomycetales (AA-Y5), Pseudozyma sp. (PH-M15), Candida sp. (MO-Y11), and Trichosporon montevideense (MO-M16) were shown to have the highest activity of lipase, protease, Mnp, and LiP, respectively. No yeast had amylase, cellulose, phytase, or laccase activity. Although only 60 isolates produced ligninolytic enzymes, 249 of the 257 yeasts could decolorize different dyes through the mechanism of biodegradation (222 isolates) or bio-sorption. The types of extracellular enzymes that the yeasts produced were significantly shaped by the types of wastewater treated. PMID:23261999

  3. Effects of Silver Sulphadiazine on Production of Extracellular Proteins by Strains of Staphylococcus Aureus Isolated from Burns Wound

    PubMed Central

    Javid Khojasteh, Vahideh; Alfakhri, Souad; Foster, Howard Anthony

    2016-01-01

    Previous studies had shown that sub-inhibitory concentrations of silver sulphadiazine (AgSD) stimulated the production of Toxic Shock Syndrome Toxin-1 in certain strains (responder strains) of Staphylococcus aureus and that protease production was also affected. No changes were detected in other strains (non-responders). Extracellular proteins from eleven responder and non-responder strains grown with and without AgSD were separated by SDS PAGE. There were three classes of response, responder strains that showed enhancement of synthesis of certain proteins, non-responder strains that showed no change and responder strains that showed a general decrease in exoprotein production in the early stages of growth. The results showed that the effects of AgSD were complex and that S. aureus strains were heterogenous with respect to their response to sub-inhibitory concentrations of AgSD. PMID:27642339

  4. Effects of Silver Sulphadiazine on Production of Extracellular Proteins by Strains of Staphylococcus Aureus Isolated from Burns Wound.

    PubMed

    Javid Khojasteh, Vahideh; Alfakhri, Souad; Foster, Howard Anthony

    2016-01-01

    Previous studies had shown that sub-inhibitory concentrations of silver sulphadiazine (AgSD) stimulated the production of Toxic Shock Syndrome Toxin-1 in certain strains (responder strains) of Staphylococcus aureus and that protease production was also affected. No changes were detected in other strains (non-responders). Extracellular proteins from eleven responder and non-responder strains grown with and without AgSD were separated by SDS PAGE. There were three classes of response, responder strains that showed enhancement of synthesis of certain proteins, non-responder strains that showed no change and responder strains that showed a general decrease in exoprotein production in the early stages of growth. The results showed that the effects of AgSD were complex and that S. aureus strains were heterogenous with respect to their response to sub-inhibitory concentrations of AgSD. PMID:27642339

  5. Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4.

    PubMed

    Dahinden, C A; Clancy, R M; Gross, M; Chiller, J M; Hugli, T E

    1985-10-01

    The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes.

  6. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways

    PubMed Central

    Luo, Jing; Kong, Jin-liang; Dong, Bi-ying; Huang, Hong; Wang, Ke; Wu, Li-hong; Hou, Chang-chun; Liang, Yue; Li, Bing; Chen, Yi-qiang

    2016-01-01

    Burgeoning antibiotic resistance and unfavorable outcomes of inflammatory injury after Pseudomonas aeruginosa infection have necessitated the development of novel agents that not only target quorum sensing (QS) but also combat inflammatory injury with the least risk of resistance. This study aimed to assess the anti-QS and anti-inflammatory activities of baicalein, a traditional herbal medicine that is widely used in the People’s Republic of China, against P. aeruginosa infection. We found that subminimum inhibitory concentrations of baicalein efficiently interfered with the QS-signaling pathway of P. aeruginosa via downregulation of the transcription of QS-regulated genes and the translation of QS-signaling molecules. This interference resulted in the global attenuation of QS-controlled virulence factors, such as motility and biofilm formation, and the secretion into the culture supernatant of extracellular virulence factors, including pyocyanin, LasA protease, LasB elastase, and rhamnolipids. Moreover, we examined the anti-inflammatory activity of baicalein and its mode of action via a P. aeruginosa-infected macrophage model to address its therapeutic effect. Baicalein reduced the P. aeruginosa-induced secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα. In addition, baicalein suppressed P. aeruginosa-induced activation of the MAPK and NFκB signal-transduction pathways in cocultured macrophages; this may be the mechanism by which baicalein inhibits the production of proinflammatory cytokines. Therefore, our study demonstrates that baicalein represents a potential treatment for P. aeruginosa infection because it clearly exhibits both antibacterial and anti-inflammatory activities. PMID:26792984

  7. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFκB signal-transduction pathways.

    PubMed

    Luo, Jing; Kong, Jin-Liang; Dong, Bi-Ying; Huang, Hong; Wang, Ke; Wu, Li-Hong; Hou, Chang-Chun; Liang, Yue; Li, Bing; Chen, Yi-Qiang

    2016-01-01

    Burgeoning antibiotic resistance and unfavorable outcomes of inflammatory injury after Pseudomonas aeruginosa infection have necessitated the development of novel agents that not only target quorum sensing (QS) but also combat inflammatory injury with the least risk of resistance. This study aimed to assess the anti-QS and anti-inflammatory activities of baicalein, a traditional herbal medicine that is widely used in the People's Republic of China, against P. aeruginosa infection. We found that subminimum inhibitory concentrations of baicalein efficiently interfered with the QS-signaling pathway of P. aeruginosa via downregulation of the transcription of QS-regulated genes and the translation of QS-signaling molecules. This interference resulted in the global attenuation of QS-controlled virulence factors, such as motility and biofilm formation, and the secretion into the culture supernatant of extracellular virulence factors, including pyocyanin, LasA protease, LasB elastase, and rhamnolipids. Moreover, we examined the anti-inflammatory activity of baicalein and its mode of action via a P. aeruginosa-infected macrophage model to address its therapeutic effect. Baicalein reduced the P. aeruginosa-induced secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα. In addition, baicalein suppressed P. aeruginosa-induced activation of the MAPK and NFκB signal-transduction pathways in cocultured macrophages; this may be the mechanism by which baicalein inhibits the production of proinflammatory cytokines. Therefore, our study demonstrates that baicalein represents a potential treatment for P. aeruginosa infection because it clearly exhibits both antibacterial and anti-inflammatory activities.

  8. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    SciTech Connect

    Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

    2011-10-07

    Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

  9. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin

    NASA Astrophysics Data System (ADS)

    Das, Manash C.; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; de, Utpal C.; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-03-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It’s antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis.

  10. Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress.

    PubMed

    Chen, Lei; Mao, Feijian; Kirumba, George Chira; Jiang, Cheng; Manefield, Mike; He, Yiliang

    2015-12-01

    Microcystis (M.) aeruginosa, one of the most common bloom-forming cyanobacteria, occurs worldwide. The Qingcaosha (QCS) Reservoir is undergoing eutrophication and faces the problem of saltwater intrusion. The aim of this study was to investigate the effects of sudden salinity changes on physiological parameters and related gene transcription in M. aeruginosa under controlled laboratory conditions. The results showed that sodium chloride (50, 200 and 500 mg L(-1) NaCl) inhibited the algal growth and decreased pigment concentrations (chlorophyll a, carotenoid and phycocyanin). Sodium chloride increased both the intracellular and extracellular microcystin contents and elevated the mcyD transcript level in M. aeruginosa. It also increased the malondialdehyde (MDA) content and caused cytomembrane damage. This damage caused the release of intracellular toxins into the culture medium. In addition, NaCl decreased the maximum electron transport rate, increased the levels of reactive oxygen species (ROS) and changed the cellular redox status. Consequently, NaCl inhibited the expression of cpcB, psbA and rbcL. Furthermore, NaCl increased the activities of superoxide dismutases (SOD), catalase (CAT), glutathione reductase (GR), and total glutathione peroxidase (GPx). The transcript levels of sod and reduced glutathione (gsh) were also increased after exposure to NaCl. Our results indicate that a sudden increase in salinity increases the production and excretion of microcystin, changes the cellular redox status, enhances the activities of antioxidant enzymes, inhibits photosynthesis, and affects transcript levels of related genes in M. aeruginosa. PMID:26232039

  11. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin

    PubMed Central

    Das, Manash C.; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; De, Utpal C.; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-01-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It’s antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis. PMID:27000525

  12. Attenuation of Pseudomonas aeruginosa biofilm formation by Vitexin: A combinatorial study with azithromycin and gentamicin.

    PubMed

    Das, Manash C; Sandhu, Padmani; Gupta, Priya; Rudrapaul, Prasenjit; De, Utpal C; Tribedi, Prosun; Akhter, Yusuf; Bhattacharjee, Surajit

    2016-01-01

    Microbial biofilm are communities of surface-adhered cells enclosed in a matrix of extracellular polymeric substances. Extensive use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant strains. Pseudomonas aeruginosa is recognised as a model biofilm forming pathogenic bacterium. Vitexin, a polyphenolic group of phytochemical with antimicrobial property, has been studied for its antibiofilm potential against Pseudomonas aeruginosa in combination with azithromycin and gentamicin. Vitexin shows minimum inhibitory concentration (MIC) at 260 μg/ml. It's antibiofilm activity was evaluated by safranin staining, protein extraction, microscopy methods, quantification of EPS and in vivo models using several sub-MIC doses. Various quorum sensing (QS) mediated phenomenon such as swarming motility, azocasein degrading protease activity, pyoverdin and pyocyanin production, LasA and LasB activity of the bacteria were also evaluated. Results showed marked attenuation in biofilm formation and QS mediated phenotype of Pseudomonas aeruginosa in presence of 110 μg/ml vitexin in combination with azithromycin and gentamicin separately. Molecular docking of vitexin with QS associated LuxR, LasA, LasI and motility related proteins showed high and reasonable binding affinity respectively. The study explores the antibiofilm potential of vitexin against P. aeruginosa which can be used as a new antibiofilm agent against microbial biofilm associated pathogenesis. PMID:27000525

  13. Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress.

    PubMed

    Chen, Lei; Mao, Feijian; Kirumba, George Chira; Jiang, Cheng; Manefield, Mike; He, Yiliang

    2015-12-01

    Microcystis (M.) aeruginosa, one of the most common bloom-forming cyanobacteria, occurs worldwide. The Qingcaosha (QCS) Reservoir is undergoing eutrophication and faces the problem of saltwater intrusion. The aim of this study was to investigate the effects of sudden salinity changes on physiological parameters and related gene transcription in M. aeruginosa under controlled laboratory conditions. The results showed that sodium chloride (50, 200 and 500 mg L(-1) NaCl) inhibited the algal growth and decreased pigment concentrations (chlorophyll a, carotenoid and phycocyanin). Sodium chloride increased both the intracellular and extracellular microcystin contents and elevated the mcyD transcript level in M. aeruginosa. It also increased the malondialdehyde (MDA) content and caused cytomembrane damage. This damage caused the release of intracellular toxins into the culture medium. In addition, NaCl decreased the maximum electron transport rate, increased the levels of reactive oxygen species (ROS) and changed the cellular redox status. Consequently, NaCl inhibited the expression of cpcB, psbA and rbcL. Furthermore, NaCl increased the activities of superoxide dismutases (SOD), catalase (CAT), glutathione reductase (GR), and total glutathione peroxidase (GPx). The transcript levels of sod and reduced glutathione (gsh) were also increased after exposure to NaCl. Our results indicate that a sudden increase in salinity increases the production and excretion of microcystin, changes the cellular redox status, enhances the activities of antioxidant enzymes, inhibits photosynthesis, and affects transcript levels of related genes in M. aeruginosa.

  14. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures.

    PubMed

    Christiansen, Torben; Nielsen, Jens

    2002-08-28

    The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates. PMID:12084482

  15. Withania somnifera attenuates acid production, acid tolerance and extra-cellular polysaccharide formation of Streptococcus mutans biofilms.

    PubMed

    Pandit, Santosh; Song, Kwang-Yeob; Jeon, Jae-Gyu

    2014-01-01

    Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal. The plant has also been used as a controlling agent for dental diseases. The aim of the present study was to evaluate the activity of the methanol extract of W. somnifera against the physiological ability of cariogenic biofilms and to identify the components of the extract. To determine the activity of the extract, assays for sucrose-dependent bacterial adherence, glycolytic acid production, acid tolerance, and extracellular polysaccharide formation were performed using Streptococcus mutans biofilms. The viability change of S. mutans biofilms cells was also determined. A phytochemical analysis of the extract was performed using TLC and LC/MS/MS. The extract showed inhibitory effects on sucrose-dependent bacterial adherence (≥ 100 μg/ml), glycolytic acid production (≥ 300 μg/ml), acid tolerance (≥ 300 μg/ml), and extracellular polysaccharide formation (≥ 300 μg/ml) of S. mutans biofilms. However, the extract did not alter the viability of S. mutans biofilms cells in all concentrations tested. Based on the phytochemical analysis, the activity of the extract may be related to the presence of alkaloids, anthrones, coumarines, anthraquinones, terpenoids, flavonoids, and steroid lactones (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy withastramonolide). These data indicate that W. somnifera may be a potential agent for restraining the physiological ability of cariogenic biofilms.

  16. EprS, an autotransporter serine protease, plays an important role in various pathogenic phenotypes of Pseudomonas aeruginosa.

    PubMed

    Kida, Y; Taira, J; Kuwano, K

    2016-02-01

    Pseudomonas aeruginosa possesses an arsenal of both cell-associated (flagella, pili, alginate, etc.) and extracellular (exotoxin A, proteases, type III secretion effectors, etc.) virulence factors. Among them, secreted proteases that damage host tissues are considered to play an important role in the pathogenesis of P. aeruginosa infections. We previously reported that EprS, an autotransporter protease of P. aeruginosa, induces host inflammatory responses through protease-activated receptors. However, little is known about the role of EprS as a virulence factor of P. aeruginosa. In this study, to investigate whether EprS participates in the pathogenicity of P. aeruginosa, we characterized various pathogenic phenotypes of the wild-type PAO1 strain and its eprS-disrupted mutant. The growth assays demonstrated that the growth of the eprS mutant was somewhat lower than that of the wild-type strain in a minimal medium containing BSA as the sole carbon and nitrogen source. Thus, these results indicate that eprS would have a role in the growth of P. aeruginosa in the presence of limited nutrients, such as a medium containing proteinaceous materials as a sole nutrient source. Furthermore, disruption of eprS resulted in a decreased production of elastase, pigments, autoinducers and surfactants, and a reduction of swimming and swarming motilities. In addition, the eprS mutant exhibited a reduction in the ability to associate with A549 cells and an attenuation of virulence in leucopenic mice as compared with the wild-type strain. Collectively, these results suggest that EprS exerts pleiotropic effects on various pathogenic phenotypes of P. aeruginosa. PMID:26678838

  17. Precursor-product relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium.

    PubMed Central

    Kessler, R E; Shockman, G D

    1979-01-01

    Exponential biosynthesis and excretion of lipoteichoic acid (LTA) during the exponential phase of growth, and continued synthesis and excretion during valine starvation of Streptococcus faecium (S. faecalis ATCC 9790), were shown. During exponential growth, extracellular LTA (LTAx) accounted for approximately 13% of the total LTA in cultures, whereas during valine starvation, this percentage increased to approximately 60% within 4 h. LTAx was present in a low-molecular-weight, apparently deacylated form, whereas intracellular (LTAi) was present primarily in an apparently high-molecular-weight, acylated and micellar form. Experiments utilizing chases of either fully equilibrated or short pulses of [14C]- or [3H]glycerol were used to demonstrate that LTAx was derived directly from LTAi. PMID:106043

  18. Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens.

    PubMed

    Salamone, P R; Wodzinski, R J

    1997-09-01

    The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.

  19. A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production

    PubMed Central

    Liu, Xing; Tremblay, Pier-Luc; Malvankar, Nikhil S.; Nevin, Kelly P.; Vargas, Madeline

    2014-01-01

    The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity. PMID:24296506

  20. Norepinephrine represses the expression of toxA and the siderophore genes in Pseudomonas aeruginosa.

    PubMed

    Li, Wang; Lyte, Mark; Freestone, Primrose P; Ajmal, Aziba; Colmer-Hamood, Jane A; Hamood, Abdul N

    2009-10-01

    Among the different extracellular virulence factors produced by Pseudomonas aeruginosa are exotoxin A (ETA) and the pyoverdine and pyochelin siderophores. Production of ETA and the siderophores requires the function of the iron-starvation sigma factor PvdS, the transcriptional activator RegA, and the AraC-activator PchR. Iron represses the production of ETA and the siderophores by repressing the expression of pvdS, regA, and pchR. PvdS regulates the expression of the ETA gene, toxA, regA, and the pyoverdine synthesis genes. The catecholamine norepinephrine enhances the growth of pathogenic bacteria by transferring iron from host-binding proteins. In this study, we elucidated the mechanism by which norepinephrine and other catecholamines induce P. aeruginosa growth. We also investigated whether norepinephrine regulates the expression of toxA and the siderophore genes, and the mechanism of this regulation. Norepinephrine enhanced the growth of P. aeruginosa by supplying iron from transferrin. This provision of iron repressed the expression of toxA, the pyoverdine genes pvdD and pvdE, and their regulators, pvdS, regA, and pchR, suggesting that norepinephrine accomplishes this repression through PvdS and PchR. Additionally, norepinephrine bypassed PvdS and supported the growth of a pvdS deletion mutant, indicating that norepinephrine transfers iron to P. aeruginosa independent of pyoverdine. Thus, norepinephrine apparently influences the pathogenesis of P. aeruginosa by affecting its pattern of growth and the production of virulence factors. PMID:19686346

  1. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa PAO1 by Ayurveda spice clove (Syzygium aromaticum) bud extract.

    PubMed

    Krishnan, Thiba; Yin, Wai-Fong; Chan, Kok-Gan

    2012-01-01

    Quorum sensing controls the virulence determinants in most proteobacteria. In this work, the hexane, chloroform and methanol extracts of an Ayurveda spice, namely clove (Syzygium aromaticum), shown anti-quorum sensing activity. Hexane and methanol extracts of clove inhibited the response of C. violaceum CV026 to exogenously supplied N-hexanoylhomoserine lactone, in turn preventing violacein production. Chloroform and methanol extracts of clove significantly reduced bioluminescence production by E. coli [pSB1075] grown in the presence of N-(3-oxododecanoyl)-L-homoserine lactone. We demonstrated that clove extract inhibited quorum sensing-regulated phenotypes in Pseudomonas aeruginosa PA01, including expression of lecA::lux (by hexane extract), swarming (maximum inhibition by methanol extract), pyocyanin (maximum inhibition by hexane extract). This study shows that the presence of natural compounds that exhibit anti-quorum sensing activity in the clove extracts may be useful as the lead of anti-infective drugs.

  2. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa PAO1 by Ayurveda spice clove (Syzygium aromaticum) bud extract.

    PubMed

    Krishnan, Thiba; Yin, Wai-Fong; Chan, Kok-Gan

    2012-01-01

    Quorum sensing controls the virulence determinants in most proteobacteria. In this work, the hexane, chloroform and methanol extracts of an Ayurveda spice, namely clove (Syzygium aromaticum), shown anti-quorum sensing activity. Hexane and methanol extracts of clove inhibited the response of C. violaceum CV026 to exogenously supplied N-hexanoylhomoserine lactone, in turn preventing violacein production. Chloroform and methanol extracts of clove significantly reduced bioluminescence production by E. coli [pSB1075] grown in the presence of N-(3-oxododecanoyl)-L-homoserine lactone. We demonstrated that clove extract inhibited quorum sensing-regulated phenotypes in Pseudomonas aeruginosa PA01, including expression of lecA::lux (by hexane extract), swarming (maximum inhibition by methanol extract), pyocyanin (maximum inhibition by hexane extract). This study shows that the presence of natural compounds that exhibit anti-quorum sensing activity in the clove extracts may be useful as the lead of anti-infective drugs. PMID:22666015

  3. Inhibition of Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa PAO1 by Ayurveda Spice Clove (Syzygium Aromaticum) Bud Extract

    PubMed Central

    Krishnan, Thiba; Yin, Wai-Fong; Chan, Kok-Gan

    2012-01-01

    Quorum sensing controls the virulence determinants in most proteobacteria. In this work, the hexane, chloroform and methanol extracts of an Ayurveda spice, namely clove (Syzygium aromaticum), shown anti-quorum sensing activity. Hexane and methanol extracts of clove inhibited the response of C. violaceum CV026 to exogenously supplied N‐hexanoylhomoserine lactone, in turn preventing violacein production. Chloroform and methanol extracts of clove significantly reduced bioluminescence production by E. coli [pSB1075] grown in the presence of N-(3-oxododecanoyl)-l-homoserine lactone. We demonstrated that clove extract inhibited quorum sensing-regulated phenotypes in Pseudomonas aeruginosa PA01, including expression of lecA::lux (by hexane extract), swarming (maximum inhibition by methanol extract), pyocyanin (maximum inhibition by hexane extract). This study shows that the presence of natural compounds that exhibit anti-quorum sensing activity in the clove extracts may be useful as the lead of anti-infective drugs. PMID:22666015

  4. Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites.

    PubMed

    Liu, Y; Cui, Y; Mukherjee, A; Chatterjee, A K

    1998-07-01

    The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of soft-rot diseases in plants and plant products. Ecc71 also produces HarpinEcc, the elicitor of hypersensitive reaction (HR) and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). OHL controls extracellular enzyme and HarpinEcc production. The levels of these enzymes, as well as the expression of hrpNEcc, the structural gene for HarpinEcc, and ohll, the gene specifying OHL synthesis, are negatively regulated by RsmaA. rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His-RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a sigma70-like promoter. In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA. rsmB' DNA hybridizes with the 259 base and the 479 base transcripts. A 3' RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho-independent terminator. The expression of rsmB-lacZ transcriptional fusions established that the rsmB' RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB' RNA is derived by processing of the primary rsmB RNA. In Ecc71, rsmB' expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrpNEcc and ohll transcripts. By contrast, a plasmid with the rsmB' DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71. The rsmB' effect also occurs in

  5. Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli▿ †

    PubMed Central

    Fisher, Adam C.; Haitjema, Charles H.; Guarino, Cassandra; Çelik, Eda; Endicott, Christine E.; Reading, Craig A.; Merritt, Judith H.; Ptak, A. Celeste; Zhang, Sheng; DeLisa, Matthew P.

    2011-01-01

    The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates. PMID:21131519

  6. Production, Purification, and Properties of Extracellular Carboxyl Esterases from Bacillus subtilis NRRL 365

    PubMed Central

    Meghji, K.; Ward, O. P.; Araujo, A.

    1990-01-01

    Bacillus subtilis NRRL 365 produced high extracellular carboxyl esterase activity in submerged culture media containing wheat bran, corn steep liquor, and salts. Supplementation of this medium with glucose reduced esterase activity to 37% of that in the unsupplemented control. Esterase activity was purified by ammonium sulfate fractionation, DEAE-Sephadex A-50 ion-exchange chromatography with sodium chloride gradient elution, and preparative polyacrylamide gel electrophoresis. The resultant purified components, esterases I and II, manifested single bands following silver staining of polyacrylamide gel electrophoresis gels and had final specific activities of 80 and 520 U/mg, respectively. Molecular weights for components I and II were 36,000 and 105,000 to 110,000, respectively. Esterases I and II both had a pH optimum of 8.0, with relative activities of 10 and 85%, respectively, at pH 9.0. Kms with p-nitrophenylacetate were 0.91 mM for esterase I and 0.67 mM for esterase II. In general, patterns of enzyme inhibition were similar for both components. Differences were observed in the relative activities of esterases I and II towards p-nitrophenyl esters of acetate, propionate, and butyrate; Activity ratios for components I and II were 100:94:48 and 100:36:23, respectively. The purified components did not hydrolyze long-chain triglycerides and did not manifest proteolytic activity. Images PMID:16348375

  7. Buckling Reduces eNOS Production and Stimulates Extracellular Matrix Remodeling in Arteries in Organ Culture.

    PubMed

    Xiao, Yangming; Liu, Qin; Han, Hai-Chao

    2016-09-01

    Artery buckling alters the fluid shear stress and wall stress in the artery but its temporal effect on vascular wall remodeling is poorly understood. The purpose of this study was to investigate the early effect of artery buckling on endothelial nitric oxide synthase (eNOS) expression and extracellular matrix remodeling. Bilateral porcine carotid arteries were maintained in an ex vivo organ culture system with and without buckling while under the same physiological pressure and flow rate for 3-7 days. Matrix metalloproteinase-2 (MMP-2), MMP-9, fibronectin, elastin, collagen I, III and IV, tissue inhibitor of metalloproteinase-2 (TIMP-2), and eNOS were determined using Western blotting and immunohistochemistry. Our results showed that MMP-2 expression level was significantly higher in buckled arteries than in the controls and higher at the inner curve than at the outer curve of buckled arteries, while collagen IV content showed an opposite trend, suggesting that artery buckling increased MMP-2 expression and collagen IV degradation in a site-specific fashion. However, no differences for MMP-9, fibronectin, elastin, collagen I, III, and TIMP-2 were observed among the outer and inner curve sides of buckled arteries and straight controls. Additionally, eNOS expression was significantly decreased in buckled arteries. These results suggest that artery buckling triggers uneven wall remodeling that could lead to development of tortuous arteries. PMID:26913855

  8. Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production.

    PubMed

    Andeer, Peter F; Learman, Deric R; McIlvin, Matt; Dunn, James A; Hansel, Colleen M

    2015-10-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants in environmental systems. A number of biotic and abiotic pathways induce the oxidation of Mn(II) to Mn oxides. Here, we use a combination of proteomic analyses and activity assays, to identify the enzyme(s) responsible for extracellular superoxide-mediated Mn oxide formation by a bacterium within the ubiquitous Roseobacter clade. We show that animal haem peroxidases (AHPs) located on the outer membrane and within the secretome are responsible for Mn(II) oxidation. These novel peroxidases have previously been implicated in direct Mn(II) oxidation by phylogenetically diverse bacteria. Yet, we show that in this Roseobacter species, AHPs mediate Mn(II) oxidation not through a direct reaction but by producing superoxide and likely also by degrading hydrogen peroxide. These findings point to a eukaryotic-like oscillatory oxidative-peroxidative enzymatic cycle by these AHPs that leads to Mn oxide formation by this organism. AHP expression appears unaffected by Mn(II), yet the large energetic investment required to produce and secrete these enzymes points to an as yet unknown physiological function. These findings are further evidence that bacterial peroxidases and secreted enzymes, in general, are unappreciated controls on the cycling of metals and reactive oxygen species (ROS), and by extension carbon, in natural systems.

  9. Impact of a Transposon Insertion in phzF2 on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa

    PubMed Central

    Phelan, Vanessa V.; Moree, Wilna J.; Aguilar, Julieta; Cornett, Dale S.; Koumoutsi, Alexandra; Noble, Suzanne M.; Pogliano, Kit; Guerrero, Carlos A.

    2014-01-01

    In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production. PMID:24532776

  10. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity

    PubMed Central

    Hassan, Mohamed M.

    2014-01-01

    mycoparasitism effect of the extracellular enzymes. PMID:26019588

  11. The gallium(III)-salicylidene acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxin production by Pseudomonas aeruginosa.

    PubMed

    Rzhepishevska, Olena; Hakobyan, Shoghik; Ekstrand-Hammarström, Barbro; Nygren, Yvonne; Karlsson, Torbjörn; Bucht, Anders; Elofsson, Mikael; Boily, Jean-François; Ramstedt, Madeleine

    2014-09-01

    Bacterial biofilms cause a range of problems in many areas and especially in health care. Biofilms are difficult to eradicate with traditional antibiotics and consequently there is a need for alternative ways to prevent and/or remove bacterial biofilms. Furthermore, the emergence of antibiotic resistance in bacteria creates a challenge to find new types of antibiotics with a lower evolutionary pressure for resistance development. One route to develop such drugs is to target the so called virulence factors, i.e. bacterial systems used when bacteria infect a host cell. This study investigates synergy effects between Ga(III) ions, previously reported to suppress biofilm formation and growth in bacteria, and salicylidene acylhydrazides (hydrazones) that have been proposed as antivirulence drugs targeting the type three secretion system used by several Gram-negative pathogens, including Pseudomonas aerugionosa, during bacterial infection of host cells. A library of hydrazones was screened for: Fe(III) binding, enhanced anti-biofilm effect with Ga(III) on P. aeruginosa, and low cytotoxicity to mammalian cells. The metal coordination for the most promising ligand, 2-Oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) with Ga(III) was investigated using extended X-ray absorption fine structure spectroscopy as well as density functional theory. The results showed that Ga(III) chelates the hydrazone with 5- and 6-membered chelating rings, and that the Ga(III)-ME0163 complex enhanced the antibiofilm effect of Ga(III) while suppressing the type three secretion system in P. aeruginosa. The latter effect was not observed for the hydrazone alone and was similar for Ga(III)-citrate and Ga(III)-ME0163 complexes, indicating that the inhibition of virulence was caused by Ga(III).

  12. Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis.

    PubMed

    Detry, Jean; Rosenbaum, Thorsten; Lütz, Stephan; Hahn, Doris; Jaeger, Karl-Erich; Müller, Michael; Eggert, Thorsten

    2006-10-01

    Two extracellular lipases from Bacillus subtilis, B. subtilis lipase A and lipase B, have been expressed in the heterologous host Escherichia coli, biochemically characterized and used for the kinetic resolution of (rac)-trans-1,2-diacetoxycyclohexane. Both enzymes were selectively acting on the (R,R)-enantiomer of the racemic substrate, highly specifically hydrolyzing only one of the two ester groups present, thus allowing the preparation of enantiopure (R,R)- and (S,S)-cyclohexane-trans-1,2-diol. The reaction conditions for the use of purified enzyme and crude cell lyophilizate were optimized and reactions in batch and repetitive batch modes were carried out on a preparative scale to yield enantiopure product (>99% enantiomeric excess). PMID:16586103

  13. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  14. Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

    PubMed Central

    Trejo-Hernández, Abigail; Andrade-Domínguez, Andrés; Hernández, Magdalena; Encarnación, Sergio

    2014-01-01

    Inter-kingdom and interspecies interactions are ubiquitous in nature and are important for the survival of species and ecological balance. The investigation of microbe-microbe interactions is essential for understanding the in vivo activities of commensal and pathogenic microorganisms. Candida albicans, a polymorphic fungus, and Pseudomonas aeruginosa, a Gram-negative bacterium, are two opportunistic pathogens that interact in various polymicrobial infections in humans. To determine how P. aeruginosa affects the physiology of C. albicans and vice versa, we compared the proteomes of each species in mixed biofilms versus single-species biofilms. In addition, extracellular proteins were analyzed. We observed that, in mixed biofilms, both species showed differential expression of virulence proteins, multidrug resistance-associated proteins, proteases and cell defense, stress and iron-regulated proteins. Furthermore, in mixed biofilms, both species displayed an increase in mutability compared with monospecific biofilms. This characteristic was correlated with the downregulation of enzymes conferring protection against DNA oxidation. In mixed biofilms, P. aeruginosa regulates its production of various molecules involved in quorum sensing and induces the production of virulence factors (pyoverdine, rhamnolipids and pyocyanin), which are major contributors to the ability of this bacterium to cause disease. Overall, our results indicate that interspecies competition between these opportunistic pathogens enhances the production of virulence factors and increases mutability and thus can alter the course of host-pathogen interactions in polymicrobial infections. PMID:24739628

  15. Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse

    PubMed Central

    Ashfaque, Mohammad; Solomon, Sushil; Pathak, Neelam

    2014-01-01

    Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5. PMID:25489168

  16. High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements.

    PubMed

    Zhang, Junjiao; Kang, Zhen; Ling, Zhenmin; Cao, Wenlong; Liu, Long; Wang, Miao; Du, Guocheng; Chen, Jian

    2013-10-01

    The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.

  17. Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis.

    PubMed

    Singh, R S; Bhermi, H K

    2008-10-01

    Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30 degrees C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30 degrees C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask.

  18. Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis.

    PubMed

    Singh, R S; Bhermi, H K

    2008-10-01

    Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30 degrees C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30 degrees C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask. PMID:18280145

  19. High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy.

    PubMed

    Gu, Lei; Zhang, Juan; Liu, Baihong; Du, Guocheng; Chen, Jian

    2015-02-01

    In this work, a combined strategy was developed to improve the production of glucose oxidase (GOD) (EC 1.1.3.4) in Pichia pastoris. One of the main challenges facing protein production by the high-density fermentation of P. pastoris is the high demand for oxygen. Another challenge is how to balance a reduction in oxygen consumption and its effects on protein production. Herein, a combined strategy involving mannitol co-feeding, two-stage methanol induction, and the co-expression of the transcriptional activator general control non-derepressible 4 (GCN4) from P. pastoris was used. A two-stage, co-feeding strategy, based on a mannitol/methanol mixture in a 3-L fermentor was used to enhance cell viability and protein production. This resulted in an increased GOD yield of 1208.2 U/mL compared with a control strain (427.6 U/mL). An increase in the copy number of the GCN4 gene enhanced the GOD yield (1634.7 U/mL) by 2.8-fold and the protein concentration (19.55 g/L) by 1.58-fold compared with the control (7.59 g/L). This strategy illustrates a way to overcome the high oxygen requirement during high-density fermentation of P. pastoris and balances the reduction of oxygen consumption and protein production. Moreover, the series of strategies presented in this work provide valuable and novel information for the industrial production of GOD.

  20. Inhibition of epithelial cell adhesion by retinoic acid. Relationship to reduced extracellular matrix production and alterations in Ca2+ levels.

    PubMed Central

    Varani, J.; Gibbs, D. F.; Inman, D. R.; Shah, B.; Fligiel, S. E.; Voorhees, J. J.

    1991-01-01

    Human squamous epithelial cells maintained in growth factor-deficient medium were examined for sensitivity to all-trans retinoic acid (retinoic acid). Under conditions of low external Ca2+ (0.15 mmol/l [millimolar]), or high external Ca2+ (1.4 mmol/l), retinoic acid stimulated proliferation. Concomitantly, cell-substrate adhesion was decreased. Enzyme-linked immunosorbent assays were used to assess production of two extracellular matrix components, ie, fibronectin and thrombospondin. In the presence of retinoic acid, production of both was decreased. Because both fibronectin and thrombospondin serve as epithelial cell adhesion factors, the decreased production of these moieties could contribute to reduced adhesion. Using 45Ca2+ to measure total cell-associated Ca2+ and the Ca2(+)-sensitive dye Indo-1 to measure intracellular free Ca2+, it was found that concentrations of retinoic acid that altered cell-substrate adhesion in the squamous epithelial cells had no effect on total, cell-associated Ca2+, but reduced intracellular free Ca2+ by 50% to 60%. Because Ca2+ is a regulator of adhesion, the ability of retinoic acid to modulate Ca2+ levels in the squamous epithelial cells may explain, in part, how retinoic acid influences their adhesiveness. Images Figure 1 PMID:2012176

  1. Neutrophil extracellular trap formation is increased in psoriasis and induces human β-defensin-2 production in epidermal keratinocytes.

    PubMed

    Hu, Stephen Chu-Sung; Yu, Hsin-Su; Yen, Feng-Lin; Lin, Chi-Ling; Chen, Gwo-Shing; Lan, Cheng-Che E

    2016-01-01

    Neutrophil extracellular traps (NETs) have been implicated in the development of certain immune-mediated diseases, but their role in psoriasis has not been clearly defined. Human β-defensin-2 (HBD-2) is an important antimicrobial peptide overexpressed in psoriasis epidermis. We evaluated whether the amount of NETs is increased in psoriasis and determined the effect of NETs on HBD-2 production in epidermal keratinocytes. Using fluorescent microscopy, we found that patients with psoriasis (n = 48) had higher amount of NETotic cells in their peripheral blood compared to healthy controls (n = 48) and patients with eczema (n = 35). Psoriasis sera showed increased ability to induce NET formation in control neutrophils but normal NET degradation ability. The amount of NETs in the peripheral blood correlated with psoriasis disease severity. NETosis was also observed in the majority (18 of 20) of psoriasis skin specimens. Furthermore, NETs induced HBD-2 mRNA and protein production in keratinocytes, and immunohistochemical analysis confirmed strong expression of HBD-2 in psoriasis lesional skin. In summary, NET formation is increased in peripheral blood and lesional skin of psoriasis patients and correlates with disease severity. Additionally, NET-induced HBD-2 production may provide a novel mechanism for the decreased susceptibility of psoriasis plaques to microbial infections. PMID:27493143

  2. Oxidation of Microcystis aeruginosa and Anabaena flos-aquae by ozone: impacts on cell integrity and chlorination by-product formation.

    PubMed

    Coral, Lucila A; Zamyadi, Arash; Barbeau, Benoit; Bassetti, Fatima J; Lapolli, Flávio R; Prévost, Michèle

    2013-06-01

    Pre-ozonation of cyanobacterial (CB) cells in raw water and inter-ozonation of settled water can cause CB cell damage. However, there is limited information about the level of lysis or changes in cell properties after ozonation, release of intracellular compounds and their contribution to the formation of disinfection by-products (DBPs). This study aims to: (1) assess the extent of the pre-ozonation effects on CB cell properties; (2) determine the CT (ozone concentration × detention time) values required for complete loss of cell viability; and (3) study the DBPs formation associated with the pre-ozonation of cyanobacterial cells in laboratorial suspensions. To these ends, both Microcystis aeruginosa and Anabaena flos-aquae suspensions were prepared at concentrations of 250,000 cells mL(-1) and 1,500,000 cells mL(-1) and were subjected to ozone dosages of 0.5, 2.0 and 4.0 mg L(-1) at pH 6 and pH 8. A quick and complete loss of viability was achieved for both CB species after exposure (CT) to ozone of <0.2 mg min L(-1), although no significant decrease in total cell numbers was observed. Maximum dissolved organic carbon (DOC) releases of 0.96 mg L(-1) and 1.63 mg L(-1) were measured after ozonation of 250,000 cells mL(-1) of M. aeruginosa and A. flos-aquae, respectively. DOC release was found to be pH and ozone dose dependent. Ozonation of CB cells increased formation of trihalomethanes (THM) and haloacetic acids (HAA), mainly for suspensions of A. flos-aquae at pH 8 (by 174% and 65% for THM and HAA respectively). Utilities considering using ozone for oxidising CB cells should weigh out the benefit of CB control with the potential increased formation of chlorinated DBPs.

  3. Production and cytotoxicity of extracellular insoluble and droplets of soluble melanin by Streptomyces lusitanus DMZ-3.

    PubMed

    Madhusudhan, D N; Mazhari, Bi Bi Zainab; Dastager, Syed G; Agsar, Dayanand

    2014-01-01

    A Streptomyces lusitanus DMZ-3 strain with potential to synthesize both insoluble and soluble melanins was detected. Melanins are quite distinguished based on their solubility for varied biotechnological applications. The present investigation reveals the enhanced production of insoluble and soluble melanins in tyrosine medium by a single culture. Streptomyces lusitanus DMZ-3 was characterized by 16S rRNA gene analysis. An enhanced production of 5.29 g/L insoluble melanin was achieved in a submerged bioprocess following response surface methodology. Combined interactive effect of temperature (50°C), pH (8.5), tyrosine (2.0 g/L), and beef extract (0.5 g/L) were found to be critical variables for enhanced production in central composite design analysis. An optimized indigenous slant culture system was an innovative approach for the successful production (264 mg/L) of pure soluble melanin from the droplets formed on the surface of the culture. Both insoluble and soluble melanins were confirmed and characterized by Chemical, reactions, UV, FTIR, and TLC analysis. First time, cytotoxic study of melanin using brine shrimps was reported. Maximum cytotoxic activity of soluble melanin was Lc50-0.40 µg/mL and insoluble melanin was Lc50-0.80 µg/mL.

  4. Production and Cytotoxicity of Extracellular Insoluble and Droplets of Soluble Melanin by Streptomyces lusitanus DMZ-3

    PubMed Central

    Madhusudhan, D. N.; Mazhari, Bi Bi Zainab; Dastager, Syed G.

    2014-01-01

    A Streptomyces lusitanus DMZ-3 strain with potential to synthesize both insoluble and soluble melanins was detected. Melanins are quite distinguished based on their solubility for varied biotechnological applications. The present investigation reveals the enhanced production of insoluble and soluble melanins in tyrosine medium by a single culture. Streptomyces lusitanus DMZ-3 was characterized by 16S rRNA gene analysis. An enhanced production of 5.29 g/L insoluble melanin was achieved in a submerged bioprocess following response surface methodology. Combined interactive effect of temperature (50°C), pH (8.5), tyrosine (2.0 g/L), and beef extract (0.5 g/L) were found to be critical variables for enhanced production in central composite design analysis. An optimized indigenous slant culture system was an innovative approach for the successful production (264 mg/L) of pure soluble melanin from the droplets formed on the surface of the culture. Both insoluble and soluble melanins were confirmed and characterized by Chemical, reactions, UV, FTIR, and TLC analysis. First time, cytotoxic study of melanin using brine shrimps was reported. Maximum cytotoxic activity of soluble melanin was Lc50-0.40 µg/mL and insoluble melanin was Lc50-0.80 µg/mL. PMID:24839603

  5. Antipathogenic potential of marine Bacillus sp. SS4 on N-acyl-homoserine-lactone-mediated virulence factors production in Pseudomonas aeruginosa (PAO1).

    PubMed

    Musthafa, K Syed; Saroja, V; Pandian, S Karutha; Ravi, A Veera

    2011-03-01

    Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5-2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33-86%) and biofilm formation (33-88%), total protease (20-65%), LasA protease (59-68%), LasB elastase (36-68%), pyocyanin (17-86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751). PMID:21451248

  6. Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

    SciTech Connect

    Hughes, Ronny C.; McFeeters, Hana; Coates, Leighton; McFeeters, Robert L.

    2014-10-15

    The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62,c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da-1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

  7. Irgasan-induced pigmentation in Serratia marcescens and Pseudomonas aeruginosa.

    PubMed

    Kranz, R G; Lynch, D L

    1979-01-01

    Two irgasan-resistant micro-organisms (P. aeruginosa and S. marcescens) were used to study the effects of various antibiotic and chemotherapeutic agents on pigment production. These agents included streptomycin, thallium acetate, polymyxin B, hexachlorophene, irgasan, prodigiosin and DMSO (dimethyl sulphoxide). Only irgasan, compared to other drugs and membrane-active agents showed the unique property of inducing pigmentation in both P. aeruginosa and S. marcescens, i.e. prodigiosin in S. marcescens and pyocyanin in P. aeruginosa.

  8. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa.

    PubMed

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  9. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

    PubMed Central

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  10. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    PubMed

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

  11. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

    PubMed Central

    Dash, Biplab Kumar; Rahman, M. Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

  12. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    PubMed

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  13. Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

    PubMed

    Arez, Bruno F; Alves, Luís; Paixão, Susana M

    2014-11-01

    The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

  14. Phytolacca americana inhibits the high glucose-induced mesangial proliferation via suppressing extracellular matrix accumulation and TGF-beta production.

    PubMed

    Jeong, Seung Il; Kim, Kang Ju; Choo, Yong Kug; Keum, Kyung Soo; Choi, Bong Kyu; Jung, Kyu Yong

    2004-02-01

    This study describes a potential of Phytolaccaceae (Phytolacca americana var.) as an inhibitor of high glucose-stimulated production of extracellular matrix (ECM) proteins and TGF-beta in cultured glomerular mesangial cells (GMCs). Raising the ambient glucose concentration for 24 hrs caused a dose-dependent increase in [3H]thymidine incorporation of GMCs, and the maximal response was achieved at 20 mM. Phytolaccaceae extracts (2.5-20 microg/ml) inhibited the high glucose-induced [3H]thymidine incorporation in a dose-dependent manner, and the concentrations tested here did not affect to the cell viability. Exposure of the GMCs to 20 mM glucose caused both ECM (collagen and fibronectin) accumulation and TGF-beta secretion, and these changes were significantly diminished by treatment of GMCs with Phytolaccaceae (10 microg/ml). Taken together, these results indicate that Phytolaccaceae inhibits the high glucose-induced GMCs proliferation partially through suppressing accumulation of ECM components and TGF-beta production, suggesting that Phytolaccaceae may be a promising agent for treating the development and progression of diabetic glomerulopathy.

  15. Analytical methods for soluble microbial products (SMP) and extracellular polymers (ECP) in wastewater treatment systems: a review.

    PubMed

    Kunacheva, Chinagarn; Stuckey, David C

    2014-09-15

    Effluents from biological processes contain a wide range of complex organic compounds, including soluble microbial products (SMP) and extracellular polymers (ECP), released during bacteria metabolism in mixed culture in bioreactors. It is important to clearly identify the primary components of SMPs and ECPs in order to understand the fundamental mechanisms of biological activity that create these compounds, and how to reduce these compounds in the effluent. In addition, these compounds constitute the main foulants in membrane bioreactors which are being used more widely around the world. A review on the extraction of ECP, characterization, and identification of SMPs and ECPs is presented, and we summarize up-to-date pretreatments and analytical methods for SMPs. Most researchers have focused more on the overall properties of SMPs and ECPs such as their concentrations, molecular weight distribution, aromaticity, hydrophobic and hydrophilic properties, biodegradability, and toxicity characteristics. Many studies on the identification of effluent SMPs show that most of these compounds were not present in the influent, such as humic acids, polysaccharides, proteins, nucleic acids, organic acids, amino acids, exocellular enzymes, structural components of cells and products of energy metabolism. A few groups of researchers have been working on the identification of compounds in SMPs using advanced analytical techniques such as GC-MS, LC-IT-TOF-MS and MALDI-TOF-MS. However, there is still considerably more work needed to be done analytically to fully understand the chemical characteristics of SMPs and ECPs.

  16. Extracellular amylase(s) production by fungi Botryodiplodia theobromae and Rhizopus oryzae grown on cassava starch residue.

    PubMed

    Ray, R C

    2004-10-01

    The fungi Botryodiplodia theobromae and Rhizopus oryzae produce extracellular amylase when grown on a liquid medium containing 2% (WN) soluble starch or cassava starch residue(CSR) (as starch equivalent), a waste generated after extraction of starch from cassava, as the sole carbon source. Using CSR as the sole carbon source, the highest amylase activity of 3.25 and 3.8 units (mg, glucose released x ml(-1) x h(-1)) were obtained in shake flask cultures during the late stationary phase of growth of B. theobromae and R. oryzae, respectively. These values were slightly lower than the values obtained using soluble starch as the carbon source. Maximum enzyme synthesis in CSR incorporated medium occurred at the growth temperature of 30 degrees C and pH 6.0. Presence of inorganic NH4+ salts like ammonium acetate and ammonium nitrate in culture medium yielded more amylase than the other nitrogen sources. Amylase(s) production in the controlled environment of a Table-Top glass Jar Fermenter (2-L capacity) was 4.8 and 5.1 units for B. theobromae and R. oryzae, respectively using CSR as the carbon substrate. It is concluded that CSR, a cheap agricultural waste obtained after starch extraction from cassava could replace soluble starch as carbon substrate for commercial production of fungal amylase(s).

  17. Heterotrophic bacterial production and extracellular enzymatic activity in sinking particulate matter in the western North Pacific Ocean.

    PubMed

    Yamada, Namiha; Fukuda, Hideki; Ogawa, Hiroshi; Saito, Hiroaki; Suzumura, Masahiro

    2012-01-01

    Heterotrophic activities on sinking particulate matter (SPM) play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP) in seawater (HBP(SW)) and SPM (HBP(SPM)) as well as potential extracellular enzyme activity (EEA) in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N), the Kuroshio Extension area (35°N), and the subtropical gyre (20°N). Depth-integrated HBP(SW) from the surface to 500 m was comparable between the locations, whereas HBP(SPM) at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC) export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase) activity, β-glucosidase (BGase) activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production. PMID:23109933

  18. High-level extracellular production of D-Psicose-3-epimerase with recombinant Escherichia coli by a two-stage glycerol feeding approach.

    PubMed

    Gu, Lei; Zhang, Juan; Liu, Baihong; Wu, Chongde; Du, Guocheng; Chen, Jian

    2013-11-01

    The aim of this study is to achieve high-level extracellular production of D-Psicose-3-epimerase (DPE) with recombinant Escherichia coli. High-level production of DPE is one of the key factors in D-Psicose production. In the present study, the gene AAL45544.1 from Agrobacterium tumefaciens str. C58 was modified by artificial synthesis for overexpression in E. coli. The total DPE activity reached 3.96 U mL(-1) after optimization of the media composition, induction temperature, and concentration of inducer. Furthermore, it was found that addition of glycine had a positive effect on the extracellular production of DPE, which reached 3.5 U mL(-1). Finally, a two-stage glycerol feeding strategy based on both the specific growth rate before induction and the amount of glycerol residues after induction was applied in a 3-L fermenter. After a series of optimal strategies in the 3-L fermenter, the total and extracellular DPE activity were 5.08- and 3.11-fold higher than that noted in the shake flask. The extracellular and intracellular DPE activity reached 10.9 and 13.2 U mL(-1), achieving 25.5 and 31.1 % conversion of D-fructose to D-psicose, respectively. The systemic strategies presented in this study provide valuable novel information for the industrial application of DPE.

  19. An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.

    PubMed

    Bao, Ru-Meng; Yang, Hong-Ming; Yu, Chang-Mei; Zhang, Wei-Fen; Tang, Jin-Bao

    2016-10-01

    Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the β-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.

  20. Regulation of extracellular polygalacturonase production in Pseudomonas solanacearum. Progress report, [May 1, 1992--April 30, 1994

    SciTech Connect

    Allen, C.

    1994-06-01

    Pseudomonas solanacearum is an economically important plant pathogen that causes bacterial wilt disease of diverse crops. The bacterium produces at least three isozymes of polygalacturonase, which degrade plant cell walls and contribute substantially to bacterial wilt disease development. The central objective of this research project is to determine how expression of these enzymes is regulated. To this end, we isolated a positive trans-acting regulator of polygalacturonase production (pehR). We have focused on further characterization of the pehR mutant pheonotype, and studies of pehR expression. Preliminary results suggest pehR also regulates bacterial motility. An investigation of two unusual tyrosine phosphoproteins in P. solanacearum is also described.

  1. Production of Extracellular Laccase from Bacillus subtilis MTCC 2414 Using Agroresidues as a Potential Substrate

    PubMed Central

    Muthukumarasamy, Narayanan P.; Jackson, Beenie; Joseph Raj, Antony; Sevanan, Murugan

    2015-01-01

    Laccases are the model enzymes for multicopper oxidases and participate in several applications such as bioremediation, biopulping, textile, and food industries. Laccase producing bacterium, Bacillus subtilis MTCC 2414, was subjected to optimization by conventional techniques and was partially purified using ammonium salt precipitation method. The agroresidue substrates used for higher yield of laccase were rice bran and wheat bran. Maximum production was achieved at temperature 30°C (270 ± 2.78 U/mL), pH 7.0 (345 ± 3.14 U/mL), and 96 h (267 ± 2.64 U/mL) of incubation. The carbon and nitrogen sources resulted in high enzyme yield at 3% sucrose (275 ± 3.11 U/mL) and 3% peptone (352.2 ± 4.32 U/mL) for rice bran and 3% sucrose (247.4 ± 3.51 U/mL) and 3% peptone (328 ± 3.33 U/mL) for wheat bran, respectively. The molecular weights of partially purified laccase were 52 kDa for rice bran and 55 kDa for wheat bran. The laccase exhibited optimal activity at 70°C (260.3 ± 6.15 U/mL), pH 9.0 (266 ± 4.02 U/mL), and metal ion CuSO4 (141.4 ± 6.64) was found to increase the production. This is the first report that delivers the higher yield of laccase produced from B. subtilis MTCC 2414 using agroresidues as a potential substrate. PMID:26451255

  2. Production of Extracellular Laccase from Bacillus subtilis MTCC 2414 Using Agroresidues as a Potential Substrate.

    PubMed

    Muthukumarasamy, Narayanan P; Jackson, Beenie; Joseph Raj, Antony; Sevanan, Murugan

    2015-01-01

    Laccases are the model enzymes for multicopper oxidases and participate in several applications such as bioremediation, biopulping, textile, and food industries. Laccase producing bacterium, Bacillus subtilis MTCC 2414, was subjected to optimization by conventional techniques and was partially purified using ammonium salt precipitation method. The agroresidue substrates used for higher yield of laccase were rice bran and wheat bran. Maximum production was achieved at temperature 30°C (270 ± 2.78 U/mL), pH 7.0 (345 ± 3.14 U/mL), and 96 h (267 ± 2.64 U/mL) of incubation. The carbon and nitrogen sources resulted in high enzyme yield at 3% sucrose (275 ± 3.11 U/mL) and 3% peptone (352.2 ± 4.32 U/mL) for rice bran and 3% sucrose (247.4 ± 3.51 U/mL) and 3% peptone (328 ± 3.33 U/mL) for wheat bran, respectively. The molecular weights of partially purified laccase were 52 kDa for rice bran and 55 kDa for wheat bran. The laccase exhibited optimal activity at 70°C (260.3 ± 6.15 U/mL), pH 9.0 (266 ± 4.02 U/mL), and metal ion CuSO4 (141.4 ± 6.64) was found to increase the production. This is the first report that delivers the higher yield of laccase produced from B. subtilis MTCC 2414 using agroresidues as a potential substrate. PMID:26451255

  3. [Formation of disinfection by-products by Microcystis aeruginosa intracellular organic matter: comparison between chlorination and bromination].

    PubMed

    Tian, Chuan; Guo, Ting-Ting; Liu, Rui-Ping; Jefferson, William; Liu, Hui-Juan; Qu, Jiu-Hui

    2013-11-01

    In order to illustrate the effects of released algal organic matter in cyanobacteria blooms on raw water quality and water treatment process, intracellular organic matter (IOM) of Microcystis aeruginosa, which is the dominant species in cyanobacteria blooms, was chosen as a precursor and characterized. In addition, the transformation of IOM and the formation of disinfection byproducts were evaluated at different pH of 6.5, 7.1 and 8.4 after chlorination or bromination, with subsequent correlation analysis. The results indicated that IOM was primarily composed of macromolecular matter, i. e. , the species with relative molecular weight of > 30 x 10(3), constituting 68.8% of dissolved organic carbon (DOC). Fluorescence excitation-emission matrix indicated that IOM was mainly composed of aromatic protein-like matter with an intensity of 92.6 AU x L x mg(-1). After reaction with chlorine or bromine, the intensity of aromatic protein-like peaks decreased sharply by 76.6% - 93.3%, and its reduction correlated well with the formation of trihalomethane (THMs, R2 = 0.81) and haloacetic acid (HAAs, R2 = 0.77). The formation of THMs and HAAs increased with the increase in pH. Compared with chlorine, bromine formed more THMs and HAAs, and tended to form highly halogenated HAAs. However, with increasing pH, the reactivity with IOM between chlorine and bromine was closer, i.e, k(OBr-IOM)/k(OCl-(IOM) < k(HOBr-IOM/k(HOCl-IOM).

  4. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity.

    PubMed Central

    Toder, D S; Ferrell, S J; Nezezon, J L; Rust, L; Iglewski, B H

    1994-01-01

    The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB. Images PMID:8132339

  5. Isolation of a mucoid alginate-producing Pseudomonas aeruginosa strain from the equine guttural pouch.

    PubMed Central

    Govan, J R; Sarasola, P; Taylor, D J; Tatnell, P J; Russell, N J; Gacesa, P

    1992-01-01

    The isolation and characterization of a mucoid, alginate-producing strain of Pseudomonas aeruginosa from a nonhuman host, namely, in chondroids from an equine guttural pouch, is reported for the first time. Pure cultures of P. aeruginosa 12534 were isolated from a 17-month-old pony mare with a history of chronic bilateral mucopurulent nasal discharge from the right guttural pouch. Transmission electron microscopy of chondroids showed mucoid P. aeruginosa growing as microcolonies within a matrix of extracellular material. On the basis of expression of the mucoid phenotype under different growth conditions, P. aeruginosa 12534 belongs to group 1 and resembles other isolates carrying the muc-23 mutation. The bulk of the extracellular material was characterized as being alginate by chemical and 1H nuclear magnetic resonance analyses, which showed that it had a composition similar to that produced by isolates of P. aeruginosa from human patients with cystic fibrosis. Images PMID:1551975

  6. A novel L-aspartate dehydrogenase from the mesophilic bacterium Pseudomonas aeruginosa PAO1: molecular characterization and application for L-aspartate production.

    PubMed

    Li, Yinxia; Kawakami, Norika; Ogola, Henry Joseph Oduor; Ashida, Hiroyuki; Ishikawa, Takahiro; Shibata, Hitoshi; Sawa, Yoshihiro

    2011-06-01

    L-aspartate dehydrogenase (EC 1.4.1.21; L: -AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative L-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for L-aspartate (L-Asp) and oxaloacetate (OAA) of 127 and 147 U mg(-1), respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T (m) value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K (m) values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The L-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of L-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of L-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of L-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.

  7. Isolation and identification of actinomycetes for production of novel extracellular glutaminase free L-asparaginase.

    PubMed

    Saxena, Akansha; Upadhyay, Ramraj; Kango, Naveen

    2015-12-01

    Over the recent years glutaminase free L-asparaginase has gained more importance due to better therapeutic properties for treatment of acute lymphoblastic leukemia. Actinomycetes are known for L-asparaginase activity. In the current study, 80 actinomycetes were isolated from various soil habitats by serial dilution technique. Presence of L-asparaginase was investigated in a total of 240 actinomycetes by tubed agar method using modified M-9 medium. A total of 165 actinomycetes were found positive for L-asparaginase activity. Among these, 57 actinomycetes producing larger zones of L-asparagine hydrolysis were further screened for their capacity to produce glutaminase-free L-asparaginase. Four L-glutaminase-free actinomycetes were found to be potential L-asparaginase producers. These actinomycetes were identified as Streptomyces cyaneus (SAP 1287, CFS 1560), S. exfoliates (CFS 1557) and S. phaeochromogenes (GS 1573) on the basis of morphological and biochemical identification studies. Maximum L-asparaginase activity (19.2 Uml(-1)) was observed in culture filtrate of S. phaeochromogenes under submerged fermentation. Results indicate that S. phaeochromogenes could be a potential source of glutaminase free L-asparaginase for commercial purpose. To the best of our knowledge, this is the first report on production of glutaminase free L-asparaginase from S. cyaneus, S. exfoliatus and S. phaeochromogenes. PMID:26742323

  8. Growth of Heterotrophic Bacteria and Algal Extracellular Products in Oligotrophic Waters

    PubMed Central

    McFeters, Gordon A.; Stuart, Sidney A.; Olson, Susan B.

    1978-01-01

    The unexpected observation of 200 to 400 coliform bacteria per 100 ml in an unpolluted pristine stream was studied within Grand Teton National Park, Wyo. The high numbers of waterborne bacteria occurred in mid- to late summer at a location where there was a coincidental bloom of an algal mat community. Periphyton samplers were used to measure the algal growth that coincided with the increase in number of bacteria. Laboratory studies followed the growth of various coliform bacteria in the supernatant obtained from a Chlorella culture isolated from the mat community. Mixed natural bacterial populations from the stream and pure cultures of water-isolated fecal and nonfecal coliforms increased by two to three orders of magnitude at 13°C when grown in the algal supernatant. Radioactive algal products were obtained by feeding an axenic Chlorella culture 14C-labeled bicarbonate under laboratory cultivation at 13°C with illumination. Radioactive organic material from the algae became incorporated into the particulate fraction of pure cultures of coliform bacteria as they reproduced and was later released as they died. PMID:16345278

  9. Extracellular bio-production and characterization of small monodispersed CdSe quantum dot nanocrystallites

    NASA Astrophysics Data System (ADS)

    Suresh, Anil K.

    2014-09-01

    Engineered nanoparticles of diverse forms are being profoundly used for various applications and demand ecologically benign synthesis processes. Conventional chemical methods employed for the syntheses of nanoparticles are environmentally unfriendly and energy intensive. Biologically inspired biofabrication approaches that utilize naturally existing microorganisms or plant extracts or biomaterials might overcome these issues. The present investigation for the first time shows the synthesis of small and monodispersed cadmium selenide nanoparticles utilizing the plant pathogenic fungus, Helminthosporum solani upon incubating with an aqueous solution of CdCl2 and SeCl4 under ambient conditions. Multiple physical characterizations involving ultraviolet-visible and photoluminescence spectroscopy, transmission electron microscopy, selected area electron diffraction and X-ray photoelectron spectroscopy confirmed the production, purity, optical and surface characteristics, crystalline nature, size and shape distributions, and elemental composition of the nanoparticles. Pluralities of the particles are monodisperse spheres with a mean diameter of 5.5 ± 2 nm, are hydrophilic, highly stable with a broad photoluminescence and 1% quantum yield. This approach provides an alternative facile route for the biofabrication of quantum dot that is reliable, environmentally friendly, and lends itself directly for the creation of fluorescent biological labels.

  10. Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis.

    PubMed

    Maehashi, Kenji; Matano, Mami; Saito, Makiko; Udaka, Shigezo

    2010-05-01

    Riboflavin-binding protein (RBP) is a glycophosphoprotein found in hen eggs. We previously identified the extraordinary characteristic of RBP in reducing bitterness. For a more detailed study on the mode of action and industrial application of this characteristic, we investigated the microbial production of recombinant RBP (rRBP). We constructed a chicken RBP gene expression vector by inserting the RBP cDNA in pNCMO2, the Escherichia coli-Brevibacillus choshinensis shuttle vector. B. choshinensis HPD31 transformants produced 0.8g/l of processed and unglycosylated RBP in a soluble form in the culture supernatant. However, the expressed RBP was partially dimerized and monomeric RBP was purified by two step anion-exchange and gel-filtration chromatographies. The purified rRBP elicited bitterness reduction against quinine and caffeine, although it largely lost its riboflavin-binding ability. These results indicated that glycosylation and riboflavin-binding ability are not essential for the bitterness reduction of RBP. In addition, we assessed the usefulness of the Brevibacillus system for the expression and secretion of RBP as a new type of bitterness inhibitor. PMID:20045733

  11. Production of extracellular biopolymers and identification of intracellular proteins and Rhizobium tropici.

    PubMed

    Oliveira, José; Figueiredo, Marcia; Silva, Marcia; Malta, Marília; Vendruscolo, Claire; Almeida, Hélio

    2012-12-01

    The objective of this study was to identify species of rhizobia (from the IPA 403 and IPA 49 isolates), to assess the physico-chemical characteristics of the biopolymers produced by these rhizobia and to determine the soluble intracellular proteins that are present in these rhizobia. The polysaccharides containing acetyl and pyruvic acid groups that were produced by different strains that had been cultivated in yeast extract mannitol (YEM) medium for 132, 144, and 168 h were evaluated for yield, viscosity, and concentration. Based on the analysis of their partial 16S rDNA sequences, both isolates were identified as Rhizobium tropici. The polymers produced in liquid YEM medium were recovered, dried and weighed to determine culture yield. Soluble intracellular proteins were identified through the techniques of 2D-PAGE and mass spectrometry for cultures that were cultivated for 168 h. The largest biopolymer yield and the highest viscosity and concentration of acetyl and pyruvic acids were obtained from the IPA 403 isolate after 168 h of culture. The proteins that were identified for the CIAT 899 isolate included elongation factor TU, a chaperone; GroE/GroEs and a putative glycosyltransferase, all of which catalyze the production of polysaccharides. For the IPA 403 strain, dinitrogenase and nitrogenase iron proteins were found. In the IPA 49 strain, glyceraldehyde-3-phosphate dehydrogenase was found along with two other proteins, the beta subunit of an electron-transferring flavoprotein and a dehydrogenase.

  12. Extracellular polysaccharides from Ascomycota and Basidiomycota: production conditions, biochemical characteristics, and biological properties.

    PubMed

    Osińska-Jaroszuk, Monika; Jarosz-Wilkołazka, Anna; Jaroszuk-Ściseł, Jolanta; Szałapata, Katarzyna; Nowak, Artur; Jaszek, Magdalena; Ozimek, Ewa; Majewska, Małgorzata

    2015-12-01

    Fungal polysaccharides (PSs) are the subject of research in many fields of science and industry. Many properties of PSs have already been confirmed and the list of postulated functions continues to grow. Fungal PSs are classified into different groups according to systematic affinity, structure (linear and branched), sugar composition (homo- and heteropolysaccharides), type of bonds between the monomers (β-(1 → 3), β-(1 → 6), and α-(1 → 3)) and their location in the cell (cell wall PSs, exoPSs, and endoPSs). Exopolysaccharides (EPSs) are most frequently studied fungal PSs but their definition, classification, and origin are still not clear and should be explained. Ascomycota and Basidiomycota fungi producing EPS have different ecological positions (saprotrophic and endophytic, pathogenic or symbiotic-mycorrhizae fungi); therefore, EPSs play different biological functions, for example in the protection against environmental stress factors and in interactions with other organisms. EPSs obtained from Ascomycota and Basidiomycota fungal cultures are known for their antioxidant, immunostimulating, antitumor, and antimicrobial properties. The major objective of the presented review article was to provide a detailed description of the state-of-the-art knowledge of the effectiveness of EPS production by filamentous and yeast Ascomycota and Basidiomycota fungi and techniques of derivation of EPSs, their biochemical characteristics, and biological properties allowing comprehensive analysis as well as indication of similarities and differences between these fungal groups. Understanding the role of EPSs in a variety of processes and their application in food or pharmaceutical industries requires improvement of the techniques of their derivation, purification, and characterization. The detailed analyses of data concerning the derivation and application of Ascomycota and Basidiomycota EPSs can facilitate development and trace the direction of application of these EPSs

  13. Large strain stimulation promotes extracellular matrix production and stiffness in an elastomeric scaffold model.

    PubMed

    D׳Amore, Antonio; Soares, Joao S; Stella, John A; Zhang, Will; Amoroso, Nicholas J; Mayer, John E; Wagner, William R; Sacks, Michael S

    2016-09-01

    Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the denovo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments.

  14. Large strain stimulation promotes extracellular matrix production and stiffness in an elastomeric scaffold model.

    PubMed

    D׳Amore, Antonio; Soares, Joao S; Stella, John A; Zhang, Will; Amoroso, Nicholas J; Mayer, John E; Wagner, William R; Sacks, Michael S

    2016-09-01

    Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the denovo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments. PMID:27344402

  15. Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004.

    PubMed

    Ali Amoozegar, Mohammad; Zahra Fatemi, Azadeh; Reza Karbalaei-Heidari, Hamid; Reza Razavi, Mohamad

    2007-01-01

    An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml(-1)). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 degrees C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 degrees C, with 18% and 41% activity reductions at temperature 50 and 70 degrees C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0-10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0-10% NaCl).

  16. Influence of hydrogel mechanical properties and mesh size on vocal fold fibroblast extracellular matrix production and phenotype

    PubMed Central

    Hahn, Mariah S.; Liao, Huimin; Munoz-Pinto, Dany; Qu, Xin; Hou, Yaping; Grunlan, Melissa A.

    2008-01-01

    Current clinical management of vocal fold (VF) scarring produces inconsistent and often suboptimal results. Researchers are investigating a number of alternative treatments for VF lamina propria (LP) scarring, including designer implant materials for functional LP regeneration. In the present study, we investigate the effects of the initial scaffold elastic modulus and mesh size on encapsulated VF fibroblast (VFF) extracellular matrix (ECM) production toward rational scaffold design. Polyethylene glycol diacrylate (PEGDA) hydrogels were selected for this study since their material properties, including mechanical properties, mesh size, degradation rate and bioactivity, can be tightly controlled and systematically modified. Porcine VFF were encapsulated in four PEGDA hydrogels with degradation half lives of ~25 days and initial elastic compressive moduli ranging from ~30 to 100 kPa and initial mesh sizes ranging from ~9 to 27 nm. After 30 days of static culture, VFF ECM production and phenotype in each formulation was assessed biochemically and histologically. Sulfated glycosaminoglycan synthesis increased in similar degree with both increasing initial modulus and decreasing initial mesh size. In contrast, elastin production decreased with increasing initial modulus but increased with decreasing initial mesh size. Both collagen deposition and the induction of a myofibroblastic phenotype depended strongly on initial mesh size but appeared largely unaffected by variations in initial modulus. The present results indicate that scaffold mesh size warrants further investigation as a critical regulator of VFF ECM synthesis. Furthermore, this study validates a systematic and controlled approach for analyzing VFF response to scaffold properties, which should aid in rational scaffold selection/design. PMID:18515199

  17. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    PubMed Central

    Bisht, Deepali; Yadav, Santosh Kumar; Darmwal, Nandan Singh

    2013-01-01

    Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization. PMID:24159311

  18. The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

    PubMed Central

    Guragain, Manita; King, Michelle M.; Williamson, Kerry S.; Pérez-Osorio, Ailyn C.; Akiyama, Tatsuya; Khanam, Sharmily

    2016-01-01

    ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in carP had a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+ and responding through its regulatory targets that

  19. N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces.

    PubMed

    Olofsson, Ann-Cathrin; Hermansson, Malte; Elwing, Hans

    2003-08-01

    N-Acetyl-L-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces. PMID:12902275

  20. N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces.

    PubMed

    Olofsson, Ann-Cathrin; Hermansson, Malte; Elwing, Hans

    2003-08-01

    N-Acetyl-L-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces.

  1. The characteristics of extracellular polymeric substances and soluble microbial products in moving bed biofilm reactor-membrane bioreactor.

    PubMed

    Duan, Liang; Jiang, Wei; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W

    2013-11-01

    The characteristics of extracellular polymeric substances (EPS) and soluble microbial products (SMP) in conventional membrane bioreactor (MBR) and in moving bed biofilm reactor-membrane bioreactors (MBBR-MBR) were investigated in long-term (170 days) experiments. The results showed that all reactors had high removal efficiency of ammonium and COD, despite very different fouling conditions. The MBBR-MBR with media fill ratio of 26.7% had much lower total membrane resistance and no obvious fouling were detected during the whole operation. In contrast, MBR and MBBR-MBR with lower and higher media fill experienced more significant fouling. Low fouling at optimum fill ratio may be due to the higher percentage of small molecular size (<1 kDa) and lower percentage of large molecular size (>100 kDa) of EPS and SMP in the reactor. The composition of EPS and SMP affected fouling due to different O-H bonds in hydroxyl functional groups, and less polysaccharides and lipids.

  2. The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

    PubMed

    Dötsch, Andreas; Eckweiler, Denitsa; Schniederjans, Monika; Zimmermann, Ariane; Jensen, Vanessa; Scharfe, Maren; Geffers, Robert; Häussler, Susanne

    2012-01-01

    In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms. PMID:22319605

  3. Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions.

    PubMed

    García-Hidalgo, Javier; Hormigo, Daniel; Prieto, María Auxiliadora; Arroyo, Miguel; de la Mata, Isabel

    2012-03-01

    The phaZ ( Sex ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T (m)) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics. PMID:21845385

  4. Extracellular polymeric substances production kinetics of 13 sludge isolates using wastewater sludge as raw material and its flocculation potential.

    PubMed

    More, Tanaji; Mahmoudi, Amine; Yan, Song; Tyagi, Rajeshwar Dayal

    2015-01-01

    The kinetics of batch fermentation of 13 extracellular polymeric substances (EPS) producing bacterial strains (9 Bacillus, 2 Serratia and 2 Yersinia) were carried out using sterilized sludge as a raw material. The most of Bacillus (µ(max): 0.11-0.27 h⁻¹), Serratia (µ(max): 0.23-0.27 h⁻¹) and Yersinia (µ(max): 0.18-0.19 h⁻¹) strains had capability to grow and produce EPS (1.36-2.12 g/L) in the sterilized sludge. In general, EPS production was mixed growth associated for all the bacterial strains cultivated independently. Bacillus sp. 7, Serratia sp. 2 and Yersinia sp. 2 produced higher concentration (1.95-2.12 g/L) of EPS than the other remaining bacterial strains. Protein and carbohydrate contents of EPS remained constant during fermentation. Broth EPS (B-EPS) exhibited high kaolin flocculation activity (≥ 75%) in most of the cases except Bacillus sp. 1, Bacillus sp. 5 and Bacillus sp. 9, respectively. In general, high flocculation activities (FAs) (≥ 75%), were attained using 1.31-1.70 mg B-EPS/g kaolin, 0.45-0.97 mg protein/g kaolin and 0.11-0.21 mg carbohydrates/g kaolin. The study suggests that further systematic exploration is required for optimizing the process of EPS production. EPS produced in the sludge can potentially be used for different water and wastewater treatments.

  5. Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

    PubMed

    Dagar, Vikas Kumar; Adivitiya; Devi, Nirmala; Khasa, Yogender Pal

    2016-10-01

    Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

  6. Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod and low glycemic short-horned sculpin.

    PubMed

    Clow, Kathy A; Short, Connie E; Driedzic, William R

    2016-05-01

    Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism

  7. NAD(P)H oxidase-dependent intracellular and extracellular O2·- production in coronary arterial myocytes from CD38 knockout mice

    PubMed Central

    Xu, Ming; Zhang, Yang; Xia, Min; Li, Xiao-Xue; Ritter, Joseph K; Zhang, Fan; Li, Pin-Lan

    2011-01-01

    Activation of NAD(P)H oxidase has been reported to produce superoxide (O2 ·-) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use NAD(P)H oxidase product, NAD+ or NADP+ to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), which mediates intracellular Ca2+ signaling. The present study was designed to test a hypothesis that CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2 ·-in mouse coronary arterial myocytes (CAMs). By fluorescent microscopic imaging, we simultaneously monitored extra- and intracellular O2 ·-production in wild-type (CD38+/+) and CD38 knockout (CD38-/-) CAMs in response to oxotremorine (OXO), a muscarinic type 1 (M1) receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2 ·-production in CAMs. Consistently, the OXO-induced intracellular O2 ·-production was markedly inhibited by CD38 shRNA or CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2 ·- production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2 ·-production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2+ concentration and restored intracellular O2 ·-production in CD38-/- CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that CD38/cADPR pathway importantly controls Nox4-mediated intracellular O2 ·-production and that CD38-dependent intracellular O2 ·-production

  8. Lactic acid bacteria protect human intestinal epithelial cells from Staphylococcus aureus and Pseudomonas aeruginosa infections.

    PubMed

    Affhan, S; Dachang, W; Xin, Y; Shang, D

    2015-01-01

    Staphylococcus aureus and Pseudomonas aeruginosa are opportunistic pathogens that cause nosocomial and food-borne infections. They promote intestinal diseases. Gastrointestinal colonization by S. aureus and P. aeruginosa has rarely been researched. These organisms spread to extra gastrointestinal niches, resulting in increasingly progressive infections. Lactic acid bacteria are Gram-positive bacteria that produce lactic acid as the major end-product of carbohydrate fermentation. These bacteria inhibit pathogen colonization and modulate the host immune response. This study aimed to investigate the effects of Lactobacillus acidophilus and Lactobacillus rhamnosus on enteric infections caused by the paradigmatic human pathogens S. aureus ATCC25923 and P. aeruginosa ATCC27853. The effect of whole cells and neutralized cell-free supernatant (CFS) of the lactobacilli on LoVo human carcinoma enterocyte (ATCC CCL-229) infection was analyzed by co-exposure, pre-exposure, and post-exposure studies. Simultaneous application of whole cells and CFS of the lactobacilli significantly eradicated enterocyte infection (P < 0.05); however, this effect was not seen when the whole cells and CFS were added after or prior to the infection (P > 0.05). This result could be attributed to interference by extracellular polymeric substances and cell surface hydrophobicity, which resulted in the development of a pathogen that did not form colonies. Furthermore, results of the plate count and LIVE/ DEAD BacLight bacterial viability staining attributed this inhibition to a non-bacteriocin-like substance, which acted independently of organic acid and H2O2 production. Based on these results, the cell-free supernatant derived from lactobacilli was concluded to restrain the development of S. aureus and P. aeruginosa enteric infections. PMID:26681052

  9. Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production.

    PubMed

    Jin, Kaiming; Zhou, Lian; Jiang, Haixia; Sun, Shuang; Fang, Yunling; Liu, Jianhua; Zhang, Xuehong; He, Ya-Wen

    2015-11-01

    The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity.

  10. Genetic linkage in Pseudomonas aeruginosa of algT and nadB: mutation in nadB does not affect NAD biosynthesis or alginate production.

    PubMed

    DeVries, C A; Hassett, D J; Flynn, J L; Ohman, D E

    1995-04-14

    The 68-min region of the chromosome of Pseudomonas aeruginosa (Pa) contains the gene algT, encoding a putative alternate sigma factor similar to sigma E in Escherichia coli, that is required for the expression of several genes in the alginate biosynthetic regulon. Sequences immediately upstream from algT were found to contain a divergently expressed open reading frame encoding a 60-kDa protein with 64 and 36% identity to the nadB gene products of E. coli and Bacillus subtilis, respectively. The nadB gene encodes L-aspartate oxidase and has been shown in several bacteria to be essential for de novo nicotinamide-adenine dinucleotide (NAD) biosynthesis. Pa nadB complemented the growth requirement for nicotinic acid in a nadB mutant strain of E. coli, suggesting that this gene encodes a functional homologue of L-aspartate oxidase. A nadB::Tn501 mutant was constructed by gene replacement in the alginate-producing strain, Pa FRD. This NadB- mutant still produced alginate and appeared normal with respect to the regulation of alginate synthesis. Interestingly, the NadB- mutant did not have an auxotrophic phenotype for nicotinic acid, indicating that this nadB was not essential for NAD biosynthesis in Pa. These results suggest the possibility that Pa has an alternate mechanism for de novo NAD biosynthesis.

  11. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

    PubMed

    Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

  12. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

    PubMed Central

    Díaz-Vegas, Alexis; Campos, Cristian A.; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

  13. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

    PubMed

    Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

  14. Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

    PubMed Central

    O'Brien, Siobhán; Hodgson, David J.; Buckling, Angus

    2014-01-01

    Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation. PMID:24898376

  15. [Effect of preozonation on disinfection by-products formation potential of algae cells and extracellular organic matter].

    PubMed

    Fang, Jing-yun; Ma, Jun; Wang, Li-ning; Zhang, Tao; Chen, Zhong-lin; Gao, Yue-hua

    2006-06-01

    Water containing Oscillatoria agardii was cultured under controlled conditions and harvested in the late log growth phase. The objective was to determine: the contribution of algae cells and algae extracellular organic matter (EOM) to the disinfection by-products formation potential (DBPFP), and the effects of preozonation including ozone dosage and preozonation time on DBPFP of algae cells and EOM and mechanism of these effects. The results show that the main trihalomethanes from both Oscillatoria cells and EOM are chloroform and bromodichloromethane, and that the main haloacetic acids are dichloroacetic acid and trichloroacetic acid. HAAFP from algae cells and EOM themselves or after preozonation followed by coagulation is more than THMFP, which shows that more attention should be paid to the control of HAA in the treatment of algae containing water. DBPFP of EOM is reduced by preozonation, and DBPFP clearly decreases with time. Compared with traditional coagulation, the dosage of 0.975 mg/L preozonation with reaction time of 10 minntes followed by coagulation can decrease DBPFP of EOM by 31% and decrease HAAFP by 52.6%, but increase THMFP by 12.5% under this experiment's condition. This result shows the major reason preozonation can control the DBPFP of EOM is that it can control HAAFP effectively. At the same time, the DBPFP of algae cells is significantly increased by preozonation, and this increase is almost a linear function of preozonation time. It can be concluded that in the real water treatment case most of algae cells should be removed intact before preozonation to control the DBPFP.

  16. Aberrant production of extracellular matrix proteins and dysfunction in kidney endothelial cells with a short duration of diabetes.

    PubMed

    Grutzmacher, Cathy; Park, SunYoung; Zhao, Yun; Morrison, Margaret E; Sheibani, Nader; Sorenson, Christine M

    2013-01-01

    Diabetic nephropathy is the most common cause of end-stage renal disease and is a major risk factor for cardiovascular disease. In the United States, microvascular complications during diabetic nephropathy contribute to high morbidity and mortality rates. However, the cell-autonomous impact of diabetes on kidney endothelial cell function requires further investigation. Male Akita/+ [autosomal dominant mutation in the insulin II gene (Ins2)] mice reproducibly develop diabetes by 4 wk of age. Here, we examined the impact a short duration of diabetes had on kidney endothelial cell function. Kidney endothelial cells were prepared from nondiabetic and diabetic mice (4 wk of diabetes) to delineate the early changes in endothelial cell function. Kidney endothelial cells from Akita/+ mice following 4 wk of diabetes demonstrated aberrant expression of extracellular matrix proteins including decreased osteopontin and increased fibronectin expression which correlated with increased α5-integrin expression. These changes were associated with the attenuation of migration and capillary morphogenesis. Kidney endothelial cells from Akita/+ mice had decreased VEGF levels but increased levels of endothelial nitric oxide synthase(eNOS) and NO, suggesting uncoupling of VEGF-mediated NO production. Knocking down eNOS expression in Akita/+ kidney endothelial cells increased VEGF expression, endothelial cell migration, and capillary morphogenesis. Furthermore, attenuation of sprouting angiogenesis of aortas from Akita/+ mice with 8 wk of diabetes was restored in the presence of the antioxidant N-acetylcysteine. These studies demonstrate that aberrant endothelial cell function with a short duration of diabetes may set the stage for vascular dysfunction and rarefaction at later stages of diabetes.

  17. Virulence of Aeromonas hydrophila to channel catfish Ictaluras punctatus fingerlings in the presence and absence of bacterial extracellular products.

    PubMed

    Pridgeon, Julia W; Klesius, Phillip H

    2011-07-12

    We investigated the virulence of three 2009 west Alabama isolates of Aeromonas hydrophila (AL09-71, AL09-72 and AL09-73) to channel catfish Ictalurus punctatus fingerlings (4.6 +/- 1.3 g) in the presence and absence of extracellular products (ECPs) from overnight bacterial culture using both bath immersion and intraperitoneal injection routes. At a concentration of 1.65 x 10(8) colony-forming units (CFU) ml(-1), AL09-73 without its ECPs killed 100% of the catfish fingerlings within 2 h by bath immersion. However, at a similar concentration, AL09-73 in the presence of its ECPs killed only 23 +/- 6% catfish fingerlings. The absence of ECPs in the bath immersion experiment also significantly (p < 0.05) increased the virulence of AL09-71, AL09-72, and AL98-C1B, a 1998 Alabama strain of A. hydrophila, suggesting that the virulence of the 4 A. hydrophila isolates was mainly due to bacterial cells, not to their overnight ECPs. Filter-sterilized ECPs failed to kill any catfish by bath immersion or injection. The virulence order of the 4 A. hydrophila isolates, by both bath immersion and intraperitoneal injection, was: AL09-73 > or = AL09-71 > AL09-72 > or = AL98-C1B. At 2 h post bath immersion, all 4 isolates of A. hydrophila were found in all tissues studied (skin, intestine, liver, spleen, kidney, gill and brain), with the highest bacteria count being in the gill and kidney.

  18. Cystic Fibrosis Transmembrane Conductance Regulator is an Epithelial Cell Receptor for Clearance of Pseudomonas aeruginosa from the Lung

    NASA Astrophysics Data System (ADS)

    Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.

    1997-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant Δ F508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

  19. Interaction of Pseudomonas aeruginosa alkaline protease and elastase with human polymorphonuclear leukocytes in vitro.

    PubMed

    Kharazmi, A; Döring, G; Høiby, N; Valerius, N H

    1984-01-01

    Little is known about the interaction of Pseudomonas aeruginosa extracellular products and human polymorphonuclear leukocytes. The present study was designed to examine the effect of alkaline protease and elastase purified from P. aeruginosa on human neutrophil function. Neutrophil chemotaxis, oxygen consumption, glucose oxidation, superoxide production, and nitro blue tetrazolium reduction were studied. It was found that alkaline protease and elastase at fairly low concentrations (0.05 and 0.0025 micrograms/ml, respectively) inhibited chemotaxis. The inhibitory effect of both enzymes was increased at higher concentrations. The chemotaxis of preincubated and washed cells was also inhibited. Alkaline protease but not elastase inhibited opsonized zymosan-stimulated neutrophil oxygen consumption, whereas neither of the enzymes had any effect on glucose oxidation and nitro blue tetrazolium-reducing activity of stimulated neutrophils. The data on superoxide production ability of the cells indicated that the cells preincubated with enzyme and washed were capable of producing superoxide equal to the amount produced by untreated cells when they were stimulated with phorbol myristate acetate or zymosan. However, when elastase was present in the reaction mixture, the reduction of cytochrome c as a measure of superoxide production was inhibited. Inhibition of neutrophil function, particularly chemotaxis, will have important bearing on the escape of the microorganism from the phagocytic defense system of the host. The role of these products in localized infections and avascular areas such as skin burns, cornea, and, at least initially, in chronic lung colonization in cystic fibrosis patients becomes important.

  20. [The comparison of selected virulence factors in Pseudomonas aeruginosa catheter isolates].

    PubMed

    Olejnízková, Katerina; Holá, Veronika

    2012-05-01

    Healthcare quality improvement brings about an increasing number of invasive diagnostic and therapeutic procedures and thus also an increasing number of high-risk patients prone to hospital infections. Pseudomonas aeruginosa is one of the most commonly isolated nosocomial species and the treatment of the infection is often long and problematic, with frequent recurrences. The pathogenesis of Pseudomonas infection is associated with a range of virulence factors. In the present study, 93 catheter isolates of Pseudomonas aeruginosa were screened for the biofilm formation, motility and secretion of selected extracellular products. A high rate of the strains tested were producers of hemolysins, LasB elastase, and pyoverdines (> 70%). The biofilm formation was detected in 80% of isolates and formation of aerated biofilm was present in 90% of isolates with a positive correlation found between the two types of biofilm formation (p = 0.00583; gamma = 0.551). All strains showed swarming motility, 95% of strains showed swimming motility, and 75% of strains showed twitching motility. Among the virulence factors studied, only pyocyanin and pyochelin were produced by a lower proportion of isolates (< 25%). A positive correlation was seen between the production of some extracellular molecules (pyochelin and pyocyanin, pyocyanin and LasB elastase, and LasB elastase and haemolysins), between biofilm formation and formation of aerated biofilm, and between formation of aerated biofilm and pigments (pyoverdine and pyocyanin) production. On the other hand, a negative correlation was found between biofilm production and LasB elastase production and between the production of biofilm under immersion and pigments (pyoverdine and pyocyanin) production. All correlations are significant at the level p = 0.05, with the correlation coefficient gamma > 0.50.

  1. [The comparison of selected virulence factors in Pseudomonas aeruginosa catheter isolates].

    PubMed

    Olejnízková, Katerina; Holá, Veronika

    2012-05-01

    Healthcare quality improvement brings about an increasing number of invasive diagnostic and therapeutic procedures and thus also an increasing number of high-risk patients prone to hospital infections. Pseudomonas aeruginosa is one of the most commonly isolated nosocomial species and the treatment of the infection is often long and problematic, with frequent recurrences. The pathogenesis of Pseudomonas infection is associated with a range of virulence factors. In the present study, 93 catheter isolates of Pseudomonas aeruginosa were screened for the biofilm formation, motility and secretion of selected extracellular products. A high rate of the strains tested were producers of hemolysins, LasB elastase, and pyoverdines (> 70%). The biofilm formation was detected in 80% of isolates and formation of aerated biofilm was present in 90% of isolates with a positive correlation found between the two types of biofilm formation (p = 0.00583; gamma = 0.551). All strains showed swarming motility, 95% of strains showed swimming motility, and 75% of strains showed twitching motility. Among the virulence factors studied, only pyocyanin and pyochelin were produced by a lower proportion of isolates (< 25%). A positive correlation was seen between the production of some extracellular molecules (pyochelin and pyocyanin, pyocyanin and LasB elastase, and LasB elastase and haemolysins), between biofilm formation and formation of aerated biofilm, and between formation of aerated biofilm and pigments (pyoverdine and pyocyanin) production. On the other hand, a negative correlation was found between biofilm production and LasB elastase production and between the production of biofilm under immersion and pigments (pyoverdine and pyocyanin) production. All correlations are significant at the level p = 0.05, with the correlation coefficient gamma > 0.50. PMID:22880261

  2. Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-{kappa}B translocation and ROS production in synoviocytes

    SciTech Connect

    Hu, Fen; Yang, Shuang; Zhao, Dan; Zhu, Shuyan; Wang, Yuxiang; Li, Junying

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Moderate extracellular acidification regulates intracellular Ca{sup 2+} mobilization. Black-Right-Pointing-Pointer Moderate acidification activates NF-{kappa}B nuclear translocation in synoviocytes. Black-Right-Pointing-Pointer Moderate acidification depresses the ROS production induced by capsaicin. Black-Right-Pointing-Pointer Moderate acidification inhibits capsaicin-caused synoviocyte death. -- Abstract: We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca{sup 2+} entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pH 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca{sup 2+} entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca{sup 2+} release from intracellular stores. The nuclear translocation of NF-{kappa}B was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-{kappa}B. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca{sup 2+} mobilization, activating NF-{kappa}B nuclear translocation and depressing ROS production.

  3. Interspecies Interaction between Pseudomonas aeruginosa and Other Microorganisms

    PubMed Central

    Tashiro, Yosuke; Yawata, Yutaka; Toyofuku, Masanori; Uchiyama, Hiroo; Nomura, Nobuhiko

    2013-01-01

    Microbes interact with each other in multicellular communities and this interaction enables certain microorganisms to survive in various environments. Pseudomonas aeruginosa is a highly adaptable bacterium that ubiquitously inhabits diverse environments including soil, marine habitats, plants and animals. Behind this adaptivity, P. aeruginosa has abilities not only to outcompete others but also to communicate with each other to develop a multispecies community. In this review, we focus on how P. aeruginosa interacts with other microorganisms. P. aeruginosa secretes antimicrobial chemicals to compete and signal molecules to cooperate with other organisms. In other cases, it directly conveys antimicrobial enzymes to other bacteria using the Type VI secretion system (T6SS) or membrane vesicles (MVs). Quorum sensing is a central regulatory system used to exert their ability including antimicrobial effects and cooperation with other microbes. At least three quorum sensing systems are found in P. aeruginosa, Las, Rhl and Pseudomonas quinolone signal (PQS) systems. These quorum-sensing systems control the synthesis of extracellular antimicrobial chemicals as well as interaction with other organisms via T6SS or MVs. In addition, we explain the potential of microbial interaction analysis using several micro devices, which would bring fresh sensitivity to the study of interspecies interaction between P. aeruginosa and other organisms. PMID:23363620

  4. Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase.

    PubMed

    El-Ghonemy, Dina Helmy; Ali, Thanaa Hamed; El-Bondkly, Ahmed Mohamed; Moharam, Maysa El-Sayed; Talkhan, Fatma Nabeeh

    2014-11-01

    Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.

  5. Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase.

    PubMed

    El-Ghonemy, Dina Helmy; Ali, Thanaa Hamed; El-Bondkly, Ahmed Mohamed; Moharam, Maysa El-Sayed; Talkhan, Fatma Nabeeh

    2014-11-01

    Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels. PMID:25119245

  6. Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180

    PubMed Central

    Borges, Gisele Martini; De Barba, Fabiana Figueredo Molin; Schiebelbein, Ana Paula; Pereira, Bruna Parmezzani; Chaves, Mariane Bonatti; Silveira, Marcia Luciane Lange; Pinho, Mauro Souza Leite; Furlan, Sandra Aparecida; Wisbeck, Elisabeth

    2013-01-01

    Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40–50 g/L) and pH values (3.0–4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (YP/S = 0.072) and maximum extracellular polysaccharide productivity (QPmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (YX/S = 0.24) and maximal biomass productivity (QXmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm3 in untreated group and 1.6 cm3 in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells. PMID:24688493

  7. The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Int...

  8. Production of extracellular proteolytic activity by Histoplasma capsulatum grown in Histoplasma-macrophage medium is limited to restriction fragment length polymorphism class 1 isolates.

    PubMed

    Zarnowski, Robert; Connolly, Patricia A; Wheat, L Joseph; Woods, Jon P

    2007-09-01

    Extracellular proteolytic activity was studied for 28 strains of Histoplasma capsulatum var. capsulatum and 2 strains of H. capsulatum var. duboisii. Secreted protease activity assessed by skim milk agarose clearance was limited solely to H. capsulatum var. capsulatum restriction fragment length polymorphism (RFLP) class 1 strains. There was a difference in proteolytic activity levels among class 1 strains. Extracellular proteolytic activity was further determined during growth of those strains in liquid medium using azodye-impregnated protein substrates. In general, the highest activities were measured when azocollagen was used, whereas azocasein and azoalbumin were cleaved less efficiently. The activity was inhibited by phenylmethylsulfonyl fluoride, 4-(2-aminoethyl) benzenesulfonyl fluoride, antipain, and chymostatin, indicating, thereby, the presence of chymotrypsin-like serine proteases. Chromatographic analyses as well as variable substrate use at different culture times revealed production of at least 2 different enzyme pools of the same serine-like protease family. Our results demonstrate a distinctive ability of RFLP class 1 isolates to produce and secrete serine proteinase-type activity. This peculiarity may be relevant to the biology and pathogenesis of this particular clade of H. capsulatum isolates. Overall, the feature of extracellular proteolytic activity production enables a convenient and unequivocal identification of RFLP class 1 isolates and, thereby, can be used in H. capsulatum strain differentiation and typing. PMID:17509799

  9. Extracellular ATP

    PubMed Central

    Chivasa, Stephen; Tomé, Daniel FA; Murphy, Alex M; Hamilton, John M; Lindsey, Keith; Carr, John P

    2009-01-01

    Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to cxontrol gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.1 Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.2 A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death. PMID:20009563

  10. Antibiofilm activity of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 against Pseudomonas aeruginosa.

    PubMed

    Kim, Yong-Guy; Lee, Jin-Hyung; Kim, Chang-Jin; Lee, Jae-Chan; Ju, Yoon Jung; Cho, Moo Hwan; Lee, Jintae

    2012-12-01

    Members of the actinomycetes family are a rich source of bioactive compounds including diverse antibiotics. This study sought to identify novel and non-toxic biofilm inhibitors from the actinomycetes library for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. After the screening of 4104 actinomycetes strains, we found that the culture spent medium (1 %, v/v) of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 inhibited P. aeruginosa biofilm formation by 90 % without affecting the growth of planktonic P. aeruginosa cells, while the spent media enhanced the swarming motility of P. aeruginosa. Global transcriptome analyses revealed that the spent medium of Streptomyces sp. BFI 230 induced expression of phenazine, pyoverdine, pyochelin synthesis genes, and iron uptake genes in P. aeruginosa. The addition of exogenous iron restored the biofilm formation and swarming motility of P. aeruginosa in the presence of the spent medium of Streptomyces sp. BFI 230, which suggests that the Streptomyces sp. BFI 230 strain interfered iron acquisition in P. aeruginosa. Experiments on solvent extraction, heat treatment, and proteinase K treatment suggested that hydrophilic compound(s), possibly extracellular peptides or proteins from Streptomyces sp. BFI 230 cause the biofilm reduction of P. aeruginosa. Together, this study indicates that actinomycetes strains have an ability to control the biofilm of P. aeruginosa. PMID:22722911

  11. Environmental optimization for production of 7, 10-dihydroxy-8(E)-octadecenoic acid from olive oil by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbial conversions of free unsaturated fatty acids often generate novel hydroxy fatty acids (HFA), which are known to have special properties such as higher viscosity and reactivity. Among microbial strains known to produce HFAs, Pseudomonas aeruginosa PR3 has been well studied to produce 7,10-d...

  12. Interactions of microbial biofilms with toxic trace metals; 1: Observation and modeling of cell growth, attachment, and production of extracellular polymer

    SciTech Connect

    Hsieh, K.M.; Murgel, G.A.; Lion, L.W.; Shuler, M.L. )

    1994-06-20

    Adsorbent surfaces in natural and engineered systems are frequently modified by bacterial attachment, growth of a biofilm, and bacterial production of extracellular polymer. Attached cells or sorbed polymers may alter the metal-binding characteristics of the supporting substratum and influence metal partitioning. The interdependent behavior of toxic trace metal partitioning and biofilm development requires description of the interaction between cell growth with its accompanying polymer production and metal speciation. In this article, the first of a two part series, a mechanistic model is developed to describe the growth of a film-forming bacterium which adheres to a substratum through the production of extracellular biopolymers. Each bacterial cell was modeled as a two-component structure consisting of active cell mass and biopolymer. The biopolymer component was further divided into cell-associated and dissolved categories to distinguish biopolymer which remained naturally bound to cell surfaces from that which did not. Use of this structured model permitted independent description of the dynamics of cell growth, and polymer production, both of which may influence trace metal behavior. Employing parameters obtained from independent experiments as well as published values, the model satisfactorily predicts experimental observations of bacterial growth, attachment and detachment, biopolymer production, and adsorption of polymer onto solid (glass) surfaces. The model simulated transient and steady-state biofilm systems equally well.

  13. Cell aggregation promotes pyoverdine-dependent iron uptake and virulence in Pseudomonas aeruginosa.

    PubMed

    Visaggio, Daniela; Pasqua, Martina; Bonchi, Carlo; Kaever, Volkhard; Visca, Paolo; Imperi, Francesco

    2015-01-01

    In Pseudomonas aeruginosa the Gac signaling system and the second messenger cyclic diguanylate (c-di-GMP) participate in the control of the switch between planktonic and biofilm lifestyles, by regulating the production of the two exopolysaccharides Pel and Psl. The Gac and c-di-GMP regulatory networks also coordinately promote the production of the pyoverdine siderophore, and the extracellular polysaccharides Pel and Psl have recently been found to mediate c-di-GMP-dependent regulation of pyoverdine genes. Here we demonstrate that Pel and Psl are also essential for Gac-mediated activation of pyoverdine production. A pel psl double mutant produces very low levels of pyoverdine and shows a marked reduction in the expression of the pyoverdine-dependent virulence factors exotoxin A and PrpL protease. While the exopolysaccharide-proficient parent strain forms multicellular planktonic aggregates in liquid cultures, the Pel and Psl-deficient mutant mainly grows as dispersed cells. Notably, artificially induced cell aggregation is able to restore pyoverdine-dependent gene expression in the pel psl mutant, in a way that appears to be independent of iron diffusion or siderophore signaling, as well as of recently described contact-dependent mechanosensitive systems. This study demonstrates that cell aggregation represents an important cue triggering the expression of pyoverdine-related genes in P. aeruginosa, suggesting a novel link between virulence gene expression, cell-cell interaction and the multicellular community lifestyle.

  14. Cell aggregation promotes pyoverdine-dependent iron uptake and virulence in Pseudomonas aeruginosa.

    PubMed

    Visaggio, Daniela; Pasqua, Martina; Bonchi, Carlo; Kaever, Volkhard; Visca, Paolo; Imperi, Francesco

    2015-01-01

    In Pseudomonas aeruginosa the Gac signaling system and the second messenger cyclic diguanylate (c-di-GMP) participate in the control of the switch between planktonic and biofilm lifestyles, by regulating the production of the two exopolysaccharides Pel and Psl. The Gac and c-di-GMP regulatory networks also coordinately promote the production of the pyoverdine siderophore, and the extracellular polysaccharides Pel and Psl have recently been found to mediate c-di-GMP-dependent regulation of pyoverdine genes. Here we demonstrate that Pel and Psl are also essential for Gac-mediated activation of pyoverdine production. A pel psl double mutant produces very low levels of pyoverdine and shows a marked reduction in the expression of the pyoverdine-dependent virulence factors exotoxin A and PrpL protease. While the exopolysaccharide-proficient parent strain forms multicellular planktonic aggregates in liquid cultures, the Pel and Psl-deficient mutant mainly grows as dispersed cells. Notably, artificially induced cell aggregation is able to restore pyoverdine-dependent gene expression in the pel psl mutant, in a way that appears to be independent of iron diffusion or siderophore signaling, as well as of recently described contact-dependent mechanosensitive systems. This study demonstrates that cell aggregation represents an important cue triggering the expression of pyoverdine-related genes in P. aeruginosa, suggesting a novel link between virulence gene expression, cell-cell interaction and the multicellular community lifestyle. PMID:26379660

  15. Pseudomonas aeruginosa biofilm formation and UV/irradiation exposure change surface and chemical structures of Pre-Production Resin Pellets

    NASA Astrophysics Data System (ADS)

    Stam, C. N.; Neal, A.; Park, S.; Mielke, R.; Tsapin, A. I.; Bhartia, R.; Salas, E.; Hug, W.; Behar, A. E.; Nadeau, J. L.

    2011-12-01

    Microbial interactions with synthetic polymers in open ocean is poorly understood. Plastics are a major and persistent contaminant of ocean waters. Many of these plastics are contaminated with toxic and synthetic chemicals that persist in the environment with minimal degradation. The purpose of this study is to look at the effects that microbial biofilm communities have on both surface and chemical structures of pre-production resin pellets (PRPs). Pseudomonas aeruignosa was grown with PRPs under multiple growth and nutrient conditions. These conditions were combined with varying lengths of UV exposures common to ocean environments. Material degradation of the PRPs and the changing surface and chemical structures of these synthetic polymers was evaluated using a combination of Fourier transform infrared spectroscopy, environmental scanning electron microscopy, scanning transmission electron microscopy, X-ray microtomography, and ArcGIS mapping. This study correlates with previous studies conducted on environmental PRP's , collected on the 2009 Project Kaisei expedition in the Subtropical Convergence Zone of the North Pacific Gyre. Further studies are needed to develop a full understanding of degradation rates of synthetic polymers in oceanic environments.

  16. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  17. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases. PMID:23084979

  18. Pseudomonas aeruginosa Genomic Structure and Diversity

    PubMed Central

    Klockgether, Jens; Cramer, Nina; Wiehlmann, Lutz; Davenport, Colin F.; Tümmler, Burkhard

    2011-01-01

    The Pseudomonas aeruginosa genome (G + C content 65–67%, size 5.5–7 Mbp) is made up of a single circular chromosome and a variable number of plasmids. Sequencing of complete genomes or blocks of the accessory genome has revealed that the genome encodes a large repertoire of transporters, transcriptional regulators, and two-component regulatory systems which reflects its metabolic diversity to utilize a broad range of nutrients. The conserved core component of the genome is largely collinear among P. aeruginosa strains and exhibits an interclonal sequence diversity of 0.5–0.7%. Only a few loci of the core genome are subject to diversifying selection. Genome diversity is mainly caused by accessory DNA elements located in 79 regions of genome plasticity that are scattered around the genome and show an anomalous usage of mono- to tetradecanucleotides. Genomic islands of the pKLC102/PAGI-2 family that integrate into tRNALys or tRNAGly genes represent hotspots of inter- and intraclonal genomic diversity. The individual islands differ in their repertoire of metabolic genes that make a large contribution to the pangenome. In order to unravel intraclonal diversity of P. aeruginosa, the genomes of two members of the PA14 clonal complex from diverse habitats and geographic origin were compared. The genome sequences differed by less than 0.01% from each other. One hundred ninety-eight of the 231 single nucleotide substitutions (SNPs) were non-randomly distributed in the genome. Non-synonymous SNPs were mainly found in an integrated Pf1-like phage and in genes involved in transcriptional regulation, membrane and extracellular constituents, transport, and secretion. In summary, P. aeruginosa is endowed with a highly conserved core genome of low sequence diversity and a highly variable accessory genome that communicates with other pseudomonads and genera via horizontal gene transfer. PMID:21808635

  19. Enzyme-linked immunosorbent assay for detection of antibodies to Pseudomonas aeruginosa exoproteins.

    PubMed

    Granström, M; Wretlind, B; Markman, B; Pavlovskis, O R; Vasil, M L

    1985-04-01

    Enzyme-linked immunosorbent assays were developed with four purified Pseudomonas aeruginosa extracellular proteins (exotoxin A, elastase, alkaline protease, and phospholipase C) to determine antibody levels in sera from healthy subjects and the serological response in patients colonized or infected with Pseudomonas aeruginosa. Five of 39 burn patients with wounds colonized by Pseudomonas aeruginosa had elevated antibody titers to alkaline protease. Response to the other antigens was found in only a few patients. Pseudomonas aeruginosa infections (septicemia, osteitis, pneumonia etc.) resulted in increased antibody levels to exotoxin A or phospholipase C in 15 of 22 patients. These findings suggest that repeated determinations of antibodies to Pseudomonas aeruginosa exotoxin A and phospholipase C might be used to monitor therapy in certain patients with osteitis and other deep Pseudomonas infections.

  20. Quorum sensing in marine snow and its possible influence on production of extracellular hydrolytic enzymes in marine snow bacterium Pantoea ananatis B9.

    PubMed

    Jatt, Abdul Nabi; Tang, Kaihao; Liu, Jiwen; Zhang, Zenghu; Zhang, Xiao-Hua

    2015-02-01

    Marine snow is a continuous shower of organic and inorganic detritus, and plays a crucial role in transporting materials from the sea surface to the deep ocean. The aims of the current study were to identify N-acyl homoserine lactone (AHL)-based quorum sensing (QS) signaling molecules directly from marine snow particles and to investigate the possible regulatory link between QS signals and extracellular hydrolytic enzymes produced by marine snow bacteria. The marine snow samples were collected from the surface water of China marginal seas. Two AHLs, i.e. 3OC6-HSL and C8-HSL, were identified directly from marine snow particles, while six different AHL signals, i.e. C4-HSL, 3OC6-HSL, C6-HSL, C10-HSL, C12-HSL and C14-HSL were produced by Pantoea ananatis B9 inhabiting natural marine snow particles. Of the extracellular hydrolytic enzymes produced by P. ananatis B9, alkaline phosphatase activity was highly enhanced in growth medium supplemented with exogenous AHL (C10-HSL), while quorum quenching enzyme (AiiA) drastically reduced the enzyme activity. To our knowledge, this is the first report revealing six different AHL signals produced by P. ananatis B9 and AHL-based QS system enhanced the extracellular hydrolytic enzyme in P. ananatis B9. Furthermore, this study first time revealing 3OC6-HSL production by Paracoccus carotinifaciens affiliated with Alphaproteobacteria. PMID:25764555

  1. Involvement of a new enzyme, glyoxal oxidase, in extracellular H/sub 2/O/sub 2/ production by Phanerochaete chrysosporium

    SciTech Connect

    Kersten, P.J.; Kirk, K.

    1987-05-01

    The importance of extracellular H/sub 2/O/sub 2/ in lignin degradation has become increasingly apparent with the recent discovery of H/sub 2/O/sub 2/-requiring ligninases produced by white-rot fungi. Here the authors describe a new H/sub 2/O/sub 2/-producing activity of Phanerochaete chrysosporium that involves extracellular oxidases able to use simple aldehyde, ..cap alpha..-hydroxycarbonyl, or..cap alpha..-dicarbonyl compounds as substrates. The activity is expressed during secondary metabolism, when the ligninases are also expressed. Analytical isoelectric focusing of the extracellular proteins, followed by activity staining, indicated that minor proteins with broad substrate specificities are responsible for the oxidase activity. Two of the oxidase substrates, glyoxal and methylglyoxal, were also identified, as their quinoxaline derivatives, in the culture fluid as secondary metabolites. The significance of these findings is discussed with respect to lignin degradation and other proposed systems for H/sub 2/O/sub 2/ production in P. chrysosporium.

  2. Quorum sensing in marine snow and its possible influence on production of extracellular hydrolytic enzymes in marine snow bacterium Pantoea ananatis B9.

    PubMed

    Jatt, Abdul Nabi; Tang, Kaihao; Liu, Jiwen; Zhang, Zenghu; Zhang, Xiao-Hua

    2015-02-01

    Marine snow is a continuous shower of organic and inorganic detritus, and plays a crucial role in transporting materials from the sea surface to the deep ocean. The aims of the current study were to identify N-acyl homoserine lactone (AHL)-based quorum sensing (QS) signaling molecules directly from marine snow particles and to investigate the possible regulatory link between QS signals and extracellular hydrolytic enzymes produced by marine snow bacteria. The marine snow samples were collected from the surface water of China marginal seas. Two AHLs, i.e. 3OC6-HSL and C8-HSL, were identified directly from marine snow particles, while six different AHL signals, i.e. C4-HSL, 3OC6-HSL, C6-HSL, C10-HSL, C12-HSL and C14-HSL were produced by Pantoea ananatis B9 inhabiting natural marine snow particles. Of the extracellular hydrolytic enzymes produced by P. ananatis B9, alkaline phosphatase activity was highly enhanced in growth medium supplemented with exogenous AHL (C10-HSL), while quorum quenching enzyme (AiiA) drastically reduced the enzyme activity. To our knowledge, this is the first report revealing six different AHL signals produced by P. ananatis B9 and AHL-based QS system enhanced the extracellular hydrolytic enzyme in P. ananatis B9. Furthermore, this study first time revealing 3OC6-HSL production by Paracoccus carotinifaciens affiliated with Alphaproteobacteria.

  3. Extracellular Production of a Novel Endo-β-Agarase AgaA from Pseudomonas vesicularis MA103 that Cleaves Agarose into Neoagarotetraose and Neoagarohexaose

    PubMed Central

    Hsu, Pang-Hung; Wei, Chien-Han; Lu, Wen-Jung; Shen, Fen; Pan, Chorng-Liang; Lin, Hong-Ting Victor

    2015-01-01

    The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. PMID:25768342

  4. Ginger extract inhibits biofilm formation by Pseudomonas aeruginosa PA14.

    PubMed

    Kim, Han-Shin; Park, Hee-Deung

    2013-01-01

    Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger's ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39-56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3'-5')-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

  5. Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

    PubMed Central

    Kim, Han-Shin; Park, Hee-Deung

    2013-01-01

    Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

  6. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

    PubMed

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation.

  7. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  8. Investigation of Pseudomonas aeruginosa quorum-sensing signaling system for identifying multiple inhibitors using molecular docking and structural analysis methodology.

    PubMed

    Soheili, Vahid; Bazzaz, Bibi Sedigheh Fazly; Abdollahpour, Nooshin; Hadizadeh, Farzin

    2015-12-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common Gram-negative bacterium in hospital-acquired infections. It causes death in many burn victims, cystic-fibrosis and neutropenic-cancer patients. It is known that P. aeruginosa biofilm maturation and production of cell-associated and extracellular virulence factors such as pyocyanin, elastase and rhamnolipids are under the control of a quorum-sensing (QS) system. Among several proteins involved in the Pseudomonas QS mechanism, LasR and PqsE play an important role in its cascade signaling system. They can cause increases in QS factors, biofilm maturation, and the production of virulence factors. Therefore, inhibition of these proteins can reduce the pathogenicity of P. aeruginosa. According to the structure of corresponding auto-inducers bound to these proteins, in silico calculations were performed with some non-steroidal anti-inflammatory drugs (NSAIDs) to estimate possible interactions and find the co-inhibitors of LasR and PqsE. The results showed that oxicams (Piroxicam and Meloxicam) can interact well with active sites of both proteins with the Ki of 119.43 nM and 4.0 μM for Meloxicam and 201.39 nM and 4.88 μM against LasR and PqsE, respectively. These findings suggested that Piroxicam and Meloxicam can be used as potential inhibitors for control of the P. aeruginosa QS signaling system and biofilm formation, and may be used in the design of multiple inhibitors.

  9. Three Pseudomonas aeruginosa strains with different protease profiles.

    PubMed

    Andrejko, Mariola; Zdybicka-Barabas, Agnieszka; Janczarek, Monika; Cytryńska, Małgorzata

    2013-01-01

    The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.

  10. Characteristics of heat-stable extracellular hemolysin from Pseudomonas aeruginosa.

    PubMed Central

    Fujita, K; Akino, T; Yoshioka, H

    1988-01-01

    Heat-stable hemolysin produced by the cellophane-agar plate method consisted of two major acidic glycolipids. One was composed of 2 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid, and the other was composed of 1 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid. The hemolysis-producing moiety of the hemolysin was shown to be the dimer of beta-hydroxydecanoic acid contained in the glycolipid molecule. Images PMID:3128485

  11. Effect of C/N ratio and media optimization through response surface methodology on simultaneous productions of intra- and extracellular inulinase and invertase from Aspergillus niger ATCC 20611.

    PubMed

    Dinarvand, Mojdeh; Rezaee, Malahat; Masomian, Malihe; Jazayeri, Seyed Davoud; Zareian, Mohsen; Abbasi, Sahar; Ariff, Arbakariya B

    2013-01-01

    The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO₃, Zn⁺², and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R²) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO₃, 1.5 mM (v/v) Zn⁺², and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.

  12. Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611

    PubMed Central

    Dinarvand, Mojdeh; Rezaee, Malahat; Masomian, Malihe; Jazayeri, Seyed Davoud; Zareian, Mohsen; Abbasi, Sahar; Ariff, Arbakariya B.

    2013-01-01

    The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R2) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry. PMID:24151605

  13. High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

    PubMed

    Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

    2016-08-01

    The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications. PMID:27151682

  14. Effect of C/N ratio and media optimization through response surface methodology on simultaneous productions of intra- and extracellular inulinase and invertase from Aspergillus niger ATCC 20611.

    PubMed

    Dinarvand, Mojdeh; Rezaee, Malahat; Masomian, Malihe; Jazayeri, Seyed Davoud; Zareian, Mohsen; Abbasi, Sahar; Ariff, Arbakariya B

    2013-01-01

    The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO₃, Zn⁺², and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R²) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO₃, 1.5 mM (v/v) Zn⁺², and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry. PMID:24151605

  15. Two distinct ferritin-like molecules in P. aeruginosa: The product of the bfrA gene is a bacterial ferritin (FtnA) not a bacterioferritin (Bfr)†€

    PubMed Central

    Yao, Huili; Jepkorir, Grace; Lovell, Scott; Nama, Pavithra V.; Weeratunga, Saroja; Battaile, Kevin P.; Rivera, Mario

    2011-01-01

    Two distinct types of ferritin-like molecules often coexist in bacteria, the heme binding bacterioferritins (Bfr) and the non-heme binding bacterial ferritins (Ftn). The early isolation of a ferritin-like molecule from P. aeruginosa suggested the possibility of a bacterioferritin assembled from two different subunits [Moore, G. R., Kadir, F. H., Al-Massad, F. K., Le Brun, N. E., Thomson, A. J., Greenwood, C., Keen, J. N. and Findlay, J. B. C. (1994) Biochem. J. 304, 493–497]. Subsequent studies demonstrated the presence of two genes coding for ferritin-like molecules in P. aeruginosa, designated bfrA and bfrB, and suggested that two distinct bacterioferritins may coexist [Ma, J.-F., Ochsner, U. A., Klotz, M. G, Nanayakkara, V. K., Howell, M. L., Johnson, Z., Posey, J. E., Vasil, M. L., Monaco, J. J., and Hassett, D. J. (1999) J. Bacteriol. 181, 3730–3742]. In this report we present structural evidence demonstrating that the product of the bfrA gene is a ferritin-like molecule not capable of binding heme which harbors a catalytically active ferroxidase center with structural properties similar to those characteristic of bacterial and archaeal Ftns and clearly distinct from the ferroxidase center typical of Bfrs. Consequently, the product of the bfrA gene in P. aeruginosa is a bacterial ferritin, which we propose should be termed Pa FtnA. These results, together with the previous characterization of the product of the bfrB gene as a genuine bacterioferritin (Pa BfrB) [Weeratunga, S. J., Lovell, S., Yao, H., Battaile, K. P., Fischer, C. J., Gee, C. E., and Rivera, M. (2010) Biochemistry 49. 1160–1175] indicate the coexistence of a bacterial ferritin (Pa FtnA) and a bacterioferritin (Pa BfrB) in P. aeruginosa. In agreement with this idea, we also obtained evidence demonstrating that release of iron from Pa BfrB and Pa FtnA is likely subject to different regulation in P. aerugionsa: Whereas the efficient release of iron stored in Pa FtnA requires only the input of

  16. Retinoic acid receptor-α signalling antagonizes both intracellular and extracellular amyloid-β production and prevents neuronal cell death caused by amyloid-β.

    PubMed

    Jarvis, C I; Goncalves, M B; Clarke, E; Dogruel, M; Kalindjian, S B; Thomas, S A; Maden, M; Corcoran, J P T

    2010-10-01

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain, neuronal cell loss and cognitive decline. We show here that retinoic acid receptor (RAR)α signalling in vitro can prevent both intracellular and extracellular Aβ accumulation. RARα signalling increases the expression of a disintegrin and metalloprotease 10, an α-secretase that processes the amyloid precursor protein into the non-amyloidic pathway, thus reducing Aβ production. We also show that RARα agonists are neuroprotective, as they prevent Aβ-induced neuronal cell death in cortical cultures. If RARα agonists are given to the Tg2576 mouse, the normal Aβ production in their brains is suppressed. In contrast, neither RARβ nor γ-agonists affect Aβ production or Aβ-mediated neuronal cell death. Therefore, RARα agonists have therapeutic potential for the treatment of AD.

  17. Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W.

    PubMed Central

    Nunn, D N; Lory, S

    1993-01-01

    Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates. Images PMID:8331069

  18. Spaceflight Effects on Virulence of Pseudomonas Aeruginosa

    NASA Astrophysics Data System (ADS)

    Broadway, S.; Goins, T.; Crandell, C.; Richards, C.; Patel, M.; Pyle, B.

    2008-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen found in the environment. It is known to infect the immunocompromised. The organism has about 25 virulence genes that play different roles in disease processes. Several exotoxin proteins may be produced, including ExoA, ExoS, ExoT and ExoY, and other virulence factors. In spaceflight, possible increased expression of P. aeruginosa virulence proteins could increase health risks for spaceflight crews who experience decreased immunity. Cultures of P. aeruginosa strains PA01 and PA103 grown on orbit on Shuttle Endeavour flight STS-123 vs. static ground controls were used for analysis. The production of ETA was quantitated using an ELISA procedure. Results showed that while flight cultures of PA103 produced slightly more ETA than corresponding ground controls, the opposite was found for PA01. While it appears that spaceflight has little effect on ETA, stimulation of other virulence factors could cause increased virulence of this organism in space flight. Similar increased virulence in spaceflight has been observed for other bacteria. This is important because astronauts may be more susceptible to opportunistic pathogens including P. aeruginosa.

  19. Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.

    PubMed

    Tielen, Petra; Rosin, Nathalie; Meyer, Ann-Kathrin; Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter

    2013-01-01

    Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.

  20. Regulatory and Metabolic Networks for the Adaptation of Pseudomonas aeruginosa Biofilms to Urinary Tract-Like Conditions

    PubMed Central

    Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter

    2013-01-01

    Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections. PMID:23967252

  1. In Vitro Analysis of Pseudomonas aeruginosa Virulence Using Conditions That Mimic the Environment at Specific Infection Sites.

    PubMed

    Colmer-Hamood, J A; Dzvova, N; Kruczek, C; Hamood, A N

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic lung infection in patients with cystic fibrosis (CF) and acute systemic infections in severely burned patients and immunocompromised patients including cancer patients undergoing chemotherapy and HIV infected individuals. In response to the environmental conditions at specific infection sites, P. aeruginosa expresses certain sets of cell-associated and extracellular virulence factors that produce tissue damage. Analyzing the mechanisms that govern the production of these virulence factors in vitro requires media that closely mimic the environmental conditions within the infection sites. In this chapter, we review studies based on media that closely resemble three in vivo conditions, the thick mucus accumulated within the lung alveoli of CF patients, the serum-rich wound bed and the bloodstream. Media resembling the CF alveolar mucus include standard laboratory media supplemented with sputum obtained from CF patients as well as prepared synthetic mucus media formulated to contain the individual components of CF sputum. Media supplemented with serum or individual serum components have served as surrogates for the soluble host components of wound infections, while whole blood has been used to investigate the adaptation of pathogens to the bloodstream. Studies using these media have provided valuable information regarding P. aeruginosa gene expression in different host environments as varying sets of genes were differentially regulated during growth in each medium. The unique effects observed indicate the essential role of these in vitro media that closely mimic the in vivo conditions in providing accurate information regarding the pathogenesis of P. aeruginosa infections. PMID:27571695

  2. Purification of Pseudomonas aeruginosa proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage.

    PubMed Central

    Kreger, A S; Gray, L D

    1978-01-01

    Extracellular proteases of three cornea-virulent strains of Pseudomonas aeruginosa were isolated by sequential ammonium sulfate precipitation, Ultrogel AcA 54 gel filtration, and flat-bed isoelectric focusing. The purity of the preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis , thin-layer isoelectric focusing in polyacrylamide gel, immunodiffusion and immunoelectrophoretic procedures, and tests for the presence of other known pseudomonal products. Light and electron microscopic examination of rabbit corneal lesions observed 4 to 6 h after the intracorneal injection of submicrogram amounts of the proteases revealed: (i) degeneration and necrosis of epithelium, endothelium, and keratocytes, (ii) infiltration, degeneration, and necrosis of polymorphonuclear leukocytes, (iii) loss of the characteristic weblike pattern, colloidal iron staining, and ruthenium red staining of the stromal proteoglycan ground substance, (iv) dispersal of strucutrally normal appearing collagen fibrils, ground substance, (iv) dispersal of structurally normal appearing collagen fibrils, and (v) accumulation of plasma proteins and fibrin in the necrotic corneas. These structural alterations are very similar to those observed previously during experimental P. aeruginosa keratitis, and this similarity supports the idea that pseudomonal proteases are responsible, at least in part, for the rapid and extensive liquefaction necrosis characteristic of pseudomonal-induced keratitis. In addition, the results support the idea that pseudomonal proteases elicit severe corneal damage by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the corneal stroma, and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure. Images PMID:415981

  3. Pseudomonas aeruginosa capability to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter

    PubMed Central

    D'Orazio, Melania; Mastropasqua, Maria Chiara; Cerasi, Mauro; Pacello, Francesca; Consalvo, Ada; Chirullo, Barbara; Mortensen, Brittany; Skaar, Eric P.; Ciavardelli, Domenico; Pasquali, Paolo; Battistoni, Andrea

    2015-01-01

    The ability of a large number of bacterial pathogens to multiply in the infected host and cause disease is dependent on their ability to express high affinity zinc importers. In many bacteria ZnuABC, a transporter of the ABC family, plays a central role in the process of zinc uptake in zinc poor environments, including the tissues of the infected host. To initiate an investigation into the relevance of the zinc uptake apparatus for Pseudomonas aeruginosa pathogenicity, we have generated a znuA mutant in the PA14 strain. We have found that this mutant strain displays a limited growth defect in zinc depleted media. The znuA mutant strain is more sensitive than the wild type strain to calprotectin-mediated growth inhibition, but both the strains are highly resistant to this zinc sequestering antimicrobial protein. Moreover, intracellular zinc content is not evidently affected by inactivation of the ZnuABC transporter. These findings suggest that P. aeruginosa is equipped with redundant mechanisms for the acquisition of zinc that might favor P. aeruginosa colonization of environments containing low levels of this metal. Nonetheless, deletion of znuA affects alginate production, reduces the activity of extracellular zinc-containing proteases, including LasA, LasB and Protease IV, and decreases the ability of P. aeruginosa to disseminate during systemic infections. These results indicate that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa and that ZnuABC also plays an important role in zinc homeostasis in this microorganism. PMID:25751674

  4. Clove bud oil reduces kynurenine and inhibits pqs A gene expression in P. aeruginosa.

    PubMed

    H, Jayalekshmi; Omanakuttan, Athira; Pandurangan, N; S Vargis, Vidhu; Maneesh, M; G Nair, Bipin; B Kumar, Geetha

    2016-04-01

    Quorum sensing (QS), a communication system involved in virulence of pathogenic bacteria like Pseudomonas aeruginosa is a promising target to combat multiple drug resistance. In vitro studies using clove bud oil (CBO) in P. aeruginosa revealed a concentration dependent attenuation of a variety of virulence factors including motility, extracellular DNA, exopolysaccharides and pigment production. Furthermore, treatment with CBO demonstrated a distinct dose-dependent reduction in biofilm formation as well as promoting dispersion of already formed biofilm, observations that were also supported by porcine skin ex vivo studies. Expression studies of genes involved in signalling systems of P. aeruginosa indicated a specific decrease in transcription of pqsA, but not in the lasI or rhlI levels. Additionally, the expression of vfr and gacA genes, involved in regulation, was also not affected by CBO treatment. CBO also influenced the PQS signalling pathway by decreasing the levels of kynurenine, an effect which was reversed by the addition of exogenous kynurenine. Though the synthesis of the signalling molecules of the Las and Rhl pathways was not affected by CBO, their activity was significantly affected, as observed by decrease in levels of their various effectors. Molecular modelling studies demonstrated that eugenol, the major component of CBO, favourably binds to the QS receptor by hydrophobic interactions as well as by hydrogen bonding with Arg61 and Tyr41 which are key amino acid residues of the LasR receptor. These results thus elucidate the molecular mechanism underlying the action of CBO and provide the basis for the identification of an attractive QS inhibitor. PMID:26821927

  5. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

  6. First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

    PubMed

    Mersni-Achour, Rachida; Imbert-Auvray, Nathalie; Huet, Valérie; Ben Cheikh, Yosra; Faury, Nicole; Doghri, Ibtissem; Rouatbi, Sonia; Bordenave, Stéphanie; Travers, Marie-Agnès; Saulnier, Denis; Fruitier-Arnaudin, Ingrid

    2014-11-01

    Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated. PMID:25252078

  7. Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

    PubMed Central

    Sarabhai, Sajal; Sharma, Prince; Capalash, Neena

    2013-01-01

    Background Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. Methods and Results Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C12HSL and C4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C4HSL. F7 also showed antagonistic activity against 3-oxo-C12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. Conclusions This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced

  8. Acquisition and role of molybdate in Pseudomonas aeruginosa.

    PubMed

    Pederick, Victoria G; Eijkelkamp, Bart A; Ween, Miranda P; Begg, Stephanie L; Paton, James C; McDevitt, Christopher A

    2014-11-01

    In microaerophilic or anaerobic environments, Pseudomonas aeruginosa utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO4 (2-)). Here, we show that molybdate acquisition in P. aeruginosa occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the modA gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of P. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth of P. aeruginosa and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.

  9. Microbial degradation of quinoline and methylquinolines. [Pseudomonas aeruginosa

    SciTech Connect

    Aislabie, J.; Bej, A.K.; Hurst, H.; Rothenburger, S.; Atlas, R.M. )

    1990-02-01

    Several bacterial cultures were isolated that are able to degrade quinoline and to transform or to degrade methylquinolines. The degradation of quinoline by strains of Pseudomonas aeruginosa QP and Pseudomonas. putida QP produced hydroxyquinolines, a transient pink compound, and other undetermined products. The quinoline-degrading strains of P. aeruginosa QP and P. putida QP hydroxylated a limited number of methylquinolines but could not degrade them, nor could they transform 2-methylquinoline, isoquinoline, or pyridine. Another pseudomonad, Pseudomonas sp. strain MQP, was isolated that could degrade 2-methylquinoline. P. aeruginosa QP was able to degrade or to transform quinoline and a few methylquinolines in a complex heterocyclic nitrogen-containing fraction of a shale oil. All of the quinoline- and methylquinoline-degrading strains have multiple plasmids including a common 250-kilobase plasmid. The 225-, 250-, and 320-kilobase plasmids of the P. aeruginosa QP strain all contained genes involved in quinoline metabolism.

  10. Acquisition and Role of Molybdate in Pseudomonas aeruginosa

    PubMed Central

    Pederick, Victoria G.; Eijkelkamp, Bart A.; Ween, Miranda P.; Begg, Stephanie L.; Paton, James C.

    2014-01-01

    In microaerophilic or anaerobic environments, Pseudomonas aeruginosa utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO42−). Here, we show that molybdate acquisition in P. aeruginosa occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the modA gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of P. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth of P. aeruginosa and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition. PMID:25172858

  11. Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks.

    PubMed

    Tutak, Wojtek; Park, Ki Ho; Vasilov, Anatoly; Starovoytov, Valentin; Fanchini, Giovanni; Cai, Shi-Qing; Partridge, Nicola C; Sesti, Federico; Chhowalla, Manish

    2009-06-24

    A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

  12. Melanin production through novel processing of proopiomelanocortin in the extracellular compartment of the auricular skin of C57BL/6 mice after UV-irradiation.

    PubMed

    Yamamoto, Hiroyuki; Yamane, Tomohiro; Iguchi, Kazuaki; Tanaka, Kiyotaka; Iddamalgoda, Arunasiri; Unno, Keiko; Hoshino, Minoru; Takeda, Atsushi

    2015-09-29

    The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1-8). Furthermore, ACTH(1-8) competed with [(125)I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1-8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1-8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

  13. Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product.

    PubMed

    Wilson, S; Drew, R

    1991-08-01

    Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.

  14. Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition.

    PubMed

    Blanka, Andrea; Düvel, Juliane; Dötsch, Andreas; Klinkert, Birgit; Abraham, Wolf-Rainer; Kaever, Volkhard; Ritter, Christiane; Narberhaus, Franz; Häussler, Susanne

    2015-01-01

    Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses. PMID:25872871

  15. VDUP1 exacerbates bacteremic shock in mice infected with Pseudomonas aeruginosa.

    PubMed

    Piao, Zheng-Hao; Kim, Mi Sun; Jeong, Mira; Yun, Sohyun; Lee, Suk Hyung; Sun, Hu-Nan; Song, Hae Young; Suh, Hyun-Woo; Jung, Haiyoung; Yoon, Suk Ran; Kim, Tae-Don; Lee, Young-Ho; Choi, Inpyo

    2012-11-01

    Vitamin-D3 upregulated protein-1 (VDUP1) is a stress response protein. Pseudomonas aeruginosa (P. aeruginosa) infection is a leading cause of death. Mice infected with live P. aeruginosa exhibit significantly decreased VDUP1 expression. However, the function of VDUP1 during P. aeruginosa-induced mouse bacteremic shock is unknown. To address the function of VDUP1 in P. aeruginosa-infected mice, we constructed a bacteremic shock model wherein both wild-type and VDUP1-deficient mice were infected intra-peritoneally with live P. aeruginosa. We found that VDUP1-deficient mice were more resistant to P. aeruginosa-induced bacteremic shock than wild-type mice, as shown by the increased survival, accelerated bacterial clearance and suppression of cytokine overproduction of the VDUP1-deficient mice. VDUP1 promoted the recruitment of neutrophils into the peritoneal cavities of infected mice. VDUP1 impeded the phagocytosis of non-opsonized P. aeruginosa via phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. P. aeruginosa infection induced the generation of reactive oxygen species (ROS), and the increased production of ROS by the peritoneal cells of VDUP1-deficient mice was advantageous in clearing the bacteria. Overall, VDUP1 aggravates bacteremic shock; thus, VDUP1 can be considered a target molecule for the inhibition of P. aeruginosa-induced bacteremic shock.

  16. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

    PubMed

    Bruns, Sandra; Kniemeyer, Olaf; Hasenberg, Mike; Aimanianda, Vishukumar; Nietzsche, Sandor; Thywissen, Andreas; Jeron, Andreas; Latgé, Jean-Paul; Brakhage, Axel A; Gunzer, Matthias

    2010-04-29

    Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest

  17. Improving production of extracellular proteases by random mutagenesis and biochemical characterization of a serine protease in Bacillus subtilis S1-4.

    PubMed

    Wang, X C; Zhao, H Y; Liu, G; Cheng, X J; Feng, H

    2016-01-01

    The feather is a valuable by-product with a huge annual yield produced by the poultry industry. Degradation of feathers by microorganisms is a prerequisite to utilize this insoluble protein resource. To improve the degrading efficiency of feathers, mutagenesis of the bacterium Bacillus subtilis S1-4 was performed. By combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine treatment for mutagenesis, a high protease-producing mutant (UMU4) of B. subtilis S1-4 was selected, which exhibited 2.5-fold higher extracellular caseinolytic activity than did the wild-type strain. UMU4 degraded chicken feathers more efficiently, particularly for the release of soluble proteins from the feathers, compared to the wild-type strain. Furthermore, an extracellular protease with a molecular weight of 45 kDa, as determined by SDS-PAGE, was purified from UMU4. Biochemical characterization indicated that the caseinolytic activity of the protease was largely inhibited by phenylmethanesulfonyl fluoride, suggesting that the purified enzyme is a serine protease. This protease was highly active over a wide range of pHs (6.0 to 12.0) and temperatures (50° to 75°C) with an optimal pH and temperature of 8.0 and 65°C, respectively. The purified enzyme exhibited good thermostability with a 72.2 min half-life of thermal denaturation at 60°C. In addition, this protease was not sensitive to heavy metal ions, surfactants, or oxidative reagents. In conclusion, strain improvement for protease production can serve as an alternative strategy to promote feather degradation. The UMU4 mutant of B. subtilis and its serine protease could be potentially used in various industries. PMID:27323184

  18. Fumigaclavine C improves concanavalin A-induced liver injury in mice mainly via inhibiting TNF-alpha production and lymphocyte adhesion to extracellular matrices.

    PubMed

    Zhao, Ying; Liu, Junyan; Wang, Jun; Wang, Lei; Yin, Hao; Tan, Renxiang; Xu, Qiang

    2004-06-01

    Fumigaclavine C, an alkaloidal metabolite, was produced by Aspergillus fumigatus (strain No. CY018). This study examined the effect of this compound on concanavalin A (Con A)-induced liver injury in mice, a T cell-dependent model of liver damage. Con A administration resulted in severe liver injury, T lymphocyte activation and a strong increment in spleen cell adhesion, as well as in tumour necrosis factor-alpha (TNF-alpha) production. Against this liver injury, the intraperitoneal administration of fumigaclavine C dose-dependently inhibited the elevation in transaminase activity, TNF-alpha production in serum and the histological changes, including inflammatory infiltration, hepatocyte necrosis and degeneration and Kupffer cell hyperplasia. In addition, this compound in-vitro also inhibited the proliferation of spleen cells induced by Con A, and reduced their IL-2 and TNF-alpha production. Moreover, the intraperitoneal administration of fumigaclavine C inhibited the potential of spleen cells isolated from the liver-injured mice to adhere to fibronectin, laminin and type IV collagen. These results suggest that the improvement of this T cell-mediated liver injury by fumigaclavine C may be related to the inhibition of lymphocyte activation, proliferation and adhesion to extracellular matrices as well as the reduction in TNF-alpha production. PMID:15231043

  19. Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production

    PubMed Central

    2012-01-01

    Background A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. Results Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail. Conclusions The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear

  20. Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp. SY16 using fed-batch fermentation.

    PubMed

    Kim, Hee-Sik; Jeon, Jong-Woon; Kim, Byung-Hyuk; Ahn, Chi-Yong; Oh, Hee-Mock; Yoon, Byung-Dae

    2006-04-01

    Candida sp. strain SY16 produces a glycolipid-type biosurfactant, mannosylerythritol lipid (MEL-SY16), which can reduce the surface tension of a culture broth from 72 to 30 dyne cm(-1) and highly emulsify hydrocarbons when cultured in soybean-oil-containing media. As such, laboratory-scale fermentation for MEL-SY16 production was performed using optimized conditions. In batch fermentation, MEL-SY16 was mainly produced during the stationary phase of growth, and the concentration of MEL-SY16 reached 37 g l(-1) after 200 h. The effect of pH control on the production of MEL-SY16 was also examined in batch fermentation. The highest production yield of MEL-SY16 was when the pH was controlled at 4.0, and the production was significantly improved compared to batch fermentation without pH control. In fed-batch fermentation, glucose and soybean oil (1:1, w/w) were used in combination as the initial carbon sources for cell growth, and soybean oil was used as the feeding carbon source during the MEL production phase. The feeding of soybean oil resulted in the disappearance of any foam and a sharp increase in the MEL production until 200 h, at which point the concentration of MEL-SY16 was 95 g l(-1). Among the investigated culture systems, the highest MEL-SY16 production and volumetric production rate were achieved with fed-batch fermentation. PMID:16133323

  1. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

    PubMed Central

    Balasubramanian, Deepak; Schneper, Lisa; Kumari, Hansi; Mathee, Kalai

    2013-01-01

    Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies. PMID:23143271

  2. Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin.

    PubMed Central

    Yasuda, H; Ajiki, Y; Koga, T; Kawada, H; Yokota, T

    1993-01-01

    Interactions between bacterial biofilms formed by Pseudomonas aeruginosa and clarithromycin, a macrolide having no anti-P. aeruginosa activity, were investigated. P. aeruginosa incubated for 10 days on membrane filters formed biofilms on the surfaces of the filters. The biofilms were characterized by dense colonizations of bacteria and thick membranous structures that covered the colonies. Treatment of the biofilms with a relatively low concentration of clarithromycin for 5 days resulted in an eradication of the membranous structures. Quantitative analysis of alginate and hexose was done to evaluate the quantity of polysaccharides in or on the biofilms. Treatment of the biofilms with clarithromycin decreased the quantity of alginate and hexose and therefore perhaps the quantity of polysaccharides as well. Eradication of the membranous structures of biofilms, or the decrease in the quantity of polysaccharides, resulted in an increase in the rate of penetration of antibiotics through bacterial biofilms. In vivo therapeutic effects of ofloxacin in the rat infection model, in which the biofilm mode of growth of P. aeruginosa is characteristic, were enhanced by oral coadministration of clarithromycin. It is suggested that clarithromycin eradicated glycocalyx produced by P. aeruginosa, or suppressed the production of glycocalyx, by unknown mechanisms and thereby enhanced the therapeutic efficacies of other antimicrobial agents against infections caused by P. aeruginosa. Images PMID:8239580

  3. Extracellular beta-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology.

    PubMed

    Barbosa, Aneli M; Giese, Ellen C; Dekker, Robert F H; Borsato, Dionísio; Briones Pérez, Ana I; Ubeda Iranzo, Juan F

    2010-09-30

    beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm. PMID:20493976

  4. Induction of cytokine production in cholesteatoma keratinocytes by extracellular high-mobility group box chromosomal protein 1 combined with DNA released by apoptotic cholesteatoma keratinocytes.

    PubMed

    Chi, Zhangcai; Wang, Zhengmin; Liang, Qiong; Zhu, Yaying; du, Qiang

    2015-02-01

    High-mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to determine HMGB-1 expression in vivo and to identify the effect of extracellular HMGB-1 in inflammatory process associated with bone destruction in cholesteatoma. We investigated the expression and location of HMGB-1 in the cholesteatoma and healthy skin using an immunofluorescence assay. We also detected apoptosis and DNA fragments in the cholesteatoma by TUNEL staining. HMGB-1 concentration in apoptotic supernatants from UV light-treated cells, culture supernatants and its translocation in cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells were measured by immunoblot analysis and immunofluorescence assay. Cultures of human cholesteatoma keratinocytes were exposed to CpG-DNA, HMGB-1, or CpG-DNA complexed to HMGB-1 for 24 h. Cytokines in the culture supernatant were measured by ELISA. In addition, levels of proinflammatory cytokines released by cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells with or without anti-HMGB-1 antibodies and supernatants from UV light-treated cells with DNase 1 were measured by enzyme-linked immunosorbent assay. The expression of HMGB-1 in cholesteatoma increased and it translocated both to the cytoplasm and extracellular space. Furthermore, the HMGB-1 concentration in supernatants increased significantly after addition of supernatants from UV light-treated cells. TNF-α and IL-1β can be induced by purified HMGB-1 combined with CpG-DNA in the cholesteatoma keratinocytes. In addition, supernatants of apoptotic cells containing HMGB-1-DNA were effective in inducing TNF-α and IL-1β secretion. This study suggested that persistent expression of extracellular HMGB-1 and DNA fragments in cholesteatoma leads to TNF-α and IL-1β production, causing bone resorption and destruction. Thus, we have

  5. Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress

    PubMed Central

    2013-01-01

    Background Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity. Results The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further. Conclusions The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee’s food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of

  6. Screening of Coprinus species for the production of extracellular peroxidase and evaluation of the enzyme for the treatment of aqueous phenol.

    PubMed

    Ikehata, K; Buchanan, I D

    2002-12-01

    The production of extracellular peroxidase by twenty-five strains of Coprinus species was investigated for the purpose of its application to the removal of phenolic and other aromatic compounds from industrial waste streams. After initial screening experiments, the production of peroxidase by three superior strains of C cinererus UAMH 4103, UAMH 7907 and IFO 30116 was monitored over a 15-day period. Peroxidase activity was detected after 3 days of growth and had reached itspeak another 6 days later. The peroxidase activity appeared to increase with a corresponding depletion of glucose concentration and rapidly declined immediately after the exhaustion of glucose. The effectiveness of the cultivated C. cinereus peroxidase (CIP) for the removal of aqueous phenol was evaluated in the presence and in the absence of additives including polyethylene glycol (PEG) and chitosan, and compared with those of purified horseradish peroxidase (HRP) and Arthromyces ramosus peroxidase (ARP). The addition of PEG and chitosan enhanced the efficiency of phenol transformation catalyzed by CIP by the factor of 1.5 and 1.3, respectively. Although the efficiency of phenol transformation was higher with CIP than those with purified HRP and ARP in the absence of addtives, its superiority diminished in the presence of PEG. This suggests that the by-products of fungal culture in the crude CIP solution, presumably polycarbohydrates and proteins, have protective effects on the enzyme against inactivation during catalytic transformation of phenol, and the addition of PEG provides small effects on further protection.

  7. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  8. Production of extra-cellular slime by Staphylococcus epidermidis during stationary phase of growth: its association with adherence to implantable devices.

    PubMed

    Bayston, R; Rodgers, J

    1990-10-01

    A method of optimising slime production produced by Staphylococcus epidermis and its quantitative assay was developed, which gave a preliminary indication of its identity and an assessment of the correlation between slime production and adherence of the organism to implants. After inducing vigorous growth in brain heart infusion broth to stationary phase, all nutrients were removed by washing and the organisms resuspended in sterile deionised water with added magnesium. After further incubation the culture was centrifuged and the supernatant reacted with alcian blue in 50 mM magnesium chloride/sodium acetate solution, and the amount of bound dye was measured spectrophotometrically at 620 nm after its resolubilisation using sodium dodecyl sulphate. Large quantities of slime were produced by some, but not all, strains. Preliminary electrophoresis of the slime showed mobility and staining similar to that of the glycosaminoglycans. Adherence was tested by growing strains in wells of tissue culture plates and aspirating the supernatant after incubation. After fixation and staining of adherent growth the amount of bound stain was determined spectrophotometrically after its elution with ethanol. In this series of organisms there was no correlation between the result of tests for adherence or production of extracellular slime, and no correlation between either of these and the clinical source of the organisms. PMID:2229437

  9. Partial characterization and optimization of production of extracellular alpha-amylase from Bacillus subtilis isolated from culturable cow dung microflora.

    PubMed

    Swain, Manas R; Kar, Shaktimay; Padmaja, Gourikutti; Ray, Ramesh C

    2006-01-01

    Studies of alpha-amylase production by Bacillus subtilis (CM3) isolated earlier from cow dung microflora, were carried out. The optimum temperature, pH and incubation period for amylase production were 50-70 degrees C, 5.0-9.0 and 36 h, respectively. Enzyme secretion was very similar in the presence of any of the carbon sources tested (soluble starch, potato starch, cassava starch, wheat flour, glucose, fructose, etc.). Yeast extract and ammonium acetate (1%) as nitrogen sources gave higher yield compared to other nitrogen sources (peptone, malt extract, casein, asparagine, glycine, beef extract), whereas ammonium chloride, ammonium sulphate and urea inhibited the enzyme activity. Addition of Ca+2 (10-40 mM) to the culture medium did not result in further improvement of enzyme production, whereas the addition of surfactants (Tween 20, Tween 40, Tween 80, and sodium lauryl sulphate) at 0.02% resulted in 2-15% increase in enzyme production. There were no significant variations in enzyme yield between shaked-flask and laboratory fermentor cultures. The purified enzyme is in two forms with molecular mass of 18.0 +/- 1 and 43.0 +/- 1 kDa in native SDS-PAGE.

  10. Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells. Importance for interleukin-1 production.

    PubMed

    Schmid-Alliana, A; Menou, L; Manié, S; Schmid-Antomarchi, H; Millet, M A; Giuriato, S; Ferrua, B; Rossi, B

    1998-02-01

    We have demonstrated previously that microtubule depolymerization by colchicine in human monocytes induces selective production of interleukin-1 (IL-1) (Manié, S., Schmid-Alliana, A., Kubar, J., Ferrua, B., and Rossi, B. (1993) J. Biol. Chem. 268, 13675-13681). Here, we provide evidence that disruption of the microtubule structure rapidly triggers extracellular signal-regulated kinase (ERK) activation, whereas it was without effect on SAPK2 activity, which is commonly acknowledged to control pro-inflammatory cytokine production. This process involves the activation of the entire cascade including Ras, Raf-1, MEK1/2, ERK1, and ERK2. Activation of ERKs is followed by their nuclear translocation. Although other SAPK congeners might be activated upon microtubule depolymerization, the activation of ERK1 and ERK2 is mandatory for IL-1 production as shown by the blocking effect of PD 98059, a specific MEK1/2 inhibitor. Additionally, we provide evidence that microtubule disruption also induces the activation of c-Src and Hck activities. The importance of Src kinases in the mediation of the colchicine effect is underscored by the fact that CP 118556, a specific inhibitor of Src-like kinase, abrogates both the colchicine-induced ERK activation and IL-1 production. This is the first evidence that ERK activation is an absolute prerequisite for induction of this cytokine. Altogether, our data lend support to a model where the status of microtubule integrity controls the level of Src activities that subsequently activate the ERK kinase cascade, thus leading to IL-1 production.

  11. Antidepressants increase glial cell line-derived neurotrophic factor production through monoamine-independent activation of protein tyrosine kinase and extracellular signal-regulated kinase in glial cells.

    PubMed

    Hisaoka, Kazue; Takebayashi, Minoru; Tsuchioka, Mami; Maeda, Natsuko; Nakata, Yoshihiro; Yamawaki, Shigeto

    2007-04-01

    Recent studies show that neuronal and glial plasticity are important for therapeutic action of antidepressants. We previously reported that antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells). Here, we found that amitriptyline, a tricyclic antidepressant, increased both GDNF mRNA expression and release, which were selectively and completely inhibited by mitogen-activated protein kinase kinase inhibitors. Indeed, treatment of amitriptyline rapidly increased extracellular signal-regulated kinase (ERK) activity, as well as p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase activities. Furthermore, different classes of antidepressants also rapidly increased ERK activity. The extent of acute ERK activation and GDNF release were significantly correlated to each other in individual antidepressants, suggesting an important role of acute ERK activation in GDNF production. Furthermore, antidepressants increased the acute ERK activation and GDNF mRNA expression in normal human astrocytes as well as C6 cells. Although 5-hydroxytryptamine (serotonin) (5-HT), but not noradrenaline or dopamine, increased ERK activation and GDNF release via 5-HT2A receptors, ketanserin, a 5-HT2A receptor antagonist, did not have any effect on the amitriptyline-induced ERK activation. Thus, GDNF production by amitriptyline was independent of monoamine. Both of the amitriptyline-induced ERK activation and GDNF mRNA expression were blocked by genistein, a general protein tyrosine kinase (PTK) inhibitor. Actually, we found that amitriptyline acutely increased phosphorylation levels of several phosphotyrosine-containing proteins. Taken together, these findings indicate that ERK activation through PTK regulates antidepressant-induced GDNF production and that the GDNF production in glial cells may be a novel action of the antidepressant, which is independent of monoamine. PMID:17210798

  12. Effects of Surfactants and Microwave-assisted Pretreatment of Orange Peel on Extracellular Enzymes Production by Aspergillus japonicus PJ01.

    PubMed

    Li, Pei-jun; Xia, Jin-lan; Shan, Yang; Nie, Zhen-yuan; Wang, Fei-ran

    2015-06-01

    The effects of surfactants and microwave pretreatment of orange peel powder (OPP) on the production of pectinase, cellulase, and xylanase by Aspergillus japonicus PJ01 in submerged fermentation were investigated. The results showed that when OPP was pretreated only by microwave (630 W, 9 min, and liquid/solid ratio 5), the activities of exo-pectinase, carboxymethyl cellulase (CMCase), xylanase, and filter paper cellulase (FPase) were increased by 11.8, 20.6, 16.2, and 24.0 %, respectively, and when OPP was pretreated by microwave at the same conditions cited above plus PEG 4000 at the concentration of 3 g/L, the activities of the above four enzymes were enhanced by 40.2, 30.3, 40.4, and 40.0 % after 84-h cultivation, respectively. It is suggested a synergistic effect between microwave and surfactant treatment in enhancing the multiextracellular enzymes production by OPP fermentation of A. japonicus PJ01. Chemical composition and Fourier transform infrared spectroscopy (FTIR) analysis displayed that the microwave pretreatment of OPP led to the decrease of hemicellulose and essential oil contents. Scanning electron microscope (SEM) showed that OPP surface after microwave pretreatment became porous and more susceptible to be invaded by A. japonicus. The results demonstrated that pretreatment of OPP by surfactant PEG 4000 and microwave irradiation as environment-friendly way was cost-effective in enhancing the multienzyme production from agricultural waste orange peel.

  13. Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, Halobacillus sp. LY6.

    PubMed

    Xin, Li; Hui-Ying, Yu; Xiao-Xue, Liu; Xiao, Sun

    2011-07-01

    A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na(2)SO(4) or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30-80°C), pH (6.0-12.0), and NaCl concentration (0-25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca(2+) and Cu(2+), but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.

  14. Effects of Surfactants and Microwave-assisted Pretreatment of Orange Peel on Extracellular Enzymes Production by Aspergillus japonicus PJ01.

    PubMed

    Li, Pei-jun; Xia, Jin-lan; Shan, Yang; Nie, Zhen-yuan; Wang, Fei-ran

    2015-06-01

    The effects of surfactants and microwave pretreatment of orange peel powder (OPP) on the production of pectinase, cellulase, and xylanase by Aspergillus japonicus PJ01 in submerged fermentation were investigated. The results showed that when OPP was pretreated only by microwave (630 W, 9 min, and liquid/solid ratio 5), the activities of exo-pectinase, carboxymethyl cellulase (CMCase), xylanase, and filter paper cellulase (FPase) were increased by 11.8, 20.6, 16.2, and 24.0 %, respectively, and when OPP was pretreated by microwave at the same conditions cited above plus PEG 4000 at the concentration of 3 g/L, the activities of the above four enzymes were enhanced by 40.2, 30.3, 40.4, and 40.0 % after 84-h cultivation, respectively. It is suggested a synergistic effect between microwave and surfactant treatment in enhancing the multiextracellular enzymes production by OPP fermentation of A. japonicus PJ01. Chemical composition and Fourier transform infrared spectroscopy (FTIR) analysis displayed that the microwave pretreatment of OPP led to the decrease of hemicellulose and essential oil contents. Scanning electron microscope (SEM) showed that OPP surface after microwave pretreatment became porous and more susceptible to be invaded by A. japonicus. The results demonstrated that pretreatment of OPP by surfactant PEG 4000 and microwave irradiation as environment-friendly way was cost-effective in enhancing the multienzyme production from agricultural waste orange peel. PMID:25920331

  15. BEEP: An assay to detect bio-energetic and envelope permeability alterations in Pseudomonas aeruginosa.

    PubMed

    Suh, Sang-Jin; Shuman, Jon; Carroll, Leslie P; Silo-Suh, Laura

    2016-06-01

    We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased screen of a transposon insertion library in P. aeruginosa strain PAO1 in order to expedite the isolation of mutants with defects in bioenergetic pathways. We successfully isolated insertion mutants that were reduced for extracellular ATP accumulation and identified the corresponding mutations that caused the phenotype. Most of the genes identified from this analysis were associated with energy metabolism and several appeared to be potentially novel bioenergetic targets. In addition, we show that treatment of P. aeruginosa strain PAO1 with antibiotics that disrupt the bacterial cell envelope leads to greater extracellular ATP accumulation. In summary, increases in extracellular ATP accumulation above wild type levels indicated a perturbation of membrane permeability while decreases in extracellular ATP accumulation indicated defects in bioenergetics. PMID:27089860

  16. Effects of limonene stress on the growth of and microcystin release by the freshwater cyanobacterium Microcystis aeruginosa FACHB-905.

    PubMed

    Hu, Xi; Liu, Yunguo; Zeng, Guangming; Hu, Xinjiang; Wang, Yaqin; Zeng, Xiaoxia

    2014-07-01

    The effects of limonene exposure on the growth of Microcystisaeruginosa and the release of toxic intracellular microcystin (MCY) were tested by evaluating the results obtained from the batch culture experiments with M. aeruginosa FACHB-905. The time series of cell as well as intracellular and extracellular MCY concentrations were evaluated during 5d of the incubation. After exposure to limonene, the number of cells gradually diminished; the net log cell reduction after 5d of the exposure was 3.0, 3.6, and 3.8log when the initial cell densities were set at 1.6×10(7), 1.1×10(6) and 4.1×10(5)cell/mL, respectively. Limonene was found to significantly influence the production and release of MCY. As the limonene exposure could inhibit the increase in the number of cells, the increase in the total MCY concentration in the medium was also inhibited. In the presence of limonene, the intracellular MCY was gradually released into the medium through a gradual reduction in the number of cells. The extracellular MCY concentration in the medium was significantly higher in the limonene-exposed samples than in the control samples, which confirmed that limonene cannot decompose the extracellular MCY.

  17. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

    PubMed Central

    Irazoqui, Javier E.; Troemel, Emily R.; Feinbaum, Rhonda L.; Luhachack, Lyly G.; Cezairliyan, Brent O.; Ausubel, Frederick M.

    2010-01-01

    The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

  18. Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments.

    PubMed

    Naqvi, Syeda M; Buckley, Conor T

    2015-12-01

    Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture-expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD-like micro-environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP-encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD-like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD-like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate

  19. Effect of environmental factors on allelopathic inhibition of Microcystis aeruginosa by berberine.

    PubMed

    Zhang, Shulin; Dai, Wei; Bi, Xiangdong; Zhang, Dajuan; Xing, Kezhi

    2013-01-01

    To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.

  20. Antimicrobial susceptibility differences among mucoid and non-mucoid Pseudomonas aeruginosa isolates

    PubMed Central

    Owlia, Parviz; Nosrati, Rahim; Alaghehbandan, Reza; Lari, Abdolaziz Rastegar

    2014-01-01

    Pseudomonas aeruginosa is one of the most important opportunistic bacteria, causing a wide variety of infections particularly in immunocompromised patients. The extracellular glycocalyx is produced in copious amounts by mucoid strains of P. aeruginosa. Mucoid and non-mucoid P. aeruginosa strains show some differences in their antimicrobial susceptibility pattern. The aim of this study was to investigate the frequency of mucoid and non-mucoid types and their antimicrobial susceptibility patterns isolated from Milad and Mostafa Khomeini Hospital in Tehran, Iran. One hundred P. aeruginosa isolates were collected which all were confirmed by conventional biochemical tests and PCR assay using specific primers for oprI and oprL lipoproteins. Mucoid and non-mucoid types of isolates were determined by culturing isolates on BHI agar containing Congo red and Muir mordant staining method. The susceptibility pattern of isolates against 23 different antibiotics was assessed using MIC sensititre susceptibility plates. Fifty of 100 of isolates were mucoid type, of which 14 isolates were from Mostafa Khomeini Hospital. Frequency of mucoid type of P. aeruginosa in Mostafa Khomeini hospital (70%) was higher than that seen in Milad hospital (45%). The statistical analysis of MICs results showed significant differences in antimicrobial resistance among mucoid and non-mucoid types (non mucoid strains showed more resistance against tested antibiotics). This may be due to the tendency of some antibiotics to attach to extracellular glycocalyx of mucoid strains. PMID:25152858

  1. Antimicrobial susceptibility differences among mucoid and non-mucoid Pseudomonas aeruginosa isolates.

    PubMed

    Owlia, Parviz; Nosrati, Rahim; Alaghehbandan, Reza; Lari, Abdolaziz Rastegar

    2014-01-01

    Pseudomonas aeruginosa is one of the most important opportunistic bacteria, causing a wide variety of infections particularly in immunocompromised patients. The extracellular glycocalyx is produced in copious amounts by mucoid strains of P. aeruginosa. Mucoid and non-mucoid P. aeruginosa strains show some differences in their antimicrobial susceptibility pattern. The aim of this study was to investigate the frequency of mucoid and non-mucoid types and their antimicrobial susceptibility patterns isolated from Milad and Mostafa Khomeini Hospital in Tehran, Iran. One hundred P. aeruginosa isolates were collected which all were confirmed by conventional biochemical tests and PCR assay using specific primers for oprI and oprL lipoproteins. Mucoid and non-mucoid types of isolates were determined by culturing isolates on BHI agar containing Congo red and Muir mordant staining method. The susceptibility pattern of isolates against 23 different antibiotics was assessed using MIC sensititre susceptibility plates. Fifty of 100 of isolates were mucoid type, of which 14 isolates were from Mostafa Khomeini Hospital. Frequency of mucoid type of P. aeruginosa in Mostafa Khomeini hospital (70%) was higher than that seen in Milad hospital (45%). The statistical analysis of MICs results showed significant differences in antimicrobial resistance among mucoid and non-mucoid types (non mucoid strains showed more resistance against tested antibiotics). This may be due to the tendency of some antibiotics to attach to extracellular glycocalyx of mucoid strains. PMID:25152858

  2. Staphylococcus aureus alters growth activity, autolysis, and antibiotic tolerance in a human host-adapted Pseudomonas aeruginosa lineage.

    PubMed

    Michelsen, Charlotte Frydenlund; Christensen, Anne-Mette Juel; Bojer, Martin Saxtorph; Høiby, Niels; Ingmer, Hanne; Jelsbak, Lars

    2014-11-01

    Interactions among members of polymicrobial infections or between pathogens and the commensal flora may determine disease outcomes. Pseudomonas aeruginosa and Staphylococcus aureus are important opportunistic human pathogens and are both part of the polymicrobial infection communities in human hosts. In this study, we analyzed the in vitro interaction between S. aureus and a collection of P. aeruginosa isolates representing different evolutionary steps of a dominant lineage, DK2, that have evolved through decades of growth in chronically infected patients. While the early adapted P. aeruginosa DK2 strains outcompeted S. aureus during coculture on agar plates, we found that later P. aeruginosa DK2 strains showed a commensal-like interaction, where S. aureus was not inhibited by P. aeruginosa and the growth activity of P. aeruginosa was enhanced in the presence of S. aureus. This effect is mediated by one or more extracellular S. aureus proteins greater than 10 kDa, which also suppressed P. aeruginosa autolysis and prevented killing by clinically relevant antibiotics through promoting small-colony variant (SCV) formation. The commensal interaction was abolished with S. aureus strains mutated in the agr quorum sensing system or in the SarA transcriptional virulence regulator, as well as with strains lacking the proteolytic subunit, ClpP, of the Clp protease. Our results show that during evolution of a dominant cystic fibrosis lineage of P. aeruginosa, a commensal interaction potential with S. aureus has developed.

  3. Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

    PubMed Central

    Sivaprakasam, Senthilkumar; Dhandapani, Balaji; Mahadevan, Surianarayanan

    2011-01-01

    Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1–6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value. PMID:24031785

  4. Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo.

    PubMed

    Vorotnikova, Ekaterina; McIntosh, Donna; Dewilde, Abiche; Zhang, Jianping; Reing, Janet E; Zhang, Li; Cordero, Kevin; Bedelbaeva, Khamilia; Gourevitch, Dimitri; Heber-Katz, Ellen; Badylak, Stephen F; Braunhut, Susan J

    2010-10-01

    Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals. PMID:20797438

  5. Drosophila melanogaster as an Animal Model for the Study of Pseudomonas aeruginosa Biofilm Infections In Vivo

    PubMed Central

    Mulcahy, Heidi; Sibley, Christopher D.; Surette, Michael G.; Lewenza, Shawn

    2011-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo. PMID:21998591

  6. Physiological framework for the regulation of quorum sensing-dependent public goods in Pseudomonas aeruginosa.

    PubMed

    Mellbye, Brett; Schuster, Martin

    2014-03-01

    Many bacteria possess cell density-dependent quorum-sensing (QS) systems that often regulate cooperative secretions involved in host-microbe or microbe-microbe interactions. These secretions, or "public goods," are frequently coregulated by stress and starvation responses. Here we provide a physiological rationale for such regulatory complexity in the opportunistic pathogen Pseudomonas aeruginosa. Using minimal-medium batch and chemostat cultures, we comprehensively characterized specific growth rate-limiting macronutrients as key triggers for the expression of extracellular enzymes and metabolites directly controlled by the las and rhl QS systems. Expression was unrelated to cell density, depended on the secreted product's elemental composition, and was induced only when the limiting nutrient was not also a building block of the product; rhl-dependent products showed the strongest response, caused by the largely las-independent induction of the regulator RhlR and its cognate signal. In agreement with the prominent role of the rhl system, slow growth inverted the las-to-rhl signal ratio, previously considered a characteristic distinguishing between planktonic and biofilm lifestyles. Our results highlight a supply-driven, metabolically prudent regulation of public goods that minimizes production costs and thereby helps stabilize cooperative behavior. Such regulation would be beneficial for QS-dependent public goods that act broadly and nonspecifically, and whose need cannot always be accurately assessed by the producing cell. Clear differences in the capacities of the las and rhl systems to integrate starvation signals help explain the existence of multiple QS systems in one cell.

  7. The Mucoid Switch in Pseudomonas aeruginosa Represses Quorum Sensing Systems and Leads to Complex Changes to Stationary Phase Virulence Factor Regulation

    PubMed Central

    Ryall, Ben; Carrara, Marta; Zlosnik, James E. A.; Behrends, Volker; Lee, Xiaoyun; Wong, Zhen; Lougheed, Kathryn E.; Williams, Huw D.

    2014-01-01

    The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung. PMID:24852379

  8. The mucoid switch in Pseudomonas aeruginosa represses quorum sensing systems and leads to complex changes to stationary phase virulence factor regulation.

    PubMed

    Ryall, Ben; Carrara, Marta; Zlosnik, James E A; Behrends, Volker; Lee, Xiaoyun; Wong, Zhen; Lougheed, Kathryn E; Williams, Huw D

    2014-01-01

    The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung. PMID:24852379

  9. Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

    PubMed

    Castellanos-Moguel, Judith; González-Barajas, Margarita; Mier, Teresa; Reyes-Montes, María Del Rocío; Aranda, Eduardo; Toriello, Conchita

    2007-03-01

    To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

  10. Production and Characterization of the Intra- and Extracellular Carbohydrates and Polymeric Substances (EPS) of Three Sea-Ice Diatom Species, and Evidence for a Cryoprotective Role for EPS.

    PubMed

    Aslam, Shazia N; Cresswell-Maynard, Tania; Thomas, David N; Underwood, Graham J C

    2012-12-01

    Diatoms and their associated extracellular polymeric substances (EPS) are major constituents of the microalgal assemblages present within sea ice. Yields and chemical composition of soluble and cell-associated polysaccharides produced by three sea-ice diatoms, Synedropsis sp., Fragilariopsis curta, and F. cylindrus, were compared. Colloidal carbohydrates (CC) contained heteropolysaccharides rich in mannose, xylose, galactose, and glucose. Synedropsis sp. CC consisted mainly of carbohydrates <8 kDa size, with relatively soluble EPS, compared to high proportions of less-soluble EPS produced by both Fragilariopsis spp. F. curta colloidal EPS contained high concentrations of amino sugars (AS). Both Fragilariopsis species had high yields of hot bicarbonate (HB) soluble EPS, rich in xylose, mannose, galactose, and fucose (and AS in F. cylindrus). All species had frustule-associated EPS rich in glucose-mannose. Nutrient limitation resulted in declines in EPS yields and in glucose content of all EPS fractions. Significant similarities between EPS fractions from cultures and different components of natural EP