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Sample records for aeruginosa pseudomonas putida

  1. Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere.

    PubMed Central

    Yeung, K H; Schell, M A; Hartel, P G

    1989-01-01

    The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere. PMID:2515805

  2. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    SciTech Connect

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-03-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers.

  3. Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida.

    PubMed

    Ambrosi, Cecilia; Leoni, Livia; Visca, Paolo

    2002-08-01

    We investigated the regulation of the psbA and pvdA pyoverdine biosynthesis genes, which encode the L-ornithine N(5)-oxygenase homologues in Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1, respectively. We demonstrate that pyoverdine(B10), as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, pyoverdine(PAO1) has no apparent role in the positive regulation of the pvdA gene. PMID:12147517

  4. Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida.

    PubMed

    Thupvong, T; Wiideman, A; Dunn, D; Oreschak, K; Jankowicz, B; Doering, J; Castignetti, D

    1999-09-01

    Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa. To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined. The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.

  5. A novel Pseudomonas aeruginosa Bacteriophage, Ab31, a Chimera Formed from Temperate Phage PAJU2 and P. putida Lytic Phage AF: Characteristics and Mechanism of Bacterial Resistance

    PubMed Central

    Latino, Libera; Essoh, Christiane; Blouin, Yann; Vu Thien, Hoang; Pourcel, Christine

    2014-01-01

    A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31) is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10) with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung. PMID:24699529

  6. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

    PubMed

    Latino, Libera; Essoh, Christiane; Blouin, Yann; Vu Thien, Hoang; Pourcel, Christine

    2014-01-01

    A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31) is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10) with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

  7. Chromium reduction in Pseudomonas putida.

    PubMed Central

    Ishibashi, Y; Cervantes, C; Silver, S

    1990-01-01

    Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells. PMID:2389940

  8. Methylmercury degradation by Pseudomonas putida V1.

    PubMed

    Cabral, Lucélia; Yu, Ri-Qing; Crane, Sharron; Giovanella, Patricia; Barkay, Tamar; Camargo, Flávio A O

    2016-08-01

    Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1. PMID:27062344

  9. Chemotaxis of Pseudomonas putida toward chlorinated benzoates

    SciTech Connect

    Harwood, C.S.; Parales, R.E.; Dispensa, M. )

    1990-05-01

    The chlorinated aromatic acids 3-chlorobenzoate and 4-chlorobenzoate are chemoattractants for Pseudomonas putida PRS2000. These compounds are detected by a chromosomally encoded chemotactic response to benzoate which is inducible by {beta}-ketoadipate, and intermediate of benzoate catabolism. Plasmid pAC27, encoding enzymes for 3-chlorobenzoate degradation, does not appear to carry genes for chemotaxis toward chlorinated compounds.

  10. Pseudomonas putida Stimulates Primordia on Agaricus bitorquis.

    PubMed

    Colauto, Nelson B; Fermor, Terry R; Eira, Augusto F; Linde, Giani A

    2016-04-01

    Casing layer is one step of Agaricus bisporus cultivation where there is a competitive environment with a high number of microorganisms and diversity interacting with mycelia. It is suggested that a minimal community of these microorganisms would be necessary to stimulate fructification. However, A. bisporus is not able to produce primordia in sterile casing layers or Petri dishes. Thus, the objective of this study was to characterize bacterial microbiota of casing layers from A. bisporus cultivation, isolate, identify and characterize the bacteria responsible for the stimulation of primordium and their action mechanism using Agaricus bitorquis as a primordium stimulation model. Bacterial and Pseudomonas spp. communities of different casing layers of A. bisporus cultivation were collected and quantified. It was concluded that Pseudomonas spp. corresponds to 75-85% of bacterial population of the casing layers in A. bisporus cultivation and among those 12% are Pseudomonas putida. Four biochemical assays were used to identify P. putida. In vitro primordium stimulation of living P. putida and non-living bacterial suspensions, after chemical or physical treatments, was tested using A. bitorquis as a primordium stimulation model. Primordium stimulation assay was registered by photographs, and micrographs of vertical cut of primordium were registered by scanning electron microscope. Interaction of living P. putida with A. bitorquis mycelia is capable of stimulating primordial instead of non-living bacterial suspensions. Stimulation of A. bitorquis primordia does not imply or is related to mycelial growth inhibition, but a hierarchical relation of primordium succession and development is suggested. PMID:26742772

  11. 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.

  12. Ethylene Glycol Metabolism by Pseudomonas putida

    PubMed Central

    Mückschel, Björn; Simon, Oliver; Klebensberger, Janosch; Graf, Nadja; Rosche, Bettina; Altenbuchner, Josef; Pfannstiel, Jens; Huber, Armin

    2012-01-01

    In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol. PMID:23023748

  13. Ethylene glycol metabolism by Pseudomonas putida.

    PubMed

    Mückschel, Björn; Simon, Oliver; Klebensberger, Janosch; Graf, Nadja; Rosche, Bettina; Altenbuchner, Josef; Pfannstiel, Jens; Huber, Armin; Hauer, Bernhard

    2012-12-01

    In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.

  14. Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440

    PubMed Central

    Schmitz, Simone; Nies, Salome; Wierckx, Nick; Blank, Lars M.; Rosenbaum, Miriam A.

    2015-01-01

    Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals. P. putida's obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming. Here, we engineered a strain of P. putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions. P. aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems (BES). We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P. aeruginosa on two inducible plasmids into P. putida KT2440. The best clone, P. putida pPhz, produced 45 mg/L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P. aeruginosa. This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P. putida KT2440. In the new strain, phenazine synthesis with supernatant concentrations up to 33 μg/mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator. P. putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 μA/cm2 anodic current density. Together, this work lays a foundation for future oxygen-limited biocatalysis with P. putida strains. PMID:25914687

  15. Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

    PubMed Central

    de Groot, A; Heijnen, I; de Cock, H; Filloux, A; Tommassen, J

    1994-01-01

    In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits. Images PMID:7905475

  16. Molecular analysis of the Pseudomonas aeruginosa regulatory genes ptxR and ptxS.

    PubMed

    Colmer, J A; Hamood, A N

    2001-09-01

    We have previously described two Pseudomonas aeruginosa genes, ptxR, which enhances toxA and pvc (the pyoverdine chromophore operon) expression, and ptxS, the first gene of the kgu operon for the utilization of 2-ketogluconate by P. aeruginosa. ptxS interferes with the effect of ptxR on toxA expression. In this study, we have utilized DNA hybridization experiments to determine the presence of ptxR and ptxS homologous sequences in several gram-negative bacteria. ptxR homologous sequences were detected in P. aeruginosa strains only, while ptxS homologous sequences were detected in P. aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens. Using Northern blot hybridization experiments and a ptxS-lacZ fusion plasmid, we have shown that P. aeruginosa ptxR and ptxS are expressed in P. putida and P. fluorescens. Additional Northern blot hybridization experiments confirmed that ptxS is transcribed in P. putida and P. fluorescens strains that carried no plasmid. The presence of a PtxS homologue in these strains was examined by DNA-gel shift experiments. Specific gel shift bands were detected when the lysates of P. aeruginosa, P. putida, and P. fluorescens were incubated with the ptxS operator site as probe. kgu-hybridizing sequences were detected in P. putida and P. fluorescens. These results suggest that (i) ptxR is present in P. aeruginosa, while ptxS is present in P. aeruginosa, P. putida, and P. fluorescens; (ii) both ptxR and ptxS are expressed in P. putida and P fluorescens; and (iii) a PtxS homologue may exist in P. putida and P. fluorescens. PMID:11683464

  17. Biological manganese oxidation by Pseudomonas putida in trickling filters.

    PubMed

    McKee, Kyle P; Vance, Cherish C; Karthikeyan, Raghupathy

    2016-01-01

    Biological oxidation has been researched as a viable alternative for treating waters with high manganese (Mn) concentrations, typically found in mine drainage or in some geological formations. In this study, laboratory-scale trickling filters were constructed to compare the Mn removal efficiency between filters inoculated with the Mn oxidizing bacteria, Pseudomonas putida, and filters without inoculation. Manganese oxidation and removal was found to be significantly greater in trickling filters with Pseudomonas putida after startup times of only 48 h. Mn oxidation in Pseudomonas putida inoculated trickling filters was up to 75% greater than non-inoculated filters. One-dimensional advective-dispersive models were formulated to describe the transport of Mn in trickling filter porous media. Based on the experimental transport parameters obtained, the model predicted that a filter depth of only 16 cm is needed to reduce influent concentration of 10 mg L(-1) to 0.05 mg L(-1).

  18. Engineering the Soil Bacterium Pseudomonas putida for Arsenic Methylation

    PubMed Central

    Chen, Jian; Qin, Jie; Zhu, Yong-Guan; de Lorenzo, Víctor

    2013-01-01

    Accumulation of arsenic has potential health risks through consumption of food. Here, we inserted the arsenite [As(III)] S-adenosylmethionine methyltransferase (ArsM) gene into the chromosome of Pseudomonas putida KT2440. Recombinant bacteria methylate inorganic arsenic into less toxic organoarsenicals. This has the potential for bioremediation of environmental arsenic and reducing arsenic contamination in food. PMID:23645194

  19. Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

    PubMed Central

    2011-01-01

    Background Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. Results Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. Conclusions A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid

  20. p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6

    PubMed Central

    O'Connor, Kevin E.; Witholt, Bernard; Duetz, Wouter

    2001-01-01

    Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage. PMID:11208791

  1. Biological production of monoethanolamine by engineered Pseudomonas putida S12.

    PubMed

    Foti, Mirjam; Médici, Rosario; Ruijssenaars, Harald J

    2013-09-10

    Pseudomonas putida S12 was engineered for the production of monoethanolamine (MEA) from glucose via the decarboxylation of the central metabolite L-serine, which is catalyzed by the enzyme L-serine decarboxylase (SDC). The host was first evaluated for its tolerance towards MEA as well as its endogenous ability to degrade this alkanolamine. Growth inhibition was observed at MEA concentrations above 100 mM, but growth was never completely arrested even at 750 mM of MEA. P. putida S12 was able to catabolize MEA in the absence of ammonia, but deletion of the eutBC genes that encode ethanolamine ammonia-lyase (EAL) enzyme sufficed to eliminate this capacity. For the biological production of MEA, the sdc genes from Arabidopsis thaliana (full-length and a truncated version) and Volvox carteri were expressed in P. putida S12. From 20 mM of glucose, negligible amounts of MEA were produced by P. putida S12 ΔeutBC expressing the sdc genes from A. thaliana and V. carteri. However, 0.07 mmol of MEA was obtained per g of cell dry weight of P. putida S12 ΔeutBC expressing the truncated variant of the A. thaliana SDC. When the medium was supplemented with L-serine (30 mM), MEA production increased to 1.25 mmol MEA g⁻¹ CDW, demonstrating that L-serine availability was limiting MEA production. PMID:23876477

  2. Bioremediation of p-Nitrophenol by Pseudomonas putida 1274 strain

    PubMed Central

    2014-01-01

    Background p-Nitrophenol (PNP) occurs as contaminants of industrial effluents and it is the most important environmental pollutant and causes significant health and environmental risks, because it is toxic to many living organisms. Nevertheless, the information regarding PNP degradation pathways and their enzymes remain limited. Objective To evaluate the efficacy of the Pseudomonas Putida 1274 for removal of PNP. Methods P. putida MTCC 1274 was obtained from MTCC Chandigarh, India and cultured in the minimal medium in the presence of PNP. PNP degradation efficiency was compared under different pH and temperature ranges. The degraded product was isolated and analyzed with different chromatographic and spectroscopic techniques. Results P. putida 1274 shows good growth and PNP degradation at 37°C in neutral pH. Acidic and alkali pH retarded the growth of P. putida as well as the PNP degradation. On the basis of specialized techniques, hydroquinone was identified as major degraded product. The pathway was identified for the biodegradation of PNP. It involved initial removal of the nitrate group and formation of hydroquinone as one of the intermediates. Conclusion Our results suggested that P. putida 1274 strain would be a suitable aspirant for bioremediation of nitro-aromatic compounds contaminated sites in the environment. PMID:24581307

  3. Pseudomonas aeruginosa biofilms in disease.

    PubMed

    Mulcahy, Lawrence R; Isabella, Vincent M; Lewis, Kim

    2014-07-01

    Pseudomonas aeruginosa is a ubiquitous organism that is the focus of intense research because of its prominent role in disease. Due to its relatively large genome and flexible metabolic capabilities, this organism exploits numerous environmental niches. It is an opportunistic pathogen that sets upon the human host when the normal immune defenses are disabled. Its deadliness is most apparent in cystic fibrosis patients, but it also is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder, surface growth on implanted biomaterials, and within hospital surface and water supplies, where it poses a host of threats to vulnerable patients (Peleg and Hooper, N Engl J Med 362:1804-1813, 2010; Breathnach et al., J Hosp Infect 82:19-24, 2012). Once established in the patient, P. aeruginosa can be especially difficult to treat. The genome encodes a host of resistance genes, including multidrug efflux pumps (Poole, J Mol Microbiol Biotechnol 3:255-264, 2001) and enzymes conferring resistance to beta-lactam and aminoglycoside antibotics (Vahdani et al., Annal Burns Fire Disast 25:78-81, 2012), making therapy against this gram-negative pathogen particularly challenging due to the lack of novel antimicrobial therapeutics (Lewis, Nature 485: 439-440, 2012). This challenge is compounded by the ability of P. aeruginosa to grow in a biofilm, which may enhance its ability to cause infections by protecting bacteria from host defenses and chemotherapy. Here, we review recent studies of P. aeruginosa biofilms with a focus on how this unique mode of growth contributes to its ability to cause recalcitrant infections.

  4. Silver against Pseudomonas aeruginosa biofilms.

    PubMed

    Bjarnsholt, Thomas; Kirketerp-Møller, Klaus; Kristiansen, Søren; Phipps, Richard; Nielsen, Anne Kirstine; Jensen, Peter Østrup; Høiby, Niels; Givskov, Michael

    2007-08-01

    Silver has been recognized for its antimicrobial properties for centuries. Most studies on the antibacterial efficacy of silver, with particular emphasis on wound healing, have been performed on planktonic bacteria. Our recent studies, however, strongly suggest that colonization of wounds involves bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa, but that the silver concentration is important. A concentration of 5-10 mug/mL silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 mug/mL) had no effect. The bactericidal concentration of silver required to eradicate the bacterial biofilm was 10-100 times higher than that used to eradicate planktonic bacteria. These observations strongly indicate that the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. It is suggested that clinicians and manufacturers of the said wound dressings consider whether they are treating wounds primarily colonized either by biofilm-forming or planktonic bacteria.

  5. Sorption of Pseudomonas putida onto differently structured kaolinite minerals

    NASA Astrophysics Data System (ADS)

    Vasiliadou, I. A.; Papoulis, D.; Chrysikopoulos, C.; Panagiotaras, D.; Karakosta, E.; Fardis, M.; Papavassiliou, G.

    2010-12-01

    The presence of bio-colloids (e.g. bacteria and viruses) in the subsurface could be attributed to the release of particles from septic tanks, broken sewer lines or from artificial recharge with treated municipal wastewater. Bio-colloid transport in the subsurface is significantly affected by sorption onto the solid matrix. Bio-colloid attachment onto mobile or suspended in the aqueous phase soil particles (e.g. clay or other minerals) also may influence their fate and transport in the subsurface. The present study focuses on the investigation of Pseudomonas (Ps.) putida sorption onto well (KGa-1) and poorly (KGa-2) crystallized kaolinite minerals. Batch experiments were carried out to determine the sorption isotherms of Ps. putida onto both types of kaolinite particles. The sorption process of Ps. putida onto KGa-1 and KGa-2 is adequately described by a Langmuir isotherm. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy as well as Nuclear Magnetic Resonance were employed to study the sorption mechanisms of Ps. putida. Experimental results indicated that KGa-2 presented higher affinity and sorption capacity than KGa-1. It was shown that electrostatic interactions and structural disorders can influence the sorption capacity of clay particles.

  6. Efficient recombinant production of prodigiosin in Pseudomonas putida

    PubMed Central

    Domröse, Andreas; Klein, Andreas S.; Hage-Hülsmann, Jennifer; Thies, Stephan; Svensson, Vera; Classen, Thomas; Pietruszka, Jörg; Jaeger, Karl-Erich; Drepper, Thomas; Loeschcke, Anita

    2015-01-01

    Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis. PMID:26441905

  7. Efficient recombinant production of prodigiosin in Pseudomonas putida.

    PubMed

    Domröse, Andreas; Klein, Andreas S; Hage-Hülsmann, Jennifer; Thies, Stephan; Svensson, Vera; Classen, Thomas; Pietruszka, Jörg; Jaeger, Karl-Erich; Drepper, Thomas; Loeschcke, Anita

    2015-01-01

    Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

  8. Antibiotic Conditioned Growth Medium of Pseudomonas Aeruginosa

    ERIC Educational Resources Information Center

    Benathen, Isaiah A.; Cazeau, Barbara; Joseph, Njeri

    2004-01-01

    A simple method to study the consequences of bacterial antibiosis after interspecific competition between microorganisms is presented. Common microorganisms are used as the test organisms and Pseudomonas aeruginosa are used as the source of the inhibitor agents.

  9. The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida.

    PubMed

    Jiménez-Fernández, Alicia; López-Sánchez, Aroa; Calero, Patricia; Govantes, Fernando

    2015-02-01

    We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

  10. Composition of Pseudomonas aeruginosa slime

    PubMed Central

    Brown, M. R. W.; Foster, J. H. Scott; Clamp, J. R.

    1969-01-01

    1. The slime produced by eight strains of Pseudomonas aeruginosa on a number of different media was demonstrated to be qualitatively the same. Small quantitative differences may be occasioned by differences in the extraction procedure, the growth medium or the strain of organism used. 2. The slime was shown to be predominantly polysaccharide with some nucleic acid material and a small amount of protein. 3. The hydrolysed polysaccharide fraction consists mainly of glucose with smaller amounts of mannose. This accounts for some 50–60% of the total slime. In addition, there is some 5% of hyaluronic acid. The nucleic acid material represents approx. 20% of the total weight, and is composed of both RNA and DNA. 4. Minor components are protein, rhamnose and glucosamine, the protein being less than 5% of the total. 5. Hyaluronic acid is produced in greater quantities from nutrient broth than from chemically defined media, and is more firmly attached to the cells than the other components. PMID:4240755

  11. Specific Gene Loci of Clinical Pseudomonas putida Isolates

    PubMed Central

    Molina, Lázaro; Udaondo, Zulema; Duque, Estrella; Fernández, Matilde; Bernal, Patricia; Roca, Amalia; de la Torre, Jesús; Ramos, Juan Luis

    2016-01-01

    Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host’s immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria. PMID:26820467

  12. Genome features of Pseudomonas putida LS46, a novel polyhydroxyalkanoate producer and its comparison with other P. putida strains.

    PubMed

    Sharma, Parveen K; Fu, Jilagamazhi; Zhang, Xiangli; Fristensky, Brian; Sparling, Richard; Levin, David B

    2014-01-01

    A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation

  13. Genome features of Pseudomonas putida LS46, a novel polyhydroxyalkanoate producer and its comparison with other P. putida strains

    PubMed Central

    2014-01-01

    A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation

  14. Amino Acid Racemization in Pseudomonas putida KT2440

    PubMed Central

    Radkov, Atanas D.

    2013-01-01

    d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the kcat/Km values with l- and d-lysine were 3 orders of magnitude greater than the kcat/Km values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher kcat/Km values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species. PMID:23995642

  15. Trace Metal Sequestration by the Manganese Oxidizing Bacterium Pseudomonas putida

    NASA Astrophysics Data System (ADS)

    Toner, B.; Manceau, A.; Marcus, M. A.; Sposito, G.

    2002-12-01

    Bacterial cells are an important source of chemically reactive surfaces in freshwater and soil environments. Pseudomonas putida strain MnB1 cells, like many gram negative bacteria, present an outer membrane studded with phosphate groups and carbohydrates as well as a billowing biofilm of extracellular polysaccharides to the surrounding microenvironment. The cell outer membrane and the biofilm possess functional groups that complex trace metals. During certain growth phases P. putida is also a manganese oxidizing bacterium, causing the cells to coat themselves in Mn(IV) oxide. Therefore, in addition to the cell outer membrane and associated biofilm, trace metals may sorb to the biogenic Mn oxide. To explore the relative contributions to trace metal sorption by the bacterial cells and biogenic Mn oxide, zinc and nickel were added to suspensions of bacterial cells with three different conditions: cells in the absence of Mn, cells in the process of Mn oxidation and cells with preformed biogenic Mn oxide. Adsorption isotherms were measured to quantify Zn and Ni sorption to P. putida in the presence and absence of biogenic Mn oxide. Zinc and Ni K-edge EXAFS spectra were measured to determine how and where the metals were binding to the bacterial cells and biogenic Mn oxide. The Zn and Ni adsorption isotherms exhibited two plateaus. The metal complexation was dependent on concentration with Zn having a higher affinity for phosphate and Ni for carboxyl functional groups. The preformed biogenic Mn oxide has high affinity for Zn and Ni and the bacterial surface contributed little to metal removal from solution under these conditions. However, if the metal is present in solution while Mn oxidation is occurring the bacterial cell surface influences greatly the overall removal of metal. Manganese oxidizing bacteria such as P. putida contribute to environmental metal sequestration by catalyzing the production of Mn oxide minerals, and the bacterial cells are themselves reactive

  16. [Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains].

    PubMed

    Grigor'eva, N V; Kondrat'eva, T F; Krasil'nikova, E N; Karavaĭko, G I

    2006-01-01

    The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187T and P. stutzeri VKM B-975T. Upon the introduction of CN- and SCN- into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH4+ was observed. Due to the high rate of their utilization, NH3, NH4+, and CNO- were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN- or SCN-. Both Pseudomonas strains decomposed SCN- via cyanate production. The cyanase activity was 0.75 micromol/(min mg protein) for P. putida strain 21 and 1.26 micromol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN- but absent in cells grown with NH4+. Strain 21 of P. putida was a more active CN- decomposer than strain 18 of P. stutzeri. Ammonium and CO2 were the terminal nitrogen and carbon products of CN- and SCN- decomposition. The terminal sulfur products of SCN- decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN- and SCN-) and sulfur (SCN-). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained.

  17. Draft Genome Sequence Analysis of a Pseudomonas putida W15Oct28 Strain with Antagonistic Activity to Gram-Positive and Pseudomonas sp. Pathogens

    PubMed Central

    Ye, Lumeng; Hildebrand, Falk; Dingemans, Jozef; Ballet, Steven; Laus, George; Matthijs, Sandra; Berendsen, Roeland; Cornelis, Pierre

    2014-01-01

    Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors. PMID:25369289

  18. Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram-positive and Pseudomonas sp. pathogens.

    PubMed

    Ye, Lumeng; Hildebrand, Falk; Dingemans, Jozef; Ballet, Steven; Laus, George; Matthijs, Sandra; Berendsen, Roeland; Cornelis, Pierre

    2014-01-01

    Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors. PMID:25369289

  19. The Accessory Genome of Pseudomonas aeruginosa

    PubMed Central

    Kung, Vanderlene L.; Ozer, Egon A.; Hauser, Alan R.

    2010-01-01

    Summary: Pseudomonas aeruginosa strains exhibit significant variability in pathogenicity and ecological flexibility. Such interstrain differences reflect the dynamic nature of the P. aeruginosa genome, which is composed of a relatively invariable “core genome” and a highly variable “accessory genome.” Here we review the major classes of genetic elements comprising the P. aeruginosa accessory genome and highlight emerging themes in the acquisition and functional importance of these elements. Although the precise phenotypes endowed by the majority of the P. aeruginosa accessory genome have yet to be determined, rapid progress is being made, and a clearer understanding of the role of the P. aeruginosa accessory genome in ecology and infection is emerging. PMID:21119020

  20. Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furfural (2-furaldehyde) is a furan formed by dehydration of pentose sugars. Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid and Coenzyme A intermediates. To identify genes involved in furan metabolism, two P. putida transposo...

  1. Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

    DOE PAGES

    Linger, Jeffrey G.; Hobdey, Sarah E.; Franden, Mary Ann; Fulk, Emily M.; Beckham, Gregg T.

    2016-02-02

    Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, wemore » engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Furthermore, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.« less

  2. Toluene Diffusion and Reaction in Unsaturated Pseudomonas putida Biofilms

    PubMed Central

    Holden, Patricia A.; Hunt, James R.; Firestone, Mary K.

    2010-01-01

    Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, we have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. We experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, we measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 × 10−7 cm2/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Our studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems. PMID:18642338

  3. Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains

    PubMed Central

    Fernández, Matilde; Porcel, Mario; de la Torre, Jesús; Molina-Henares, M. A.; Daddaoua, Abdelali; Llamas, María A.; Roca, Amalia; Carriel, Victor; Garzón, Ingrid; Ramos, Juan L.; Alaminos, Miguel; Duque, Estrella

    2015-01-01

    Pseudomonas putida strains are ubiquitous in soil and water but have also been reported as opportunistic human pathogens capable of causing nosocomial infections. In this study we describe the multilocus sequence typing of four P. putida strains (HB13667, HB8234, HB4184, and HB3267) isolated from in-patients at the Besançon Hospital (France). The four isolates (in particular HB3267) were resistant to a number of antibiotics. The pathogenicity and virulence potential of the strains was tested ex vivo and in vivo using different biological models: human tissue culture, mammalian tissues, and insect larvae. Our results showed a significant variability in the ability of the four strains to damage the host; HB13667 did not exhibit any pathogenic traits, HB4184 caused damage only ex vivo in human tissue cultures, and HB8234 had a deleterious effect in tissue culture and in vivo on rat skin, but not in insect larvae. Interestingly, strain HB3267 caused damage in all the model systems studied. The putative evolution of these strains in medical environments is discussed. PMID:26379646

  4. Biofilm formation-defective mutants in Pseudomonas putida.

    PubMed

    López-Sánchez, Aroa; Leal-Morales, Antonio; Jiménez-Díaz, Lorena; Platero, Ana I; Bardallo-Pérez, Juan; Díaz-Romero, Alberto; Acemel, Rafael D; Illán, Juan M; Jiménez-López, Julia; Govantes, Fernando

    2016-07-01

    Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.

  5. Investigation of plasmid-induced growth defect in Pseudomonas putida.

    PubMed

    Mi, Jia; Sydow, Anne; Schempp, Florence; Becher, Daniela; Schewe, Hendrik; Schrader, Jens; Buchhaupt, Markus

    2016-08-10

    Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant. PMID:27287537

  6. Toxicity of titanium dioxide nanoparticles on Pseudomonas putida.

    PubMed

    Combarros, R G; Collado, S; Díaz, M

    2016-03-01

    The increasing use of engineered nanoparticles (NPs) in industrial and household applications will very likely lead to the release of such materials into the environment. As wastewater treatment plants (WWTPs) are usually the last barrier before the water is discharged into the environment, it is important to understand the effects of these materials in the biotreatment processes, since the results in the literature are usually contradictory. We proposed the use of flow cytometry (FC) technology to obtain conclusive results. Aqueous solutions of TiO2 nanoparticles (0-2 mg mL(-1)) were used to check its toxicity effect using Pseudomonas putida as simplified model of real sludge over room light. Physiological changes in P. putida from viable to viable but non-culturable cells were observed by flow cytometry in presence of TiO2. The damaged and dead cell concentrations were below 5% in all cases under study. Both FSC and SSC parameter increased with TiO2 dose dependent manner, indicating nanoparticles uptake by the bacteria. The biological removal of salicylic acid (SA) was also significantly impacted by the presence of TiO2 in the medium reducing the efficiency. The use of FC allows also to develop and fit segregated kinetic models, giving the impact of TiO2 nanoparticles in the physiological subpopulations growth and implications for SA removal.

  7. Toluene diffusion and reaction in unsaturated Pseudomonas putida biofilms

    SciTech Connect

    Holden, P.A.; Hunt, J.R.; Firestone, M.K.

    1997-12-20

    Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, the authors have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. They experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, the authors measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 {times} 10{sup {minus}7} cm{sup 2}/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Their studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems.

  8. Biodegradation of nitrobenzene through a hybrid pathway in Pseudomonas putida

    SciTech Connect

    Jung, K.H.; Lee, J.Y.; Kim, H.S.

    1995-12-20

    The biodegradation of nitrobenzene was attempted by using Pseudomonas putida TB 103 which possesses the hybrid pathway combining the tod and the tol pathways. Analysis of the metabolic flux of nitrobenzene through the hybrid pathway indicated that nitrobenzene was initially oxidized to cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene by toluene dioxygenase in the tod pathway and then channeled into the tol pathway, leading to the complete biodegradation of nitrobenzene. A crucial metabolic step redirecting the metabolic flux of nitrobenzene from the tod to the tol pathway was determined from the genetic and biochemical studies on the enzymes involved in the tol pathway. From these results, it was found that toluate-cis-glycol dehydrogenase could convert cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene to catechol in the presence of NAD{sup +} with liberation of nitrite and the reduced form of NAD{sup +} (NADH) into the medium.

  9. Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment.

    PubMed

    Remold, Susanna K; Purdy-Gibson, Megan E; France, Michael T; Hundley, Thomas C

    2015-01-01

    By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively). Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.

  10. Pseudomonas aeruginosa in Healthcare Settings

    MedlinePlus

    ... becoming more difficult to treat because of increasing antibiotic resistance. Selecting the right antibiotic usually requires that a ... to help educate people about Pseudomonas infections, and antibiotic resistance, and to encourage prevention activities and healthy behaviors ...

  11. Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10

    PubMed Central

    Buyer, Jeffrey S.; Sikora, Lawrence J.; Kratzke, Marian G.

    1990-01-01

    Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 × 10−12 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores. PMID:16348116

  12. Integrated foam fractionation for heterologous rhamnolipid production with recombinant Pseudomonas putida in a bioreactor.

    PubMed

    Beuker, Janina; Steier, Anke; Wittgens, Andreas; Rosenau, Frank; Henkel, Marius; Hausmann, Rudolf

    2016-03-01

    Heterologeous production of rhamnolipids in Pseudomonas putida is characterized by advantages of a non-pathogenic host and avoidance of the native quorum sensing regulation in Pseudomonas aeruginosa. Yet, downstream processing is a major problem in rhamnolipid production and increases in complexity at low rhamnolipid titers and when using chemical foam control. This leaves the necessity of a simple concentrating and purification method. Foam fractionation is an elegant method for in situ product removal when producing microbial surfactants. However, up to now in situ foam fractionation is nearly exclusively reported for the production of surfactin with Bacillus subtilis. So far no cultivation integrated foam fractionation process for rhamnolipid production has been reported. This is probably due to excessive bacterial foam enrichment in that system. In this article a simple integrated foam fractionation process is reported for heterologous rhamnolipid production in a bioreactor with easily manageable bacterial foam enrichments. Rhamnolipids were highly concentrated in the foam during the cultivation process with enrichment factors up to 200. The described process was evaluated at different pH, media compositions and temperatures. Foam fractionation processes were characterized by calculating procedural parameter including rhamnolipid and bacterial enrichment, rhamnolipid recovery, YX/S, YP/X, and specific as well as volumetric productivities. Comparing foam fractionation parameters of the rhamnolipid process with the surfactin process a high effectiveness of the integrated foam fractionation for rhamnolipid production was demonstrated.

  13. Pseudomonas aeruginosa Dose-Response and Bathing Water Infection

    EPA Science Inventory

    Pseudomonas aeruginosa is the most commonly identified opportunistic pathogen associated with pool acquired bather disease. To better understand why this microorganism poses this protracted problem we recently appraised P. aeruginosa pool risk management. Much is known about the ...

  14. Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors.

    PubMed

    Reymond, Jean-Louis; Bergmann, Myriam; Darbre, Tamis

    2013-06-01

    Synthetic glycopeptide dendrimers composed of a branched oligopeptide tree structure appended with glycosidic groups at its multiple N-termini were investigated for binding to the Pseudomonas aeruginosa lectins LecB and LecA. These lectins are partly responsible for the formation of antibiotic resistant biofilms in the human pathogenic bacterium P. aeruginosa, which causes lethal airway infections in immune-compromised and cystic fibrosis patients. Glycopeptide dendrimers with high affinity to the lectins were identified by screening of combinatorial libraries. Several of these dendrimers, in particular the LecB specific glycopeptide dendrimers FD2 and D-FD2 and the LecA specific glycopeptide dendrimers GalAG2 and GalBG2, also efficiently block P. aeruginosa biofilm formation and induce biofilm dispersal in vitro. Structure-activity relationship and structural studies are reviewed, in particular the observation that multivalency is essential to the anti-biofilm effect in these dendrimers.

  15. Maintenance of chromosome structure in Pseudomonas aeruginosa

    PubMed Central

    Rybenkov, Valentin V.

    2014-01-01

    Replication and segregation of genetic information is an activity central to the well-being of all living cells. Concerted mechanisms have evolved that ensure that each cellular chromosome is replicated once and only once per cell cycle and then faithfully segregated into daughter cells. Despite remarkable taxonomic diversity, these mechanisms are largely conserved across eubacteria, although species specific distinctions can often be noted. Here, we provide an overview of the current state of knowledge about maintenance of the chromosome structure in Pseudomonas aeruginosa. We focus on global chromosome organization and its dynamics during DNA replication and cell division. Special emphasis is made on contrasting these activities in P. aeruginosa and other bacteria. Among unique P. aeruginosa features are the presence of two distinct autonomously replicating sequences and multiple condensins, which suggests existence of novel regulatory mechanisms. PMID:24863732

  16. Developing an international Pseudomonas aeruginosa reference panel

    PubMed Central

    De Soyza, Anthony; Hall, Amanda J; Mahenthiralingam, Eshwar; Drevinek, Pavel; Kaca, Wieslaw; Drulis-Kawa, Zuzanna; Stoitsova, Stoyanka R; Toth, Veronika; Coenye, Tom; Zlosnik, James E A; Burns, Jane L; Sá-Correia, Isabel; De Vos, Daniel; Pirnay, Jean-Paul; Kidd, Timothy J; Reid, David; Manos, Jim; Klockgether, Jens; Wiehlmann, Lutz; Tümmler, Burkhard; McClean, Siobhán; Winstanley, Craig

    2013-01-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents. PMID:24214409

  17. Transport of C5 dicarboxylate compounds by Pseudomonas putida.

    PubMed Central

    Edwards, W V; Sando, J J; Hartline, R A

    1979-01-01

    Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate. PMID:479107

  18. Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

    PubMed Central

    Paliwal, Vasundhara; Raju, Sajan C.; Modak, Arnab; Phale, Prashant S.; Purohit, Hemant J.

    2014-01-01

    Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNAGly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation. PMID:24475028

  19. Draft Genome Sequence of Medium-Chain-Length Polyhydroxyalkanoate-Producing Pseudomonas putida Strain LS46.

    PubMed

    Sharma, Parveen K; Fu, Jilagamazhi; Zhang, Xiangli; Fristensky, Brian W; Davenport, Karen; Chain, Patrick S G; Sparling, Richard; Levin, David B

    2013-04-18

    We describe the draft genome sequence of Pseudomonas putida strain LS46, a novel isolate that synthesizes medium-chain-length polyhydroxyalkanoates. The draft genome of P. putida LS46 consists of approximately 5.86 million bp, with a G+C content of 61.69%. A total of 5,316 annotated genes and 5,219 coding sequences (CDS) were identified.

  20. Complete Genome Sequence of the p-Nitrophenol-Degrading Bacterium Pseudomonas putida DLL-E4

    PubMed Central

    Hu, Xiaojun; Wang, Jue; Wang, Fei; Chen, Qiongzhen; Huang, Yan

    2014-01-01

    The first complete genome sequence of a p-nitrophenol (PNP)-degrading bacterium is reported here. Pseudomonas putida DLL-E4, a Gram-negative bacterium isolated from methyl-parathion-polluted soil, can utilize PNP as the sole carbon and nitrogen source. P. putida DLL-E4 has a 6,484,062 bp circular chromosome that contains 5,894 genes, with a G+C content of 62.46%. PMID:24948765

  1. Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida

    SciTech Connect

    Wu X.; van der Lelie D.; Monchy, S.; Taghavi, S.; Zhu, W.; Ramos, J.

    2011-03-01

    Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands.

  2. Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida

    PubMed Central

    Wu, Xiao; Monchy, Sébastien; Taghavi, Safiyh; Zhu, Wei; Ramos, Juan; van der Lelie, Daniel

    2011-01-01

    Pseudomonas putida is a gram-negative rod-shaped gammaproteobacterium that is found throughout various environments. Members of the species P. putida show a diverse spectrum of metabolic activities, which is indicative of their adaptation to various niches, which includes the ability to live in soils and sediments contaminated with high concentrations of heavy metals and organic contaminants. Pseudomonas putida strains are also found as plant growth-promoting rhizospheric and endophytic bacteria. The genome sequences of several P. putida species have become available and provide a unique tool to study the specific niche adaptation of the various P. putida strains. In this review, we compare the genomes of four P. putida strains: the rhizospheric strain KT2440, the endophytic strain W619, the aromatic hydrocarbon-degrading strain F1 and the manganese-oxidizing strain GB-1. Comparative genomics provided a powerful tool to gain new insights into the adaptation of P. putida to specific lifestyles and environmental niches, and clearly demonstrated that horizontal gene transfer played a key role in this adaptation process, as many of the niche-specific functions were found to be encoded on clearly defined genomic islands. PMID:20796030

  3. Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6

    SciTech Connect

    Spain, J.C.; Gibson, D.T.

    1988-06-01

    The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.

  4. 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.

  5. Pseudomonas Aeruginosa: Resistance to the Max

    PubMed Central

    Poole, Keith

    2011-01-01

    Pseudomonas aeruginosa is intrinsically resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism. Resistance to multiple classes of antimicrobials (multidrug resistance) in particular is increasingly common in P. aeruginosa, with a number of reports of pan-resistant isolates treatable with a single agent, colistin. Acquired resistance in this organism is multifactorial and attributable to chromosomal mutations and the acquisition of resistance genes via horizontal gene transfer. Mutational changes impacting resistance include upregulation of multidrug efflux systems to promote antimicrobial expulsion, derepression of ampC, AmpC alterations that expand the enzyme's substrate specificity (i.e., extended-spectrum AmpC), alterations to outer membrane permeability to limit antimicrobial entry and alterations to antimicrobial targets. Acquired mechanisms contributing to resistance in P. aeruginosa include β-lactamases, notably the extended-spectrum β-lactamases and the carbapenemases that hydrolyze most β-lactams, aminoglycoside-modifying enzymes, and 16S rRNA methylases that provide high-level pan-aminoglycoside resistance. The organism's propensity to grow in vivo as antimicrobial-tolerant biofilms and the occurrence of hypermutator strains that yield antimicrobial resistant mutants at higher frequency also compromise anti-pseudomonal chemotherapy. With limited therapeutic options and increasing resistance will the untreatable P. aeruginosa infection soon be upon us? PMID:21747788

  6. Pseudomonas aeruginosa endophthalmitis masquerading as chronic uveitis

    PubMed Central

    Nagaraj, Kalpana Badami; Jayadev, Chaitra

    2013-01-01

    A 65-year-old male presented with decreased vision in the left eye of 15-day duration after having undergone an uneventful cataract surgery 10 months back. He had been previously treated with systemic steroids for recurrent uveitis postoperatively on three occasions in the same eye. B-scan ultrasonography showed multiple clumplike echoes suggestive of vitreous inflammation. Aqueous tap revealed Pseudomonas aeruginosa sensitive to ciprofloxacin. The patient was treated with intravitreal ciprofloxacin and vancomycin along with systemic ciprofloxacin with good clinical response. Even a virulent organism such as P.aeruginosa can present as a chronic uveitis, which, if missed, can lead to a delay in accurate diagnosis and appropriate management. PMID:23803484

  7. Pseudomonas aeruginosa ventilator-associated pneumonia management

    PubMed Central

    Ramírez-Estrada, Sergio; Borgatta, Bárbara; Rello, Jordi

    2016-01-01

    Ventilator-associated pneumonia is the most common infection in intensive care unit patients associated with high morbidity rates and elevated economic costs; Pseudomonas aeruginosa is one of the most frequent bacteria linked with this entity, with a high attributable mortality despite adequate treatment that is increased in the presence of multiresistant strains, a situation that is becoming more common in intensive care units. In this manuscript, we review the current management of ventilator-associated pneumonia due to P. aeruginosa, the most recent antipseudomonal agents, and new adjunctive therapies that are shifting the way we treat these infections. We support early initiation of broad-spectrum antipseudomonal antibiotics in present, followed by culture-guided monotherapy de-escalation when susceptibilities are available. Future management should be directed at blocking virulence; the role of alternative strategies such as new antibiotics, nebulized treatments, and vaccines is promising. PMID:26855594

  8. Risk assessment of Pseudomonas aeruginosa in water.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

    from ingesting P. aeruginosa in drinking water is low. The risk is slightly higher if the subject is taking an antibiotic resisted by P. aeruginosa. The fact that individuals on ampicillin are more susceptible to Pseudomonas gastrointestinal infection probably results from suppression of normal intestinal flora, which would allow Pseudomonas to colonize. The process of estimating risk was significantly constrained because of the absence of specific (quantitative) occurrence data for Pseudomonas. Sensitivity analysis shows that the greatest source of variability/uncertainty in the risk assessment is from the density distribution in the exposure rather than the dose-response or water consumption distributions. In summary, two routes appear to carry the greatest health risks from contacting water contaminated with P. aeruginosa (1) skin exposure in hot tubs and (2) lung exposure from inhaling aerosols.

  9. Physical Forces Shape Group Identity of Swimming Pseudomonas putida Cells

    PubMed Central

    Espeso, David R.; Martínez-García, Esteban; de Lorenzo, Víctor; Goñi-Moreno, Ángel

    2016-01-01

    The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving toward each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e., intolerance to mix in time and space with otherwise identical others) has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end, we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces—not genetic or metabolic programs. PMID

  10. Physical Forces Shape Group Identity of Swimming Pseudomonas putida Cells

    PubMed Central

    Espeso, David R.; Martínez-García, Esteban; de Lorenzo, Víctor; Goñi-Moreno, Ángel

    2016-01-01

    The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving toward each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e., intolerance to mix in time and space with otherwise identical others) has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end, we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces—not genetic or metabolic programs.

  11. The Pseudomonas aeruginosa Proteome during Anaerobic Growth‡

    PubMed Central

    Wu, Manhong; Guina, Tina; Brittnacher, Mitchell; Nguyen, Hai; Eng, Jimmy; Miller, Samuel I.

    2005-01-01

    Isotope-coded affinity tag analysis and two-dimensional gel electrophoresis followed by tandem mass spectrometry were used to identify Pseudomonas aeruginosa proteins expressed during anaerobic growth. Out of the 617 proteins identified, 158 were changed in abundance during anaerobic growth compared to during aerobic growth, including proteins whose increased expression was expected based on their role in anaerobic metabolism. These results form the basis for future analyses of alterations in bacterial protein content during growth in various environments, including the cystic fibrosis airway. PMID:16291692

  12. Siderotyping of fluorescent pseudomonads: characterization of pyoverdines of Pseudomonas fluorescens and Pseudomonas putida strains from Antarctica.

    PubMed

    Meyer, J M; Stintzi, A; Coulanges, V; Shivaji, S; Voss, J A; Taraz, K; Budzikiewicz, H

    1998-11-01

    Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens 10CW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour to each other, whereas the fifth strain, P. fluorescens 51W, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 10CW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared to be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines. PMID:9846748

  13. Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level.

    PubMed

    Silge, Anja; Schumacher, Wilm; Rösch, Petra; Da Costa Filho, Paulo A; Gérard, Cédric; Popp, Jürgen

    2014-07-01

    The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis.

  14. Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level.

    PubMed

    Silge, Anja; Schumacher, Wilm; Rösch, Petra; Da Costa Filho, Paulo A; Gérard, Cédric; Popp, Jürgen

    2014-07-01

    The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis. PMID:24958608

  15. Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

    PubMed

    Asif, Huma; Studholme, David J; Khan, Asifullah; Aurongzeb, M; Khan, Ishtiaq A; Azim, M Kamran

    2016-01-01

    We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches. PMID:27560648

  16. Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard

    PubMed Central

    Asif, Huma; Studholme, David J.; Khan, Asifullah; Aurongzeb, M.; Khan, Ishtiaq A.; Azim, M. Kamran

    2016-01-01

    Abstract We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches. PMID:27560648

  17. Acute toxic effects of three pesticides on Pseudomonas putida monitored by microcalorimeter.

    PubMed

    Chen, Hui-Lun; Yao, Jun; Wang, Fei; Bramanti, Emilia; Maskow, Thomas; Zaray, Gyula

    2009-02-01

    A series of calorimetric experiments were performed to investigate the toxic effects of beta-cypermethrin (BCP), bensulfuron-methyl (BSM) and prometryne (PM) on Pseudomonas putida (P. putida). The metabolic action of P. putida on the three pesticides was studied by obtaining power-time curves. The growth of P. putida was inhibited completely in each case when the concentrations of pesticides were up to 80 micro g mL(- 1). The relationships between the inhibitory ratio (k) and doses of contaminants were approximately linear for the three pesticides. The total heat dissipated per milliliter (Q(total)) for the pesticides decreased during the course of the experiment. The OD(600) of P. putida growth in the absence and presence of pesticides was also obtained. The power-time curves of P. putida growth coincided with its turbidity curves. This elucidates that microcalorimetric method agrees well with the routine microbiological method. Among these three pesticides, BSM was found to be the most toxic with an IC(50) of 19.24 micro g mL(- 1) against P. putida. PM exhibited moderate virulence with an IC(50) of 27.86 micro g mL(- 1) and BCP had the lowest toxicity with an IC(50) of 39.64 micro g mL(- 1). PMID:19130374

  18. Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

    PubMed Central

    Domenech, Carlos Eduardo; Otero, Lisandro Horacio; Beassoni, Paola Rita; Lisa, Angela Teresita

    2011-01-01

    Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O)2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure. PMID:21915373

  19. Comparative genomic and functional analyses: unearthing the diversity and specificity of nematicidal factors in Pseudomonas putida strain 1A00316

    PubMed Central

    Guo, Jing; Jing, Xueping; Peng, Wen-Lei; Nie, Qiyu; Zhai, Yile; Shao, Zongze; Zheng, Longyu; Cai, Minmin; Li, Guangyu; Zuo, Huaiyu; Zhang, Zhitao; Wang, Rui-Ru; Huang, Dian; Cheng, Wanli; Yu, Ziniu; Chen, Ling-Ling; Zhang, Jibin

    2016-01-01

    We isolated Pseudomonas putida (P. putida) strain 1A00316 from Antarctica. This bacterium has a high efficiency against Meloidogyne incognita (M. incognita) in vitro and under greenhouse conditions. The complete genome of P. putida 1A00316 was sequenced using PacBio single molecule real-time (SMRT) technology. A comparative genomic analysis of 16 Pseudomonas strains revealed that although P. putida 1A00316 belonged to P. putida, it was phenotypically more similar to nematicidal Pseudomonas fluorescens (P. fluorescens) strains. We characterized the diversity and specificity of nematicidal factors in P. putida 1A00316 with comparative genomics and functional analysis, and found that P. putida 1A00316 has diverse nematicidal factors including protein alkaline metalloproteinase AprA and two secondary metabolites, hydrogen cyanide and cyclo-(l-isoleucyl-l-proline). We show for the first time that cyclo-(l-isoleucyl-l-proline) exhibit nematicidal activity in P. putida. Interestingly, our study had not detected common nematicidal factors such as 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin in P. putida 1A00316. The results of the present study reveal the diversity and specificity of nematicidal factors in P. putida strain 1A00316. PMID:27384076

  20. Cryptic transposable phages of Pseudomonas aeruginosa

    SciTech Connect

    Krylov, V.N.; Mit`kina, L.N.; Pleteneva, E.A.; Aleshin, V.V.

    1995-11-01

    Frequencies of nucleotide sequences homologous to phage transposons (PT) of two species, D3112 and B3, were assessed in genomes of natural Pseudomonas aeruginosa strains by the dot-blot hybridization method. These strains were incapable of liberating viable phages on a lawn of the PA01 standard indicator strain of P. aeruginosa. It was shown that the homologies detected belong to two groups, high and intermediate, with respect to homology level. Homology patterns were classified as high when they provided signals comparable to those for hybridization in a positive control; patterns were classified as intermediate when the hybridization level was higher than the background level, but lower than in the positive control. Homologous PT sequences were designated as cryptic PT. Intact cryptic PT prophages were shown to exist in genomes of particular natural strains manifesting a higher level of hybridization. However, the growth of these phages was limited by the restriction system of strain PA01. It is possible to isolate strains maintaining the growth of some cryptic PT. These strains differed from P. aeruginosa with respect to the specificity of the restriction and modification system. Nevertheless, in most cases, the attempt to identify a novel host capable of maintaining growth of a cryptic PT failed. Natural strains often carry cryptic PT related to both known PT species, D3112 and B3. The frequency of cryptic PT is extremely high, reaching 30% in strains with a high level of homology only and up to 50% in all strains exhibiting homology. This high PT frequency is assumed to be associated with the considerable variation of P. aeruginosa. 15 refs., 1 fig., 2 tabs.

  1. 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

  2. ACTIVE EFFLUX OF ORGANIC SOLVENTS BY PSEUDOMONAS PUTIDA S12 IS INDUCED BY SOLVENTS

    EPA Science Inventory

    Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the...

  3. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis.

    PubMed

    Shirakawa, Márcia Aiko; Cincotto, Maria Alba; Atencio, Daniel; Gaylarde, Christine C; John, Vanderley M

    2011-04-01

    The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials. PMID:24031661

  4. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

    PubMed Central

    Chang, Philip Lee; Yen, Teh Fu

    1985-01-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed. PMID:16346956

  5. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock.

    PubMed

    Chang, P L; Yen, T F

    1985-12-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed.

  6. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

    PubMed Central

    Shirakawa, Márcia Aiko; Cincotto, Maria Alba; Atencio, Daniel; Gaylarde, Christine C.; John, Vanderley M.

    2011-01-01

    The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials. PMID:24031661

  7. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis.

    PubMed

    Shirakawa, Márcia Aiko; Cincotto, Maria Alba; Atencio, Daniel; Gaylarde, Christine C; John, Vanderley M

    2011-04-01

    The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

  8. Genome sequence of the plant growth-promoting rhizobacterium Pseudomonas putida S11.

    PubMed

    Ponraj, Paramasivan; Shankar, Manoharan; Ilakkiam, Devaraj; Rajendhran, Jeyaprakash; Gunasekaran, Paramasamy

    2012-11-01

    Here we report the genome sequence of a plant growth-promoting rhizobacterium, Pseudomonas putida S11. The length of the draft genome sequence is approximately 5,970,799 bp, with a G+C content of 62.4%. The genome contains 6,076 protein-coding sequences.

  9. Draft Genome Sequences of Four Hospital-Associated Pseudomonas putida Isolates

    PubMed Central

    Marsh, Jane W.; Ezeonwuka, Chinelo D.; Pasculle, Anthony W.; Pacey, Marissa P.; Querry, Ashley M.; Muto, Carlene A.; Harrison, Lee H.

    2016-01-01

    We present here the draft genome sequences of four Pseudomonas putida isolates belonging to a single clone suspected for nosocomial transmission between patients and a bronchoscope in a tertiary hospital. The four genome sequences belong to a single lineage but contain differences in their mobile genetic elements. PMID:27688339

  10. Draft Genome Sequences of Four Hospital-Associated Pseudomonas putida Isolates.

    PubMed

    Mustapha, Mustapha M; Marsh, Jane W; Ezeonwuka, Chinelo D; Pasculle, Anthony W; Pacey, Marissa P; Querry, Ashley M; Muto, Carlene A; Harrison, Lee H

    2016-01-01

    We present here the draft genome sequences of four Pseudomonas putida isolates belonging to a single clone suspected for nosocomial transmission between patients and a bronchoscope in a tertiary hospital. The four genome sequences belong to a single lineage but contain differences in their mobile genetic elements. PMID:27688339

  11. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    ERIC Educational Resources Information Center

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students a chance…

  12. Complete Genome of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida BIRD-1

    SciTech Connect

    Matilla, M.A.; van der Lelie, D.; Pizarro-Tobias, P.; Roca, A.; Fernandez, M.; Duque, E.; Molina, L.; Wu, X.; Gomez, M. J.; Segura, A.; Ramos, J.-L.

    2011-03-01

    We report the complete sequence of the 5.7-Mbp genome of Pseudomonas putida BIRD-1, a metabolically versatile plant growth-promoting rhizobacterium that is highly tolerant to desiccation and capable of solubilizing inorganic phosphate and iron and of synthesizing phytohormones that stimulate seed germination and plant growth.

  13. Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94.

    PubMed

    Mendes, Sónia; Pereira, Luciana; Batista, Carlos; Martins, Lígia O

    2011-10-01

    Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi-bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.

  14. Manganese (Mn) Oxidation Increases Intracellular Mn in Pseudomonas putida GB-1

    PubMed Central

    Banh, Andy; Chavez, Valarie; Doi, Julia; Nguyen, Allison; Hernandez, Sophia; Ha, Vu; Jimenez, Peter; Espinoza, Fernanda; Johnson, Hope A.

    2013-01-01

    Bacterial manganese (Mn) oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS). Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection. PMID:24147089

  15. Vesiculation from Pseudomonas aeruginosa under SOS

    PubMed Central

    Maredia, Reshma; Devineni, Navya; Lentz, Peter; Dallo, Shatha F.; Yu, JiehJuen; Guentzel, Neal; Chambers, James; Arulanandam, Bernard; Haskins, William E.; Weitao, Tao

    2012-01-01

    Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity. PMID:22448133

  16. Cotransport of TiO2 nanoparticles and Pseudomonas putida in porous media

    NASA Astrophysics Data System (ADS)

    Zaharis, Ioannis; Manariotis, Ioannis D.; Chrysikopoulos, Constantinos V.

    2015-04-01

    The scope of this study was to investigate the cotransport of Pseudomonas putida and TiO2 nanoparticles (NPs) in porous media. Flowthrough experiments were conducted in glass columns with diameter of 2.5 cm and length of 30 cm, packed with 2-mm diameter spherical glass beads. Anatase TiO2 NPs solutions were prepared in distilled water of at two different concentrations: 5 and 50 mg/L. The concentration of P. putida solutions varied from 105 to 109 cfu/mL. Initially, transport experiments were conducted separately for P. putida and TiO2 NPs. Subsequently, TiO2 and P. putida cotransport experiments were conducted. The concentration of TiO2 NPs was measured by a fluorescence spectrophotometer and P. putida concentration was determined by plate counts on agar plates and optical density measurements. All experiments were conducted with two different flow rates: 1 and 2 mL/min. The transport experiments with P. putida exhibited similar transport behavior with the tracer (NaBr) indicating that there was not considerable retention. The mass recovery of P. putida was close to 100% in all of the transport experiments conducted. However, the transport experiments with TiO2 NPs suggested that a significant portion of the NPs was retained in the column. Based on the cotransport experimental data, it is evident that the transport of P. putida was not significantly affected by the presence of TiO2. It should be noted that the mass recovery of NPs in the transport and costransport experiments was between 40 and 60%.

  17. Isolation of oxidase-negative Pseudomonas aeruginosa from sputum culture.

    PubMed Central

    Hampton, K D; Wasilauskas, B L

    1979-01-01

    Two isolates of Pseudomonas aeruginosa lacking characteristic indophenol oxidase were recovered from a sputum specimen. A discussion of the characteristic biochemical tests and antibiograms along with a possible explanation for this phenomenon is presented. PMID:225349

  18. Two unusual pilin sequences from different isolates of Pseudomonas aeruginosa.

    PubMed Central

    Pasloske, B L; Sastry, P A; Finlay, B B; Paranchych, W

    1988-01-01

    The pilin genes of two Pseudomonas aeruginosa strains isolated from two different patients with cystic fibrosis were cloned and sequenced. The predicted protein sequences of these two pilins had several unusual features compared with other published P. aeruginosa pilin sequences. PMID:2841299

  19. Heavy metal tolerance genes alter cellular thermodynamics in Pseudomonas putida and river Pseudomonas spp. and influence amebal predation.

    PubMed

    McTee, Michael R; Gibbons, Sean M; Feris, Kevin; Gordon, Nathan S; Gannon, James E; Ramsey, Philip W

    2013-10-01

    Predation rates were measured for two Acanthamoeba castellanii strains feeding on metal-tolerant and metal-sensitive strains of Pseudomonas putida and compared with cellular thermodynamic data. Predation rates by A. castellanii strain ATCC 30010 correlated with cell volume of the prey. To explore whether this observation could be environmentally relevant, pseudomonad species were isolated from a pristine and a metal-contaminated river and were paired based on phylogenetic and physiological relatedness. Then, cellular thermodynamics and predation rates were measured on the most similar pseudomonad pair. Under cadmium stress, the strain from contaminated river sediments, Pseudomonas sp. CF150, exited metabolic dormancy faster than its pair from pristine sediments, Pseudomonas sp. N9, but consumed available resources less efficiently (more energy was lost as heat). Predation rates by both strains of ameba were greater on Pseudomonas sp. CF150 than on Pseudomonas sp. N9 at the highest cadmium concentration. PMID:23895438

  20. Expression analysis of the fpr (ferredoxin-NADP{sup +} reductase) gene in Pseudomonas putida KT2440

    SciTech Connect

    Lee, Yunho; Pena-Llopis, Samuel; Kang, Yoon-Suk; Shin, Hyeon-Dong; Demple, Bruce; Madsen, Eugene L.; Jeon, Che Ok; Park, Woojun . E-mail: wpark@korea.ac.kr

    2006-01-27

    The ferredoxin-NADP{sup +} reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440.

  1. Redundancy in putrescine catabolism in solvent tolerant Pseudomonas putida S12.

    PubMed

    Bandounas, Luaine; Ballerstedt, Hendrik; de Winde, Johannes H; Ruijssenaars, Harald J

    2011-06-10

    Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines have also been implicated in stressed cells. Previous transcriptomics results of P. putida strains producing an aromatic compound, or being exposed to the solvent toluene, indicated differentially expressed genes involved in polyamine transport and metabolism. Therefore, the metabolism of the polyamine, putrescine was investigated in P. putida S12, as no putrescine degradation pathways have been described for this strain. Via transcriptome analysis various, often redundant, putrescine-induced genes were identified as being potentially involved in putrescine catabolism via oxidative deamination and transamination. A series of knockout mutants were constructed in which up to six of these genes were sequentially deleted, and although putrescine degradation was affected in some of these mutants, complete elimination of putrescine degradation in P. putida S12 was not achieved. Evidence was found for the presence of an alternative pathway for putrescine degradation involving γ-glutamylation. The occurrence of multiple putrescine degradation routes in the solvent-tolerant P. putida S12 is indicative of the importance of controlling polyamine homeostasis, as well as of the high metabolic flexibility exhibited by this microorganism.

  2. Toxicity of graphene oxide on growth and metabolism of Pseudomonas putida.

    PubMed

    Combarros, R G; Collado, S; Díaz, M

    2016-06-01

    The increasing consumption of graphene derivatives leads to greater presence of these materials in wastewater treatment plants and ecological systems. The toxicity effect of graphene oxide (GO) on the microbial functions involved in the biological wastewater treatment process is studied, using Pseudomonas putida and salicylic acid (SA) as bacterial and pollutant models. A multiparametric flow cytometry (FC) method has been developed to measure the metabolic activity and viability of P. putida in contact with GO. A continuous reduction in the percentages of viable cells and a slight increase, lower than 5%, in the percentages of damaged and dead cells, suggest that P. putida in contact with GO loses the membrane integrity but preserves metabolic activity. The growth of P. putida was strongly inhibited by GO, since 0.05mgmL(-1) of GO reduced the maximum growth by a third, and the inhibition was considerably greater for GO concentrations higher than 0.1mgmL(-1). The specific SA removal rate decreased with GO concentration up to 0.1mgmL(-1) indicating that while GO always reduces the growth of P. putida, for concentrations higher than 0.1mgmL(-1), it also reduces its activity. Similar behaviour is observed using simulated urban and industrial wastewaters, the observed effects being more acute in the industrial wastewaters.

  3. Ni2+ removal and recovery from electroplating effluent by Pseudomonas putida 5-x cell biomass.

    PubMed

    Wang, L; Chua, H; Wong, P K; Lo, W H; Yu, P H F

    2003-03-01

    Ni2+ and Cu2+ are the major heavy metal ions in electroplating wastewater of Hong Kong. In the present study, Pseudomonas putida 5-x cell biomass was used to remove Ni2+ from electroplating effluent. Ni2+ adsorption capacity of P. putida 5-x cell biomass cultured in sulphate-limiting medium was found to be minimum in early logarithmic growth phase, and maximum of 28.1 mg g(-1) in late stationary growth phase. Pretreated cells by 0.1 mol L(-1) HCl could greatly enhance the Ni2+ adsorption capacity of cell biomass from 28.1 to 36.7 mg g(-1) and had no significant effect on biomass loss. The adsorption process of P. putida 5-x fresh cells and pretreated cell all could be expressed with Freundlich isotherm. TEM analyses indicated that acidic pretreatment degraded the superficial layer-capsule outside of the fresh cell to improve the adsorption capacity of cell to Ni2+. The Ni2+ bound by P. putida 5-x cell biomass could be efficiently recovered using 0.1 mol L(-1) HCl, and the cell biomass could be reused at least five cycles for Ni2+ removal and recovery with 93% above removal efficiency and 98% above recovery rate. Owing to the Cu2+ presented in electroplating wastewater inhibiting Ni2+ adsorption process by P. putida 5-x cell biomass, two-stage biosorption processes should be designed to remove and recover Cu2+ and Ni2+ sequentially from electroplating effluent.

  4. The Regulatory Network of Pseudomonas aeruginosa

    PubMed Central

    2011-01-01

    Background Pseudomonas aeruginosa is an important bacterial model due to its metabolic and pathogenic abilities, which allow it to interact and colonize a wide range of hosts, including plants and animals. In this work we compile and analyze the structure and organization of an experimentally supported regulatory network in this bacterium. Results The regulatory network consists of 690 genes and 1020 regulatory interactions between their products (12% of total genes: 54% sigma and 16% of transcription factors). This complex interplay makes the third largest regulatory network of those reported in bacteria. The entire network is enriched for activating interactions and, peculiarly, self-activation seems to occur more prominent for transcription factors (TFs), which contrasts with other biological networks where self-repression is dominant. The network contains a giant component of 650 genes organized into 11 hierarchies, encompassing important biological processes, such as, biofilms formation, production of exopolysaccharide alginate and several virulence factors, and of the so-called quorum sensing regulons. Conclusions The study of gene regulation in P. aeruginosa is biased towards pathogenesis and virulence processes, all of which are interconnected. The network shows power-law distribution -input degree -, and we identified the top ten global regulators, six two-element cycles, the longest paths have ten steps, six biological modules and the main motifs containing three and four elements. We think this work can provide insights for the design of further studies to cover the many gaps in knowledge of this important bacterial model, and for the design of systems strategies to combat this bacterium. PMID:22587778

  5. Ambroxol interferes with Pseudomonas aeruginosa quorum sensing.

    PubMed

    Lu, Qi; Yu, Jialin; Yang, Xiqiang; Wang, Jiarong; Wang, Lijia; Lin, Yayin; Lin, Lihua

    2010-09-01

    The mucolytic agent ambroxol has been reported to interfere with the formation of Pseudomonas aeruginosa-derived biofilms in addition to reducing alginate production by undefined mechanisms. Since quorum sensing is a key regulator of virulence and biofilm formation, we examined the effects of ambroxol on P. aeruginosa PAO1 wild-type bacterial clearance rates, adhesion profiles and biofilm formation compared with the quorum sensing-deficient, double-mutant strains DeltalasR DeltarhlR and DeltalasI DeltarhlI. Data presented in this report demonstrated that ambroxol treatment reduced survival rates of the double-mutant strains compared with the wild-type strain in a dose-dependent manner even though the double-mutants had increased adhesion in the presence of ambroxol compared with the wild-type strain. The PAO1 wild-type strain produced a significantly thicker biofilm (21.64+/-0.57 microm) compared with the biofilms produced by the DeltalasR DeltarhlR (7.36+/-0.2 microm) and DeltalasI DeltarhlI (6.62+/-0.31 microm) isolates. Ambroxol treatment reduced biofilm thickness, increased areal porosity, and decreased the average diffusion distance and textual entropy of wild-type and double-mutant strains. However, compared with the double-mutant strains, the changes observed for the wild-type strain were more clearly defined. Finally, ambroxol exhibited significant antagonistic quorum-sensing properties, suggesting that it could be adapted for use clinically in the treatment of cystic fibrosis and to reduce biofilm formation and in the colonisation of indwelling devices. PMID:20580207

  6. Ambroxol interferes with Pseudomonas aeruginosa quorum sensing.

    PubMed

    Lu, Qi; Yu, Jialin; Yang, Xiqiang; Wang, Jiarong; Wang, Lijia; Lin, Yayin; Lin, Lihua

    2010-09-01

    The mucolytic agent ambroxol has been reported to interfere with the formation of Pseudomonas aeruginosa-derived biofilms in addition to reducing alginate production by undefined mechanisms. Since quorum sensing is a key regulator of virulence and biofilm formation, we examined the effects of ambroxol on P. aeruginosa PAO1 wild-type bacterial clearance rates, adhesion profiles and biofilm formation compared with the quorum sensing-deficient, double-mutant strains DeltalasR DeltarhlR and DeltalasI DeltarhlI. Data presented in this report demonstrated that ambroxol treatment reduced survival rates of the double-mutant strains compared with the wild-type strain in a dose-dependent manner even though the double-mutants had increased adhesion in the presence of ambroxol compared with the wild-type strain. The PAO1 wild-type strain produced a significantly thicker biofilm (21.64+/-0.57 microm) compared with the biofilms produced by the DeltalasR DeltarhlR (7.36+/-0.2 microm) and DeltalasI DeltarhlI (6.62+/-0.31 microm) isolates. Ambroxol treatment reduced biofilm thickness, increased areal porosity, and decreased the average diffusion distance and textual entropy of wild-type and double-mutant strains. However, compared with the double-mutant strains, the changes observed for the wild-type strain were more clearly defined. Finally, ambroxol exhibited significant antagonistic quorum-sensing properties, suggesting that it could be adapted for use clinically in the treatment of cystic fibrosis and to reduce biofilm formation and in the colonisation of indwelling devices.

  7. Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid

    PubMed Central

    Fernández, Matilde; Niqui-Arroyo, José Luis; Conde, Susana; Duque, Estrella

    2012-01-01

    In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [14C]naphthalene, measured as the evolution of 14CO2, while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil. PMID:22582075

  8. Draft Genome Sequence of Caprolactam-Degrading Pseudomonas putida Strain SJ3

    PubMed Central

    Hong, Sung-Jun; Park, Gun-Seok; Khan, Abdur Rahim; Jung, Byung Kown; Park, Yeong-Jun; Yoo, Na-Kyung; Lee, Changhee; Park, Choi Kyu

    2015-01-01

    Pseudomonas putida strain SJ3, which possesses caprolactam-degrading ability, was isolated from dyeing industry wastewater in Daegu, Republic of Korea. Here, we describe the draft genome sequence and annotation of the strain. The 5,596,765-bp-long genome contains 4,293 protein-coding genes and 68 RNA genes with 61.70% G+C content. PMID:26205864

  9. Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

    PubMed Central

    Cadieux, J E; Kuzio, J; Milazzo, F H; Kropinski, A M

    1983-01-01

    Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C. Images PMID:6409883

  10. Spaceflight promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Kim, Wooseong; Tengra, Farah K; Young, Zachary; Shong, Jasmine; Marchand, Nicholas; Chan, Hon Kit; Pangule, Ravindra C; Parra, Macarena; Dordick, Jonathan S; Plawsky, Joel L; Collins, Cynthia H

    2013-01-01

    Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight. PMID:23658630

  11. Poly‐3‐hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies

    PubMed Central

    Arias, Sagrario; Sandoval, Angel; Arcos, Mario; Cañedo, Librada M.; Maestro, Beatriz; Sanz, Jesús M.; Naharro, Germán; Luengo, José M.

    2008-01-01

    Summary The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium‐chain‐length poly‐hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC‐phaC1)or only phaC2(P. putida U Δpha pMC‐phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC‐phaC1strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5‐phenylvaleric, 6‐phenylhexanoic, 7‐phenylheptanoic, 8‐phenyloctanoic or 9‐phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6‐phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC‐phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3‐hydroxy‐5‐phenylvaleryl‐CoA. The possible influence of the putative three‐dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed. PMID:21261834

  12. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation.

  13. Nosocomial infections due to Pseudomonas aeruginosa: review of recent trends.

    PubMed

    Cross, A; Allen, J R; Burke, J; Ducel, G; Harris, A; John, J; Johnson, D; Lew, M; MacMillan, B; Meers, P

    1983-01-01

    The role of Pseudomonas aeruginosa in nosocomial infections occurring since 1975 is reviewed. Data from the National Nosocomial Infections Study conducted by the Centers for Disease Control, from individual medical centers, and from the literature were used to compare the relative frequency of occurrence of nosocomial infection caused by P. aeruginosa with that of infection caused by other gram-negative bacilli. The relative frequency of P. aeruginosa as a nosocomial pathogen has increased, although wide variations are seen among individual medical centers. P. aeruginosa continues to be a major pathogen among patients with immunosuppression, cystic fibrosis, malignancy, and trauma. While Staphylococcus aureus has become the predominant pathogen in some large burn centers, P. aeruginosa is the most important gram-negative pathogen. Periodic review of the epidemiology of P. aeruginosa infection is warranted in view of the changing incidence of infection caused by this organism.

  14. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation. PMID:27392247

  15. Biosorption of aluminum through the use of non-viable biomass of Pseudomonas putida.

    PubMed

    Boeris, Paola Sabrina; Agustín, María Del Rosario; Acevedo, Diego Fernando; Lucchesi, Gloria Inés

    2016-10-20

    Living and non-living biomass of Pseudomonas putida A (ATCC 12633) was used as biosorbent for the removing of Al(3+) from aqueous solutions. The process was stable with time, efficient at pH 4.3 and between 15°C and 42°C. Two isotherms models were applied to describe the interaction between the biosorbent and Al(3+). Non-living biomass of P. putida A (ATCC 12633) was found to be the most efficient at adsorbing Al(3+) with a maximum sorption capacity of 0.55mg Al(3+)/gr adsorbent and with 36×10(5) binding sites of Al(3+)/microorganisms. Infrared spectroscopy analysis shows that the biosorbent present some vibrational band of functional groups that change in presence of Al(3+): hydroxyl, carboxyl and phosphate. Considering that Al(3+) binds to the phosphate group of phosphatidylcholine, non-viable biomass of P. putida PB01 (mutant lacking phosphatidylcholine) was used. Aluminum adsorption of the parental strain was 30 times higher than values registered in P. putida PB01 (36×10(5) sites/microorganism vs 1.2×10(5) sites/microorganism, respectively). This result evidenced that the absence of phosphatidylcholine significantly affected the availability of the binding sites and consequently the efficiency of the biomass to adsorb Al(3+). PMID:27485814

  16. Biosorption of aluminum through the use of non-viable biomass of Pseudomonas putida.

    PubMed

    Boeris, Paola Sabrina; Agustín, María Del Rosario; Acevedo, Diego Fernando; Lucchesi, Gloria Inés

    2016-10-20

    Living and non-living biomass of Pseudomonas putida A (ATCC 12633) was used as biosorbent for the removing of Al(3+) from aqueous solutions. The process was stable with time, efficient at pH 4.3 and between 15°C and 42°C. Two isotherms models were applied to describe the interaction between the biosorbent and Al(3+). Non-living biomass of P. putida A (ATCC 12633) was found to be the most efficient at adsorbing Al(3+) with a maximum sorption capacity of 0.55mg Al(3+)/gr adsorbent and with 36×10(5) binding sites of Al(3+)/microorganisms. Infrared spectroscopy analysis shows that the biosorbent present some vibrational band of functional groups that change in presence of Al(3+): hydroxyl, carboxyl and phosphate. Considering that Al(3+) binds to the phosphate group of phosphatidylcholine, non-viable biomass of P. putida PB01 (mutant lacking phosphatidylcholine) was used. Aluminum adsorption of the parental strain was 30 times higher than values registered in P. putida PB01 (36×10(5) sites/microorganism vs 1.2×10(5) sites/microorganism, respectively). This result evidenced that the absence of phosphatidylcholine significantly affected the availability of the binding sites and consequently the efficiency of the biomass to adsorb Al(3+).

  17. Effect of Pseudomonas putida on Growth and Anthocyanin Pigment in Two Poinsettia (Euphorbia pulcherrima) Cultivars

    PubMed Central

    Zulueta-Rodriguez, Ramon; Cordoba-Matson, Miguel Victor; Hernandez-Montiel, Luis Guillermo; Murillo-Amador, Bernardo; Rueda-Puente, Edgar; Lara, Liliana

    2014-01-01

    Pseudomonas putida is plant growth promoting rhizobacteria (PGPR) that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble) using three strains of P. putida, as well as a mixture of the three (MIX). Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4), volume of roots (45 versus 35 cm3), number of leaves (78 versus 58), and area of leaf (1,788 versus 1,331 cm2), except for the number of flowers (8.8 versus 11.6). To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars. PMID:25097888

  18. Effect of Pseudomonas putida on growth and anthocyanin pigment in two poinsettia (Euphorbia pulcherrima) cultivars.

    PubMed

    Zulueta-Rodriguez, Ramon; Cordoba-Matson, Miguel Victor; Hernandez-Montiel, Luis Guillermo; Murillo-Amador, Bernardo; Rueda-Puente, Edgar; Lara, Liliana

    2014-01-01

    Pseudomonas putida is plant growth promoting rhizobacteria (PGPR) that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble) using three strains of P. putida, as well as a mixture of the three (MIX). Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4), volume of roots (45 versus 3  cm(3)), number of leaves (78 versus 58), and area of leaf (1,788 versus 1,331 cm(2)), except for the number of flowers (8.8 versus 11.6). To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars.

  19. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  20. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-06-16

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA.

  1. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  2. Identification and Characterization of an N-Acylhomoserine Lactone-Dependent Quorum-Sensing System in Pseudomonas putida Strain IsoF

    PubMed Central

    Steidle, Anette; Allesen-Holm, Marie; Riedel, Kathrin; Berg, Gabriele; Givskov, Michael; Molin, Søren; Eberl, Leo

    2002-01-01

    Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptional ppuA::luxAB fusion we demonstrate that expression of ppuA is AHL dependent. Furthermore, transcription of the AHL synthase ppuI is shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking the ppu gene cluster indicated that the four genes form an island in the suhB-PA3819 intergenic region of the currently sequenced P. putida strain KT2440. Moreover, we provide evidence that the ppu genes are not present in other AHL-producing P. putida strains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both a ppuI and a ppuA mutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development. PMID:12450862

  3. Metal Inhibition of Growth and Manganese Oxidation in Pseudomonas putida GB-1

    NASA Astrophysics Data System (ADS)

    Pena, J.; Sposito, G.

    2009-12-01

    Biogenic manganese oxides (MnO2) are ubiquitous nanoparticulate minerals that contribute to the adsorption of nutrient and toxicant metals, the oxidative degradation of various organic compounds, and the respiration of metal-reducing bacteria in aquatic and terrestrial environments. The formation of these minerals is catalyzed by a diverse and widely-distributed group of bacteria and fungi, often through the enzymatic oxidation of aqueous Mn(II) to Mn(IV). In metal-impacted ecosystems, toxicant metals may alter the viability and metabolic activity of Mn-oxidizing organisms, thereby limiting the conditions under which biogenic MnO2 can form and diminishing their potential as adsorbent materials. Pseudomonas putida GB-1 (P. putida GB-1) is a model Mn-oxidizing laboratory culture representative of freshwater and soil biofilm-forming bacteria. Manganese oxidation in P. putida GB-1 occurs via two single-electron-transfer reactions, involving a multicopper oxidase enzyme found on the bacterial outer membrane surface. Near the onset of the stationary phase of growth, dark brown MnO2 particles are deposited in a matrix of bacterial cells and extracellular polymeric substances, thus forming heterogeneous biomineral assemblages. In this study, we assessed the influence of various transition metals on microbial growth and manganese oxidation capacity in a P. putida GB-1 culture propagated in a nutrient-rich growth medium. The concentration-response behavior of actively growing P. putida GB-1 cells was investigated for Fe, Co, Ni, Cu and Zn at pH ≈ 6 in the presence and absence of 1 mM Mn. Toxicity parameters such as EC0, EC50 and Hillslope, and EC100 were obtained from the sigmoidal concentration-response curves. The extent of MnO2 formation in the presence of the various metal cations was documented 24, 50, 74 and 104 h after the metal-amended medium was inoculated. Toxicity values were compared to twelve physicochemical properties of the metals tested. Significant

  4. Subtilase SprP exerts pleiotropic effects in Pseudomonas aeruginosa.

    PubMed

    Pelzer, Alexander; Polen, Tino; Funken, Horst; Rosenau, Frank; Wilhelm, Susanne; Bott, Michael; Jaeger, Karl-Erich

    2014-02-01

    The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ∆sprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms. PMID:24376018

  5. Effects of ambroxol on alginate of mature Pseudomonas aeruginosa biofilms.

    PubMed

    Li, Fang; Yu, Jialin; Yang, Hua; Wan, Zhenyan; Bai, Dan

    2008-07-01

    Biofilm-forming bacteria Pseudomonas aeruginosa is a common pathogen in mechanically ventilated newborns, which can cause life-threatening infections. Alginate of mucoid Pseudomonas aeruginosa biofilms is considered an important virulence factor which contributes to the resistance to antibiotics. Traditionally, ambroxol is widely used in newborns with lung problems as a mucolytic agent and antioxidant agent as well. And there are few studies that demonstrated the anti-biofilm activity of ambroxol. In this study, we found that ambroxol can affect the structure of mucoid Pseudomonas aeruginosa biofilms. Further, we found that ambroxol reduces the production of alginate, the expression of the important genes and the activity of key enzyme guanosine diphospho-D-mannose dehydrogenase (GDP-mannose dehydrogenase; GMD) which were involved in alginate biosynthesis.

  6. Chromosomal Locus for Cadmium Resistance in Pseudomonas putida Consisting of a Cadmium-Transporting ATPase and a MerR Family Response Regulator

    PubMed Central

    Lee, Seon-Woo; Glickmann, Eric; Cooksey, Donald A.

    2001-01-01

    Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharactarized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species. PMID:11282588

  7. Response of plant-colonizing pseudomonads to hydrogen peroxide. [Pseudomonas putida

    SciTech Connect

    Katsuwon, J.; Anderson, A.J. )

    1989-11-01

    Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H{sub 2}O{sub 2}) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H{sub 2}O{sub 2} was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 {mu}M) of H{sub 2}O{sub 2}. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.

  8. Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa.

    PubMed Central

    Donald, L J; Molgat, G F; Duckworth, H W

    1989-01-01

    The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted. Images PMID:2507528

  9. Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa.

    PubMed

    Frangipani, Emanuela; Bonchi, Carlo; Minandri, Fabrizia; Imperi, Francesco; Visca, Paolo

    2014-09-01

    Gallium (Ga) is an iron mimetic that has successfully been repurposed for antibacterial chemotherapy. To improve the antibacterial potency of Ga on Pseudomonas aeruginosa, the effect of complexation with a variety of siderophores and synthetic chelators was tested. Ga complexed with the pyochelin siderophore (at a 1:2 ratio) was more efficient than Ga(NO3)3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga entrance into the cell through the pyochelin translocon.

  10. Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa.

    PubMed

    Frangipani, Emanuela; Bonchi, Carlo; Minandri, Fabrizia; Imperi, Francesco; Visca, Paolo

    2014-09-01

    Gallium (Ga) is an iron mimetic that has successfully been repurposed for antibacterial chemotherapy. To improve the antibacterial potency of Ga on Pseudomonas aeruginosa, the effect of complexation with a variety of siderophores and synthetic chelators was tested. Ga complexed with the pyochelin siderophore (at a 1:2 ratio) was more efficient than Ga(NO3)3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga entrance into the cell through the pyochelin translocon. PMID:24957826

  11. Toxic effect of environmentally relevant concentration of silver nanoparticles on environmentally beneficial bacterium Pseudomonas putida.

    PubMed

    Khan, S Sudheer; Ghouse, Syed Shabin; Chandran, Preethy

    2015-07-01

    Silver nanoparticles (Ag NPs) are being increasingly used in many consumer products owing to their excellent antimicrobial properties. The continuous use of Ag NPs in consumer products will lead to environmental release. The present study evaluated the toxic effects and the possible underlying mechanism of Ag NPs on Pseudomonas putida. Ag NP exposure inhibited growth of the cells. Increased lipid peroxidation occurred coincident with suppression of the antioxidant defense system. Ag NP exposure caused reactive oxygen species (ROS) production, glutathione depletion and inactivation of the antioxidant enzyme superoxide dismutase, catalase and glutathione reductase. The addition of superoxide dismutase or pretreatment of P. putida with N-acetyl cysteine that quenches ROS reduced toxicity of the NPs. PMID:25627470

  12. Biofiltration of ethyl acetate by Pseudomonas putida immobilized on walnut shell.

    PubMed

    Zare, Hossein; Najafpour, Ghasem; Rahimnejad, Mostafa; Tardast, Ali; Gilani, Saeedeh

    2012-11-01

    A biofilter packed with walnut shells was used to eliminate ethyl acetate from an air stream. The shells treated with NaOH were used as medium for immobilization of Pseudomonas putida PTCC 1694. At an empty bed residence time (EBRT) of 60s, a removal efficiency of 99% was achieved at inlet concentrations lower than 430ppm of ethyl acetate. The removal efficiency decreased below 80% with an increase in inlet concentration of ethyl acetate. When the EBRT was increased to 75 s, the removal efficiency remained above 80% even though the inlet loading rate was increased to 421g/m(3)h. Michaelis-Menten type and zero-order diffusion limited models were employed and the predicted data perfectly matched the experimental data. Thus P. putida immobilized on walnut shell has potential for the removal of ethyl acetate from air streams.

  13. Biotransformation of 6,6-Dimethylfulvene by Pseudomonas putida RE213

    PubMed Central

    Eaton, R. W.; Selifonov, S. A.

    1996-01-01

    The biotransformation of 6,6-dimethylfulvene [5-(1-methylethylidene)-1,3-cyclopentadiene], a nonaromatic C(inf5) carbocyclic analog of isopropylbenzene, was examined by using Pseudomonas putida RE213, a Tn5-generated dihydrodiol-accumulating mutant of the isopropylbenzene-degrading strain P. putida RE204. 6,6-Dimethylfulvene was converted to a single chiral product identified as (+)-(1R,2S)-cis-1,2-dihydroxy-5-(1-methylethylidene)-3-cyclopentene. This isopropylbenzene 2,3-dioxygenase-catalyzed transformation demonstrates the potential of bacterial arene dioxygenases for the direct conversion of cyclopentadienylidene compounds to homochiral C(inf5) carbocyclic cis-diols for use in enantiocontrolled organic syntheses. PMID:16535266

  14. Induction of the tod operon by trichloroethylene in Pseudomonas putida TVA8

    SciTech Connect

    Shingleton, J.T.; Applegate, B.M.; Nagel, A.C.; Bienkowski, P.R.; Sayler, G.S.

    1998-12-01

    Bioluminescence, mRNA levels, and toluene degradation rates in Pseudomonas putida TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE). TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations. Compared to uninduced TVA8 cultures, todC1 mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures. Compared to uninduced P. putida F1 cultures, todC1 mRNA levels increased 4,4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures. Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures.

  15. Regulation of Hydroxylation and Nitroreduction Pathways during Metabolism of the Neonicotinoid Insecticide Imidacloprid by Pseudomonas putida.

    PubMed

    Lu, Tian-Qi; Mao, Shi-Yun; Sun, Shi-Lei; Yang, Wen-Long; Ge, Feng; Dai, Yi-Jun

    2016-06-22

    Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism. PMID:27230024

  16. Biofiltration of ethyl acetate by Pseudomonas putida immobilized on walnut shell.

    PubMed

    Zare, Hossein; Najafpour, Ghasem; Rahimnejad, Mostafa; Tardast, Ali; Gilani, Saeedeh

    2012-11-01

    A biofilter packed with walnut shells was used to eliminate ethyl acetate from an air stream. The shells treated with NaOH were used as medium for immobilization of Pseudomonas putida PTCC 1694. At an empty bed residence time (EBRT) of 60s, a removal efficiency of 99% was achieved at inlet concentrations lower than 430ppm of ethyl acetate. The removal efficiency decreased below 80% with an increase in inlet concentration of ethyl acetate. When the EBRT was increased to 75 s, the removal efficiency remained above 80% even though the inlet loading rate was increased to 421g/m(3)h. Michaelis-Menten type and zero-order diffusion limited models were employed and the predicted data perfectly matched the experimental data. Thus P. putida immobilized on walnut shell has potential for the removal of ethyl acetate from air streams. PMID:22940351

  17. Metabolism of chlorofluorocarbons and polybrominated compounds by pseudomonas putida G786(pHG-2) via an engineered metabolic pathway

    SciTech Connect

    Hur, H.G.; Sadowsky, M.J.; Wackett, L.P.

    1994-11-01

    Polyhalogenated EPA Priority Pollutants are among the most toxic and persistent of the xenobiotic compounds found in the environment. In those instances when biodegradation does occure, it is typically via reductive dechlorination reactions in anaerobic sediments. These reactions are very slow and difficult to study. In this study, cytochrome P-450{sub cam} from Pseudomonas putida G786 and toluene dioxygenase from P. putida F1 were used to catalyze consecutive cometabolic dehalogenation reactions. New halogenated substrates for both were identified. The results demonstrate the metabolism of polybrominated compounds and chlorofluoroalkanes via the engineered metabolic pathway in P. putida G786(pHG-2). 26 refs., 5 figs., 2 tabs.

  18. Die-off and survival of Pseudomonas aeruginosa in freshwater.

    PubMed

    de Vicente, A; Aviles, M; Borrego, J J; Romero, P

    1988-03-01

    Studies of the survival of Pseudomonas aeruginosa in freshwater, in situ and in the laboratory, were carried out. A die-off of P. aeruginosa very similar to those of the microbial indicators of fecal pollution, especially to the coliforms, was observed from the results obtained by in situ experiments. The laboratory studies show that the factors tested which exert the greatest effect on the survival of P. aeruginosa in freshwater are the luminous radiations and non-filtrable biotic factors. Furthermore, a negative effect on the viability of this microorganism in freshwater is observed when sewage is added. PMID:3131996

  19. 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

  20. 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

  1. Cytosine chemoreceptor McpC in Pseudomonas putida F1 also detects nicotinic acid.

    PubMed

    Parales, Rebecca E; Nesteryuk, Vasyl; Hughes, Jonathan G; Luu, Rita A; Ditty, Jayna L

    2014-12-01

    Soil bacteria are generally capable of growth on a wide range of organic chemicals, and pseudomonads are particularly adept at utilizing aromatic compounds. Pseudomonads are motile bacteria that are capable of sensing a wide range of chemicals, using both energy taxis and chemotaxis. Whilst the identification of specific chemicals detected by the ≥26 chemoreceptors encoded in Pseudomonas genomes is ongoing, the functions of only a limited number of Pseudomonas chemoreceptors have been revealed to date. We report here that McpC, a methyl-accepting chemotaxis protein in Pseudomonas putida F1 that was previously shown to function as a receptor for cytosine, was also responsible for the chemotactic response to the carboxylated pyridine nicotinic acid.

  2. Elastase Deficiency Phenotype of Pseudomonas aeruginosa Canine Otitis Externa Isolates

    PubMed Central

    Petermann, Shana R.; Doetkott, Curt; Rust, Lynn

    2001-01-01

    Pseudomonas aeruginosa veterinary isolates were assayed for elastase and total matrix protease activity. The elastase activity of canine ear isolates was much less than that of strain PAO1 and that of all other veterinary isolates (P < 0.0001). The results indicate that canine ear isolates have a distinct elastase phenotype. PMID:11329471

  3. Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa.

    PubMed

    Leoni, L; Orsi, N; de Lorenzo, V; Visca, P

    2000-03-01

    In Pseudomonas aeruginosa, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes, pvdS, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from Pseudomonas fluorescens, PfrI and PupI from Pseudomonas putida, and FecI from Escherichia coli, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of P. aeruginosa is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, alpha(2)betabeta' subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the pvdA gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (pvd) promoters was significantly lower in E. coli overexpressing the multicopy pvdS gene than in wild-type P. aeruginosa PAO1 carrying the single gene copy, and pvd::lacZ transcriptional fusions were silent in both pfrI (the pvdS homologue) and pfrA (a positive regulator of pseudobactin biosynthetic genes) mutants of P. putida WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from E. coli lacked the ability to generate in vitro transcripts from the pvdA promoter. These observations suggest that at least one additional positive regulator could be required for full activity of

  4. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida

    SciTech Connect

    Irie, S.; Doi, S.; Yorifuji, T.; Takagi, M.; Yano, K.

    1987-11-01

    The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.

  5. Global Pseudomonas aeruginosa biodiversity as reflected in a Belgian river.

    PubMed

    Pirnay, Jean-Paul; Matthijs, Sandra; Colak, Huri; Chablain, Patrice; Bilocq, Florence; Van Eldere, Johan; De Vos, Daniel; Zizi, Martin; Triest, Ludwig; Cornelis, Pierre

    2005-07-01

    The biodiversity of the bacterium Pseudomonas aeruginosa in an aquatic environment (the Woluwe River, Brussels, Belgium) was analysed. Surface water was sampled bimonthly over a 1-year period (2000-2001) at seven sites evenly dispersed over the river. Total bacterial counts were performed and P. aeruginosa strains were isolated on a selective medium. A weighed out sample of 100 randomly chosen presumptive P. aeruginosa isolates was further analysed. A set of data consisting of the nucleotide sequence of the oprL gene, a DNA-based fingerprint (amplified fragment length polymorphism, AFLP), serotype, pyoverdine type and antibiogram (MICs of 21 clinically relevant antibiotics) was assembled. These data were integrated with those previously obtained for 73 P. aeruginosa clinical and environmental isolates collected across the world. The combined results were analysed and compared using biological data analysis software. Our findings indicate a positive relationship between the extent of pollution and the prevalence of P. aeruginosa. Surprisingly, the Woluwe River P. aeruginosa community was almost as diverse as the global P. aeruginosa population. Indeed, the Woluwe River harboured members of nearly all successful clonal complexes. With the exception of one multidrug-resistant (MDR) strain, belonging to a ubiquitous and clinically relevant serotype O11 clone, antibiotic resistance levels were relatively low. These findings illustrate the significance of river water as a reservoir and source of distribution of potentially pathogenic P. aeruginosa strains and could have repercussions on antinosocomial infection strategies.

  6. Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa.

    PubMed

    Green, S K; Schroth, M N; Cho, J J; Kominos, S K; Vitanza-jack, V B

    1974-12-01

    Pseudomonas aeruginosa was detected in 24% of the soil samples but in only 0.13% of the vegetable samples from various agricultural areas of California. The distribution of pyocin types of soil and vegetable isolates was similar to that of clinical strains, and three of the soil isolates were resistant to carbenicillin. Pseudomonas aeruginosa multiplied in lettuce and bean under conditions of high temperature and high relative humidity (27 C and 80-95% relative humidity) but declined when the temperature and humidity were lowered (16 C, 55-75% relative humidity). The results suggest that soil is a reservior for P. aeruginosa and that the bacterium has the capacity to colonize plants during favorable conditions of temperature and moisture. PMID:4217591

  7. 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

  8. Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa.

    PubMed

    Zhou, Su; Yin, Hua; Tang, Shaoyu; Peng, Hui; Yin, Donggao; Yang, Yixuan; Liu, Zehua; Dang, Zhi

    2016-05-01

    Proliferation of cyanobacteria in aquatic ecosystems has caused water security problems throughout the world. Our preliminary study has showed that Pseudomonas aeruginosa can inhibit the growth of cyanobacterium, Microcystis aeruginosa. In order to explore the inhibitory mechanism of P. aeruginosa on the cell growth and synthesis of intracellular substances of M. aeruginosa, concentrations of Chlorophyll-a, intracellular protein, carbohydrate, enzyme activities and ion metabolism of M. aeruginosa, were investigated. The results indicated that 83.84% algicidal efficiency of P. aeruginosa was achieved after treatment for 7 days. The strain inhibited the reproduction of M. aeruginosa by impeding the synthesis of intracellular protein and carbohydrate of cyanobacterium, and only a very small part of intracellular protein and carbohydrate was detected after exposure to P. aeruginosa for 5 days. P. aeruginosa caused the alteration of intracellular antioxidant enzyme activity of M. aeruginosa, such as catalase, peroxidase. The accumulation of malondialdehyde aggravated membrane injury after treatment for 3 days. P. aeruginosa also affected the ion metabolism of cyanobacteria. The release of Na(+) and Cl(-) was significantly enhanced while the uptake of K(+), Ca(2+), Mg(2+), NO3(-) and SO4(2)(-) decreased. Surface morphology and intracellular structure of cyanobacteria and bacterial cells changed dramatically over time as evidenced by electron microscope (SEM) and transmission electron microscope (TEM) analysis. These results revealed that the algicidal activity of P. aeruginosa was primarily due to the fermentation liquid of P. aeruginosa that impeded the synthesis of intracellular protein and carbohydrate, and damaged the cell membrane through membrane lipid peroxidation.

  9. Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa.

    PubMed

    Zhou, Su; Yin, Hua; Tang, Shaoyu; Peng, Hui; Yin, Donggao; Yang, Yixuan; Liu, Zehua; Dang, Zhi

    2016-05-01

    Proliferation of cyanobacteria in aquatic ecosystems has caused water security problems throughout the world. Our preliminary study has showed that Pseudomonas aeruginosa can inhibit the growth of cyanobacterium, Microcystis aeruginosa. In order to explore the inhibitory mechanism of P. aeruginosa on the cell growth and synthesis of intracellular substances of M. aeruginosa, concentrations of Chlorophyll-a, intracellular protein, carbohydrate, enzyme activities and ion metabolism of M. aeruginosa, were investigated. The results indicated that 83.84% algicidal efficiency of P. aeruginosa was achieved after treatment for 7 days. The strain inhibited the reproduction of M. aeruginosa by impeding the synthesis of intracellular protein and carbohydrate of cyanobacterium, and only a very small part of intracellular protein and carbohydrate was detected after exposure to P. aeruginosa for 5 days. P. aeruginosa caused the alteration of intracellular antioxidant enzyme activity of M. aeruginosa, such as catalase, peroxidase. The accumulation of malondialdehyde aggravated membrane injury after treatment for 3 days. P. aeruginosa also affected the ion metabolism of cyanobacteria. The release of Na(+) and Cl(-) was significantly enhanced while the uptake of K(+), Ca(2+), Mg(2+), NO3(-) and SO4(2)(-) decreased. Surface morphology and intracellular structure of cyanobacteria and bacterial cells changed dramatically over time as evidenced by electron microscope (SEM) and transmission electron microscope (TEM) analysis. These results revealed that the algicidal activity of P. aeruginosa was primarily due to the fermentation liquid of P. aeruginosa that impeded the synthesis of intracellular protein and carbohydrate, and damaged the cell membrane through membrane lipid peroxidation. PMID:26866757

  10. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

    PubMed

    Espinosa-Urgel, M; Salido, A; Ramos, J L

    2000-05-01

    Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

  11. Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

    PubMed

    Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

    2016-01-01

    Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. PMID:26617065

  12. Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand▿†

    PubMed Central

    Ebert, Birgitta E.; Kurth, Felix; Grund, Marcel; Blank, Lars M.; Schmid, Andreas

    2011-01-01

    Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae. The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds. PMID:21803911

  13. Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp.

    SciTech Connect

    Park, H.S.; Lim, S.J.; Chang, Y.K.; Kim, H.S.; Livingston, A.G.

    1999-03-01

    A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs of coculture of two microbial strains was attempted. Pseudomonas putida HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and {sup 1}H nuclear magnetic resonance analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs, Rhodococcus sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed.

  14. Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid

    SciTech Connect

    Ramos, J.L.; Duque, E.; Ramos-Gonzalez, M.-I. )

    1991-01-01

    Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grown on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25{degree}C than at 37{degree}C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.

  15. Pseudomonas putida KT2440 response to nickel or cobalt induced stress by quantitative proteomics.

    PubMed

    Ray, Prasun; Girard, Vincent; Gault, Manon; Job, Claudette; Bonneu, Marc; Mandrand-Berthelot, Marie-Andrée; Singh, Surya S; Job, Dominique; Rodrigue, Agnès

    2013-01-01

    Nickel and cobalt are obligate nutrients for the gammaproteobacteria but when present at high concentrations they display toxic effects. These two metals are present in the environment, their origin being either from natural sources or from industrial use. In this study, the effect of inhibitory concentrations of Ni or Co was assessed on the soil bacterium Pseudomonas putida KT2440 using a proteomic approach. The identification of more than 400 spots resulted in the quantification of 160 proteins that underwent significant variations in cells exposed to Co and Ni. This analysis allowed us to depict the cellular response of P. putida cells toward metallic stress. More precisely, the parallel comparison of the two proteomes showed distinct responses of P. putida to Ni or Co toxicity. The most striking effect of Co was revealed by the accumulation of several proteins involved in the defense against oxidative damage, which include proteins involved in the detoxification of the reactive oxygen species, superoxides and peroxides. The up-regulation of the genes encoding these enzymes was confirmed using qRT-PCR. Interestingly, in the Ni-treated samples, sodB, encoding superoxide dismutase, was up-regulated, indicating the apparition of superoxide radicals due to the presence of Ni. However, the most striking effect of Ni was the accumulation of several proteins involved in the synthesis of amino acids. The measurement of the amount of amino acids in Ni-treated cells revealed a strong accumulation of glutamate. PMID:23235558

  16. Metabolomics reveals the physiological response of Pseudomonas putida KT2440 (UWC1) after pharmaceutical exposure.

    PubMed

    Currie, Felicity; Broadhurst, David I; Dunn, Warwick B; Sellick, Christopher A; Goodacre, Royston

    2016-04-01

    Human pharmaceuticals have been detected in wastewater treatment plants, rivers, and estuaries throughout Europe and the United States. It is widely acknowledged that there is insufficient information available to determine whether prolonged exposure to low levels of these substances is having an impact on the microbial ecology in such environments. In this study we attempt to measure the effects of exposing cultures of Pseudomonas putida KT2440 (UWC1) to six pharmaceuticals by looking at differences in metabolite levels. Initially, we used Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate analysis to discriminate between cell cultures exposed to different pharmaceuticals. This suggested that on exposure to propranolol there were significant changes in the lipid complement of P. putida. Metabolic profiling with gas chromatography-mass spectrometry (GC-MS), coupled with univariate statistical analyses, was used to identify endogenous metabolites contributing to discrimination between cells exposed to the six drugs. This approach suggested that the energy reserves of exposed cells were being expended and was particularly evident on exposure to propranolol. Adenosine triphosphate (ATP) concentrations were raised in P. putida exposed to propranolol. Increased energy requirements may be due to energy dependent efflux pumps being used to remove propranolol from the cell. PMID:26932201

  17. Toluene dioxygenase expression correlates with trichloroethylene degradation capacity in Pseudomonas putida F1 cultures.

    PubMed

    Liu, Jianbo; Amemiya, Takashi; Chang, Qing; Qian, Yi; Itoh, Kiminori

    2012-09-01

    Trichloroethylene (TCE) is extensively used in commercial applications, despite its risk to human health via soil and groundwater contamination. The stability of TCE, which is a useful characteristic for commercial application, makes it difficult to remove it from the environment. Numerous studies have demonstrated that TCE can be effectively removed from the environment using bioremediation. Pseudomonas putida F1 is capable of degrading TCE into less hazardous byproducts via the toluene dioxygenase pathway (TOD). Unfortunately, these bioremediation systems are not self-sustaining, as the degradation capacity declines over time. Fortunately, the replacement of metabolic co-factors is sufficient in many cases to maintain effective TCE degradation. Thus, monitoring systems must be developed to predict when TCE degradation rates are likely to decline. Herein, we show evidence that tod expression levels correlate with the ability of P. putida F1 to metabolize TCE in the presence of toluene. Furthermore, the presence of toluene improves the replication of P. putida F1, even when TCE is present at high concentration. These findings may be applied to real world applications to decide when the bioremediation system requires supplementation with aromatic substrates, in order to maintain maximum TCE removal capacity.

  18. Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes.

    PubMed

    Benedetti, Ilaria; de Lorenzo, Víctor; Nikel, Pablo I

    2016-01-01

    Bacterial biofilms outperform planktonic counterparts in whole-cell biocatalysis. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 is ruled by a regulatory network controlling the levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for designing a genetic device that over-runs the synthesis or degradation of c-di-GMP--thus making P. putida to form biofilms at user's will. For this purpose, the transcription of either yedQ (diguanylate cyclase) or yhjH (c-di-GMP phoshodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting strain was subsequently endowed with a synthetic operon and tested for 1-chlorobutane biodegradation. Upon addition of cyclohexanone to the culture medium, the thereby designed P. putida cells formed biofilms displaying high dehalogenase activity. These results show that the morphologies and physical forms of whole-cell biocatalysts can be genetically programmed while purposely designing their biochemical activity.

  19. Metabolic Engineering of Pseudomonas putida KT2440 to Produce Anthranilate from Glucose

    PubMed Central

    Kuepper, Jannis; Dickler, Jasmin; Biggel, Michael; Behnken, Swantje; Jäger, Gernot; Wierckx, Nick; Blank, Lars M.

    2015-01-01

    The Pseudomonas putida KT2440 strain was engineered in order to produce anthranilate (oAB, ortho-aminobenzoate), a precursor of the aromatic amino acid tryptophan, from glucose as sole carbon source. To enable the production of the metabolic intermediate oAB, the trpDC operon encoding an anthranilate phosphoribosyltransferase (TrpD) and an indole-3-glycerol phosphate synthase (TrpC), were deleted. In addition, the chorismate mutase (pheA) responsible for the conversion of chorismate over prephenate to phenylpyruvate was deleted in the background of the deletion of trpDC to circumvent a potential drain of precursor. To further increase the oAB production, a feedback insensitive version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by the aroGD146N gene and an anthranilate synthase (trpES40FG) were overexpressed separately and simultaneously in the deletion mutants. With optimized production conditions in a tryptophan-limited fed-batch process a maximum of 1.54 ± 0.3 g L-1 (11.23 mM) oAB was obtained with the best performing engineered P. putida KT2440 strain (P. putida ΔtrpDC pSEVA234_aroGD146N_trpES40FG). PMID:26635771

  20. Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid.

    PubMed Central

    Ramos, J L; Duque, E; Ramos-Gonzalez, M I

    1991-01-01

    Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils. PMID:2036014

  1. Metabolic Engineering of Pseudomonas putida KT2440 to Produce Anthranilate from Glucose.

    PubMed

    Kuepper, Jannis; Dickler, Jasmin; Biggel, Michael; Behnken, Swantje; Jäger, Gernot; Wierckx, Nick; Blank, Lars M

    2015-01-01

    The Pseudomonas putida KT2440 strain was engineered in order to produce anthranilate (oAB, ortho-aminobenzoate), a precursor of the aromatic amino acid tryptophan, from glucose as sole carbon source. To enable the production of the metabolic intermediate oAB, the trpDC operon encoding an anthranilate phosphoribosyltransferase (TrpD) and an indole-3-glycerol phosphate synthase (TrpC), were deleted. In addition, the chorismate mutase (pheA) responsible for the conversion of chorismate over prephenate to phenylpyruvate was deleted in the background of the deletion of trpDC to circumvent a potential drain of precursor. To further increase the oAB production, a feedback insensitive version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by the aroG (D146N) gene and an anthranilate synthase (trpE (S40F) G) were overexpressed separately and simultaneously in the deletion mutants. With optimized production conditions in a tryptophan-limited fed-batch process a maximum of 1.54 ± 0.3 g L(-1) (11.23 mM) oAB was obtained with the best performing engineered P. putida KT2440 strain (P. putida ΔtrpDC pSEVA234_aroG (D146N) _trpE (S40F) G). PMID:26635771

  2. Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

    PubMed Central

    Duque, Estrella; Fernández, Matilde; Molina-Santiago, Carlos; Roca, Amalia; Porcel, Mario; de la Torre, Jesús; Segura, Ana; Plesiat, Patrick; Jeannot, Katy; Ramos, Juan-Luis

    2014-01-01

    Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts. PMID:24465371

  3. Repression of Pseudomonas putida phenanthrene-degrading activity by plant root extracts and exudates.

    PubMed

    Rentz, Jeremy A; Alvarez, Pedro J J; Schnoor, Jerald L

    2004-06-01

    The phenanthrene-degrading activity (PDA) of Pseudomonas putida ATCC 17484 was repressed after incubation with plant root extracts of oat (Avena sativa), osage orange (Maclura pomifera), hybrid willow (Salix alba x matsudana), kou (Cordia subcordata) and milo (Thespesia populnea) and plant root exudates of oat (Avena sativa) and hybrid poplar (Populus deltoides x nigra DN34). Total organic carbon content of root extracts ranged from 103 to 395 mg l(-1). Characterization of root extracts identified acetate (not detectable to 8.0 mg l(-1)), amino acids (1.7-17.3 mg l(-1)) and glucose (1.6-14.0 mg l(-1)), indicating a complex mixture of substrates. Repression was also observed after exposure to potential root-derived substrates, including organic acids, glucose (carbohydrate) and glutamate (amino acid). Carbon source regulation (e.g. catabolite repression) was apparently responsible for the observed repression of P. putida PDA by root extracts. However, we showed that P. putida grows on root extracts and exudates as sole carbon and energy sources. Enhanced growth on root products may compensate for partial repression, because larger microbial populations are conducive to faster degradation rates. This would explain the commonly reported increase in phenanthrene removal in the rhizosphere.

  4. 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.

  5. Electrochemically monitoring the antibiotic susceptibility of Pseudomonas aeruginosa biofilms.

    PubMed

    Webster, Thaddaeus A; Sismaet, Hunter J; Chan, I-ping J; Goluch, Edgar D

    2015-11-01

    The condition of cells in Pseudomonas aeruginosa biofilms was monitored via the electrochemical detection of the electro-active virulence factor pyocyanin in a fabricated microfluidic growth chamber coupled with a disposable three electrode cell. Cells were exposed to 4, 16, and 100 mg L(-1) colistin sulfate after overnight growth. At the end of testing, the measured maximum peak current (and therefore pyocyanin concentration) was reduced by approximately 68% and 82% in P. aeruginosa exposed to 16 and 100 mg L(-1) colistin sulfate, respectively. Samples were removed from the microfluidic chamber, analyzed for viability using staining, and streaked onto culture plates to confirm that the P. aeruginosa cells were affected by the antibiotics. The correlation between electrical signal drop and the viability of P. aeruginosa cells after antibiotic exposure highlights the usefulness of this approach for future low cost antibiotic screening applications.

  6. 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

  7. Incidence and persistence of Pseudomonas aeruginosa in whirlpools.

    PubMed Central

    Price, D; Ahearn, D G

    1988-01-01

    Pseudomonas aeruginosa was isolated from seven commercial and two residential whirlpools that were treated with halogens. None of the commercial whirlpools was constantly maintained at appropriate disinfection levels. Superchlorination or the draining, cleaning, disinfection, and refilling of whirlpools markedly reduced densities of P. aeruginosa in whirlpool water, but the bacterial populations were rapidly reestablished (less than 10(3) cells per ml) when disinfectant concentrations decreased below recommended levels (chlorine, 3.0 ppm [3.0 micrograms/ml]; bromine, 6.0 ppm). P. aeruginosa in the water was replenished from various sources, such as hoses used to fill the whirlpool and the biofilm in the filter and piping of the whirlpool systems. Daily monitoring and adjustment of chemical characteristics (regardless of bather load) were essential for controlling densities of P. aeruginosa. Images PMID:3141463

  8. Entner-Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1.

    PubMed

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-08-01

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

  9. Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11.

    PubMed

    Ponraj, Paramasivan; Shankar, Manoharan; Ilakkiam, Devaraj; Rajendhran, Jeyaprakash; Gunasekaran, Paramasamy

    2013-06-01

    Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd(-) and S11 PqqF(-) by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd(-) and S11 PqqF(-) was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES-NaOH (pH 7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd(-) and S11 PqqF(-). Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.

  10. Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

    PubMed Central

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-01-01

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a “sulfoglycolytic” pathway, in addition to the classical glycolytic (Embden–Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner–Doudoroff pathway for glucose-6-phosphate: It involves an NAD+-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)+-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium. PMID:26195800

  11. Entner-Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1.

    PubMed

    Felux, Ann-Katrin; Spiteller, Dieter; Klebensberger, Janosch; Schleheck, David

    2015-08-01

    Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a "sulfoglycolytic" pathway, in addition to the classical glycolytic (Embden-Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner-Doudoroff pathway for glucose-6-phosphate: It involves an NAD(+)-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)(+)-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium. PMID:26195800

  12. Pseudomonas aeruginosa colonization in patients with spinal cord injuries.

    PubMed Central

    Gilmore, D S; Bruce, S K; Jimenez, E M; Schick, D G; Morrow, J W; Montgomerie, J Z

    1982-01-01

    The prevalence of Pseudomonas aeruginosa colonization of patients with spinal cord injury was studied annually from 1976 to 1980. The urethra, perineum, rectum, drainage bag, and urine of patients on the spinal cord injury service were cultured. A total of 224 men and 32 women were studied. Most patients were managed with an external urinary collection system or padding, with or without intermittent catheterization. P. aeruginosa was cultured from one or more body sites (urethra, perineum, or rectum) in 65% of men and 18% of women. Drainage bags on the beds were frequently colonized with P. aeruginosa (73%). Significant bacteriuria with P. aeruginosa was present in 19% of the men and 13% of the women. P. aeruginosa colonization of body sites in men was closely associated with the use of an external urinary collection system. Significantly greater urethral and perineal colonization was found in men using an external urinary collection system. P. aeruginosa serotype 11 was the predominant serotype for the first 3 years, and the number of patients colonized with serotype 11 increased with length of hospital stay. The prevalence of serotype 11 significantly decreased in the last 2 years. The antibiotic susceptibility of the strains of P. aeruginosa isolated from these patients did not change in the 5 years, except that there was increasing susceptibility to carbenicillin in later years. This increasing susceptibility to carbenicillin was a reflection of a decreased prevalence of serotype 11 in these patients, since serotype 11 was more resistant than other serotypes to carbenicillin. PMID:6818251

  13. Pseudomonas aeruginosa: assessment of risk from drinking water.

    PubMed

    Hardalo, C; Edberg, S C

    1997-01-01

    Pseudomonas aeruginosa is an ubiquitous environmental bacterium. It can be recovered, often in high numbers, in common food, especially vegetables. Moreover, it can be recovered in low numbers in drinking water. A small percentage of clones of P. aeruginosa possesses the required number of virulence factors to cause infection. However, P. aeruginosa will not proliferate on normal tissue but requires previously organs. Further narrowing the risk to human health is that only certain specific hosts are at risk, including patients with profound neutropenia, cystic fibrosis, severe burns, and those subject to foreign device installation. Other than these very well-defined groups, the general population is refractory to infection with P. aeruginosa. Because of its ubiquitous nature, it is not only not practical to eliminate P. aeruginosa from our food and drinking water, but attempts to do so would produce disinfection byproducts more hazardous than the species itself. Moreover, because there is no readily available sensitive and specific means to detect and identify P. aeruginosa available in the field, any potential regulation governing its control would not have a defined laboratory test measure of outcome. Accordingly, attempts to regulate P. aeruginosa in drinking water would not yield public health protection benefits and could, in fact, be counterproductive in this regard.

  14. Proteomic analysis of keratitis-associated Pseudomonas aeruginosa

    PubMed Central

    Sewell, Abby; Dunmire, Jeffrey; Wehmann, Michael; Rowe, Theresa

    2014-01-01

    Purpose To compare the proteomic profile of a clinical isolate of Pseudomonas aeruginosa (P. aeruginosa) obtained from an infected cornea of a contact lens wearer and the laboratory strain P. aeruginosa ATCC 10145. Methods Antibiotic sensitivity, motility, biofilm formation, and virulence tests were performed using standard methods. Whole protein lysates were analyzed with liquid chromatography/ tandem mass spectrometry (LC-MS/MS) in triplicate, and relative protein abundances were determined with spectral counting. The G test followed by a post hoc Holm-Sidak adjustment was used for the statistical analyses to determine significance in the differential expression of proteins between the two strains. Results A total of 687 proteins were detected. One-hundred thirty-three (133) proteins were significantly different between the two strains. Among these, 13 were upregulated, and 16 were downregulated in the clinical strain compared to ATCC 10145, whereas 57 were detected only in the clinical strain. The upregulated proteins are associated with virulence and pathogenicity. Conclusions Proteins detected at higher levels in the clinical strain of P. aeruginosa were proteins known to be virulence factors. These results confirm that the keratitis-associated P. aeruginosa strain is pathogenic and expresses a higher number of virulence factors compared to the laboratory strain ATCC 10145. Identification of the protein profile of the corneal strain of P. aeruginosa in this study will aid in elucidating novel intervention strategies for reducing the burden of P. aeruginosa infection in keratitis. PMID:25221424

  15. Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

    PubMed

    Cheng, Lei; Yin, Sheng; Chen, Min; Sun, Baoguo; Hao, Shuai; Wang, Chengtao

    2016-08-01

    As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13 mg L(-1) after 24 h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas. PMID:27154464

  16. Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains

    PubMed Central

    Lindmeyer, Martin; Jahn, Michael; Vorpahl, Carsten; Müller, Susann; Schmid, Andreas; Bühler, Bruno

    2015-01-01

    Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed. PMID:26483771

  17. Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa.

    PubMed

    Enouf, J; Laurence, F; Farrugia, G; Blanchard, P; Robert-Gero, M

    1976-10-11

    The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa. 0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida. The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the smae concentration of the inhibitor. --P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture. --MAMP has no effect on P. alcaligenes. The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNAmet from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

  18. Mechanisms of solvent resistance mediated by interplay of cellular factors in Pseudomonas putida.

    PubMed

    Ramos, Juan-Luis; Sol Cuenca, Maria; Molina-Santiago, Carlos; Segura, Ana; Duque, Estrella; Gómez-García, María R; Udaondo, Zulema; Roca, Amalia

    2015-07-01

    A number of microorganisms have the ability to thrive in the presence of a range of toxic solvents. Tolerance to these chemicals is a multifactorial process, meaning that bacterial cells use a set of physiological and gene expression changes to overcome the damage imparted by these chemicals. This review focuses mainly on issues related to tolerance to aromatic hydrocarbons and butanol in Pseudomonas, although other microorganisms are also discussed. Pseudomonas putida strains contain a circular chromosome of approximately 6 Mbp which encodes about 5300 genes. A combination of physiological and biochemical assays, a genome-wide collection of mutants and several omics approaches have provided useful information to help identify functions involved in solvent tolerance in P. putida. The solvent response involves fine-tuning of lipid fluidity to adjust membrane functions including impermeabilization, activation of a general stress-response system, increased energy generation and induction of specific efflux pumps that extrude solvents to the medium. These responses are modulated at the transcriptional level by local and global regulators as well as by a number of sRNAs whose levels fluctuate with the presence of solvents in the environment. Taken as a whole these regulatory inputs orchestrate the complex network of metabolic responses observed after solvent addition. PMID:25934123

  19. [Removal of toluene waste gas by Pseudomonas putida with a bio-trickling filter].

    PubMed

    Zhang, Shu-Jing; Li, Jian; Li, Yi-Li; Jin, Yu-Quan; Sun, Li

    2007-08-01

    In transient conditions close to the industrialized application situation, the removal of toluene was investigated with a lab-scale bio-trickling filter inoculated with pure bacterial culture (Pseudomonas putida). The start-up process and the ability of resisting different toluene loading in the steady state on the performance of the bio-trickling filter were studied. The microstructure of biofilm in the filter was also observed. With inlet concentration range from 544 to 1044 mg x m(-3) at the temperature ranging from 17 to 26 degrees C, the removal efficiency of toluene was almost 100% at the residence time of 54 s and 43.2 s. The maximum volumetric removal loading of 105.35 g x (m3 x h)(-1) was achieved. The results indicate that it was feasible to remove toluene by Pseudomonas putida which had not be acclimated by toluene. In the steady state, the bio-trickling filter had a high flexibility for the load change and the removal efficiency of the reactor was not influenced by the variance of residence time and inlet concentration. The rapid increase of biofilm can be controlled by adjusting the interval of nutrition liquid accession. There were some changes in bacterial community, and lots of micro-pore existed in the biofilm. It was proved that the absorption of the biofilm was an important precondition for the biodegradation of toluene.

  20. Crystal Structure of the Pseudomonas aeruginosa Virulence Factor Regulator

    SciTech Connect

    Cordes, Timothy J.; Worzalla, Gregory A.; Ginster, Aaron M.; Forest, Katrina T.

    2012-09-07

    Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prvents the growth of P. aeruginosa.

  1. Draft Genome Sequence of Pseudomonas putida BW11M1, a Banana Rhizosphere Isolate with a Diversified Antimicrobial Armamentarium

    PubMed Central

    Swings, Toon; Michiels, Jan; Gross, Harald; De Mot, René

    2016-01-01

    In this study, we report the draft genome of Pseudomonas putida BW11M1, a banana rhizosphere isolate producing various antimicrobial compounds, including a lectin-like bacteriocin, an R-type tailocin, the cyclic lipopeptide xantholysin, and the fatty acid–derived pseudopyronine. PMID:27081131

  2. Draft Genome Sequence of Pseudomonas putida JLR11, a Facultative Anaerobic 2,4,6-Trinitrotoluene Biotransforming Bacterium.

    PubMed

    Pascual, Javier; Udaondo, Zulema; Molina, Lazaro; Segura, Ana; Esteve-Núñez, Abraham; Caballero, Antonio; Duque, Estrella; Ramos, Juan Luis; van Dillewijn, Pieter

    2015-09-03

    We report the draft genome sequence of Pseudomonas putida JLR11, a facultative anaerobic bacterium that has been studied in detail for its capacity to use the explosive 2,4,6-trinitrotoluene (TNT) as a nitrogen source. The sequence confirms the mechanisms used by this versatile strain to reduce and assimilate nitrogen from TNT.

  3. Draft Genome Sequence of Pseudomonas putida JLR11, a Facultative Anaerobic 2,4,6-Trinitrotoluene Biotransforming Bacterium

    PubMed Central

    Pascual, Javier; Udaondo, Zulema; Molina, Lazaro; Segura, Ana; Esteve-Núñez, Abraham; Caballero, Antonio; Duque, Estrella; Ramos, Juan Luis

    2015-01-01

    We report the draft genome sequence of Pseudomonas putida JLR11, a facultative anaerobic bacterium that has been studied in detail for its capacity to use the explosive 2,4,6-trinitrotoluene (TNT) as a nitrogen source. The sequence confirms the mechanisms used by this versatile strain to reduce and assimilate nitrogen from TNT. PMID:26337875

  4. Complete genome sequence of solvent-tolerant Pseudomonas putida S12 including megaplasmid pTTS12.

    PubMed

    Kuepper, J; Ruijssenaars, H J; Blank, L M; de Winde, J H; Wierckx, N

    2015-04-20

    Pseudomonas putida S12 is a solvent-tolerant gamma-proteobacterium with an extensive track record for production of industrially relevant chemicals. Here we report the annotated complete genome sequence of this organism, including the megaplasmid pTTS12 which encodes many of the unique features of the S12 strain. PMID:25746905

  5. 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

  6. Characterization of Molecular Mechanisms Controlling fabAB Transcription in Pseudomonas aeruginosa

    PubMed Central

    Schweizer, Herbert P.; Choi, Kyoung-Hee

    2012-01-01

    Background The FabAB pathway is one of the unsaturated fatty acid (UFA) synthesis pathways for Pseudomonas aeruginosa. It was previously noted that this operon was upregulated in biofilms and repressed by exogenous UFAs. Deletion of a 30 nt fabA upstream sequence, which is conserved in P. aeruginosa, P. putida, and P. syringae, led to a significant decrease in fabA transcription, suggesting positive regulation by an unknown positive regulatory mechanism. Methods/Principal Findings Here, genetic and biochemical approaches were employed to identify a potential fabAB activator. Deletion of candidate genes such as PA1611 or PA1627 was performed to determine if any of these gene products act as a fabAB activator. However, none of these genes were involved in the regulation of fabAB transcription. Use of mariner-based random mutagenesis to screen for fabA activator(s) showed that several genes encoding unknown functions, rpoN and DesA may be involved in fabA regulation, but probably via indirect mechanisms. Biochemical attempts performed did fail to isolate an activator of fabAB operon. Conclusion/Significance The data suggest that fabA expression might not be regulated by protein-binding, but by a distinct mechanism such as a regulatory RNA-based mechanism. PMID:23056212

  7. Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase.

    PubMed Central

    Gill, J F; Deretic, V; Chakrabarty, A M

    1986-01-01

    Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot- blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22. Images PMID:2426246

  8. 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

  9. Effects of norspermidine on Pseudomonas aeruginosa biofilm formation and eradication.

    PubMed

    Qu, Lin; She, Pengfei; Wang, Yangxia; Liu, Fengxia; Zhang, Di; Chen, Lihua; Luo, Zhen; Xu, Huan; Qi, Yong; Wu, Yong

    2016-06-01

    Biofilms are defined as aggregation of single cell microorganisms and associated with over 80% of all the microbial infections. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen capable of leading to various infections in immunocompromised people. Recent studies showed that norspermidine, a kind of polyamine, prevented and disrupted biofilm formation by some Gram-negative bacterium. In this study, the effects of norspermidine on P. aeruginosa biofilm formation and eradication were tested. Microtiter plate combined with crystal violet staining was used to study the effects of norspermidine on P. aeruginosa initial attachment, then we employed SEM (scanning electron microscope), qRT-PCR, and QS-related virulence factor assays to investigate how norspermidine prevent biofilm formation by P. aeruginosa. We reported that high-dose norspermidine had bactericide effect on P. aeruginosa, and norspermidine began to inhibit biofilm formation and eradicate 24-h mature biofilm at concentration of 0.1 and 1 mmol/L, respectively, probably by preventing cell-surface attachment, inhibiting swimming motility, and downregulating QS-related genes expression. To investigate the potential utility of norspermidine in preventing device-related infections, we found that catheters immersed with norspermidine were effective in eradicating mature biofilm. These results suggest that norspermidine could be a potent antibiofilm agent for formulating strategies against P. aeruginosa biofilm. PMID:26817804

  10. Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections

    PubMed Central

    Cigana, Cristina; Lorè, Nicola Ivan; Riva, Camilla; De Fino, Ida; Spagnuolo, Lorenza; Sipione, Barbara; Rossi, Giacomo; Nonis, Alessandro; Cabrini, Giulio; Bragonzi, Alessandra

    2016-01-01

    Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies. PMID:26883959

  11. 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

  12. COMPARATIVE TAXONOMY OF CRYSTALLOGENIC STRAINS OF PSEUDOMONAS AERUGINOSA AND PSEUDOMONAS CHLORORAPHIS

    PubMed Central

    Haynes, William C.; Rhodes, Lenora J.

    1962-01-01

    Haynes, William C. (Northern Utilization Research and Development Division, Peoria, Ill.) and Lenora J. Rhodes. Comparative taxonomy of crystallogenic strains of Pseudomonas aeruginosa and Pseudomonas chlororaphis. J. Bacteriol. 84:1080–1084. 1962.—Only 11 of 39 strains received in the Agricultural Research Service Culture Collection under the designation Pseudonomas chlororaphis proved to be authentic; 28 were typical, pyocyanogenic strains of P. aeruginosa. The reason for this disproportionately high rate of misidentification apparently arises from an erroneous belief that the ability to produce green and yellow crystals of chlororaphin and oxychlororaphin is confined to P. chlororaphis. The ability of many strains of P. aeruginosa to do likewise is not well known. Inasmuch as the characteristic is not unique to P. chlororaphis, other criteria are required to distinguish crystallogenic strains of these species. After a taxonomic comparison of 18 strains of P. chlororaphis and 47 crystallogenic strains of P. aeruginosa, it was determined that there are three main distinctions: (i) P. aeruginosa grows well at 42 C but fails to grow upon serial transfer at 5 C, whereas P. chlororaphis fails to grow at 42 C, but grows well at 5 C: (ii) most strains of P. aeruginosa produce pyocyanin, whereas P. chlororaphis strains do not; (iii) P. aeruginosa cells possess only one or two polar flagella, whereas P. chlororaphis usually has at least four, sometimes as many as eight, polar flagella. PMID:13963593

  13. Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa.

    PubMed Central

    Hungerer, C; Troup, B; Römling, U; Jahn, D

    1995-01-01

    The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to open reading frames located in the 5' region of enterobacterial hemA genes. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined

  14. Membrane proteomes of Pseudomonas aeruginosa and Acinetobacter baumannii.

    PubMed

    Dé, E; Cosette, P; Coquet, L; Siroy, A; Alexandre, S; Duncan, A; Naudin, B; Rihouey, C; Schaumann, A; Junter, G A; Jouenne, T

    2011-12-01

    Acinetobacter baumannii and Pseudomonas aeruginosa are known for their intrinsic resistance to antibiotics. Between mechanisms involved in this resistance, diminished expression of outer membrane proteins and up-regulation of efflux pumps play an important role. The characterization of membrane proteins is consequently necessary because of their importance in the antibiotic resistance but also in virulence. This review presents proteomic investigations aiming to describe the protein content of the membranes of these two bacterial species. PMID:19942379

  15. Membrane proteomes of Pseudomonas aeruginosa and Acinetobacter baumannii.

    PubMed

    Dé, E; Cosette, P; Coquet, L; Siroy, A; Alexandre, S; Duncan, A; Naudin, B; Rihouey, C; Schaumann, A; Junter, G A; Jouenne, T

    2011-12-01

    Acinetobacter baumannii and Pseudomonas aeruginosa are known for their intrinsic resistance to antibiotics. Between mechanisms involved in this resistance, diminished expression of outer membrane proteins and up-regulation of efflux pumps play an important role. The characterization of membrane proteins is consequently necessary because of their importance in the antibiotic resistance but also in virulence. This review presents proteomic investigations aiming to describe the protein content of the membranes of these two bacterial species.

  16. Structure of type II dehydroquinase from Pseudomonas aeruginosa

    PubMed Central

    Reiling, Scott; Kelleher, Alan; Matsumoto, Monica M.; Robinson, Gonteria; Asojo, Oluwatoyin A.

    2014-01-01

    Pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. Ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for P. aeruginosa but are absent in mammals. One such enzyme, type II dehydroquinase (DHQase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. This step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aromatic metabolites. The recombinant expression, purification and crystal structure of catalytically active DHQase from P. aeruginosa (PaDHQase) are presented. Cubic crystals belonging to space group F23, with unit-cell parameters a = b = c = 125.39 Å, were obtained by vapor diffusion in sitting drops and the structure was refined to an R factor of 16% at 1.74 Å resolution. PaDHQase is a prototypical type II DHQase with the classical flavodoxin-like α/β topology. PMID:25372814

  17. Isolation of an iron-binding compound from Pseudomonas aeruginosa.

    PubMed Central

    Cox, C D; Graham, R

    1979-01-01

    An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 X 10(-6) M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid). PMID:104968

  18. Haemolytic uraemic syndrome associated with Pseudomonas aeruginosa sepsis.

    PubMed

    Narayanan, Parameswaran; Rustagi, Rashi S; Sivaprakasam, Prabha; Subramanian, Mahadevan; Parameswaran, Sreejith; Mandal, Jharna; Kaplan, B S

    2013-11-01

    Haemolytic uraemic syndrome (HUS) is a recognized complication of infection with Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae type 1. Infections with other micro-organisms, especially Streptococcus pneumoniae, have been cited as causes of HUS. In addition, influenza virus and other viruses may rarely be associated with this syndrome. A 2-year-old girl presented with severe Pseudomonas aeruginosa sepsis with renal failure and ecthyma gangrenosum. Further investigations revealed features of HUS. She was managed with antibiotics and other supportive measures including peritoneal dialysis, and subsequently made a full recovery. A possible role of neuraminidase in the pathogenesis of P. aeruginosa-associated HUS was proposed. This is the first reported case of P. aeruginosa sepsis leading to HUS.

  19. Singly Flagellated Pseudomonas aeruginosa Chemotaxes Efficiently by Unbiased Motor Regulation

    PubMed Central

    Cai, Qiuxian; Li, Zhaojun; Ouyang, Qi

    2016-01-01

    ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that has long been known to chemotax. More recently, it has been established that chemotaxis is an important factor in the ability of P. aeruginosa to make biofilms. Genes that allow P. aeruginosa to chemotax are homologous with genes in the paradigmatic model organism for chemotaxis, Escherichia coli. However, P. aeruginosa is singly flagellated and E. coli has multiple flagella. Therefore, the regulation of counterclockwise/clockwise flagellar motor bias that allows E. coli to efficiently chemotax by runs and tumbles would lead to inefficient chemotaxis by P. aeruginosa, as half of a randomly oriented population would respond to a chemoattractant gradient in the wrong sense. How P. aeruginosa regulates flagellar rotation to achieve chemotaxis is not known. Here, we analyze the swimming trajectories of single cells in microfluidic channels and the rotations of cells tethered by their flagella to the surface of a variable-environment flow cell. We show that P. aeruginosa chemotaxes by symmetrically increasing the durations of both counterclockwise and clockwise flagellar rotations when swimming up the chemoattractant gradient and symmetrically decreasing rotation durations when swimming down the chemoattractant gradient. Unlike the case for E. coli, the counterclockwise/clockwise bias stays constant for P. aeruginosa. We describe P. aeruginosa’s chemotaxis using an analytical model for symmetric motor regulation. We use this model to do simulations that show that, given P. aeruginosa’s physiological constraints on motility, its distinct, symmetric regulation of motor switching optimizes chemotaxis. PMID:27048795

  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. 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.

  2. MexXY multidrug efflux system of Pseudomonas aeruginosa

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2012-01-01

    Anti-pseudomonas aminoglycosides, such as amikacin and tobramycin, are used in the treatment of Pseudomonas aeruginosa infections. However, their use is linked to the development of resistance. During the last decade, the MexXY multidrug efflux system has been comprehensively studied, and numerous reports of laboratory and clinical isolates have been published. This system has been increasingly recognized as one of the primary determinants of aminoglycoside resistance in P. aeruginosa. In P. aeruginosa cystic fibrosis isolates, upregulation of the pump is considered the most common mechanism of aminoglycoside resistance. Non-fermentative Gram-negative pathogens possessing very close MexXY orthologs such as Achromobacter xylosoxidans and various Burkholderia species (e.g., Burkholderia pseudomallei and B. cepacia complexes), but not B. gladioli, are intrinsically resistant to aminoglycosides. Here, we summarize the properties (e.g., discovery, mechanism, gene expression, clinical significance) of the P. aeruginosa MexXY pump and other aminoglycoside efflux pumps such as AcrD of Escherichia coli, AmrAB-OprA of B. pseudomallei, and AdeABC of Acinetobacter baumannii. MexXY inducibility of the PA5471 gene product, which is dependent on ribosome inhibition or oxidative stress, is noteworthy. Moreover, the discovery of the cognate outer membrane component (OprA) of MexXY in the multidrug-resistant clinical isolate PA7, serotype O12 deserves special attention. PMID:23233851

  3. Reduction of PCN biosynthesis by NO in Pseudomonas aeruginosa.

    PubMed

    Gao, Lei; Zhang, Yuying; Wang, Yan; Qiao, Xinhua; Zi, Jing; Chen, Chang; Wan, Yi

    2016-08-01

    Pyocyanin (PCN), a virulence factor synthesized by Pseudomonas aeruginosa, plays an important role during clinical infections. There is no study of the effect of nitric oxide (NO) on PCN biosynthesis. Here, the effect of NO on PCN levels in Pseudomonas aeruginosa strain PAO1, a common reference strain, was tested. The results showed that the NO donor sodium nitroprusside (SNP) can significantly reduce PCN levels (82.5% reduction at 60μM SNP). Furthermore, the effect of endogenous NO on PCN was tested by constructing PAO1 nor (NO reductase gene) knockout mutants. Compared to the wild-type strain, the Δnor strain had a lower PCN (86% reduction in Δnor). To examine whether the results were universal with other P. aeruginosa strains, we collected 4 clinical strains from a hospital, tested their PCN levels after SNP treatment, and obtained similar results, i.e., PCN biosynthesis was inhibited by NO. These results suggest that NO treatment may be a new strategy to inhibit PCN biosynthesis and could provide novel insights into eliminating P. aeruginosa virulence as a clinical goal.

  4. Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.

    PubMed Central

    Klinger, J D; Tandler, B; Liedtke, C M; Boat, T F

    1984-01-01

    We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections. Images PMID:6568227

  5. Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM

    PubMed Central

    Peng, Yanjie; He, Yanhui; Wu, Zhansheng; Lu, Jianjiang; Li, Chun

    2014-01-01

    The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K2HPO4, 0.19 g/L MnSO4 and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K2HPO4, 0.17 g/L MnSO4 and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 1013 cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198. PMID:25763026

  6. Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes

    PubMed Central

    Garmendia, Junkal; De Las Heras, Aitor; Galvão, Teca Calcagno; De Lorenzo, Víctor

    2008-01-01

    Summary Although different biological approaches for detection of anti‐personnel mines and other unexploded ordnance (UXO) have been entertained, none of them has been rigorously documented thus far in the scientific literature. The industrial 2,4,6 trinitrotoluene (TNT) habitually employed in the manufacturing of mines is at all times tainted with a small but significant proportion of the more volatile 2,4 dinitrotoluene (2,4 DNT) and other nitroaromatic compounds. By using mutation‐prone PCR and DNA sequence shuffling we have evolved in vitro and selected in vivo variants of the effector recognition domain of the toluene‐responsive XylR regulator of the soil bacterium Pseudomonas putida that responds to mono‐, bi‐ and trinitro substituted toluenes. Re‐introduction of such variants in P. putida settled the transcriptional activity of the cognate promoters (Po and Pu) as a function of the presence of nitrotoluenes in the medium. When strains bearing transcriptional fusions to reporters with an optical output (luxAB, GFP) were spread on soil spotted with nitrotoluenes, the signal triggered by promoter activation allowed localization of the target compounds on the soil surface. Our data provide a proof of concept that non‐natural transcription factors evolved to respond to nitroaromatics can be engineered in soil bacteria and inoculated on a target site to pinpoint the presence of explosives. This approach thus opens new ways to tackle this gigantic humanitarian problem. PMID:21261843

  7. The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

    PubMed Central

    de Vrind, J. P. M.; Brouwers, G. J.; Corstjens, P. L. A. M.; den Dulk, J.; de Vrind-de Jong, E. W.

    1998-01-01

    A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed. PMID:9758767

  8. Biodegradation of 1-allyloxy-4-propoxybenzene by selected strains of Pseudomonas putida.

    PubMed

    Ebrahimi, Parisa; Plettner, Erika

    2014-02-01

    Dialkoxybenzenes constitute a class of organic compounds with anti feeding and oviposition effects on the cabbage looper, Trichoplusia ni. Among them, 1-allyloxy-4-propoxybenzene has the highest feeding deterrence activity and potential for development as commercial insect control agent. To develop this compound, its fate in the environment needs to be studied. The fate of organic compounds in the environment depends on their biodegradability in the soil. We present results of laboratory biodegradation experiments of 1-allyloxy-4-propoxybenzene with three strains of Pseudomonas putida. Two of the three strains of P. putida tested were able to metabolize 1-allyloxy-4-propoxybenzene. Both strains required induction of the catabolic pathway. Specifically, strain ATCC 17453 (which contains the CAM plasmid) metabolized 1-allyloxy-4-propoxybenzene by first dealkylating. This gave both possible monoalkoxy phenols after five days, followed by dihydroquinone after 8 days. In vitro tests with CYP101A1 (cytochrome P450cam, a camphor hydroxylase), revealed that the dealkylation is catalyzed by this enzyme.

  9. Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors.

    PubMed

    Chung, Tsuey-Ping; Wu, Pei-Chen; Juang, Ruey-Shin

    2004-07-20

    The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.

  10. Pseudomonas putida response in membrane bioreactors under salicylic acid-induced stress conditions.

    PubMed

    Collado, Sergio; Rosas, Irene; González, Elena; Gutierrez-Lavin, Antonio; Diaz, Mario

    2014-02-28

    Starvation and changing feeding conditions are frequently characteristics of wastewater treatment plants. They are typical causes of unsteady-state operation of biological systems and provoke cellular stress. The response of a membrane bioreactor functioning under feed-induced stress conditions is studied here. In order to simplify and considerably amplify the response to stress and to obtain a reference model, a pure culture of Pseudomonas putida was selected instead of an activated sludge and a sole substrate (salicylic acid) was employed. The system degraded salicylic acid at 100-1100mg/L with a high level of efficiency, showed rapid acclimation without substrate or product inhibition phenomena and good stability in response to unsteady states caused by feed variations. Under starvation conditions, specific degradation rates of around 15mg/gh were achieved during the adaptation of the biomass to the new conditions and no biofilm formation was observed during the first days of experimentation using an initial substrate to microorganisms ratio lower than 0.1. When substrate was added to the reactor as pulses resulting in rapidly changing concentrations, P. putida growth was observed only for substrate to microorganism ratios higher than 0.6, with a maximum YX/S of 0.5g/g. Biofilm development under changing feeding conditions was fast, biomass detachment only being significant for biomass concentrations on the membrane surface that were higher than 16g/m(2).

  11. A Pseudomonas putida Strain Genetically Engineered for 1,2,3-Trichloropropane Bioremediation

    PubMed Central

    Samin, Ghufrana; Pavlova, Martina; Arif, M. Irfan; Postema, Christiaan P.; Damborsky, Jiri

    2014-01-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. PMID:24973068

  12. Robustness of Pseudomonas putida KT2440 as a host for ethanol biosynthesis.

    PubMed

    Nikel, Pablo I; de Lorenzo, Víctor

    2014-12-25

    Expansion of the burgeoning biofuels agenda involves not only the design of suitable genetic and metabolic devices but also their deployment into suitable hosts that can endure the stress brought about by the products themselves. The microorganisms that are easiest to genetically manipulate for these endeavors (e.g. Escherichia coli) are often afflicted by an undesirable sensitivity to the very product that they are engineered to synthesize. In this context, we have examined the resistance to the stress arising from ethanol synthesis and/or its addition to cultures of recombinant Pseudomonas putida, using as a benchmark the same trait in an E. coli strain. To this end, ethanologenic strains of these two species were constructed by functionally expressing pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) from Zymomonas mobilis. Recombinants were compared under anoxic conditions as ethanol producers, and cell survival, stress resistance, and phenotypic stability were quantified in each case. P. putida consistently outperformed E. coli in every ethanol tolerance test conducted - whether the alcohol was produced endogenously or added exogenously. These results highlight the value of this bacterium as a microbial cell factory for the production of biofuels owing to its naturally pre-evolved ability to withstand different kinds of chemical stresses.

  13. The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1

    SciTech Connect

    Vrind, J.P.M. de; Brouwers, G.J.; Corstijens, P.L.A.M.; Dulk, J. den; Vrind-de Jong, E.W. de

    1998-10-01

    A Pseudomonas putida strain, strain GB-1, oxidizes Mn{sup 2+} to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn{sup 2+} oxidation and/or secretion of the Mn{sup 2+}-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn{sup 2+} oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn{sup 2+}-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn{sup 2+} oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.

  14. Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida.

    PubMed

    Ito, Fumihiro; Tamiya, Takayuki; Ohtsu, Iwao; Fujimura, Makoto; Fukumori, Fumiyasu

    2014-12-01

    Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles. The KTΔclpB mutant showed the same heat shock response (HSR) as the wild-type, both in terms of heat-shock protein (Hsp) synthesis (other than ClpB) and in hsp gene expression; however, the mutant was quite sensitive to high temperatures and was unable to disaggregate into thermo-mediated protein aggregates, indicating that ClpB is important for cell survival after heat stress and essential for solubilization of protein aggregates. On the other hand, the KTΔdnaJ mutant was temperature-sensitive, and formed more protein aggregates (especially of high molecular weight) upon heat stress than did KT. P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ(32) activity and quantity, as well as the mRNA levels of hsp genes.

  15. Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440.

    PubMed

    Nikel, Pablo I; Kim, Juhyun; de Lorenzo, Víctor

    2014-01-01

    While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in the shift between customary C sources (e.g. glucose or succinate) to the polyol. Transcriptomic results were contrasted with key enzymatic activities under the same culture conditions. Cognate expression profiles revealed that genes encoding enzymes of the Entner-Doudoroff route and other catabolic pathways, e.g. the gluconate and 2-ketogluconate loops, were significantly downregulated on glycerol. Yet, the compound simultaneously elicited a gluconeogenic response that indicated an efficient channelling of C skeletons back to biomass build-up through the glyoxylate shunt rather than energization of the cells through downwards pathways, i.e. tricarboxylic acid cycle and oxidative phosphorylation. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favouring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other C sources.

  16. Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida

    PubMed Central

    Ito, Fumihiro; Tamiya, Takayuki; Ohtsu, Iwao; Fujimura, Makoto; Fukumori, Fumiyasu

    2014-01-01

    Molecular chaperones function in various important physiological processes. Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles. The KTΔclpB mutant showed the same heat shock response (HSR) as the wild-type, both in terms of heat-shock protein (Hsp) synthesis (other than ClpB) and in hsp gene expression; however, the mutant was quite sensitive to high temperatures and was unable to disaggregate into thermo-mediated protein aggregates, indicating that ClpB is important for cell survival after heat stress and essential for solubilization of protein aggregates. On the other hand, the KTΔdnaJ mutant was temperature-sensitive, and formed more protein aggregates (especially of high molecular weight) upon heat stress than did KT. P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ32 activity and quantity, as well as the mRNA levels of hsp genes. PMID:25303383

  17. Cd adsorption onto Pseudomonas putida in the presence and absence of extracellular polymeric substances

    NASA Astrophysics Data System (ADS)

    Ueshima, Masato; Ginn, Brian R.; Haack, Elizabeth A.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.

    2008-12-01

    The role of bacterial extracellular polymeric substances (EPS) in metal adsorption was determined by studying Cd adsorption onto the gram-negative bacterial species Pseudomonas putida with and without enzymatic removal of EPS from the biomass material. A range of experimental approaches were used to characterize the Cd adsorption reactions, including bulk proton and Cd adsorption measurements, FTIR spectroscopy, and fluorescence microscopy. The proton-reactivities of the biomass samples with EPS are not significantly different from those obtained for EPS-free biomass. Similarly, the presence of EPS does not significantly affect the extent of Cd removal from solution by the biomass on a mass-normalized basis, based on bulk Cd adsorption measurements conducted as a function of pH, nor does it appear to strongly affect the Cd-binding groups as observed by FTIR. However, fluorescence microscopy indicates that Cd, although concentrated on cell walls, is also bound to some extent to EPS. Together, the results from this study suggest that the P. putida EPS can bind significant concentrations of Cd from solution, and that the nature and mass-normalized extent of the binding is similar to that of the cell wall. Therefore, the EPS-bearing systems do not exhibit enhanced mass-normalized removal of Cd from solution relative to the EPS-free systems. The presence of the EPS effectively increases the viability of cells exposed to aqueous Cd, likely due to sequestration of the Cd away from the cells due to Cd-EPS binding.

  18. Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

    PubMed Central

    Parales, R E; Harwood, C S

    1992-01-01

    beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified. Images PMID:1624453

  19. Production of toluene cis-glycol by Pseudomonas putida in glucose fed-batch culture

    SciTech Connect

    Jenkins, R.O.; Stephens, G.M.; Dalton, H.

    1987-05-01

    Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene cis-glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25 h) of the process or the final concentration of TCG achieved. The rates of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. The results suggest that TCG is the mediator of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluene dioxygenase activity and a marked decrease in culture viability.

  20. Bypasses in intracellular glucose metabolism in iron-limited Pseudomonas putida.

    PubMed

    Sasnow, Samantha S; Wei, Hua; Aristilde, Ludmilla

    2016-02-01

    Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion. PMID:26377487

  1. Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa

    SciTech Connect

    Saiman, L.; Cacalano, G.; Prince, A. )

    1990-08-01

    Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

  2. Genetic and Functional Diversity of Pseudomonas aeruginosa Lipopolysaccharide

    PubMed Central

    Lam, Joseph S.; Taylor, Véronique L.; Islam, Salim T.; Hao, Youai; Kocíncová, Dana

    2011-01-01

    Lipopolysccharide (LPS) is an integral component of the Pseudomonas aeruginosa cell envelope, occupying the outer leaflet of the outer membrane in this Gram-negative opportunistic pathogen. It is important for bacterium–host interactions and has been shown to be a major virulence factor for this organism. Structurally, P. aeruginosa LPS is composed of three domains, namely, lipid A, core oligosaccharide, and the distal O antigen (O-Ag). Most P. aeruginosa strains produce two distinct forms of O-Ag, one a homopolymer of D-rhamnose that is a common polysaccharide antigen (CPA, formerly termed A band), and the other a heteropolymer of three to five distinct (and often unique dideoxy) sugars in its repeat units, known as O-specific antigen (OSA, formerly termed B band). Compositional differences in the O units among the OSA from different strains form the basis of the International Antigenic Typing Scheme for classification via serotyping of different strains of P. aeruginosa. The focus of this review is to provide state-of-the-art knowledge on the genetic and resultant functional diversity of LPS produced by P. aeruginosa. The underlying factors contributing to this diversity will be thoroughly discussed and presented in the context of its contributions to host–pathogen interactions and the control/prevention of infection. PMID:21687428

  3. The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa.

    PubMed

    Toll-Riera, Macarena; San Millan, Alvaro; Wagner, Andreas; MacLean, R Craig

    2016-05-01

    Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs.

  4. A Network Biology Approach to Denitrification in Pseudomonas aeruginosa

    PubMed Central

    Arat, Seda; Bullerjahn, George S.; Laubenbacher, Reinhard

    2015-01-01

    Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO2), nitric oxide (NO) and nitrous oxide (N2O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O2), nitrate (NO3), and phosphate (PO4) suggests that PO4 concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO4 on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N2O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide. PMID:25706405

  5. Infectious conjunctivitis caused by Pseudomonas aeruginosa isolated from a bathroom

    PubMed Central

    2013-01-01

    Background The elucidation of the routes of transmission of a pathogen is crucial for the prevention of infectious diseases caused by bacteria that are not a resident in human tissue. The purpose of this report is to describe a case of suture-related conjunctivitis caused by Pseudomonas aeruginosa for which we identified the transmission route using pulsed-field gel electrophoresis (PFGE). Case presentation A 38-year-old man, who had undergone surgery for glaucoma 2 years ago previously, presented with redness, discomfort, and mucopurulent discharge in the right eye. A 9–0 silk suture had been left on the conjunctiva. A strain of P. aeruginosa was isolated from a culture obtained from the suture, and the patient was therefore diagnosed with suture-related conjunctivitis caused by P. aeruginosa. The conjunctivitis was cured by the application of an antimicrobial ophthalmic solution and removal of the suture. We used PFGE to survey of the indoor and outdoor environments around the patient’s house and office in order to elucidate the route of transmission of the infection. Three strains of P. aeruginosa were isolated from the patient’s indoor environment, and the isolate obtained from the patient’s bathroom was identical to that from the suture. Conclusion The case highlights the fact that an indoor environmental strain of P. aeruginosa can cause ocular infections. PMID:23815865

  6. 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.

  7. Pseudomonas aeruginosa Virulence and Therapy: Evolving Translational Strategies

    PubMed Central

    Veesenmeyer, Jeffrey L.; Lisboa, Thiago; Rello, Jordi

    2009-01-01

    Structured abstract Objective Although most reviews of Pseudomonas aeruginosa therapeutics focus on antibiotics currently in use or in the pipeline, we review evolving translational strategies aimed at using virulence factor antagonists as adjuvant therapies. Data Source Current literature regarding P. aeruginosa virulence determinants and approaches that target them, with an emphasis on type III secretion, quorum-sensing, biofilms, and flagella. Data Extraction and Synthesis P. aeruginosa remains one of the most important pathogens in nosocomial infections, with high associated morbidity and mortality. Its predilection to develop resistance to antibiotics and expression of multiple virulence factors contributes to the frequent ineffectiveness of current therapies. Among the many P. aeruginosa virulence determinants that impact infections, type III secretion, quorum sensing, biofilm formation, and flagella have been the focus of much recent investigation. Here we review how increased understanding of these important bacterial structures and processes has enabled the development of novel approaches to inhibit each. These promising translational strategies may lead to the development of adjuvant therapies capable of improving outcomes. Conclusions Adjuvant therapies directed against virulence factors have the potential to improve outcomes in P. aeruginosa infections. PMID:19325463

  8. [Resistance to antibiotics in Pseudomonas aeruginosa in Colombian hospitals].

    PubMed

    Villa, Lina M; Cortés, Jorge A; Leal, Aura L; Meneses, Andrés; Meléndez, Martha P

    2013-12-01

    Pseudomonas aeruginosa infections cause high morbidity and mortality. We performed a descriptive analysis of the rates of antibiotic resistance in isolates of P. aeruginosa in 33 hospitals enrolled in a surveillance network in Colombia. The study was conducted between January 2005 and December 2009 .9905 isolates of P. aeruginosa were identified, (4.9% of all strains). In intensive care units (ICU) P. aeruginosa showed an overall resistance to aztreonam, cefepime , ceftazidime, imipenem, meropenem , and piperacillin / tazobactam of 31.8% , 23.9% , 24.8%, 22.5%, 20.3% and 22.3%, respectively. Resistance rates increased for piperacillin/tazobactam, cefepime, and imipenem; remained unchanged for meropenem; and decreased for aminoglycosides, quinolones and ceftazidime. Resistance to one, two and three or more families of antibiotics was found in 17%, 12.5%, and 32.1%, respectively. In samples collected from the wards, the resistance rate was lower but usually over 10%. Antibiotic resistance in P. aeruginosa isolates in hospitalized patients and particularly in those admitted to ICUs in Colombia is high.

  9. The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa

    PubMed Central

    Wagner, Andreas; MacLean, R. Craig

    2016-01-01

    Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs. PMID:27149698

  10. A network biology approach to denitrification in Pseudomonas aeruginosa

    DOE PAGES

    Arat, Seda; Bullerjahn, George S.; Laubenbacher, Reinhard

    2015-02-23

    Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO₂), nitric oxide (NO) and nitrous oxide (N₂O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O₂), nitrate (NO₃),more » and phosphate (PO₄) suggests that PO₄ concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO₄ on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N₂O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.« less

  11. Variation in chlorobenzoate catabolism by Pseudomonas putida P111 as a consequence of genetic alterations

    SciTech Connect

    Brenner, V.; Focht, D.D. ); Hernandez, B.S. )

    1993-09-01

    Chlorobenzoates are key intermediates in the degradative pathways of polychlorinated biphenyls and benzoate herbicides. Bacteria that cometabolize these pollutants generally accumulate chlorobenzoates because they are not able to grow on them. Special interest has been focused on ortho-chlorobenzoates because they are more refractory to biodegradation. In all of these studies the enzyme responsible for the first attack on the ortho-chlorobenzoates possesses minimal or negligible activity with meta- or para-chlorobenzoates. This study reports evidence for the existence of two separate benzoate dioxygenases in Pseudomonas putida P111 and for the transpostional nature of the clc operon, on the basis of genetic investigations of different phenotypic variants of this strain. 42 refs., 4 figs., 1 tab.

  12. The purification and characterization of 4-ethylphenol methylenehydroxylase, a flavocytochrome from Pseudomonas putida JD1.

    PubMed

    Reeve, C D; Carver, M A; Hopper, D J

    1989-10-15

    The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium. PMID:2556994

  13. c-Type Cytochromes and Manganese Oxidation in Pseudomonas putida MnB1

    PubMed Central

    Caspi, Ron; Tebo, Bradley M.; Haygood, M. G.

    1998-01-01

    Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation. PMID:9758766

  14. Oxidative stress in bacteria (Pseudomonas putida) exposed to nanostructures of silicon carbide.

    PubMed

    Borkowski, Andrzej; Szala, Mateusz; Kowalczyk, Paweł; Cłapa, Tomasz; Narożna, Dorota; Selwet, Marek

    2015-09-01

    Silicon carbide (SiC) nanostructures produced by combustion synthesis can cause oxidative stress in the bacterium Pseudomonas putida. The results of this study showed that SiC nanostructures damaged the cell membrane, which can lead to oxidative stress in living cells and to the loss of cell viability. As a reference, micrometric SiC was also used, which did not exhibit toxicity toward cells. Oxidative stress was studied by analyzing the activity of peroxidases, and the expression of the glucose-6-phosphate dehydrogenase gene (zwf1) using real-time PCR and northern blot techniques. Damage to nucleic acid was studied by isolating and hydrolyzing plasmids with the formamidopyrimidine [fapy]-DNA glycosylase (also known as 8-oxoguanine DNA glycosylase) (Fpg), which is able to detect damaged DNA. The level of viable microbial cells was investigated by propidium iodide and acridine orange staining.

  15. Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida)

    SciTech Connect

    Sokol, W.

    1987-12-01

    The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. The equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.

  16. Characterization of Pseudooxynicotine Amine Oxidase of Pseudomonas putida S16 that Is Crucial for Nicotine Degradation

    PubMed Central

    Hu, Haiyang; Wang, Weiwei; Tang, Hongzhi; Xu, Ping

    2015-01-01

    Pseudooxynicotine amine oxidase (Pnao) is essential to the pyrrolidine pathway of nicotine degradation of Pseudomonas putida strain S16, which is significant for the detoxification of nicotine, through removing the CH3NH2 group. However, little is known about biochemical mechanism of this enzyme. Here, we characterized its properties and biochemical mechanism. Isotope labeling experiments provided direct evidence that the newly introduced oxygen atom in 3-succinoylsemialdehyde-pyridine is derived from H2O, but not from O2. Pnao was very stable at temperatures below 50 °C; below this temperature, the enzyme activity increased as temperature rose. Site-directed mutagenesis studies showed that residue 180 is important for its thermal stability. In addition, tungstate may enhance the enzyme activity, which has rarely been reported before. Our findings make a further understanding of the crucial Pnao in nicotine degradation. PMID:26634650

  17. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    PubMed

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures. PMID:27333909

  18. c-Type cytochromes and manganese oxidation in Pseudomonas putida MnB1

    SciTech Connect

    Caspi, R.; Tebo, B.M.; Haygood, M.G.

    1998-10-01

    Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. The authors used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and they characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.

  19. Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida).

    PubMed

    Sokol, W

    1987-12-01

    The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.

  20. [The role of mineral phosphorus compounds in naphthalene biodegradation by Pseudomonas putida].

    PubMed

    Puntus, I F; Ryazanova, L P; Zvonarev, A N; Funtikova, T V; Kulakovskaya, T V

    2015-01-01

    The effect of phosphate concentration in the culture medium on the growth and naphthalene degradation by Pseudomonas putida BS 3701 was studied. The limiting concentration of phosphate was 0.4 mM and 0.1 mM under cultivation in media with naphthalene and glucose, respectively The phosphate deficiency correlated with a decrease in the activities of naphthalene dioxygenase and salicylate hydroxylase and with salicylate accumulation in the culture medium. We suggest that this fact indicates the impaired regulation of gene expression of "upper" and "lower" pathways of naphthalene oxidation. Under naphthalene degradation, the cells accumulated three times more inorganic polyphosphates as compared with the consumption of glucose. The involvement of polyphosphates in the regulation of naphthalene metabolism has been considered.

  1. Characterization of Pseudooxynicotine Amine Oxidase of Pseudomonas putida S16 that Is Crucial for Nicotine Degradation.

    PubMed

    Hu, Haiyang; Wang, Weiwei; Tang, Hongzhi; Xu, Ping

    2015-12-04

    Pseudooxynicotine amine oxidase (Pnao) is essential to the pyrrolidine pathway of nicotine degradation of Pseudomonas putida strain S16, which is significant for the detoxification of nicotine, through removing the CH3NH2 group. However, little is known about biochemical mechanism of this enzyme. Here, we characterized its properties and biochemical mechanism. Isotope labeling experiments provided direct evidence that the newly introduced oxygen atom in 3-succinoylsemialdehyde-pyridine is derived from H2O, but not from O2. Pnao was very stable at temperatures below 50 °C; below this temperature, the enzyme activity increased as temperature rose. Site-directed mutagenesis studies showed that residue 180 is important for its thermal stability. In addition, tungstate may enhance the enzyme activity, which has rarely been reported before. Our findings make a further understanding of the crucial Pnao in nicotine degradation.

  2. Why Does the Healthy Cornea Resist Pseudomonas aeruginosa Infection?

    PubMed Central

    Evans, David J.; Fleiszig, Suzanne M. J.

    2013-01-01

    Purpose To provide our perspective on why the cornea is resistant to infection based on our research results with Pseudomonas aeruginosa. Perspective We focus on our current understanding of the interplay between bacteria, tear fluid and the corneal epithelium that determine health as the usual outcome, and propose a theoretical model for how contact lens wear might change those interactions to enable susceptibility to P. aeruginosa infection. Methods Use of “null-infection” in vivo models, cultured human corneal epithelial cells, contact lens-wearing animal models, and bacterial genetics help to elucidate mechanisms by which P. aeruginosa survive at the ocular surface, adheres, and traverses multilayered corneal epithelia. These models also help elucidate the molecular mechanisms of corneal epithelial innate defense. Results and Discussion Tear fluid and the corneal epithelium combine to make a formidable defense against P. aeruginosa infection of the cornea. Part of that defense involves the expression of antimicrobials such as β-defensins, the cathelicidin LL-37, cytokeratin-derived antimicrobial peptides, and RNase7. Immunomodulators such as SP-D and ST2 also contribute. Innate defenses of the cornea depend in part on MyD88, a key adaptor protein of TLR and IL-1R signaling, but the basal lamina represents the final barrier to bacterial penetration. Overcoming these defenses involves P. aeruginosa adaptation, expression of the type three secretion system, proteases, and P. aeruginosa biofilm formation on contact lenses. Conclusion After more than two decades of research focused on understanding how contact lens wear predisposes to P. aeruginosa infection, our working hypothesis places blame for microbial keratitis on bacterial adaptation to ocular surface defenses, combined with changes to the biochemistry of the corneal surface caused by trapping bacteria and tear fluid against the cornea under the lens. PMID:23601656

  3. Understanding butanol tolerance and assimilation in Pseudomonas putida BIRD-1: an integrated omics approach.

    PubMed

    Cuenca, María del Sol; Roca, Amalia; Molina-Santiago, Carlos; Duque, Estrella; Armengaud, Jean; Gómez-Garcia, María R; Ramos, Juan L

    2016-01-01

    Pseudomonas putida BIRD-1 has the potential to be used for the industrial production of butanol due to its solvent tolerance and ability to metabolize low-cost compounds. However, the strain has two major limitations: it assimilates butanol as sole carbon source and butanol concentrations above 1% (v/v) are toxic. With the aim of facilitating BIRD-1 strain design for industrial use, a genome-wide mini-Tn5 transposon mutant library was screened for clones exhibiting increased butanol sensitivity or deficiency in butanol assimilation. Twenty-one mutants were selected that were affected in one or both of the processes. These mutants exhibited insertions in various genes, including those involved in the TCA cycle, fatty acid metabolism, transcription, cofactor synthesis and membrane integrity. An omics-based analysis revealed key genes involved in the butanol response. Transcriptomic and proteomic studies were carried out to compare short and long-term tolerance and assimilation traits. Pseudomonas putida initiates various butanol assimilation pathways via alcohol and aldehyde dehydrogenases that channel the compound to central metabolism through the glyoxylate shunt pathway. Accordingly, isocitrate lyase - a key enzyme of the pathway - was the most abundant protein when butanol was used as the sole carbon source. Upregulation of two genes encoding proteins PPUBIRD1_2240 and PPUBIRD1_2241 (acyl-CoA dehydrogenase and acyl-CoA synthetase respectively) linked butanol assimilation with acyl-CoA metabolism. Butanol tolerance was found to be primarily linked to classic solvent defense mechanisms, such as efflux pumps, membrane modifications and control of redox state. Our results also highlight the intensive energy requirements for butanol production and tolerance; thus, enhancing TCA cycle operation may represent a promising strategy for enhanced butanol production. PMID:26986205

  4. Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1

    PubMed Central

    Parker, Dorothy L.; Lee, Sung-Woo; Geszvain, Kati; Davis, Richard E.; Gruffaz, Christelle; Meyer, Jean-Marie; Torpey, Justin W.; Tebo, Bradley M.

    2014-01-01

    When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III). PMID:24847318

  5. Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1.

    PubMed

    Parker, Dorothy L; Lee, Sung-Woo; Geszvain, Kati; Davis, Richard E; Gruffaz, Christelle; Meyer, Jean-Marie; Torpey, Justin W; Tebo, Bradley M

    2014-01-01

    When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III). PMID:24847318

  6. Modeling of TCE and Toluene Toxicity to Pseudomonas putida F1

    NASA Astrophysics Data System (ADS)

    Singh, R.; Olson, M. S.

    2009-12-01

    Prediction of viable bacterial distribution with respect to contaminants is important for efficient bioremediation of contaminated ground-water aquifers, particularly those contaminated with residual NAPLs. While bacterial motility and chemotaxis may help situate bacteria close to high concentrations of contaminant thereby enhancing bioremediation, prolonged exposure to high concentrations of contaminates is toxic to contaminant-degrading bacteria. The purpose of this work is to model the toxicity of trichloroethylene and toluene to Pseudomonas putida F1. The Live/Dead® bacterial viability assay was used to determine the toxic effect of chemical contaminants on the viability of P. putida F1 in a sealed zero head-space experimental environment. Samples of bacterial suspensions were exposed to common ground-water pollutants, TCE and toluene, for different durations. Changes in live and dead cell populations were monitored over the course of experiments using fluorescence microscopy. Data obtained from these toxicity experiments were fit to simple linear and exponential bacterial decay models using non-linear regression to describe loss of bacterial viability. TCE toxicity to P. putida F1 was best described with an exponential decay model (Figure 1a), with a decay constant kTCE = 0.025 h-4.95 (r2 = 0.956). Toluene toxicity showed a marginally better fit to the linear decay model (Figure 1b) (r2 = 0.971), with a decay constant ktoluene = 0.204 h-1. Best-fit model parameters obtained for both TCE and toluene were used to predict bacterial viability in toxicity experiments with higher contaminant concentrations and matched well with experimental data. Results from this study can be used to predict bacterial accumulation and viability near NAPL sources, and thus may be helpful in improving bioremediation performance assessment of contaminated sites. Figure 1: Survival ratios (S = N/No) of P. putida F1 in TCE- (a) and toluene- (b) stressed samples (observed (

  7. 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.

  8. INHIBITION OF VIRULENCE FACTORS OF PSEUDOMONAS AERUGINOSA BY DICLOFENAC SODIUM.

    PubMed

    Abbas, Hisham A

    2015-01-01

    Resistance of Pseudomonas aeruginosa to antibiotics is a major problem. Targeting virulence factors is an alternative option to avoid the emergence of resistance to antibiotics. The effect of sub-inhibitory concentration of diclofenac sodium on the production of virulence factors of P. aeruginosa was investigated. The virulence factors included protease, haemolysin, pyocyanin and pyoverdin, in addition to pathogenic behaviors such as swimming and twitching motilities and biofilm formation. Diclofenac sodium showed significant inhibition of virulence factors as compared to the control. Diclofenac sodium decreased twitching and swimming motilities by 29.27% and 45.36%, respectively. The percentage of inhibition of pyocyanin by diclofenac sodium was 42.32%. On the other hand, pyoverdin was inhibited to a lesser extent (36.72%). Diclofenac sodium reduced protease by 52.58% and biofilm formation by 58.37%. Moreover, haemolytic activity in the presence of diclofenac sodium was 15.64% as compared to the control (100% haemolytic activity). The inhibitory activities may be due to inhibition of quorum sensing that regulates the expression of virulence factors. This study suggests the potential for the use of diclofenac sodium as an anti-virulence agent in the treatment of Pseudomonas aeruginosa infections.

  9. INHIBITION OF VIRULENCE FACTORS OF PSEUDOMONAS AERUGINOSA BY DICLOFENAC SODIUM.

    PubMed

    Abbas, Hisham A

    2015-01-01

    Resistance of Pseudomonas aeruginosa to antibiotics is a major problem. Targeting virulence factors is an alternative option to avoid the emergence of resistance to antibiotics. The effect of sub-inhibitory concentration of diclofenac sodium on the production of virulence factors of P. aeruginosa was investigated. The virulence factors included protease, haemolysin, pyocyanin and pyoverdin, in addition to pathogenic behaviors such as swimming and twitching motilities and biofilm formation. Diclofenac sodium showed significant inhibition of virulence factors as compared to the control. Diclofenac sodium decreased twitching and swimming motilities by 29.27% and 45.36%, respectively. The percentage of inhibition of pyocyanin by diclofenac sodium was 42.32%. On the other hand, pyoverdin was inhibited to a lesser extent (36.72%). Diclofenac sodium reduced protease by 52.58% and biofilm formation by 58.37%. Moreover, haemolytic activity in the presence of diclofenac sodium was 15.64% as compared to the control (100% haemolytic activity). The inhibitory activities may be due to inhibition of quorum sensing that regulates the expression of virulence factors. This study suggests the potential for the use of diclofenac sodium as an anti-virulence agent in the treatment of Pseudomonas aeruginosa infections. PMID:27328521

  10. Adherence of Pseudomonas aeruginosa to contact lenses

    SciTech Connect

    Miller, M.J.

    1988-01-01

    The purpose of this research was to examined the interactions of P. aeruginosa with hydrogel contact lenses and other substrata, and characterize adherence to lenses under various physiological and physicochemical conditions. Isolates adhered to polystyrene, glass, and hydrogel lenses. With certain lens types, radiolabeled cells showed decreased adherence with increasing water content of the lenses, however, this correlation with not found for all lenses. Adherence to rigid gas permeable lenses was markedly greater than adherence to hydrogels. Best adherence occurred near pH 7 and at a sodium chloride concentration of 50 mM. Passive adhesion of heat-killed cells to hydrogels was lower than the adherence obtained of viable cells. Adherence to hydrogels was enhanced by mucin, lactoferrin, lysozyme, IgA, bovine serum albumin, and a mixture of these macromolecules. Adherence to coated and uncoated lenses was greater with a daily-wear hydrogel when compared with an extended-wear hydrogel of similar polymer composition. Greater adherence was attributed to a higher concentration of adsorbed macromolecules on the 45% water-content lens in comparison to the 55% water-content lens.

  11. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

    PubMed Central

    Ángeles, Martínez-Toledo; Refugio, Rodríguez-Vázquez

    2013-01-01

    In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = −0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = −0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil. PMID:24294259

  12. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil.

    PubMed

    Ángeles, Martínez-Toledo; Refugio, Rodríguez-Vázquez

    2013-01-01

    In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil. PMID:24294259

  13. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil.

    PubMed

    Ángeles, Martínez-Toledo; Refugio, Rodríguez-Vázquez

    2013-01-01

    In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.

  14. Crude glycerol as feedstock for the sustainable production of p-hydroxybenzoate by Pseudomonas putida S12.

    PubMed

    Verhoef, Suzanne; Gao, Nisi; Ruijssenaars, Harald J; de Winde, Johannes H

    2014-01-25

    Crude glycerol is a promising renewable feedstock in bioconversion processes for the production of fuels and chemicals. Impurities present in crude glycerol can however, negatively impact the fermentation process. Successful crude glycerol utilization requires robust microbial production hosts that tolerate and preferably, can utilize such impurities. We investigated utilization of crude, unpurified glycerol as a substrate for the production of aromatic compounds by solvent tolerant Pseudomonas putida S12. In high-cell density fed-batch fermentations, P. putida S12 surprisingly performed better on crude glycerol than on purified glycerol. By contrast, growth of Escherichia coli was severely compromised under these high cell density cultivation conditions on crude glycerol. For P. putida S12 the biomass-to-substrate yield, maximum biomass production rate and substrate uptake rate were consistently higher on crude glycerol. Moreover, production of p-hydroxybenzoate by engineered P. putida S12palB5 on crude glycerol showed a 10% yield improvement over production on purified glycerol. P. putida S12 is a favorable host for bioconversion processes utilizing crude glycerol as a substrate. Its intrinsic stress-tolerance properties provide the robustness required for efficient growth and metabolism on this renewable substrate. PMID:23999132

  15. The Role of CzcRS Two-Component Systems in the Heavy Metal Resistance of Pseudomonas putida X4

    PubMed Central

    Liu, Pulin; Chen, Xi; Huang, Qiaoyun; Chen, Wenli

    2015-01-01

    The role of different czcRS genes in metal resistance and the cross-link between czcRS and czcCBA in Pseudomonas putida X4 were studied to advance understanding of the mechanisms by which P. putida copes with metal stress. Similar to P. putida KT2440, two complete czcRS1 and czcRS2 two-component systems, as well as a czcR3 without the corresponding sensing component were amplified in P. putida X4. The histidine kinase genes czcS1 and czcS2 were inactivated and fused to lacZ by homologous recombination. The lacZ fusion assay revealed that Cd2+ and Zn2+ caused a decrease in the transcription of czcRS1, whereas Cd2+ treatment enhanced the transcription of czcRS2. The mutation of different czcRSs showed that all czcRSs are necessary to facilitate full metal resistance in P. putida X4. A putative gene just downstream of czcR3 is related to metal ion resistance, and its transcription was activated by Zn2+. Data from quantitative real-time polymerase chain reaction (qRT-PCR) strongly suggested that czcRSs regulate the expression of czcCBA, and a cross-link exists between different czcRSs. PMID:26225958

  16. Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings

    PubMed Central

    Brandenburg, Kenneth S.; Calderon, Diego F.; Kierski, Patricia R.; Brown, Amanda L.; Shah, Nihar M.; Abbott, Nicholas L.; Schurr, Michael J.; Murphy, Christopher J.; McAnulty, Jonathan F.; Czuprynski, Charles J.

    2016-01-01

    Chronic non-healing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building upon prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the 3-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing. PMID:26342168

  17. Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings.

    PubMed

    Brandenburg, Kenneth S; Calderon, Diego F; Kierski, Patricia R; Brown, Amanda L; Shah, Nihar M; Abbott, Nicholas L; Schurr, Michael J; Murphy, Christopher J; McAnulty, Jonathan F; Czuprynski, Charles J

    2015-01-01

    Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.

  18. Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings.

    PubMed

    Brandenburg, Kenneth S; Calderon, Diego F; Kierski, Patricia R; Brown, Amanda L; Shah, Nihar M; Abbott, Nicholas L; Schurr, Michael J; Murphy, Christopher J; McAnulty, Jonathan F; Czuprynski, Charles J

    2015-01-01

    Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing. PMID:26342168

  19. Pseudomonas aeruginosa PAO1 Kills Caenorhabditis elegans by Cyanide Poisoning

    PubMed Central

    Gallagher, Larry A.; Manoil, Colin

    2001-01-01

    In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium. PMID:11591663

  20. [Sodium houttuyfonate inhibits virulence related motility of Pseudomonas aeruginosa].

    PubMed

    Wu, Da-qiang; Huang, Wei-feng; Duan, Qiang-jun; Cheng, Hui-juan; Wang, Chang-zhong

    2015-04-01

    Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic. PMID:26281603

  1. Adaptation of Aerobically Growing Pseudomonas aeruginosa to Copper Starvation▿ †

    PubMed Central

    Frangipani, Emanuela; Slaveykova, Vera I.; Reimmann, Cornelia; Haas, Dieter

    2008-01-01

    Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA′-′lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases. PMID:18708503

  2. Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition

    PubMed Central

    Peek, Mary E.; Bhatnagar, Abhinav; McCarty, Nael A.; Zughaier, Susu M.

    2012-01-01

    Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs. PMID:22973307

  3. The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa.

    PubMed

    Wegele, Rosalina; Tasler, Ronja; Zeng, Yuhong; Rivera, Mario; Frankenberg-Dinkel, Nicole

    2004-10-29

    For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.

  4. 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

  5. Adaptation of aerobically growing Pseudomonas aeruginosa to copper starvation.

    PubMed

    Frangipani, Emanuela; Slaveykova, Vera I; Reimmann, Cornelia; Haas, Dieter

    2008-10-01

    Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases. PMID:18708503

  6. Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa.

    PubMed

    Muller, Jocelyn Fraga; Ghosh, Sudeshna; Ikuma, Kaoru; Stevens, Ann M; Love, Nancy G

    2015-11-01

    Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment.

  7. Flagellation of Pseudomonas aeruginosa in newly divided cells

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Lee, Calvin; Anda, Jaime; Wong, Gerard

    2015-03-01

    For monotrichous bacteria, Pseudomonas aeruginosa, after cell division, one daughter cell inherits the old flagellum from its mother cell, and the other grows a new flagellum during or after cell division. It had been shown that the new flagellum grows at the distal pole of the dividing cell when the two daughter cells haven't completely separated. However, for those daughter cells who grow new flagella after division, it still remains unknown at which pole the new flagellum will grow. Here, by combining our newly developed bacteria family tree tracking techniques with genetic manipulation method, we showed that for the daughter cell who did not inherit the old flagellum, a new flagellum has about 90% chances to grow at the newly formed pole. We proposed a model for flagellation of P. aeruginosa.

  8. [Profiles of resistance to aminosides of Pseudomonas aeruginosa].

    PubMed

    Lesage, D; Delisle-Mizon, F; Vergez, P; Daguet, G

    1987-05-01

    Among all Gram-negative bacilli, Pseudomonas aeruginosa is one of the most resistant to aminoglycosides. Five hundred and seventeen P. aeruginosa strains were studied. Isolates came from three Paris hospitals. Reference strains were provided by P. Courvalin and A. Philippon. The following aminoglycosides were used: streptomycin (S), spectinomycin (Sp), kanamycin (K), neomycin (N), gentamicin (G), sisomicin (Ss), netilmicin (Nt), tobramycin (T), amikacin (A), habekacin (H). The in vitro activity of antibiotics was evaluated by the standardized disk agar diffusion test. Distribution of inhibition zone diameters among susceptible strains were represented by histograms. Resistance frequency to aminoglycosides was: G: 61.5%, Ss: 38.1%, T: 35.8%, Nt: 58.2%, A: 15.5%, Seven resistance patterns were identified: G: 3%, G Ss: 3%, G Nt: 8%, G Ss Nt: 7%, G Ss T: 5%, G Ss T Nt: 53%, G Ss T Nt A: 21%. Hypothesis about resistance mechanisms and interpretation of disk agar diffusion test are discussed.

  9. Mass Spectrometry Analysis of Pseudomonas aeruginosa Treated With Azithromycin

    PubMed Central

    Phelan, Vanessa V.; Fang, Jinshu; Dorrestein, Pieter C.

    2015-01-01

    In microbiology, changes in specialized metabolite production (cell-to-cell signaling metabolites, virulence factors and natural products) are measured using phenotypic assays. However, advances in mass spectrometry based techniques including imaging mass spectrometry (IMS) now allow researchers to directly visualize the production of specialized metabolites from microbial colony biofilms. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the effect of the macrolide antibiotic azithromycin (AZM) on colony biofilms of Pseudomonas aeruginosa. While previous research suggested that AZM may inhibit cell-to-cell signaling of P. aeruginosa and thereby reducing pathogenicity, we observed no clear decrease in specialized metabolite production. PMID:25801585

  10. [Water used for hemodialysis equipment: where is Pseudomonas aeruginosa?].

    PubMed

    Ducki, Sébastien; Francini, Nicolas; Blech, Marie-Françoise

    2005-05-01

    The water used in dilution of the dialysis solutions constitutes an essential element of the efficiency and the safety of this therapeutics. Water must be specifically treated, and some technical rules must be respected, such as disinfection of the equipment for water treatment, to guarantee a satisfying level for whole the installation. This article reports the investigations, which were led to find the spring of Pseudomonas aeruginosa which contamined in a recurring way the water feeding dialysis equipment. The observation of samples'chronology and an analysis of the sanitary pad suggested a contamination during disinfection. Sample of residual water from the pump used for the injection of Dialox identified this reservoir as origin of the contamination. To stop this contamination by P. aeruginosa, a pump maintenance revision and purges of the system were used.

  11. Mass Spectrometry Analysis of Pseudomonas aeruginosa Treated with Azithromycin

    NASA Astrophysics Data System (ADS)

    Phelan, Vanessa V.; Fang, Jinshu; Dorrestein, Pieter C.

    2015-06-01

    In microbiology, changes in specialized metabolite production (cell-to-cell signaling metabolites, virulence factors, and natural products) are measured using phenotypic assays. However, advances in mass spectrometry-based techniques including imaging mass spectrometry (IMS) now allow researchers to directly visualize the production of specialized metabolites from microbial colony biofilms. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the effect of the macrolide antibiotic azithromycin (AZM) on colony biofilms of Pseudomonas aeruginosa. Although previous research suggested that AZM may inhibit cell-to-cell signaling of P. aeruginosa and thereby reduce pathogenicity, we observed no clear decrease in specialized metabolite production.

  12. Regulation of the Mandelate Pathway in Pseudomonas aeruginosa

    PubMed Central

    Rosenberg, S. L.

    1971-01-01

    The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate → benzoylformate → benzaldehyde → benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the β-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzymes, benzoylformate decarboxylase and benzaldehyde dehydrogenase, are both induced by benzoylformate. The same benzaldehyde dehydrogenase, or one very similar to it, is also induced by β-ketoadipate, an intermediate in the subsequent metabolism of benzoate. This dehydrogenase may also be induced by adipate or a metabolite of adipate. These conclusions have been drawn from the physiological and genetic properties of wild-type P. aeruginosa strains and from the study of mutants lacking the second and third enzyme activities. PMID:5003176

  13. [Use od ozone for disinfection of ships' system of water supply contaminated by Pseudomonas aeruginosa].

    PubMed

    Rakhmanin, Iu A; Strikalenko, T V; Mokienko, A V; Stoianova, N V; Gutsel', Iu I

    1990-11-01

    Experimental substantiation is given of the use of ozone in doses, recommended for disinfection of water and ship water supply systems infected by Pseudomonas aeruginosa. The positive effect of ozonation of water supply systems infected by Pseudomonas aeruginosa was confirmed by results of field testing on ships of the Black sea marine steam-navigation.

  14. Revised structures of the pyoverdins from Pseudomonas putida CFBP 2461 and from Pseudomonas fluorescens CFBP 2392.

    PubMed

    Beiderbeck, H; Taraz, K; Meyer, J M

    1999-12-01

    Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, D- and L-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins. PMID:10816733

  15. The Approach to Pseudomonas aeruginosa in Cystic Fibrosis.

    PubMed

    Talwalkar, Jaideep S; Murray, Thomas S

    2016-03-01

    There is a high prevalence of Pseudomonas aeruginosa in patients with cystic fibrosis and clear epidemiologic links between chronic infection and morbidity and mortality exist. Prevention and early identification of infection are critical, and stand to improve with the advent of new vaccines and laboratory methods. Once the organism is identified, a variety of treatment options are available. Aggressive use of antipseudomonal antibiotics is the standard of care for acute pulmonary exacerbations in cystic fibrosis, and providers must take into account specific patient characteristics when making treatment decisions related to antibiotic selection, route and duration of administration, and site of care.

  16. Antibiotic susceptibility of clinical isolates of Pseudomonas aeruginosa.

    PubMed

    Cervantes-Vega, C; Chavez, J; Rodriguez, M G

    1986-01-01

    Three hundred and twenty two clinical isolates of Pseudomonas aeruginosa collected in Morelia, México, were analyzed for in vitro susceptibility to five antibiotics by agar dilution tests. Antibiotic resistance was shown by 50% of total isolates. Frequencies of resistance were: streptomycin, 47%; gentamicin, 13%; tobramycin, 8%; and carbenicillin, 7%; no amikacin resistance was found. The more common resistance patterns were streptomycin, gentamicin-streptomycin, and tobramycin-gentamicin-streptomycin. Resistance to either tobramycin, gentamicin or carbenicillin was found mainly in pyocin type 10 isolates. The proportion of antibiotic resistant isolates ranged from 37 to 75% in four hospitals, and amounted 24% in three clinical laboratories.

  17. Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

    PubMed Central

    Chen, Y H; Hancock, R E; Mishell, R I

    1980-01-01

    Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens. Images Fig. 2 PMID:6769818

  18. Nanoindentation of Pseudomonas aeruginosa bacterial biofilm using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Baniasadi, Mahmoud; Xu, Zhe; Gandee, Leah; Du, Yingjie; Lu, Hongbing; Zimmern, Philippe; Minary-Jolandan, Majid

    2014-12-01

    Bacterial biofilms are a source of many chronic infections. Biofilms and their inherent resistance to antibiotics are attributable to a range of health issues including affecting prosthetic implants, hospital-acquired infections, and wound infection. Mechanical properties of biofilm, in particular, at micro- and nano-scales, are governed by microstructures and porosity of the biofilm, which in turn may contribute to their inherent antibiotic resistance. We utilize atomic force microscopy (AFM)-based nanoindentation and finite element simulation to investigate the nanoscale mechanical properties of Pseudomonas aeruginosa bacterial biofilm. This biofilm was derived from human samples and represents a medically relevant model.

  19. Genomic analysis of the role of RNase R in the turnover of Pseudomonas putida mRNAs.

    PubMed

    Fonseca, Pilar; Moreno, Renata; Rojo, Fernando

    2008-09-01

    RNase R is a 3'-5' highly processive exoribonuclease that can digest RNAs with extensive secondary structure. We analyzed the global effect of eliminating RNase R on the Pseudomonas putida transcriptome and the expression of the rnr gene under diverse conditions. The absence of RNase R led to increased levels of many mRNAs, indicating that it plays an important role in mRNA turnover.

  20. Infections with Pseudomonas aeruginosa in patients with cystic fibrosis.

    PubMed

    Tümmler, B; Bosshammer, J; Breitenstein, S; Brockhausen, I; Gudowius, P; Herrmann, C; Herrmann, S; Heuer, T; Kubesch, P; Mekus, F; Römling, U; Schmidt, K D; Spangenberg, C; Walter, S

    1997-02-01

    The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid

  1. Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440.

    PubMed

    Matthijs, Sandra; Laus, Georges; Meyer, Jean-Marie; Abbaspour-Tehrani, Kourosch; Schäfer, Mathias; Budzikiewicz, Herbert; Cornelis, Pierre

    2009-12-01

    Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida. PMID:19459056

  2. The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition‐related alterations

    PubMed Central

    Rühl, Jana; Hein, Eva‐Maria; Hayen, Heiko; Schmid, Andreas; Blank, Lars M.

    2012-01-01

    Summary Microorganisms, such as Pseudomonas putida, utilize specific physical properties of cellular membrane constituents, mainly glycerophospholipids, to (re‐)adjust the membrane barrier to environmental stresses. Building a basis for membrane composition/function studies, we inventoried the glycerophospholipids of different Pseudomonas and challenged membranes of growing cells with n‐butanol. Using a new high‐resolution liquid chromatography/mass spectrometry (LC/MS) method, 127 glycerophospholipid species [e.g. phosphatidylethanolamine PE(32:1)] with up to five fatty acid combinations were detected. The glycerophospholipid inventory consists of 305 distinct glycerophospholipids [e.g. PE(16:0/16:1)], thereof 14 lyso‐glycerophospholipids, revealing conserved compositions within the four investigated pseudomonads P. putida KT2440, DOT‐T1E, S12 and Pseudomonas sp. strain VLB120. Furthermore, we addressed the influence of environmental conditions on the glycerophospholipid composition of Pseudomonas via long‐time exposure to the sublethal n‐butanol concentration of 1% (v/v), focusing on: (i) relative amounts of glycerophospholipid species, (ii) glycerophospholipid head group composition, (iii) fatty acid chain length, (iv) degree of saturation and (v) cis/trans isomerization of unsaturated fatty acids. Observed alterations consist of changing head group compositions and for the solvent‐sensitive strain KT2440 diminished fatty acid saturation degrees. Minor changes in the glycerophospholipid composition of the solvent‐tolerant strains P. putida S12 and Pseudomonas sp. VLB120 suggest different strategies of the investigated Pseudomonas to maintain the barrier function of cellular membranes. PMID:21895997

  3. Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.

    PubMed

    Rodríguez-Herva, José Juan; Duque, Estrella; Molina-Henares, María Antonia; Navarro-Avilés, Gloria; Van Dillewijn, Pieter; De La Torre, Jesús; Molina-Henares, Antonio J; La Campa, Ana Sánchez-de; Ran, F Ann; Segura, Ana; Shingler, Victoria; Ramos, Juan-Luis

    2010-06-01

    Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region. PMID:23766109

  4. The functional structure of central carbon metabolism in Pseudomonas putida KT2440.

    PubMed

    Sudarsan, Suresh; Dethlefsen, Sarah; Blank, Lars M; Siemann-Herzberg, Martin; Schmid, Andreas

    2014-09-01

    What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

  5. Potential of Pseudomonas putida PCI2 for the Protection of Tomato Plants Against Fungal Pathogens.

    PubMed

    Pastor, Nicolás; Masciarelli, Oscar; Fischer, Sonia; Luna, Virginia; Rovera, Marisa

    2016-09-01

    Tomato is one of the most economically attractive vegetable crops due to its high yields. Diseases cause significant losses in tomato production worldwide. We carried out Polymerase Chain Reaction studies to detect the presence of genes encoding antifungal compounds in the DNA of Pseudomonas putida strain PCI2. We also used liquid chromatography-electrospray tandem mass spectrometry to detect and quantify the production of compounds that increase the resistance of plants to diseases from culture supernatants of PCI2. In addition, we investigated the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in PCI2. Finally, PCI2 was used for inoculation of tomato seeds to study its potential biocontrol activity against Fusarium oxysporum MR193. The obtained results showed that no fragments for the encoding genes of hydrogen cyanide, pyoluteorin, 2,4-diacetylphloroglucinol, pyrrolnitrin, or phenazine-1-carboxylic acid were amplified from the DNA of PCI2. On the other hand, PCI2 produced salicylic acid and jasmonic acid in Luria-Bertani medium and grew in a culture medium containing ACC as the sole nitrogen source. We observed a reduction in disease incidence from 53.33 % in the pathogen control to 30 % in tomato plants pre-inoculated with PCI2 as well as increases in shoot and root dry weights in inoculated plants, as compared to the pathogenicity control. This study suggests that inoculation of tomato seeds with P. putida PCI2 increases the resistance of plants to root rot caused by F. oxysporum and that PCI2 produces compounds that may be involved at different levels in increasing such resistance. Thus, PCI2 could represent a non-contaminating management strategy potentially applicable in vegetable crops such as tomato. PMID:27246499

  6. Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.

    PubMed

    Rodríguez-Herva, José Juan; Duque, Estrella; Molina-Henares, María Antonia; Navarro-Avilés, Gloria; Van Dillewijn, Pieter; De La Torre, Jesús; Molina-Henares, Antonio J; La Campa, Ana Sánchez-de; Ran, F Ann; Segura, Ana; Shingler, Victoria; Ramos, Juan-Luis

    2010-06-01

    Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

  7. Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida*

    PubMed Central

    Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M.; de Lorenzo, Víctor

    2011-01-01

    The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida. PMID:21239488

  8. The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4

    PubMed Central

    Chen, Qiongzhen; Tu, Hui; Luo, Xue; Zhang, Biying; Huang, Fei; Li, Zhoukun; Wang, Jue; Shen, Wenjing; Wu, Jiale; Cui, Zhongli

    2016-01-01

    Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2), para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR). It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study. PMID:27191401

  9. Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

    PubMed

    Ramalho, Rita; Cunha, Joaquim; Teixeira, Paula; Gibbs, Paul A

    2002-03-01

    Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water. PMID:11777584

  10. Effects of Cobalt on Manganese Oxidation by Pseudomonas putida MnB1

    NASA Astrophysics Data System (ADS)

    Pena, J.; Bargar, J.; Sposito, G.

    2005-12-01

    The oxidation of Mn(II) in the environment is thought to occur predominantly through biologically mediated pathways. During the stationary phase of growth, the well-characterized freshwater and soil bacterium Pseudomonas putida MnB1 oxidizes soluble Mn(II) to a poorly crystalline layer type Mn(IV) oxide. These Mn oxide particles (2 - 5 nm thickness) are deposited in a matrix of extracellular polymeric substances (EPS) surrounding the cell, creating a multi-component system distinct from commonly studied synthetic Mn oxides. Accurate characterization of the reactivity of these biomineral assemblages is essential to understanding trace metal biogeochemistry in natural waters and sediments. Moreover, these biogenic oxides may potentially be used for the remediation of surface and ground waters impacted by mining, industrial pollution, and other anthropogenic activities. In this study, we consider the interactions between Co, P. putida MnB1, and its biogenic Mn oxide. Cobalt is a redox-active transition metal which exists in the environment as Co(II) and Co(III). While Co is not generally found in the environment at toxic concentrations, it may be released as a byproduct of mining activities (e.g. levels of up to 20 μM are found in Pinal Creek, AZ, a stream affected by copper mining). In addition, the radionuclide 60Co, formed by neutron activation in nuclear reactors, is of concern at Department of Energy sites, such as that at Hanford, and has several industrial applications, including radiotherapy. We address the following questions: Do high levels of Co inhibit enzymatic processes such as Mn(II) oxidation? Can the multicopper oxidase enzyme involved in Mn(II) oxidation facilitate Co(II) oxidation? Lastly, does the organic matter surrounding the oxides affect Co or Mn oxide reactivity? These issues were approached via wet chemical analysis, synchrotron radiation X-ray diffraction (SR-XRD), and extended X-ray absorption fine structure (EXAFS) spectroscopy. In the

  11. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    PubMed Central

    2012-01-01

    Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l) with relative decolorization values of 91.2% (3 h) and 97.1% (18 h), as well as high activity to AR18 (1 g/l) by 80.5% (3 h) and 89.0% (18 h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h

  12. Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440

    PubMed Central

    2013-01-01

    Background Accumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations. Results Cells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a PrpoS → rpoS‘-’lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria. Conclusions While polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and

  13. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel.

    PubMed

    Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

    2016-08-11

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work.

  14. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel

    PubMed Central

    Arivett, Brock A.; Ream, Dave C.; Fiester, Steven E.; Kidane, Destaalem

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  15. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel.

    PubMed

    Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  16. Comparative proteomic analysis reveals mechanistic insights into Pseudomonas putida F1 growth on benzoate and citrate

    PubMed Central

    2013-01-01

    Pseudomonas species are capable to proliferate under diverse environmental conditions and thus have a significant bioremediation potential. To enhance our understanding of their metabolic versatility, this study explores the changes in the proteome and physiology of Pseudomonas putida F1 resulting from its growth on benzoate, a moderate toxic compound that can be catabolized, and citrate, a carbon source that is assimilated through central metabolic pathways. A series of repetitive batch cultivations were performed to ensure a complete adaptation of the bacteria to each of these contrasting carbon sources. After several growth cycles, cell growth stabilized at the maximum level and exhibited a reproducible growth profile. The specific growth rates measured for benzoate (1.01 ± 0.11 h-1) and citrate (1.11 ± 0.12 h-1) were similar, while a higher yield was observed for benzoate (0.6 and 0.3 g cell mass per g of benzoate and citrate, respectively), reflecting the different degrees of carbon reduction in the two substrates. Comparative proteomic analysis revealed an enrichment of several oxygenases/dehydrogenases in benzoate-grown cells, indicative of the higher carbon reduction of benzoate. Moreover, the upregulation of all 14 proteins implicated in benzoate degradation via the catechol ortho-cleavage pathway was observed, while several stress-response proteins were increased to aid cells to cope with benzoate toxicity. Unexpectedly, citrate posed more challenges than benzoate in the maintenance of pH homeostasis, as indicated by the enhancement of the Na+/H+ antiporter and carbonic anhydrase. The study provides important mechanistic insights into Pseudomonas adaptation to varying carbon sources that are of great relevance to bioremediation efforts. PMID:24156539

  17. Modulation of biofilms of Pseudomonas aeruginosa by quinolones.

    PubMed Central

    Yassien, M; Khardori, N; Ahmedy, A; Toama, M

    1995-01-01

    The interaction between four fluoroquinolones (ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin) and biofilms of Pseudomonas aeruginosa in wells of microtiter plates and on segments of vascular catheters were studied in an in vitro model of vascular catheter colonization. Subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of the fluoroquinolones reduced the adherence of P. aeruginosa to 30 to 33, 44 to 47, and 61 to 67% of that of controls, respectively. The addition of high concentrations of the fluoroquinolones (12.5 and 400 micrograms/ml) to preformed biofilms (grown for 48 h at 37 degrees C) decreased the adherence of P. aeruginosa to 69 to 77 and 39 to 60% of that of controls, respectively. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of fluoroquinolones reduced the number of adherent bacteria to 21 to 23, 40 to 46, and 55 to 70% of that of the controls, respectively. Scanning electron microscopy demonstrated a significant reduction in glycocalyx formation and adherent bacteria in the presence of pefloxacin at one-half to one-eight of the MIC. Vascular catheter segments precolonized with P. aeruginosa for 24 h and exposed to the fluoroquinolones at 4 to 25 times the MIC (50 micrograms/ml) for 2 h showed <5% growth of adherent cells compared with controls. No adherent organisms were cultured in the presence of 8 to 50 times the MIC (100 micrograms/ml). Scanning electron microscopy studies of preformed biofilms exposed to pefloxacin verified the results obtained by culture. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adherence of P. aeruginosa to plastic surfaces and vascular catheters. Clinically achievable concentrations of fluoroquinolones (50 to 100 micrograms/ml) were able to eradicate preformed biofilms on vascular catheters. PMID:8619580

  18. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

    PubMed

    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p<0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections. PMID:25704369

  19. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

    PubMed

    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p<0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections.

  20. Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation

    PubMed Central

    Baumgarten, Thomas; Sperling, Stefanie; Seifert, Jana; von Bergen, Martin; Steiniger, Frank; Wick, Lukas Y.

    2012-01-01

    Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context. PMID:22752175

  1. Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF.

    PubMed

    Moor, Hanna; Teppo, Annika; Lahesaare, Andrio; Kivisaar, Maia; Teras, Riho

    2014-12-01

    Bacteria form biofilm as a response to a number of environmental signals that are mediated by global transcription regulators and alarmones. Here we report the involvement of the global transcription regulator Fis in Pseudomonas putida biofilm formation through regulation of lapA and lapF genes. The major component of P. putida biofilm is proteinaceous and two large adhesive proteins, LapA and LapF, are known to play a key role in its formation. We have previously shown that Fis overexpression enhances P. putida biofilm formation. In this study, we used mini-Tn5 transposon mutagenesis to select potential Fis-regulated genes involved in biofilm formation. A total of 90 % of the studied transposon mutants carried insertions in the lap genes. Since our experiments showed that Fis-enhanced biofilm is mostly proteinaceous, the amounts of LapA and LapF from P. putida cells lysates were quantified using SDS-PAGE. Fis overexpression increases the quantity of LapA 1.6 times and decreases the amount of LapF at least 4 times compared to the wild-type cells. The increased LapA expression caused by Fis overexpression was confirmed by FACS analysis measuring the amount of LapA-GFP fusion protein. Our results suggest that the profusion of LapA in the Fis-overexpressed cells causes enhanced biofilm formation in mature stages of P. putida biofilm and LapF has a minor role in P. putida biofilm formation.

  2. Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa

    PubMed Central

    Arts, Isabelle S.; Ball, Geneviève; Leverrier, Pauline; Garvis, Steven; Nicolaes, Valérie; Vertommen, Didier; Ize, Bérengère; Tamu Dufe, Veronica; Messens, Joris; Voulhoux, Romé; Collet, Jean-François

    2013-01-01

    ABSTRACT Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. PMID:24327342

  3. 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

  4. PA3297 Counteracts Antimicrobial Effects of Azithromycin in Pseudomonas aeruginosa.

    PubMed

    Tan, Hao; Zhang, Lu; Weng, Yuding; Chen, Ronghao; Zhu, Feng; Jin, Yongxin; Cheng, Zhihui; Jin, Shouguang; Wu, Weihui

    2016-01-01

    Pseudomonas aeruginosa causes acute and chronic infections in human. Its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Among the alternatives is the unconventional usage of conventional antibiotics, of which the macrolide antibiotic azithromycin (AZM) provides a paradigmatic example. AZM therapy is associated with a small but consistent improvement in respiratory function of cystic fibrosis patients suffering from chronic P. aeruginosa infection. Besides immunomodulating activities, AZM represses bacterial genes involved in virulence, quorum sensing, biofilm formation, and motility, all of which are due to stalling of ribosome and depletion of cellular tRNA pool. However, how P. aeruginosa responds to and counteracts the effects of AZM remain elusive. Here, we found that deficiency of PA3297, a gene encoding a DEAH-box helicase, intensified AZM-mediated bacterial killing, suppression of pyocyanin production and swarming motility, and hypersusceptibility to hydrogen peroxide. We demonstrated that expression of PA3297 is induced by the interaction between AZM and ribosome. Importantly, mutation of PA3297 resulted in elevated levels of unprocessed 23S-5S rRNA in the presence of AZM, which might lead to increased susceptibility to AZM-mediated effects. Our results revealed one of the bacterial responses in counteracting the detrimental effects of AZM. PMID:27014238

  5. Aerobic biodegradation pathway for Remazol Orange by Pseudomonas aeruginosa.

    PubMed

    Sarayu, K; Sandhya, S

    2010-02-01

    Removal of azo dyes from effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are mutagenic and carcinogenic. Pseudomonas aeruginosa grew well in the presence of Remazol Orange (RO) and was able to decolorize and degrade it. In the present study, the decolorization and degradation efficiency using single culture P. aeruginosa with RO and textile wastewaters is studied. The elucidation of decolorization pathway for P. aeruginosa is of special interest. The degradation pathway and the metabolic products formed during the degradation were also predicted with the help of high performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analysis. The data show the cleavage of the azo dye RO to form both methyl metanilic acid and 4-aminobenzoic acid after decolorization and finally to oxidation forms benzoic acid, alkenes, aldehydes, and alkynes. The organism was able to decolorize the dye RO and wastewater effectively to the maximum of 82.4% and 62%, respectively.

  6. 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

  7. 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

  8. 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.

  9. Distinct synergistic action of piperacillin and methylglyoxal against Pseudomonas aeruginosa.

    PubMed

    Mukherjee, Sayanti; Chaki, Shaswati; Das, Sukhen; Sen, Saswati; Dutta, Samir Kr; Dastidar, Sujata G

    2011-07-01

    The dicarbonyl compound methylglyoxal is a natural constituent of Manuka honey produced from Manuka flowers in New Zealand. It is known to possess both anticancer and antibacterial activity. Such observations prompted to investigate the ability of methylglyoxal as a potent drug against multidrug resistant Pseudomonas aeruginosa. A total of 12 test P. aeruginosa strains isolated from various hospitals were tested for their resistances against many antibiotics, most of which are applied in the treatment of P. aeruginosa infections. Results revealed that the strains were resistant to many drugs at high levels, only piperacillin, carbenicillin, amikacin and ciprofloxacin showed resistances at comparatively lower levels. Following multiple experimentations it was observed that methylglyoxal was also antimicrobic against all the strains at comparable levels. Distinct and statistically significant synergism was observed between methylglyoxal and piperacillin by disc diffusion tests when compared with their individual effects. The fractional inhibitory concentration index of this combination evaluated by checkerboard analysis, was 0.5, which confirmed synergism between the pair. Synergism was also noted when methylglyoxal was combined with carbenicillin and amikacin. PMID:21800506

  10. Pseudomonas aeruginosa immunotype 5 polysaccharide-toxin A conjugate vaccine.

    PubMed Central

    Cryz, S J; Furer, E; Sadoff, J C; Germanier, R

    1986-01-01

    Polysaccharide (PS) derived from Pseudomonas aeruginosa immunotype 5 lipopolysaccharide was covalently coupled to toxin A by reductive amination with adipic acid dihydrazide as a spacer molecule. The resulting PS-toxin A conjugate was composed of 27.5% PS and 72.5% toxin A. The conjugate was composed of heterogeneous high-molecular-weight species, all of which possessed an Mr greater than 670,000. The conjugate was nontoxic for mice and nonpyrogenic at a dose of 50 micrograms/kg of body weight when intravenously administered to rabbits. Immunization of rabbits with the conjugate evoked both an antilipopolysaccharide immunoglobulin G (IgG) and an anti-toxin A IgG response. Anticonjugate IgG was capable of neutralizing the cytotoxic effect of toxin A. Immunization of mice with the conjugate increased the mean lethal dose from 4.5 X 10(1) P. aeruginosa for control mice to 9.6 X 10(5) P. aeruginosa for vaccinated mice. Similarly, immunization raised the mean lethal dose for toxin A from 0.2 to 4.67 micrograms per mouse. PMID:3082756

  11. Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

    PubMed Central

    2015-01-01

    The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue. Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal of P. aeruginosa and other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases in P. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal. PMID:26260455

  12. 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.

  13. PA3297 Counteracts Antimicrobial Effects of Azithromycin in Pseudomonas aeruginosa

    PubMed Central

    Tan, Hao; Zhang, Lu; Weng, Yuding; Chen, Ronghao; Zhu, Feng; Jin, Yongxin; Cheng, Zhihui; Jin, Shouguang; Wu, Weihui

    2016-01-01

    Pseudomonas aeruginosa causes acute and chronic infections in human. Its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Among the alternatives is the unconventional usage of conventional antibiotics, of which the macrolide antibiotic azithromycin (AZM) provides a paradigmatic example. AZM therapy is associated with a small but consistent improvement in respiratory function of cystic fibrosis patients suffering from chronic P. aeruginosa infection. Besides immunomodulating activities, AZM represses bacterial genes involved in virulence, quorum sensing, biofilm formation, and motility, all of which are due to stalling of ribosome and depletion of cellular tRNA pool. However, how P. aeruginosa responds to and counteracts the effects of AZM remain elusive. Here, we found that deficiency of PA3297, a gene encoding a DEAH-box helicase, intensified AZM-mediated bacterial killing, suppression of pyocyanin production and swarming motility, and hypersusceptibility to hydrogen peroxide. We demonstrated that expression of PA3297 is induced by the interaction between AZM and ribosome. Importantly, mutation of PA3297 resulted in elevated levels of unprocessed 23S-5S rRNA in the presence of AZM, which might lead to increased susceptibility to AZM-mediated effects. Our results revealed one of the bacterial responses in counteracting the detrimental effects of AZM. PMID:27014238

  14. Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems.

    PubMed

    Singh, Braj R; Singh, Brahma N; Singh, Akanksha; Khan, Wasi; Naqvi, Alim H; Singh, Harikesh B

    2015-01-01

    Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling. PMID:26347993

  15. Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems

    PubMed Central

    Singh, Braj R.; Singh, Brahma N.; Singh, Akanksha; Khan, Wasi; Naqvi, Alim H.; Singh, Harikesh B.

    2015-01-01

    Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling. PMID:26347993

  16. 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

  17. Response surface methodology for cadmium biosorption on Pseudomonas aeruginosa.

    PubMed

    Ahmady-Asbchin, Salman

    2016-01-01

    In this research the effects of various physicochemical factors on Cd(2+) biosorption such as initial metal concentration, pH and contact exposure time were studied. This study has shown a Cd(2+) biosorption, equilibrium time of about 5 min for Pseudomonas aeruginosa and the adsorption equilibrium data were well described by Langmuir equation. The maximum capacity for biosorption has been extrapolated to 0.56 mmol.g(-1) for P. aeruginosa. The thermodynamic properties ΔG(0), ΔH(0), and ΔS(0) of Cd(2+) for biosorption were analyzed by the equilibrium constant value obtained from experimented data at different temperatures. The results show that biosorption of Cd(2+) by P. aeruginosa are endothermic and spontaneous with ΔH value of 36.35 J.mol(-1). By response surface methodology, the quadratic model has adequately described the experimental data based on the adjusted determination coefficient (R(2) = 0.98). The optimum conditions for maximum uptake onto the biosorbent were established at 0.5 g.l(-1) biosorbent concentration, pH 6 for the aqueous solution, and a temperature of 30 °C. PMID:27232396

  18. Mechanism of azithromycin inhibition of HSL synthesis in Pseudomonas aeruginosa.

    PubMed

    Zeng, Jianming; Zhang, Ni; Huang, Bin; Cai, Renxin; Wu, Binning; E, Shunmei; Fang, Chengcai; Chen, Cha

    2016-04-14

    Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of nosocomial infections. Unfortunately, P. aeruginosa has low antibiotic susceptibility due to several chromosomally encoded antibiotic resistance genes. Hence, we carried out mechanistic studies to determine how azithromycin affects quorum sensing and virulence in P. aeruginosa. lasI and rhlI single and double mutants were constructed. We then undertook a quantitative approach to determine the optimal concentration of azithromycin and culture time that can affect the expression of HSLs. Furthermore, based on the above results, the effect on quorum sensing was analyzed at a transcriptional level. It was found that 2 μg/mL azithromycin caused a 79% decrease in 3-oxo-C12-HSL secretion during cultivation, while C4-HSL secretion was strongly repressed in the early stages. Azithromycin acts on ribosomes; to determine whether this can elicit alternative modes of gene expression, transcriptional regulation of representative virulence genes was analyzed. We propose a new relationship for lasI and rhlI: lasI acts as a cell density sensor, and rhlI functions as a fine-tuning mechanism for coordination between different quorum sensing systems.

  19. 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

  20. Evolutionary genomics of epidemic and nonepidemic strains of Pseudomonas aeruginosa

    PubMed Central

    Dettman, Jeremy R.; Rodrigue, Nicolas; Aaron, Shawn D.; Kassen, Rees

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen of humans and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Prolonged infection of the respiratory tract can lead to adaptation of the pathogen to the CF lung environment. To examine the general patterns of adaptation associated with chronic infection, we obtained genome sequences from a collection of P. aeruginosa isolated from airways of patients with CF. Our analyses support a nonclonal epidemic population structure, with a background of unique, recombining genotypes, and the rare occurrence of successful epidemic clones. We present unique genome sequence evidence for the intercontinental spread of an epidemic strain shared between CF clinics in the United Kingdom and North America. Analyses of core and accessory genomes identified candidate genes and important functional pathways associated with adaptive evolution. Many genes of interest were involved in biological functions with obvious roles in this pathosystem, such as biofilm formation, antibiotic metabolism, pathogenesis, transport, reduction/oxidation, and secretion. Key factors driving the adaptive evolution of this pathogen within the host appear to be the presence of oxidative stressors and antibiotics. Regions of the accessory genome unique to the epidemic strain were enriched for genes in transporter families that efflux heavy metals and antibiotics. The epidemic strain was significantly more resistant than nonepidemic strains to three different antibiotics. Multiple lines of evidence suggest that selection imposed by the CF lung environment has a major influence on genomic evolution and the genetic characteristics of P. aeruginosa isolates causing contemporary infection. PMID:24324153

  1. Quorum sensing and policing of Pseudomonas aeruginosa social cheaters.

    PubMed

    Wang, Meizhen; Schaefer, Amy L; Dandekar, Ajai A; Greenberg, E Peter

    2015-02-17

    The bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that uses a quorum sensing signal cascade to activate expression of dozens of genes when sufficient population densities have been reached. Quorum sensing controls production of several key virulence factors, including secreted proteases such as elastase. Cooperating groups of bacteria growing on protein are susceptible to social cheating by quorum-sensing defective mutants. A possible way to restrict cheater emergence is by policing where cooperators produce costly goods to sanction or punish cheats. The P. aeruginosa LasR-LasI quorum sensing system controls genes including those encoding proteases and also those encoding a second quorum-sensing system, the RhlR-RhlI system, which controls numerous genes including those for cyanide production. By using RhlR quorum sensing mutants and cyanide synthesis mutants, we show that cyanide production is costly and cyanide-producing cooperators use cyanide to punish LasR-null social cheaters. Cooperators are less susceptible to cyanide than are LasR mutants. These experiments demonstrate policing in P. aeruginosa, provide a mechanistic understanding of policing, and show policing involves the cascade organization of the two quorum sensing systems in this bacterium.

  2. Effect of Human Burn Wound Exudate on Pseudomonas aeruginosa Virulence

    PubMed Central

    Gonzalez, Manuel R.; Fleuchot, Betty; Lauciello, Leonardo; Jafari, Paris; Applegate, Lee Ann; Raffoul, Wassim; Que, Yok-Ai

    2016-01-01

    ABSTRACT Burn wound sepsis is currently the main cause of morbidity and mortality after burn trauma. Infections by notorious pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii impair patient recovery and can even lead to fatality. In this study, we investigated the effect of burn wound exudates (BWEs) on the virulence of those pathogens. BWEs were collected within 7 days after burn trauma from 5 burn patients. We first monitored their effect on pathogen growth. In contrast to A. baumannii and S. aureus, P. aeruginosa was the only pathogen able to grow within these human fluids. Expression of typical virulence factors such as pyocyanin and pyoverdine was even enhanced compared the levels seen with standard laboratory medium. A detailed chemical composition analysis of BWE was performed, which enabled us to determine the major components of BWE and underline the metabolic modifications induced by burn trauma. These data are essential for the development of an artificial medium mimicking the burn wound environment and the establishment of an in vitro system to analyze the initial steps of burn wound infections. IMPORTANCE Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P. aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P. aeruginosa. The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the

  3. Effect of Human Burn Wound Exudate on Pseudomonas aeruginosa Virulence.

    PubMed

    Gonzalez, Manuel R; Fleuchot, Betty; Lauciello, Leonardo; Jafari, Paris; Applegate, Lee Ann; Raffoul, Wassim; Que, Yok-Ai; Perron, Karl

    2016-01-01

    Burn wound sepsis is currently the main cause of morbidity and mortality after burn trauma. Infections by notorious pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii impair patient recovery and can even lead to fatality. In this study, we investigated the effect of burn wound exudates (BWEs) on the virulence of those pathogens. BWEs were collected within 7 days after burn trauma from 5 burn patients. We first monitored their effect on pathogen growth. In contrast to A. baumannii and S. aureus, P. aeruginosa was the only pathogen able to grow within these human fluids. Expression of typical virulence factors such as pyocyanin and pyoverdine was even enhanced compared the levels seen with standard laboratory medium. A detailed chemical composition analysis of BWE was performed, which enabled us to determine the major components of BWE and underline the metabolic modifications induced by burn trauma. These data are essential for the development of an artificial medium mimicking the burn wound environment and the establishment of an in vitro system to analyze the initial steps of burn wound infections. IMPORTANCE Microbial infection of severe burn wounds is currently a major medical challenge. Of the infections by bacteria able to colonize such injuries, those by Pseudomonas aeruginosa are among the most severe, causing major delays in burn patient recovery or leading to fatal issues. In this study, we investigated the growth properties of several burn wound pathogens in biological fluids secreted from human burn wounds. We found that P. aeruginosa strains were able to proliferate but not those of the other pathogens tested. In addition, burn wound exudates (BWEs) stimulate the expression of virulence factors in P. aeruginosa. The chemical composition analysis of BWEs enabled us to determine the major components of these fluids. These data are essential for the development of an artificial medium mimicking the burn wound

  4. Use of an ultraviolet light at point-of-dispense faucet to eliminate Pseudomonas aeruginosa.

    PubMed

    Gerba, Charles P

    2015-05-01

    Tap water is believed to be a significant source of Pseudomonas aeruginosa in health care environments. This study evaluated an ultraviolet (UV) light point-of-dispense water treatment system for control of P aeruginosa. No P aeruginosa was detected in 30 different water dispensers in which the UV light device had been operating for 1-34 months. In comparison, P aeruginosa was found in other taps that did not feature this UV light system. PMID:25721063

  5. Use of an ultraviolet light at point-of-dispense faucet to eliminate Pseudomonas aeruginosa.

    PubMed

    Gerba, Charles P

    2015-05-01

    Tap water is believed to be a significant source of Pseudomonas aeruginosa in health care environments. This study evaluated an ultraviolet (UV) light point-of-dispense water treatment system for control of P aeruginosa. No P aeruginosa was detected in 30 different water dispensers in which the UV light device had been operating for 1-34 months. In comparison, P aeruginosa was found in other taps that did not feature this UV light system.

  6. Small-molecule inhibition of choline catabolism in Pseudomonas aeruginosa and other aerobic choline-catabolizing bacteria.

    PubMed

    Fitzsimmons, Liam F; Flemer, Stevenson; Wurthmann, A Sandy; Deker, P Bruce; Sarkar, Indra Neil; Wargo, Matthew J

    2011-07-01

    Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.

  7. Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

    PubMed

    Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

    2015-06-01

    Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

  8. Pseudomonas putida KT2442 as a platform for the biosynthesis of polyhydroxyalkanoates with adjustable monomer contents and compositions.

    PubMed

    Tripathi, Lakshmi; Wu, Lin-Ping; Dechuan, Meng; Chen, Jinchun; Wu, Qiong; Chen, Guo-Qiang

    2013-08-01

    The β-oxidation weakened Pseudomonas putida were established as a platform for the production of polyhydroxyalkanoates (PHA) with adjustable monomer compositions and micro-structures. When mutant P. putida KTOYO6ΔC (phaPCJA.c) was cultivated on mixtures of sodium butyrate and sodium hexanoate (C4:C6), random copolymers of P(3HB-co-3HHx) consisting of 3-hydroxybutyrate (3HB), 3-hydroxyhexanoate (3HHx), were accumulated with 3HHx content ranged from 19 mol% to 75 mol%. While recombinant P. putida KTQQ20 grown on mixtures of sodium hexanoate and decanoic acid (C6:C10), produced random copolymers of P(3HHx-co-3HD) consisting of 3-hydroxyhexanoate (3HHx) and 3-hydroxydecanoate (3HD), the monomer fraction of 3HHx ranged from 16 mol% to 63 mol%. The comonomer compositions were easily regulated by varying the fatty acid concentrations. P. putida KTQQ20 produced a novel diblock copolymer P3HHx-b-P(3HD-co-3HDD) consisting of 49 mol% P3HHx and 51 mol% P(3HD-co-3HDD) [35.25 mol% 3HDD (3-hydroxydodecanoate)], which was characterized by (13)C NMR, HMBC NMR, DSC, GPC and universal testing machine.

  9. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production

    PubMed Central

    Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2014-01-01

    As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

  10. IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase.

    PubMed

    Ilves, Heili; Hõrak, Rita; Teras, Riho; Kivisaar, Maia

    2004-03-01

    Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445-5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA-defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase. PMID:15009901

  11. Simultaneous chromium reduction and phenol degradation in a coculture of Escherichia coli ATCC 33456 and Pseudomonas putida DMP-1.

    PubMed Central

    Shen, H; Wang, Y T

    1995-01-01

    In a defined coculture of a Cr(VI) reducer, Escherichia coli ATCC 33456, and a phenol degrader, Pseudomonas putida DMP-1, simultaneous reduction of Cr(VI) and degradation of phenol was observed. When Cr(VI) was present in the coculture, quantitative transformation of Cr(VI) into Cr(III) proceeded with simultaneous degradation of phenol. Cr(VI) reduction was correlated to phenol degradation in the coculture as demonstrated by a regression analysis of the cumulative Cr(VI) reduction and the cumulative phenol degradation. Both the rate and extent of Cr(VI) reduction and phenol degradation were significantly influenced by the population composition of the coculture. Although Cr(VI) reduction occurred as a result of E. coli metabolism, the rate of phenol degradation by P. putida may become a rate-limiting factor for Cr(VI) reduction at a low population ratio of P. putida to E. coli. Phenol degradation by P. putida was very susceptible to the presence of Cr(VI), whereas Cr(VI) reduction by E. coli was significantly influenced by phenol only when phenol was present at high concentrations (> 9 mM). PMID:7618887

  12. Functionalized polyanilines disrupt Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

    PubMed

    Gizdavic-Nikolaidis, Marija R; Pagnon, Joanne C; Ali, Naseem; Sum, Reuben; Davies, Noel; Roddam, Louise F; Ambrose, Mark

    2015-12-01

    The purpose of the present study was to investigate the antimicrobial effects of functionalized polyanilines (fPANIs) against stationary phase cells and biofilms of Pseudomonas aeruginosa and Staphylococcus aureus using homopolymer of sulfanilic acid (poly-SO3H) as a model. The chemically synthesized poly-SO3H was characterized using Fourier Transform Infra-Red (FTIR) and Ultraviolet-Visible (UV-Vis) spectroscopies. The molecular weight (Mw) and elemental analysis of homopolymer poly-SO3H were also examined. We found that poly-SO3H was bactericidal against stationary phase cells of P. aeruginosa and S. aureus at a concentration of 20 mgml(-1). Surprisingly, we discovered that the same concentration (20 mgml(-1)) of poly-SO3H significantly disrupted and killed bacterial cells present in pre-established forty-eight hour static biofilms of these organisms, as shown by crystal violet and bacterial live/dead fluorescence staining assays. In support of these data, poly-SO3H extensively diminished the expression of bacterial genes related to biofilm formation in stationary phase cells of P. aeruginosa, and seemed to greatly reduce the amount of the quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) able to be recovered from biofilms of this organism. Furthermore, we found that poly-SO3H was able to effectively penetrate and kill cells in biofilms formed by the P. aeruginosa (AESIII) isolate derived from the sputum of a cystic fibrosis patient. Taken together, the results of the present study emphasise the broad antimicrobial activities of fPANI, and suggest that they could be developed further and used in some novel ways to construct medical devices and/or industrial equipment that are refractory to colonization by biofilm-forming bacteria. PMID:26496473

  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. Functionalized polyanilines disrupt Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

    PubMed

    Gizdavic-Nikolaidis, Marija R; Pagnon, Joanne C; Ali, Naseem; Sum, Reuben; Davies, Noel; Roddam, Louise F; Ambrose, Mark

    2015-12-01

    The purpose of the present study was to investigate the antimicrobial effects of functionalized polyanilines (fPANIs) against stationary phase cells and biofilms of Pseudomonas aeruginosa and Staphylococcus aureus using homopolymer of sulfanilic acid (poly-SO3H) as a model. The chemically synthesized poly-SO3H was characterized using Fourier Transform Infra-Red (FTIR) and Ultraviolet-Visible (UV-Vis) spectroscopies. The molecular weight (Mw) and elemental analysis of homopolymer poly-SO3H were also examined. We found that poly-SO3H was bactericidal against stationary phase cells of P. aeruginosa and S. aureus at a concentration of 20 mgml(-1). Surprisingly, we discovered that the same concentration (20 mgml(-1)) of poly-SO3H significantly disrupted and killed bacterial cells present in pre-established forty-eight hour static biofilms of these organisms, as shown by crystal violet and bacterial live/dead fluorescence staining assays. In support of these data, poly-SO3H extensively diminished the expression of bacterial genes related to biofilm formation in stationary phase cells of P. aeruginosa, and seemed to greatly reduce the amount of the quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) able to be recovered from biofilms of this organism. Furthermore, we found that poly-SO3H was able to effectively penetrate and kill cells in biofilms formed by the P. aeruginosa (AESIII) isolate derived from the sputum of a cystic fibrosis patient. Taken together, the results of the present study emphasise the broad antimicrobial activities of fPANI, and suggest that they could be developed further and used in some novel ways to construct medical devices and/or industrial equipment that are refractory to colonization by biofilm-forming bacteria.

  15. Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols

    SciTech Connect

    Bartels, I.; Knackmuss, H.J.; Reineke, W.

    1984-03-01

    The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K/sub 2/) were 1.62 x 10/sup -3/ sec/sup -1/ for 3-chlorocatechol and 2.38 x 10/sup -3/ sec/sup -1/ for 3-fluorocatechol. The inhibitor constants (K/sub i/) were 23 ..mu..M for 3-chlorocatechol and 17 ..mu..M for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoci acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation. 64 references.

  16. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204

    SciTech Connect

    Eaton, R.W.; Timmis, K.N.

    1986-10-01

    A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene ..-->.. 2,3-dihydro-2,3-dihydroxyisopropylbenzene ..-->.. 3-isopropylcatechol ..-->.. 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate ..-->.. isobutyrate + 2-oxopent-4-enoate ..-->.. amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.

  17. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.

    PubMed

    Araújo, Simara Semíramis de; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite; Whitman, Christian P; Johnson, William H; Aparicio, Ricardo; Nagem, Ronaldo Alves Pinto

    2015-08-01

    The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s(-1)) values were determined at presumed NAD(+) saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway.

  18. Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida.

    PubMed

    Duque, Estrella; García, Vanina; de la Torre, Jesús; Godoy, Patricia; Bernal, Patricia; Ramos, Juan-Luis

    2004-10-01

    The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.

  19. Regulation of the pcaIJ genes for aromatic acid degradation in Pseudomonas putida.

    PubMed Central

    Parales, R E; Harwood, C S

    1993-01-01

    Six of the genes encoding enzymes of the beta-ketoadipate pathway for benzoate and 4-hydroxybenzoate degradation in Pseudomonas putida are organized into at least three separate transcriptional units. As an initial step to defining this pca regulon at the molecular level, lacZ fusions were made with the pcaI and pcaJ genes, which encode the two subunits of beta-ketoadipate:succinyl-coenzyme A transferase, the enzyme catalyzing the next-to-last step in the beta-ketoadipate pathway. Fusion analyses showed that pcaI and pcaJ constitute an operon which requires beta-ketoadipate or its nonmetabolizable analog, adipate, as well as the pcaR regulatory gene for induction. The pcaIJ promoter is likely to be a sigma 70-type promoter; it has a sigma 70-type consensus sequence and did not require the alternative sigma factor, RpoN, for induction. Deletion analysis of the promoter region of a pcaI-lacZ transcriptional fusion indicated that no specific DNA sequences upstream of the -35 region were required for full induction. This implies that the binding site for the activator protein, PcaR, is unusually close to the transcriptional start site of pcaIJ. PMID:8376330

  20. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

    PubMed

    Thuptimdang, Pumis; Limpiyakorn, Tawan; McEvoy, John; Prüß, Birgit M; Khan, Eakalak

    2015-06-15

    This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms. PMID:25756827

  1. Electricity Generation and Wastewater Treatment of Oil Refinery in Microbial Fuel Cells Using Pseudomonas putida

    PubMed Central

    Majumder, Dip; Maity, Jyoti Prakash; Tseng, Min-Jen; Nimje, Vanita Roshan; Chen, Hau-Ren; Chen, Chien-Cheng; Chang, Young-Fo; Yang, Tsui-Chu; Chen, Chen-Yen

    2014-01-01

    Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm2 in the third cycle with a maximum current density of 0.015 mA/cm2 in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10−2% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation. PMID:25247576

  2. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7.

    PubMed

    Araújo, Simara Semíramis de; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite; Whitman, Christian P; Johnson, William H; Aparicio, Ricardo; Nagem, Ronaldo Alves Pinto

    2015-08-01

    The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s(-1)) values were determined at presumed NAD(+) saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway. PMID:26032336

  3. Electricity generation and wastewater treatment of oil refinery in microbial fuel cells using Pseudomonas putida.

    PubMed

    Majumder, Dip; Maity, Jyoti Prakash; Tseng, Min-Jen; Nimje, Vanita Roshan; Chen, Hau-Ren; Chen, Chien-Cheng; Chang, Young-Fo; Yang, Tsui-Chu; Chen, Chen-Yen

    2014-09-22

    Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm² in the third cycle with a maximum current density of 0.015 mA/cm² in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10⁻²% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation.

  4. Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

    PubMed Central

    Chavarría, Max; Kleijn, Roelco J.; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2012-01-01

    ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. PMID:22434849

  5. Near-zero growth kinetics of Pseudomonas putida deduced from proteomic analysis.

    PubMed

    Panikov, Nicolai S; Mandalakis, Manolis; Dai, Shujia; Mulcahy, Lawrence R; Fowle, William; Garrett, Wendy S; Karger, Barry L

    2015-01-01

    Intensive microbial growth typically observed in laboratory rarely occurs in nature. Because of severe nutrient deficiency, natural populations exhibit near-zero growth (NZG). There is a long-standing controversy about sustained NZG, specifically whether there is a minimum growth rate below which cells die or whether cells enter a non-growing maintenance state. Using chemostat with cell retention (CCR) of Pseudomonas putida, we resolve this controversy and show that under NZG conditions, bacteria differentiate into growing and VBNC (viable but not non-culturable) forms, the latter preserving measurable catabolic activity. The proliferating cells attained a steady state, their slow growth balanced by VBNC production. Proteomic analysis revealed upregulated (transporters, stress response, self-degrading enzymes and extracellular polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic enzymes) proteins in the CCR versus batch culture. Based on these profiles, we identified intracellular processes associated with NZG and generated a mathematical model that simulated the observations. We conclude that NZG requires controlled partial self-digestion and deep reconfiguration of the metabolic machinery that results in the biosynthesis of new products and development of broad stress resistance. CCR allows efficient on-line control of NZG including VBNC production. A well-nuanced understanding of NZG is important to understand microbial processes in situ and for optimal design of environmental technologies.

  6. New methods for the isolation and characterization of biofilm-persistent mutants in Pseudomonas putida.

    PubMed

    López-Sánchez, Aroa; Jiménez-Fernández, Alicia; Calero, Patricia; Gallego, Laura D; Govantes, Fernando

    2013-10-01

    Here we describe two new methods for the genetic characterization of bacterial biofilm development. First, we have designed a microtitre dish-based approach for high-throughput screening of Pseudomonas putida mutants showing increased biofilm under dispersal conditions. Using this method, nine such biofilm-persistent mutants, bearing transposon insertions in four loci: lapG, bifA, mvaB and dksA, were isolated. Second, we have developed a serial dilution-based scheme to monitor biofilm development and dispersal in microtitre dish wells in a simple, time-efficient and reproducible manner. Using this method, we showed that (i) mutants in bifA and dksA do not undergo starvation-induced biofilm dispersal in LB or minimal medium, (ii) a mvaB mutant does not disperse the biofilm in LB, but shows a normal dispersal response in minimal medium, and (iii) unlike the lapG mutant, the bifA, mvaB and dksA mutants do not show an increase in biofilm production. The procedures shown here are useful tools for the identification of previously uncharacterized biofilm-related genes and considerably simplify the characterization of biofilm growth phenotypes.

  7. Quantitative analysis of chemotaxis towards toluene by Pseudomonas putida in a convection-free microfluidic device.

    PubMed

    Wang, Xiaopu; Atencia, Javier; Ford, Roseanne M

    2015-05-01

    Chemotaxis has been shown to be beneficial for the migration of soil-inhabiting bacteria towards industrial chemical pollutants, which they degrade. Many studies have demonstrated the importance of this microbial property under various circumstances; however, few quantitative analyses have been undertaken to measure the two essential parameters that characterize the chemotaxis of bioremediation bacteria: the chemotactic sensitivity coefficient χ(0) and the chemotactic receptor constant K(c). The main challenge to determine these parameters is that χ(0) and K(c) are coupled together in non-linear mathematical models used to evaluate them. In this study we developed a method to accurately measure these parameters for Pseudomonas putida in the presence of toluene, an important pollutant in groundwater contamination. Our approach uses a multilayer microfluidic device to expose bacteria to a convection-free linear chemical gradient of toluene that is stable over time. The bacterial distribution within the gradient is measured in terms of fluorescence intensity, and is then used to fit the parameters Kc and χ(0) with mathematical models. Critically, bacterial distributions under chemical gradients at two different concentrations were used to solve for both parameters independently. To validate the approach, the chemotaxis parameters of Escherichia coli strains towards α-methylaspartate were experimentally derived and were found to be consistent with published results from related work.

  8. Electricity generation and wastewater treatment of oil refinery in microbial fuel cells using Pseudomonas putida.

    PubMed

    Majumder, Dip; Maity, Jyoti Prakash; Tseng, Min-Jen; Nimje, Vanita Roshan; Chen, Hau-Ren; Chen, Chien-Cheng; Chang, Young-Fo; Yang, Tsui-Chu; Chen, Chen-Yen

    2014-01-01

    Microbial fuel cells (MFCs) represent a novel platform for treating wastewater and at the same time generating electricity. Using Pseudomonas putida (BCRC 1059), a wild-type bacterium, we demonstrated that the refinery wastewater could be treated and also generate electric current in an air-cathode chamber over four-batch cycles for 63 cumulative days. Our study indicated that the oil refinery wastewater containing 2213 mg/L (ppm) chemical oxygen demand (COD) could be used as a substrate for electricity generation in the reactor of the MFC. A maximum voltage of 355 mV was obtained with the highest power density of 0.005 mW/cm² in the third cycle with a maximum current density of 0.015 mA/cm² in regard to the external resistor of 1000 Ω. A maximum coulombic efficiency of 6 × 10⁻²% was obtained in the fourth cycle. The removal efficiency of the COD reached 30% as a function of time. Electron transfer mechanism was studied using cyclic voltammetry, which indicated the presence of a soluble electron shuttle in the reactor. Our study demonstrated that oil refinery wastewater could be used as a substrate for electricity generation. PMID:25247576

  9. Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

    PubMed

    Lieder, Sarah; Jahn, Michael; Koepff, Joachim; Müller, Susann; Takors, Ralf

    2016-01-01

    Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress.

  10. Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier.

    PubMed

    Peper, Stephanie; Kara, Selin; Long, Wei Sing; Liese, Andreas; Niemeyer, Bernd

    2011-08-01

    If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

  11. Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida.

    PubMed

    Yousef-Coronado, Fátima; Travieso, María L; Espinosa-Urgel, Manuel

    2008-11-01

    Mechanisms governing biofilm formation have generated considerable interest in recent years, yet comparative analyses of processes for bacterial establishment on abiotic and biotic surfaces are still limited. In this report we have expanded previous information on the genetic determinants required for colonization of plant surfaces by Pseudomonas putida populations and analyzed their correlation with biofilm formation processes on abiotic surfaces. Insertional mutations affecting flagellar genes or the synthesis and transport of the large adhesin LapA lead to decreased adhesion to seeds and biofilm formation on abiotic surfaces. The latter also causes reduced fitness in the rhizosphere. Decreased seed adhesion and altered biofilm formation kinetics are observed in mutants affected in heme biosynthesis and a gene that might participate in oxidative stress responses, whereas a mutant in a gene involved in cytochrome oxidase assembly is affected in the bacterium-plant interaction but not in bacterial establishment on abiotic surfaces. Finally, a mutant altered in lipopolysaccharide biosynthesis is impaired in seed and root colonization but seems to initiate attachment to plastic faster than the wild type. This variety of phenotypes reflects the complexity of bacterial adaptation to sessile life, and the partial overlap between mechanisms leading to biofilm formation on abiotic and biotic surfaces.

  12. Pseudomonas putida biofilm dynamics following a single pulse of silver nanoparticles.

    PubMed

    Mallevre, Florian; Fernandes, Teresa F; Aspray, Thomas J

    2016-06-01

    Pseudomonas putida mono-species biofilms were exposed to silver nanoparticles (Ag NPs) in artificial wastewater (AW) under hydrodynamic conditions. Specifically, 48 h old biofilms received a single pulse of Ag NPs at 0, 0.01, 0.1, 1, 10 and 100 mg L(-1) for 24 h in confocal laser scanning microscopy (CLSM) compatible flow-cells. The biofilm dynamics (in terms of morphology, viability and activity) were characterised at 48, 72 and 96 h. Consistent patterns were found across flow-cells and experiments at 48 h. Dose dependent impacts of NPs were then shown at 72 h on biofilm morphology (e.g. biomass, surface area and roughness) from 0.01 mg L(-1). The microbial viability was not altered below 10 mg L(-1) Ag NPs. The activity (based on the d-glucose utilisation) was impacted by concentrations of Ag NPs equal and superior to 10 mg L(-1). Partial recovery of morphology, viability and activity were finally observed at 96 h. Comparatively, exposure to Ag salt resulted in ca. one order of magnitude higher toxicity when compared to Ag NPs. Consequently, the use of a continuous culture system and incorporation of a recovery stage extends the value of biofilm assays beyond the standard acute toxicity assessment. PMID:27031799

  13. Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms

    PubMed Central

    Cárcamo-Oyarce, Gerardo; Lumjiaktase, Putthapoom; Kümmerli, Rolf; Eberl, Leo

    2015-01-01

    The term ‘quorum sensing’ (QS) is generally used to describe the phenomenon that bacteria release and perceive signal molecules to coordinate cooperative behaviour in response to their population size. QS-based communication has therefore been considered a social trait. Here we show that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced in young biofilms of Pseudomonas putida and act mainly as self-regulatory signals rather than inducing neighbouring cells. We demonstrate that QS induces the expression of putisolvin biosurfactants that are not public goods, thereby triggering asocial motility of induced cells out of microcolonies. Phenotypic heterogeneity is most prominent in the early stages of biofilm development, whereas at later stages behaviour patterns across cells become more synchronized. Our findings broaden our perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behaviour. PMID:25592773

  14. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440.

    PubMed

    Molina-Henares, M Antonia; García-Salamanca, Adela; Molina-Henares, A Jesús; de la Torre, Jesús; Herrera, M Carmen; Ramos, Juan L; Duque, Estrella

    2009-01-01

    Pseudomonas putida KT2440 is a non-pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini-Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene-encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB-like genes are present in the host chromosome. PMID:21261884

  15. Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86.

    PubMed

    Shrivastava, Rahul; Basu, Aditya; Phale, Prashant S

    2011-08-01

    Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD(+) dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD(+), while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K (m) value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K (m) and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.

  16. Synergistic effect of Pseudomonas putida and Bacillus amyloliquefaciens ameliorates drought stress in chickpea (Cicer arietinum L.).

    PubMed

    Kumar, Manoj; Mishra, Sankalp; Dixit, Vijaykant; Kumar, Manoj; Agarwal, Lalit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-01-01

    Two plant growth promoting rhizobacteria (PGPR) Pseudomonas putida NBRIRA and Bacillus amyloliquefaciens NBRISN13 with ability to tolerate abiotic stress along with multiple PGP traits like ACC deaminase activity, minerals solubilisation, hormones production, biofilm formation, siderophore activity were evaluated for their synergistic effect to ameliorate drought stress in chickpea. Earlier we have reported both the strains individually for their PGP attributes and stress amelioration in host plants. The present study explains in detail the possibilities and benefits of utilizing these 2 PGPR in consortium for improving the chickpea growth under control and drought stressed condition. In vitro results clearly demonstrate that both the PGPR strains are compatible to each other and their synergistic growth enhances the PGP attributes. Greenhouse experiments were conducted to evaluate the effect of inoculation of both strains individually and consortia in drought tolerant and sensitive cultivars (BG362 and P1003). The growth parameters were observed significantly higher in consortium as compared to individual PGPR. Colonization of both PGPR in chickpea rhizosphere has been visualized by using gfp labeling. Apart from growth parameters, defense enzymes, soil enzymes and microbial diversity were significantly modulated in individually PGPR and in consortia inoculated plants. Negative effects of drought stress has been ameliorated and apparently seen by higher biomass and reversal of stress indicators in chickpea cultivars treated with PGPR individually or in consortia. Findings from the present study demonstrate that synergistic application has better potential to improve plant growth promotion under drought stress conditions.

  17. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon

    SciTech Connect

    Zylstra, G.J.; McCombie, W.R.; Gibson, D.T.; Finette, B.A.

    1988-06-01

    Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is metabolized through the well-established meta pathway for catechol degradation. The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene, dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF). The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism. A combination of transposon mutagenesis of the PpF1 chromosome, was well as the analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon. The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.

  18. Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

    PubMed

    Lieder, Sarah; Jahn, Michael; Koepff, Joachim; Müller, Susann; Takors, Ralf

    2016-01-01

    Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress. PMID:26299279

  19. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

    PubMed

    Thuptimdang, Pumis; Limpiyakorn, Tawan; McEvoy, John; Prüß, Birgit M; Khan, Eakalak

    2015-06-15

    This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

  20. Evaluation of Zosteric Acid for Mitigating Biofilm Formation of Pseudomonas putida Isolated from a Membrane Bioreactor System

    PubMed Central

    Polo, Andrea; Foladori, Paola; Ponti, Benedetta; Bettinetti, Roberta; Gambino, Michela; Villa, Federica; Cappitelli, Francesca

    2014-01-01

    This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (−97%) and thickness (−50%), and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition. PMID:24879523

  1. Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

    PubMed Central

    Raaijmakers, J M; Bitter, W; Punte, H L; Bakker, P A; Weisbeek, P J; Schippers, B

    1994-01-01

    For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain. Images PMID:8017914

  2. Continued transmission of Pseudomonas aeruginosa from a wash hand basin tap in a critical care unit.

    PubMed

    Garvey, M I; Bradley, C W; Tracey, J; Oppenheim, B

    2016-09-01

    Pseudomonas aeruginosa is an important nosocomial pathogen, colonizing hospital water supplies including taps and sinks. We report a cluster of P. aeruginosa acquisitions during a period of five months from tap water to patients occupying the same burns single room in a critical care unit. Pseudomonas aeruginosa cultured from clinical isolates from four different patients was indistinguishable from water strains by pulsed-field gel electrophoresis. Water outlets in critical care may be a source of P. aeruginosa despite following the national guidance, and updated guidance and improved control measures are needed to reduce the risks of transmission to patients.

  3. Calcium Causes Multimerization of the Large Adhesin LapF and Modulates Biofilm Formation by Pseudomonas putida

    PubMed Central

    Martínez-Gil, Marta; Romero, Diego; Kolter, Roberto

    2012-01-01

    LapF is a large secreted protein involved in microcolony formation and biofilm maturation in Pseudomonas putida. Its C-terminal domain shows the characteristics of proteins secreted through a type I secretion system and includes a predicted calcium binding motif. We provide experimental evidence of specific binding of Ca2+ to the purified C-terminal domain of LapF (CLapF). Calcium promotes the formation of large aggregates, which disappear in the presence of the calcium chelator EGTA. Immunolocalization of LapF also shows the tendency of this protein to accumulate in vivo in certain extracellular regions. These findings, along with results showing that calcium influences biofilm formation, lead us to propose a model in which P. putida cells interact with each other via LapF in a calcium-dependent manner during the development of biofilms. PMID:23042991

  4. Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing.

    PubMed

    Miyamoto, Tatsuki; Kato, Yuka; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2016-01-01

    Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

  5. The contribution of proteomics to the unveiling of the survival strategies used by Pseudomonas putida in changing and hostile environments.

    PubMed

    Moreno, Renata; Rojo, Fernando

    2013-10-01

    Pseudomonas putida is a ubiquitous, metabolically very versatile, Gram-negative bacterium adapted to habitats as diverse as soil, water and the rhizosphere. Most strains are nonpathogenic, many are used as experimental models, and many others have biotechnological applications in the areas of agriculture, bioremediation, biocatalysis, and the production of bioplastics. This review summarizes the contribution of proteomic technologies to our understanding of how P. putida responds to different carbon sources, how it adapts to living at suboptimal temperatures or attached to surfaces, and how it responds to the presence of toxic compounds such as aromatic molecules and heavy metals. The examples described illustrate the value of proteomics in furthering our knowledge of the physiology and behavior of bacteria, knowledge that is important for understanding how they behave in their natural habitats and for optimizing their behavior in biotechnological applications. PMID:23625785

  6. Bioremoval of Basic Violet 3 and Acid Blue 93 by Pseudomonas putida and its adsorption isotherms and kinetics.

    PubMed

    Arunarani, A; Chandran, Preethy; Ranganathan, B V; Vasanthi, N S; Sudheer Khan, S

    2013-02-01

    Basic Violet 3 and Acid Blue 93 are the most important group of synthetic colourants extensively used in textile industries for dyeing cotton, wool, silk and nylon. Release of these dye pollutants in to the environment adversely affects the human health and aquatic organisms. The present study we used Pseudomonas putida MTCC 4910 for the adsorptive removal of Basic Violet 3 and Acid Blue 93 from the aqueous solutions. The pH (4-9) and NaCl concentrations (1mM-1M) did not influence the adsorption process. The equilibrium adsorption process fitted well to Freundlich model than Langmuir model. The kinetics of adsorption fitted well by pseudo-second-order. Thus in the present study an attempt has been made to exploit the dye removal capability of P. putida MTCC 4910, and it was found to be an efficient microbe that could be used for bio removal of dyes from textile effluents.

  7. Global Regulation of Food Supply by Pseudomonas putida DOT-T1E▿ †

    PubMed Central

    Daniels, Craig; Godoy, Patricia; Duque, Estrella; Molina-Henares, M. Antonia; de la Torre, Jesús; del Arco, José María; Herrera, Carmen; Segura, Ana; Guazzaroni, M. Eugenia; Ferrer, Manuel; Ramos, Juan Luis

    2010-01-01

    Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δcrc, Δcrp, ΔcyoB, and ΔptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine

  8. Pseudomonas aeruginosa KUCD1, a possible candidate for cadmium bioremediation

    PubMed Central

    Sinha, Sangram; Mukherjee, Samir Kumar

    2009-01-01

    A cadmium (8 mM) resistant Pseudomonas aeruginosa strain KUCd1 exhibiting high Cd accumulation under in vitro aerobic condition has been reported. The isolate showed a significant ability to remove more than 75% and 89% of the soluble cadmium during the active growth phase from the growth medium and from Cd-amended industrial wastewater under growth supportive condition. Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDXS) suggest the presence of Cd in the cells from mid stationary phase. The cell fractionation study revealed membrane and periplasm to be the major accumulating site in this strain. The chemical nature of the accumulated Cd was studied by X-ray powder diffraction analysis. PMID:24031411

  9. Decrease of Pseudomonas aeruginosa biofilm formation by food waste materials.

    PubMed

    Maderova, Zdenka; Horska, Katerina; Kim, Sang-Ryoung; Lee, Chung-Hak; Pospiskova, Kristyna; Safarikova, Mirka; Safarik, Ivo

    2016-01-01

    The formation of bacterial biofilm on various surfaces has significant negative economic effects. The aim of this study was to find a simple procedure to decrease the Pseudomonas aeruginosa biofilm formation in a water environment by using different food waste biological materials as signal molecule adsorbents. The selected biomaterials did not reduce the cell growth but affected biofilm formation. Promising biomaterials were magnetically modified in order to simplify manipulation and facilitate their magnetic separation. The best biocomposite, magnetically modified spent grain, exhibited substantial adsorption of signal molecules and decreased the biofilm formation. These results suggest that selected food waste materials and their magnetically responsive derivatives could be applied to solve biofilm problems in water environment. PMID:27148715

  10. Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

    PubMed Central

    Biggs, Matthew B.; Papin, Jason A.

    2013-01-01

    Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool. PMID:24147108

  11. Decrease of Pseudomonas aeruginosa biofilm formation by food waste materials.

    PubMed

    Maderova, Zdenka; Horska, Katerina; Kim, Sang-Ryoung; Lee, Chung-Hak; Pospiskova, Kristyna; Safarikova, Mirka; Safarik, Ivo

    2016-01-01

    The formation of bacterial biofilm on various surfaces has significant negative economic effects. The aim of this study was to find a simple procedure to decrease the Pseudomonas aeruginosa biofilm formation in a water environment by using different food waste biological materials as signal molecule adsorbents. The selected biomaterials did not reduce the cell growth but affected biofilm formation. Promising biomaterials were magnetically modified in order to simplify manipulation and facilitate their magnetic separation. The best biocomposite, magnetically modified spent grain, exhibited substantial adsorption of signal molecules and decreased the biofilm formation. These results suggest that selected food waste materials and their magnetically responsive derivatives could be applied to solve biofilm problems in water environment.

  12. Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

    PubMed

    Silva, Lívia V; Galdino, Anna Clara M; Nunes, Ana Paula F; dos Santos, Kátia R N; Moreira, Beatriz M; Cacci, Luciana C; Sodré, Cátia L; Ziccardi, Mariangela; Branquinha, Marta H; Santos, André L S

    2014-11-01

    Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-β-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 μg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0

  13. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.

    PubMed Central

    Møller, S; Pedersen, A R; Poulsen, L K; Arvin, E; Molin, S

    1996-01-01

    As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe. The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy, demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending from the surface into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furthermore, measurements of growth rate-related parameters for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was not degrading toluene at a maximal rate in the biofilm environment. Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated. This calculation indicated that P. putida was responsible for a significant part (65%) of the toluene degraded by the entire community. PMID:8953734

  14. Labeling of pseudomonas aeruginosa with In-111-oxine

    SciTech Connect

    Bettin, K.M.; Gerding, D.N.; O'Connor, M.J.; Forstrom, L.A.; Shafer, R.B.

    1984-01-01

    Labeling of live bacteria with gamma emitting radioisotope provides a useful tool for the experimental in vivo tracking of bacteria in various body organs of animals. The authors labeled a serum resistant strain of Pseudomonas aeruginosa (ATCC number27853) with In-111-oxine. P. aeruginosa streaked heavily on ten blood agar plates, was grown overnight, and suspended in 50 ml of saline using sterile cotton swabs. The suspension was sonicated for 3 minutes at 40 watts with a small probe, 500 ..mu..Ci of commercially prepared In-111-oxine added and the bacteria incubated at 37/sup 0/C for 2.5 hours. The labeled bacteria were centrifuged and washed once with saline and resuspended to a final volume of 50 ml in saline. The labeled Pseudomonas, 10/sup 9/-10/sup 10/ cfu/ml, retained 120-190 ..mu..Ci of cell-bound In-111. In vitro studies showed good retention of the In-111 label in saline at 37/sup 0/C (75-85% cell-bound radioactivity at 1 hour) and in canine blood at 37/sup 0/C (30-55% cell-bound radioactivity at 1 hour). The loss of cell-associated radioactivity in blood, with a corresponding decrease in the number of viable organisms, is probably a result of phagocyte-mediated killing of the organisms and subsequent release of the label. The labeled bacteria have been used successfully for sequential imaging in experimental animals to track bacteria injected into blood and the biliary tree.

  15. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution. PMID:26957519

  16. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution.

  17. Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1

    PubMed Central

    Suriyanarayanan, Tanujaa; Periasamy, Saravanan; Lin, Miao-Hsia; Ishihama, Yasushi; Swarup, Sanjay

    2016-01-01

    Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal. PMID:27701473

  18. 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.

  19. Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa.

    PubMed

    Persat, Alexandre; Inclan, Yuki F; Engel, Joanne N; Stone, Howard A; Gitai, Zemer

    2015-06-16

    Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

  20. Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione.

    PubMed

    Cheluvappa, Rajkumar; Shimmon, Ronald; Dawson, Michael; Hilmer, Sarah N; Le Couteur, David G

    2008-01-01

    Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37 degrees C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O(2)-dependent. The formation of H(2)O(2) as an intermediate and the thiol group in GSH seem to be important in this reaction. PMID:18797520

  1. Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

    PubMed Central

    Craven, R; Montie, T C

    1985-01-01

    The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers. PMID:3932326

  2. Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

    PubMed Central

    Cezairliyan, Brent; Vinayavekhin, Nawaporn; Grenfell-Lee, Daniel; Yuen, Grace J.; Saghatelian, Alan; Ausubel, Frederick M.

    2013-01-01

    Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches. PMID:23300454

  3. [New Virulent Bacteriophages Active against Multiresistant Pseudomonas aeruginosa Strains].

    PubMed

    Balarjishvili, N Sh; Kvachadze, L I; Kutateladze, M I; Meskhi, T Sh; Pataridze, T K; Berishvili, T A; Tevdoradze, E Sh

    2015-01-01

    The sensitivity of 512 newly isolated Pseudomonas aeruginosa clinical strains to six classes of anti-microbial preparations has been studied. Antibiotic-resistant strains were selected and genotyped. Three new virulent bacteriophages of the families Myoviridae and Podoviridae were isolated against these strains. The parameters of the intracellular phage development cycle were established, and the influence of inactivating factors (temperature, pH, and UV exposure) on phage viability was studied. The molecular weight of the phage genome was determined. Phage DNA restriction analysis and polyacrylamide gel electrophoresis in the presence of envelope protein SDS were carried out. The plating efficacy of phages on 28 genetically distant antibiotic-resistant P. aeruginosa strains was studied. It was established that 26 of them were lysed by phages with a high efficacy. The range of antibacterial action of the studied phages and their mixtures on 427 multi-drug-resistant clinical isolates was assessed. It is shown that including these phages in one multicomponent preparation enhanced their lytic activity. PMID:26859962

  4. Glycosylation Substrate Specificity of Pseudomonas aeruginosa 1244 Pilin*S

    PubMed Central

    Horzempa, Joseph; Comer, Jason E.; Davis, Sheila A.; Castric, Peter

    2008-01-01

    The β-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the “tail” region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur. PMID:16286455

  5. Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site

    PubMed Central

    Comer, Jason E.; Marshall, Mark A.; Blanch, Vincent J.; Deal, Carolyn D.; Castric, Peter

    2002-01-01

    Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay. PMID:12010970

  6. A molecular mechanism that stabilizes cooperative secretions in Pseudomonas aeruginosa

    PubMed Central

    Kim, Wook

    2010-01-01

    Summary Bacterial populations frequently act as a collective by secreting a wide range of compounds necessary for cell-cell communication, host colonization and virulence. However, how such behaviors avoid exploitation by spontaneous ‘cheater’ mutants that use but do not contribute to secretions remains unclear. We investigate this question using Pseudomonas aeruginosa swarming, a collective surface motility requiring massive secretions of rhamnolipid biosurfactants. We first show that swarming is immune to the evolution of rhlA− ‘cheaters’. We then demonstrate that P. aeruginosa resists cheating through metabolic prudence: wild-type cells secrete biosurfactants only when the cost of their production and impact on individual fitness is low, therefore preventing non-secreting strains from gaining an evolutionary advantage. Metabolic prudence works because the carbon-rich biosurfactants are only produced when growth is limited by another growth limiting nutrient, the nitrogen source. By genetically manipulating a strain to produce the biosurfactants constitutively we show that swarming becomes cheatable: a non-producing strain rapidly outcompetes and replaces this obligate cooperator. We argue that metabolic prudence, which may first evolve as a direct response to cheating or simply to optimize growth, can explain the maintenance of massive secretions in many bacteria. More generally, prudent regulation is a mechanism to stabilize cooperation. PMID:21166901

  7. Fructooligosacharides reduce Pseudomonas aeruginosa PAO1 pathogenicity through distinct mechanisms.

    PubMed

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed.

  8. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

    PubMed Central

    Hentzer, Morten; Wu, Hong; Andersen, Jens Bo; Riedel, Kathrin; Rasmussen, Thomas B.; Bagge, Niels; Kumar, Naresh; Schembri, Mark A.; Song, Zhijun; Kristoffersen, Peter; Manefield, Mike; Costerton, John W.; Molin, Søren; Eberl, Leo; Steinberg, Peter; Kjelleberg, Staffan; Høiby, Niels; Givskov, Michael

    2003-01-01

    Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response. PMID:12881415

  9. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

    PubMed

    Chan, Benjamin K; Sistrom, Mark; Wertz, John E; Kortright, Kaitlyn E; Narayan, Deepak; Turner, Paul E

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  10. Inactivation of Pseudomonas aeruginosa biofilm by dense phase carbon dioxide.

    PubMed

    Mun, Sungmin; Jeong, Jin-Seong; Kim, Jaeeun; Lee, Youn-Woo; Yoon, Jeyong

    2009-01-01

    Dense phase carbon dioxide (DPCD) is one of the most promising techniques available to control microorganisms as a non-thermal disinfection method. However, no study on the efficiency of biofilm disinfection using DPCD has been reported. The efficiency of DPCD in inactivating Pseudomonas aeruginosa biofilm, which is known to have high antimicrobial resistance, was thus investigated. P. aeruginosa biofilm, which was not immersed in water but was completely wet, was found to be more effectively inactivated by DPCD treatment, achieving a 6-log reduction within 7 min. The inactivation efficiency increased modestly with increasing pressure and temperature. This study also reports that the water-unimmersed condition is one of the most important operating parameters in achieving efficient biofilm control by DPCD treatment. In addition, observations by confocal laser scanning microscopy revealed that DPCD treatment not only inactivated biofilm cells on the glass coupons but also caused detachment of the biofilm following weakening of its structure as a result of the DPCD treatment; this is an added benefit of DPCD treatment.

  11. Mechanical destruction of pseudomonas aeruginosa biofilms by ultrasound exposure

    NASA Astrophysics Data System (ADS)

    Xu, Jin; Bigelow, Timothy A.; Halverson, Larry J.; Middendorf, Jill; Rusk, Ben

    2012-10-01

    Medical implants are prone to colonization by bacterial biofilms, which are highly resistant to antibiotics. Normally, surgery is required to replace the infected implant. One promising non-invasive treatment option is to destroy the biofilm with high-intensity focused ultrasound (HIFU) exposure. In our study, Pseudomonas aeruginosa bacterial biofilms were grown on graphite disks in a flow chamber for three days prior to exposing them to ultrasound pulses of varying duration or burst period. The pulses were 20 cycles in duration at a frequency of 1.1 MHz from a spherically focused transducer (f/1, 63 mm focal length), creating peak compressional and rarefactional pressures at the disk surface of 30 and 13 MPa, respectively. P. aeruginosa were tagged with GFP and cells killed by HIFU were visualized using propidium iodide, which permeates membranes of dead cells, to aid determining the extent of biofilm destruction and whether cells are alive or dead. Our results indicate that a 30-s exposure and 6-ms pulse period or those combinations with the same number of pulses, were sufficient to destroy the biofilm and to kill the remaining cells. Reducing the number of pulses decreased biofilm destruction, leaving more dead and live bacteria on the surface.

  12. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa

    PubMed Central

    Chan, Benjamin K.; Sistrom, Mark; Wertz, John E.; Kortright, Kaitlyn E.; Narayan, Deepak; Turner, Paul E.

    2016-01-01

    Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections. PMID:27225966

  13. Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity.

    PubMed

    Bandara, H M H N; Nguyen, D; Mogarala, S; Osiñski, M; Smyth, H D C

    2015-01-01

    Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.

  14. Characterization of the Polymyxin B Resistome of Pseudomonas aeruginosa

    PubMed Central

    Fernández, Lucía; Álvarez-Ortega, Carolina; Wiegand, Irith; Olivares, Jorge; Kocíncová, Dana; Lam, Joseph S.; Martínez, José Luis

    2013-01-01

    Multidrug resistance in Pseudomonas aeruginosa is increasingly becoming a threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial therapy. In many such cases, polymyxins are the only available option, although as their utilization increases so does the isolation of resistant strains. In this study, we screened a comprehensive PA14 mutant library to identify genes involved in changes of susceptibility to polymyxin B in P. aeruginosa. Surprisingly, our screening revealed that the polymyxin B resistome of this microorganism is fairly small. Thus, only one resistant mutant and 17 different susceptibility/intrinsic resistance determinants were identified. Among the susceptible mutants, a significant number carried transposon insertions in lipopolysaccharide (LPS)-related genes. LPS analysis revealed that four of these mutants (galU, lptC, wapR, and ssg) had an altered banding profile in SDS-polyacrylamide gels and Western blots, with three of them exhibiting LPS core truncation and lack of O-antigen decoration. Further characterization of these four mutants showed that their increased susceptibility to polymyxin B was partly due to increased basal outer membrane permeability. Additionally, these mutants also lacked the aminoarabinose-substituted lipid A species observed in the wild type upon growth in low magnesium. Overall, our results emphasize the importance of LPS integrity and lipid A modification in resistance to polymyxins in P. aeruginosa, highlighting the relevance of characterizing the genes that affect biosynthesis of cell surface structures in this pathogen to follow the evolution of peptide resistance in the clinic. PMID:23070157

  15. Mucin Promotes Rapid Surface Motility in Pseudomonas aeruginosa

    PubMed Central

    Yeung, Amy T. Y.; Parayno, Alicia; Hancock, Robert E. W.

    2012-01-01

    ABSTRACT An important environmental factor that determines the mode of motility adopted by Pseudomonas aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin. P. aeruginosa is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming, P. aeruginosa was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (las and rhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.” PMID:22550036

  16. Emergence of colistin resistant Pseudomonas aeruginosa at Tabriz hospitals, Iran

    PubMed Central

    Goli, Hamid Reza; Nahaei, Mohammad Reza; Ahangarzadeh Rezaee, Mohammad; Hasani, Alka; Samadi Kafil, Hossein; Aghazadeh, Mohammad

    2016-01-01

    Background and Objectives: The prevalence of multidrug resistant Pseudomonas aeruginosa is the main reason of new drugs resurgence such as colistin. The main objectives of this study were to determine the antibiotic resistance pattern and the rate of colistin resistance along with its correlation with overexpression of MexAB-OprM and MexXY-OprM efflux pumps among P. aeruginosa isolates. Materials and Methods: Hundred clinical isolates were collected from 100 patients during 6 months in 2014. Susceptibility to the eight antibiotics was investigated using Kirby-Bauer and agar dilution methods. The Quantitative Real-time PCR was used to determine the expression levels of efflux genes. Results: Resistance rates to various antibiotics were as follows: ticarcillin (73%), ciprofloxacin (65%), aztreonam (60%), ceftazidime (55%), gentamicin (55%), imipenem (49%), piperacillin/tazobactam (34%) and colistin (2%). In disk diffusion method, only two isolates were non susceptible to colistin, however in agar dilution method the two isolates were confirmed as resistant and two others were intermediate resistant. Sixty eight (68%) isolates were multi-drug resistant and 10 isolates were susceptible to all tested antibiotics. Both colistin resistant isolates showed overexpression of both efflux pumps, but two intermediate resistant isolates exhibited reduction of efflux genes expression. Conclusions: Emergence of colistin resistance is increasing in P. aeruginosa indicating great challenge in the treatment of infections caused by MDR strains of this organism in Iran. ParRS may promote either induced or constitutive resistance to colistin through the activation of distinct mechanisms such as MDR efflux pumps, and LPS modification. PMID:27092226

  17. [Virulence factors in Pseudomonas aeruginosa: mechanisms and modes of regulation].

    PubMed

    Ben Haj Khalifa, Anis; Moissenet, Didier; Vu Thien, Hoang; Khedher, Mohamed

    2011-01-01

    Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium's virulence depends on a large number of cell-associated and extracellular factors. The virulence factors play an important pathological role in the colonization, the survival of the bacteria and the invasion of tissues. There are two types of virulence factors: (1) factors involved in the acute infection: these factors are either on the surface of P. aeruginosa, either secreted. The pili allow adherence to the epithelium. The exoenzyme S and other adhesins reinforce the adherence to epithelial cells. The exotoxin A is responsible of tissue necrosis. Phospholipase C is a thermolabile haemolysin. The pathogenic role of exoenzyme S is attributable to the disruption of normal cytoskeletal organization, the destruction of immunoglobulin G and A, leads to depolymerization of actin filaments and contributes to the resistance to macrophages. P. aeruginosa produces at least four proteases causing bleeding and tissue necrosis; (2) factors involved in the chronic infection: siderophores (pyoverdin and pyochelin), allow the bacteria to multiply in the absence of ferrous ions. The strains isolated from patients with cystic fibrosis have a pseudocapsule of alginate that protects the bacterium from phagocytosis, dehydration and antibiotics. Moreover, it improves adherence to epithelial cells forming a biofilm. Two different types of regulation systems control the expression of the majority of these virulence factors: the two-component transcriptional regulatory system and the quorum sensing system. These two mechanisms are necessary to the survival and the proliferation of this microorganism in the host. PMID:21896403

  18. Comparison of UVB and UVC irradiation disinfection efficacies on Pseudomonas Aeruginosa (P. aeruginosa) biofilm

    NASA Astrophysics Data System (ADS)

    Argyraki, A.; Markvart, M.; Nielsen, Anne; Bjarnsholt, T.; Bjørndal, L.; Petersen, P. M.

    2016-04-01

    Disinfection routines are important in all clinical applications. The uprising problem of antibiotic resistance has driven major research efforts towards alternative disinfection approaches, involving light-based solutions. Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium that can cause skin, soft tissue, lungs, kidney and urinary tract infections. Moreover, it can be found on and in medical equipment causing often cross infections in hospitals. The objective of this study was to test the efficiency, of two different light-based disinfection treatments, namely UVB and UVC irradiation, on P. aeruginosa biofilms at different growth stages. In our experiments a new type of UV light emitting diodes (LEDs) were used to deliver UV irradiation on the biofilms, in the UVB (296nm) and UVC (266nm) region. The killing rate was studied as a function of dose for 24h grown biofilms. The dose was ramped from 72J/m2 to 10000J/m2. It was shown that UVB irradiation was more effective than UVC irradiation in inactivating P. aeruginosa biofilms. No colony forming units (CFU) were observed for the UVB treated biofilms when the dose was 10000 J/m2 (CFU in control sample: 7.5 x 104). UVB irradiation at a dose of 20000J/m2 on mature biofilms (72h grown) resulted in a 3.9 log killing efficacy. The fact that the wavelength of 296nm exists in daylight and has such disinfection ability on biofilms gives new perspectives for applications within disinfection at hospitals.

  19. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    EPA Science Inventory

    BACKGROUND: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexis...

  20. Lichen secondary metabolite evernic acid as potential quorum sensing inhibitor against Pseudomonas aeruginosa.

    PubMed

    Gökalsın, Barış; Sesal, Nüzhet Cenk

    2016-09-01

    Cystic Fibrosis is a genetic disease and it affects the respiratory and digestive systems. Pseudomonas aeruginosa infections in Cystic Fibrosis are presented as the main cause for high mortality and morbidity rates. Pseudomonas aeruginosa populations can regulate their virulence gene expressions via the bacterial communication system: quorum sensing. Inhibition of quorum sensing by employing quorum sensing inhibitors can leave the bacteria vulnerable. Therefore, determining natural sources to obtain potential quorum sensing inhibitors is essential. Lichens have ethnobotanical value for their medicinal properties and it is possible that their secondary metabolites have quorum sensing inhibitor properties. This study aims to investigate an alternative treatment approach by utilizing lichen secondary metabolite evernic acid to reduce the expressions of Pseudomonas aeruginosa virulence factors by inhibiting quorum sensing. For this purpose, fluorescent monitor strains were utilized for quorum sensing inhibitor screens and quantitative reverse-transcriptase PCR analyses were conducted for comparison. Results indicate that evernic acid is capable of inhibiting Pseudomonas aeruginosa quorum sensing systems.

  1. Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens

    PubMed Central

    Cigna, Jérémy; Raoul des Essarts, Yannick; Mondy, Samuel; Hélias, Valérie; Beury-Cirou, Amélie

    2015-01-01

    Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya. PMID:25635023

  2. Draft Genome Sequences of Pseudomonas fluorescens Strains PA4C2 and PA3G8 and Pseudomonas putida PA14H7, Three Biocontrol Bacteria against Dickeya Phytopathogens.

    PubMed

    Cigna, Jérémy; Raoul des Essarts, Yannick; Mondy, Samuel; Hélias, Valérie; Beury-Cirou, Amélie; Faure, Denis

    2015-01-29

    Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya.

  3. Variations in properties of L-forms of Pseudomonas aeruginosa.

    PubMed Central

    Bertolani, R; Elberg, S S; Ralston, D

    1975-01-01

    In a study of the pathogenic potentials of Pseudomonas L-forms, three unstable L-forms were derived by carbenicillin inductionfrom a mouse virulent strain of Pseudomonas aeruginosa, Rosenthal 180. One L-form, induced on a sucrose-stabilized medium, grew more slowly and differed in a number of properties from two other L-forms induced on a medium supported with polyvinylpyrilidone. After adaptation to a common liquid medium, the three L-forms differed with respect to colonial shape on solid medium, growth rate, certain biochemical properties, antibiotic sensitivities and antigenic surface, and virulence for mice. The L-form may revert in vitro to a serotype different from that of the parent culture. The revertant may acquire new antibiotic resistances and sensitivities in the absence of previous exposure to the drugs and enhanced resistance to the L-inducing agent. The three L-forms showed a characteristically lower, but wide, range of virulence than did the parental form. Though death of mice was accompanied by reversion of the L-forms in vivo to the bacterial form, reversion in vivo was not necessary for virulence of L-forms. Modification of residual cell wall antigens accompanied the induction of each L-form as determined by type-specific antisera. Images PMID:803921

  4. Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Hammond, John H.; Dolben, Emily F.; Smith, T. Jarrod; Bhuju, Sabin

    2015-01-01

    ABSTRACT In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections. IMPORTANCE Pseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently

  5. New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

    PubMed

    Santos, Ana; Mendes, Sónia; Brissos, Vânia; Martins, Lígia O

    2014-03-01

    This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology. PMID:23820555

  6. New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

    PubMed

    Santos, Ana; Mendes, Sónia; Brissos, Vânia; Martins, Lígia O

    2014-03-01

    This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

  7. Characterization of the Biogenic Mn-Oxide Produced by Pseudomonas putida Strain MnB1

    NASA Astrophysics Data System (ADS)

    Villalobos, M.; Bargar, J.; Sposito, G.

    2001-12-01

    Mn-oxide nanoparticles are common and highly reactive materials in the environment. They occur as dispersed colloids, in nodules, and as coatings having high specific surface areas and, thus, high surface reactivity. Hence, they are believed to play a major role in the fate and transport of contaminant and nutrient species in the environment. Most of these oxides are believed to be microbial in origin, and a wide array of Mn(II) oxidizing bacteria exists in almost all natural aqueous environments. However, little is known about the structures, characteristics and reactivities of these biogenic oxides. The goal of this research was to identify the Mn oxide product from a strain of the fresh water bacterial species Pseudomonas putida, and to characterize it along with analogous synthetic Mn oxides: two different MnIII/MnIV oxides identified as birnessites, and the MnIV oxide, δ -MnO2. These synthetic phases were defined as potential models based on comparison to X-ray absorption and diffraction spectra from a large number of Mn(II/III/IV) references. Characterization of biotic and abiotic Mn oxides was performed with respect to: morphology, surface area, Mn composition, structure, and surface reactivity. In this fashion, randomly-stacked hexagonal birnessite of low crystallinity was identified as a close synthetic analog to the biogenic oxide, making it suitable for reference and comparison purposes, as well as for reactivity prediction studies. This synthetic product is distinct from monoclinic birnessite, but showed some similarities to Mn oxide minerals of very low crystallinity previously identified as vernadite (δ -MnO2). This latter mineral has been identified in the past as comparable to the biogenic oxide produced by the marine Bacillus sp. strain SG-1, which suggests similarities in the biological Mn(II) oxidation processes across natural environments and bacterial species.

  8. Mechanism for Biotransformation of Nonylphenol Polyethoxylates to Xenoestrogens in Pseudomonas putida

    PubMed Central

    John, Dominic M.; White, Graham F.

    1998-01-01

    A strain of Pseudomonas putida isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEOx, where x is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEOav6, NPEOav9, and NPEOav20 (mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO2). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEOx. Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEOx mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEOx-degrading activity was inducible by substrate, and cell extracts of NPEOav9-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO8), producing sequentially, under either aerobic or anaerobic conditions, AEO7, AEO6, AEO5, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEOav9 or AEO8 under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEOx and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEOx−1, which then undergoes further cycles of the same reaction until x = 2. PMID:9721266

  9. Protective role of glycerol against benzene stress: insights from the Pseudomonas putida proteome.

    PubMed

    Bhaganna, Prashanth; Bielecka, Agata; Molinari, Gabriella; Hallsworth, John E

    2016-05-01

    Chemical activities of hydrophobic substances can determine the windows of environmental conditions over which microbial systems function and the metabolic inhibition of microorganisms by benzene and other hydrophobes can, paradoxically, be reduced by compounds that protect against cellular water stress (Bhaganna et al. in Microb Biotechnol 3:701-716, 2010; Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a). We hypothesized that this protective effect operates at the macromolecule structure-function level and is facilitated, in part at least, by genome-mediated adaptations. Based on proteome profiling of the soil bacterium Pseudomonas putida, we present evidence that (1) benzene induces a chaotrope-stress response, whereas (2) cells cultured in media supplemented with benzene plus glycerol were protected against chaotrope stress. Chaotrope-stress response proteins, such as those involved in lipid and compatible-solute metabolism and removal of reactive oxygen species, were increased by up to 15-fold in benzene-stressed cells relative to those of control cultures (no benzene added). By contrast, cells grown in the presence of benzene + glycerol, even though the latter grew more slowly, exhibited only a weak chaotrope-stress response. These findings provide evidence to support the hypothesis that hydrophobic substances induce a chaotropicity-mediated water stress, that cells respond via genome-mediated adaptations, and that glycerol protects the cell's macromolecular systems. We discuss the possibility of using compatible solutes to mitigate hydrocarbon-induced stresses in lignocellulosic biofuel fermentations and for industrial and environmental applications. PMID:26612269

  10. Influence of oxygen transfer on Pseudomonas putida effects on growth rate and biodesulfurization capacity.

    PubMed

    Escobar, S; Rodriguez, A; Gomez, E; Alcon, A; Santos, V E; Garcia-Ochoa, Felix

    2016-04-01

    The growth rate and desulfurization capacity accumulated by the cells during the growth of Pseudomonas putida KTH2 under different oxygen transfer conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different agitation conditions. During the culture, several magnitudes associated to growth, such as the specific growth rate, the dissolved oxygen concentration and the carbon source consumption have been measured. Experimental results indicate that cultures are influenced by the fluid dynamic conditions into the bioreactor. An increase in the stirrer speed from 400 to 700 rpm has a positive influence on the cell growth rate. Nevertheless, the increase of agitation from 700 to 2000 rpm hardly has any influence on the growth rate. The effect of fluid dynamics on the cells development of the biodesulfurization (BDS) capacity of the cells during growth is different. The activities of the intracellular enzymes involved in the 4S pathway change with dissolved oxygen concentration. The enzyme activities have been evaluated in cells at several growth time and different hydrodynamic conditions. An increase of the agitation from 100 to 300 rpm has a positive influence on the development of the overall BDS capacity of the cells during growth. This capacity shows a decrease for higher stirrer speeds and the activity of the enzymes monooxygenases DszC and DszA decreases dramatically. The highest value of the activity of DszB enzyme was obtained with cells cultured at 100 rpm, while this activity decreases when the stirrer speed was increased higher than this value. PMID:26762940

  11. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7

    PubMed Central

    de Araújo, Simara Semíramis; Neves, Cíntia Mara Leal; Guimarães, Samuel Leite; Whitman, Christian P.; Johnson, William H.; Aparicio, Ricardo; Nagem, Ronaldo Alves Pinto

    2016-01-01

    The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase required for conversion of 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD+. NahI is in one family of the NAD(P)+-dependent aldehyde dehydrogenase superfamily (ALDH8). In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress at 12 ºC as an N-terminal hexa-histidine-tagged fusion protein (6xHis-NahI). After the soluble protein was purified by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were also evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted for the recombinant enzyme and, for the first time, KM (1.3 ± 0.3 μM) and kcat (0.9 s−1) values were determined for this enzyme (at presumed NAD+ saturation). NahI is highly specific for its biological substrate (2-hydroxymuconate semialdehyde) and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway. PMID:26032336

  12. Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

    PubMed Central

    Morawski, B; Casy, G; Illaszewicz, C; Griengl, H; Ribbons, D W

    1997-01-01

    Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies. PMID:9190820

  13. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

    PubMed Central

    Eaton, R W; Timmis, K N

    1986-01-01

    A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4. Images PMID:3019995

  14. Removal of Pseudomonas putida biofilm and associated extracellular polymeric substances from stainless steel by alkali cleaning.

    PubMed

    Antoniou, Katerina; Frank, Joseph F

    2005-02-01

    Alkali (NaOH)-based compounds are commonly used in the food industry to clean food contact surfaces. However, little information is available on the ability of alkali and alkali-based cleaning compounds to remove extracellular polymeric substances (EPS) produced by biofilm bacteria. The objectives of this study were to determine the temperature and NaOH concentration necessary to remove biofilm EPS from stainless steel under turbulent flow conditions (clean-in-place simulation) and to determine the ability of a commercial alkaline cleaner to remove biofilm EPS from stainless steel when applied under static conditions without heat. Biofilms were produced by growing Pseudomonas putida on stainless steel for 72 h at 25 degrees C in a 1:10 dilution of Trypticase soy broth. The biofilms were treated using NaOH at concentrations of 1.28 to 6.0% and temperatures ranging from 66 to 70 degrees C. Other biofilms were treated with commercial alkaline cleaner at 25 or 4 degrees C for 1 to 30 min. Removal of EPS was determined by direct microscopic observation of samples stained with fluorescent-labeled peanut agglutinin lectin. Treatment with 1.2% NaOH at 66 degrees C for 3 min was insufficient to remove biofilm EPS. A minimum of 2.5% NaOH at 66 degrees C and 2.0% NaOH at 68 degrees C for 3 min were both effective for EPS removal. Commercial alkaline cleaner removed over 99% of biofilm EPS within 1 min at 4 and 25 degrees C under static conditions. Selection of appropriated cleaning agent formulation and use at recommended concentrations and temperatures is critical for removal of biofilm EPS from stainless steel. PMID:15726969

  15. Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane.

    PubMed

    Noda, Ken-ichi; Watanabe, Kimiko; Maruhashi, Kenji

    2003-07-01

    Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.

  16. [Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

    PubMed

    Mulyukin, A L; Kozlova, A N; Sorokin, V V; Suzina, N E; Cherdyntseva, T A; Kotova, I B; Gaponov, A M; Tutel'yan, A V; El'-Registan, G I

    2015-01-01

    Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 μg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent

  17. [Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

    PubMed

    Mulyukin, A L; Kozlova, A N; Sorokin, V V; Suzina, N E; Cherdyntseva, T A; Kotova, I B; Gaponov, A M; Tutel'yan, A V; El'-Registan, G I

    2015-01-01

    Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 μg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent

  18. Photodynamic antimicrobial therapy to inhibit pseudomonas aeruginosa of corneal isolates (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Durkee, Heather A.; Relhan, Nidhi; Arboleda, Alejandro; Halili, Francisco; De Freitas, Carolina; Alawa, Karam; Aguilar, Mariela C.; Amescua, Guillermo; Miller, Darlene; Parel, Jean-Marie

    2016-03-01

    Keratitis associated with Pseudomonas aeruginosa is difficult to manage. Treatment includes antibiotic eye drops, however, some strains of Pseudomonas aeruginosa are resistant. Current research efforts are focused on finding alternative and adjunct therapies to treat multi-drug resistant bacteria. One promising alternate technique is photodynamic therapy (PDT). The purpose of this study was to evaluate the effect of riboflavin- and rose bengal-mediated PDT on Pseudomonas aeruginosa keratitis isolates in vitro. Two isolates (S+U- and S-U+) of Pseudomonas aeruginosa were derived from keratitis patients and exposed to five experimental groups: (1) Control (dark, UV-A irradiation, 525nm irradiation); (2) 0.1% riboflavin (dark, UV-A irradiation); and (3) 0.1% rose bengal, (4) 0.05% rose bengal and (5) 0.01% rose bengal (dark, 525nm irradiation). Three days after treatment, in dark conditions of all concentration of riboflavin and rose bengal showed no inhibition in both S+U- and S-U+ strains of Pseudomonas aeruginosa. In 0.1% and 0.05% rose bengal irradiated groups, for both S+U- and S-U+ strains, there was complete inhibition of bacterial growth in the central 50mm zone corresponding to the diameter of the green light source. These in vitro results suggest that rose bengal photodynamic therapy may be an effective adjunct treatment for Pseudomonas aeruginosa keratitis.

  19. Genome Sequence of Pseudomonas aeruginosa Strain LCT-PA41, with Changed Metabolism after Space Flight.

    PubMed

    Liu, Chao; Hu, Juan; Fang, Xiangqun; Zhang, Duchao; Chang, De; Wang, Junfeng; Li, Tianzhi; Guo, Yinhua; Dai, Wenkui; Liu, Changting

    2014-01-09

    To explore the effects of space flight on microorganisms, Pseudomonas aeruginosa ATCC 27853 was sent into orbit for 398 h on the spacecraft ShenZhou VIII. Here, we present the draft genome sequence of the P. aeruginosa strain LCT-PA41, determined after space flight.

  20. Draft Genome Sequence of Pseudomonas aeruginosa Strain RB, a Bacterium Capable of Synthesizing Cadmium Selenide Nanoparticles.

    PubMed

    Ayano, Hiroyuki; Kuroda, Masashi; Soda, Satoshi; Ike, Michihiko

    2014-01-01

    Pseudomonas aeruginosa strain RB is a bacterium capable of synthesizing cadmium selenide (CdSe) nanoparticles and was isolated from a soil sample. Here, we present the draft genome sequence of P. aeruginosa strain RB. To the best of our knowledge, this is the first report of a draft genome of a CdSe-synthesizing bacterium.

  1. The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 is transferred via a novel Type IV pilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments including humans is in part due to its large and diverse genomic repertoi...

  2. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    PubMed Central

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. PMID:27194047

  3. Pseudomonas aeruginosa septic arthritis of knee after intra-articular ozone injection.

    PubMed

    Seyman, Derya; Ozen, Nevgun Sepin; Inan, Dilara; Ongut, Gozde; Ogunc, Dilara

    2012-07-01

    We describe a case of septic arthritis caused by Pseudomonas aeruginosa in an immunocompetent patient following intra-articular ozone injection into the knee. To the best of our knowledge, and after considering the current literature,we believe this case is unique as no other reports of septic arthritis caused by P. aeruginosa following intra-articular ozone injection has been made.

  4. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-05-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa.

  5. Genome Sequence of Pseudomonas aeruginosa Strain LCT-PA41, with Changed Metabolism after Space Flight

    PubMed Central

    Liu, Chao; Hu, Juan; Fang, Xiangqun; Zhang, Duchao; Chang, De; Wang, Junfeng; Li, Tianzhi; Guo, Yinhua; Dai, Wenkui

    2014-01-01

    To explore the effects of space flight on microorganisms, Pseudomonas aeruginosa ATCC 27853 was sent into orbit for 398 h on the spacecraft ShenZhou VIII. Here, we present the draft genome sequence of the P. aeruginosa strain LCT-PA41, determined after space flight. PMID:24407638

  6. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa.

    PubMed

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-05-19

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa.

  7. Comparative in vitro activities of newer quinolones against Pseudomonas species and Xanthomonas maltophilia isolated from patients with cancer.

    PubMed Central

    Rolston, K V; Messer, M; Ho, D H

    1990-01-01

    The in vitro susceptibilities of three Pseudomonas species (Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens) and Xanthomonas maltophilia to quinolone antimicrobial agents were determined. Several newer agents, particularly PD117558, PD117596, PD127391, sparfloxacin (AT-4140), A-56620, and temafloxacin, were active against Pseudomonas species. X. maltophilia isolates were generally less susceptible than were Pseudomonas isolates but were inhibited by some of the newer quinolones. PMID:2285297

  8. Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

    PubMed

    Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

    2014-07-01

    Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

  9. A 90-Kilobase Conjugative Chromosomal Element Coding for Biphenyl and Salicylate Catabolism in Pseudomonas putida KF715

    PubMed Central

    Nishi, Akito; Tominaga, Kiyomi; Furukawa, Kensuke

    2000-01-01

    The biphenyl and salicylate metabolic pathways in Pseudomonas putida KF715 are chromosomally encoded. The bph gene cluster coding for the conversion of biphenyl to benzoic acid and the sal gene cluster coding for the salicylate meta-pathway were obtained from the KF715 genomic cosmid libraries. These two gene clusters were separated by 10-kb DNA and were highly prone to deletion when KF715 was grown in nutrient medium. Two types of deletions took place at the region including only the bph genes (ca. 40 kb) or at the region including both the bph and sal genes (ca. 70 kb). A 90-kb DNA region, including both the bph and sal genes (termed the bph-sal element), was transferred by conjugation from KF715 to P. putida AC30. Such transconjugants gained the ability to grow on biphenyl and salicylate as the sole sources of carbon. The bph and sal element was located on the chromosome of the recipient. The bph-sal element in strain AC30 was also highly prone to deletion; however, it could be mobilized to the chromosome of P. putida KT2440 and the two deletion mutants of KF715. PMID:10715002

  10. Protein as Chemical Cue: Non-Nutritional Growth Enhancement by Exogenous Protein in Pseudomonas putida KT2440

    PubMed Central

    Joshi, Hiren; Dave, Rachna; Venugopalan, Vayalam P.

    2014-01-01

    Research pertaining to microbe-microbe and microbe-plant interactions has been largely limited to small molecules like quorum sensing chemicals. However, a few recent reports have indicated the role of complex molecules like proteins and polysaccharides in microbial communication. Here we demonstrate that exogenous proteins present in culture media can considerably accelerate the growth of Pseudomonas putida KT2440, even when such proteins are not internalized by the cells. The growth enhancement is observed when the exogenous protein is not used as a source of carbon or nitrogen. The data show non-specific nature of the protein inducing growth; growth enhancement was observed irrespective of the protein type. It is shown that growth enhancement is mediated via increased siderophore secretion in response to the exogenous protein, leading to better iron uptake. We highlight the ecological significance of the observation and hypothesize that exogenous proteins serve as chemical cues in the case of P.putida and are perceived as indicator of the presence of competitors in the environment. It is argued that enhanced siderophore secretion in response to exogenous protein helps P.putida establish numerical superiority over competitors by way of enhanced iron assimilation and quicker utilization of aromatic substrates. PMID:25117434

  11. Experimental and kinetic study on the cometabolic biodegradation of phenol and 4-chlorophenol by psychrotrophic Pseudomonas putida LY1.

    PubMed

    Wang, Qing; Li, Yi; Li, Jing; Wang, Yuming; Wang, Chao; Wang, Peifang

    2015-01-01

    This study investigated the kinetics of phenol and 4-chlorophenol (4-CP) biodegradation by a cold-adapted bacteria, Pseudomonas putida LY1, isolated from Songhua River sediment. The results showed that P. putida LY1 cannot grow on 4-CP as a sole carbon source. P. putida LY1 had the potential to cometabolic biodegrade phenol and 4-CP in a wide range of temperature (varying from 5 to 35 °C) with the optimal temperature around 25 °C. Mixture of phenol and 4-CP were completely removed at two 4-CP concentrations (15 and 40 mg/L) over a wide range of phenol (20-400 mg/L) concentrations, whereby the ratio of 4-CP/biomass (S 2/X) was lower than 0.03. The kinetic models of cometabolic biodegradation of phenol and 4-CP were proposed, considering the growth and nongrowth substrate inhibition. These models successfully simulate the processes of cometabolic degradation of phenol and 4-CP.

  12. cumA, a Gene Encoding a Multicopper Oxidase, Is Involved in Mn2+ Oxidation in Pseudomonas putida GB-1

    PubMed Central

    Brouwers, Geert-Jan; de Vrind, Johannes P. M.; Corstjens, Paul L. A. M.; Cornelis, Pierre; Baysse, Christine; de Vrind-de Jong, Elisabeth W.

    1999-01-01

    Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002. PMID:10103278

  13. Degradation of Tectilon Yellow 2G by hybrid technique: combination of sonolysis and biodegradation using mutant Pseudomonas putida.

    PubMed

    Srinivasan, Raman; Kathiravan, Mathur Nadarajan; Gopinath, Kannappan Panchamoorthy

    2011-02-01

    Degradation of Tectilon Yellow 2G (TY2G), an azo dye has been studied by hybrid technique involving pretreatment by sonochemical method and further biological treatment by Pseudomonas putida mutant. Pretreatment experiments were carried out by sonolysis of the dye solution at different concentrations (100-1000 mg/L). Wild type Gram-negative P. putida species isolated from the textile effluent contaminated soil, which was found to be effective towards dye degradation, has been acclimatized so as to consume TY2G as the sole source of nutrition. Mutant strain was obtained from the acclimatized species by random mutagenesis using the chemical mutagen ethidium bromide for various time intervals (6-30 min). The optimum mutagenesis exposure time for obtaining the most efficient species for dye degradation was found to be 18 min. An efficient mutant strain P. putida ACT 1 has been isolated and was used for growth experiments. The mutant strain showed a better growth compared to the wild strain. The substrate utilization kinetics has been modeled using Monod and Haldane model equations of which the Haldane model provided a better fit. The enzyme kinetics of the mutant and wild species was obtained using Michaelis-Menten equation. The mutated species showed better enzyme kinetics towards the degradation of TY2G.

  14. Effect of the introduction of the nitrogen-fixing bacteria Pseudomonas putida 23 on the nitrogen balance in soil

    NASA Astrophysics Data System (ADS)

    Shabayev, V. P.

    2010-04-01

    The inoculation of red beets with the nitrogen-fixing bacteria Pseudomonas putida 23 increased the activity of the nitrogen fixation in the rhizosphere of the plants grown on meadow soil in the central part of the Oka River floodplain. The yield of the red beets and the uptake by plants of nitrogen from the soil and from the 15N-labeled nitrogen fertilizer applied on the trial microplot increased significantly. A statistically significant additional fixation of nitrogen from the atmosphere and a positive balance of nitrogen in the soil-plant system without significant changes in the bulk content of the soil nitrogen after the plant growing were found in a greenhouse experiment with the application of P. putida. It can be supposed that the excessive nitrogen determined in this system is related to the incorporation into plants of atmospheric nitrogen fixed in the rhizosphere of the inoculated plants. The application of P. putida 23 makes it possible to decrease the rates of NPK fertilizer by two times without losses in the yield of red beets.

  15. Bioconversion of Styrene to Poly(hydroxyalkanoate) (PHA) by the New Bacterial Strain Pseudomonas putida NBUS12

    PubMed Central

    Tan, Giin-Yu Amy; Chen, Chia-Lung; Ge, Liya; Li, Ling; Tan, Swee Ngin; Wang, Jing-Yuan

    2015-01-01

    Styrene is a toxic pollutant commonly found in waste effluents from plastic processing industries. We herein identified and characterized microorganisms for bioconversion of the organic eco-pollutant styrene into a valuable biopolymer medium-chain-length poly(hydroxyalkanoate) (mcl-PHA). Twelve newly-isolated styrene-degrading Pseudomonads were obtained and partial phaC genes were detected by PCR in these isolates. These isolates assimilated styrene to produce mcl-PHA, forming PHA contents between 0.05±0.00 and 23.10±3.25% cell dry mass (% CDM). The best-performing isolate was identified as Pseudomonas putida NBUS12. A genetic analysis of 16S rDNA and phaZ genes revealed P. putida NBUS12 as a genetically-distinct strain from existing phenotypically-similar bacterial strains. This bacterium achieved a final biomass of 1.28±0.10 g L−1 and PHA content of 32.49±2.40% CDM. The extracted polymer was mainly comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanoate (C8 ), 3-hydroxydecanoate (C10 ), 3-hydroxydodecanoate (C12 ), and 3-hydroxytetradecanoate (C14 ) monomers at a ratio of 2:42:1257:17:1. These results collectively suggested that P. putida NBUS12 is a promising candidate for the biotechnological conversion of styrene into mcl-PHA. PMID:25740622

  16. Suppressing Erwinia carotovora pathogenicity by projecting N-acyl homoserine lactonase onto the surface of Pseudomonas putida cells.

    PubMed

    Li, Qianqian; Ni, Hong; Meng, Shan; He, Yan; Yu, Ziniu; Li, Lin

    2011-12-01

    N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/ aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect. PMID:22210621

  17. Determination of copper binding in Pseudomonas putida CZ1 by chemical modifications and X-ray absorption spectroscopy.

    PubMed

    Chen, XinCai; Shi, JiYan; Chen, YingXu; Xu, XiangHua; Chen, LiTao; Wang, Hui; Hu, TianDou

    2007-03-01

    Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1. PMID:17021877

  18. Biosorption of copper(II) and zinc(II) from aqueous solution by Pseudomonas putida CZ1.

    PubMed

    Chen, Xin Cai; Wang, Yuan Peng; Lin, Qi; Shi, Ji Yan; Wu, Wei Xiang; Chen, Ying Xu

    2005-12-10

    To study Pseudomonas putida CZ1, having high tolerance to copper and zinc on the removal of toxic metals from aqueous solutions, the biosorption of Cu(II) and Zn(II) by living and nonliving P. putida CZ1 were studied as functions of reaction time, initial pH of the solution and metal concentration. It was found that the optimum pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5, respectively, for Cu(II) removal. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration increased. The adsorption data with respect to both metals provide an excellent fit to the Langmuir isotherm. The binding capacity of living cells is significantly higher than that of nonliving cells at tested conditions. It demonstrated that about 40-50% of the metals were actively taken up by P. putida CZ1, with the remainder being passively bound to the bacterium. Moreover, desorption efficiency of Cu(II) and Zn(II) by living cells was 72.5 and 45.6% under 0.1M HCl and it was 95.3 and 83.8% by nonliving cells, respectively. It may be due to Cu(II) and Zn(II) uptake by the living cells enhanced by intracellular accumulation. PMID:16289732

  19. Role of the Pseudomonas quinolone signal (PQS) in sensitising Pseudomonas aeruginosa to UVA radiation.

    PubMed

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

    2015-01-01

    One of the main stress factors that bacteria face in the environment is solar ultraviolet-A (UVA) radiation, which leads to lethal effects through oxidative damage. The aim of this work was to investigate the role of 2-heptyl-3-hydroxi-4-quinolone (the Pseudomonas quinolone signal or PQS) in the response of Pseudomonas aeruginosa to UVA radiation. PQS is an intercellular quorum sensing signal associated to membrane vesicles which, among other functions, regulates genes related to iron acquisition, forms stable complexes with iron and participates in oxidative phenomena. UVA exposure of the wild-type PAO1 strain and a pqsA mutant unable to produce PQS revealed a sensitising role for this signal. Research into the mechanism involved in this phenomenon revealed that catalase, an essential factor in the UVA defence, is not related to PQS-mediated UVA sensitivity. Absorption of UVA by PQS produced its own photo-degradation, oxidation of the probe 2',7'- dichlorodihydrofluorescein and generation of singlet oxygen and superoxide anion, suggesting that this signal could be acting as an endogenous photosensitiser. The results presented in this study could explain the high sensitivity to UVA of P. aeruginosa when compared to enteric bacteria. PMID:25535873

  20. Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

    PubMed

    Hur, H G; Sadowsky, M J; Wackett, L P

    1994-11-01

    The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.

  1. Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

    PubMed

    Wang, Weiwei; Xu, Ping; Tang, Hongzhi

    2015-01-01

    Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

  2. Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

    PubMed

    Wang, Weiwei; Xu, Ping; Tang, Hongzhi

    2015-01-01

    Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds. PMID:26574178

  3. Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste

    PubMed Central

    Wang, Weiwei; Xu, Ping; Tang, Hongzhi

    2015-01-01

    Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds. PMID:26574178

  4. Voltammetric profiling of redox-active metabolites expressed by Pseudomonas aeruginosa for diagnostic purposes.

    PubMed

    Seviour, T; Doyle, L E; Lauw, S J L; Hinks, J; Rice, S A; Nesatyy, V J; Webster, R D; Kjelleberg, S; Marsili, E

    2015-03-01

    In Pseudomonas aeruginosa, chemical deconvolution of the pyocyanin voltammetric signal allows its expression to be observed simultaneously with the quorum sensing molecule Pseudomonas quinolone signal (PQS). Such analysis has revealed that PQS might protect pyocyanin from self-oxidation, but also exert a pro-oxidative effect on pyocyanin under oxidative conditions to produce additional redox metabolites. PMID:25650009

  5. 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.

  6. Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis

    PubMed Central

    Lomholt, J A; Kilian, M

    2003-01-01

    Aim: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance. Methods: Ciprofloxacin susceptibility was tested by an agar dilution method; genomic DNA corresponding to the quinolone target genes gyrA and parC, and the regulatory genes mexR and nfxB controlling drug efflux systems, was amplified by PCR and sequenced; multilocus enzyme electrophoresis was performed to examine the genetic relation among resistant strains. Results: Three out of 90 keratitis isolates (3.3%), one from the United Kingdom and two from India, exhibited MIC values of 16 mg/l or 32 mg/l. The UK isolate had a mutation in gyrA (Thr83Ile), whereas the two Indian isolates showed mutations in both gyrA (Thr83Ile) and parC (Ser87Leu). The remaining isolates from keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l. Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains. Conclusions: The vast majority of eye isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant. Mutations in more than one target gene were associated with higher MIC values. PMID:14507757

  7. 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

  8. Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms

    PubMed Central

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed. PMID:24465697

  9. A re-examination of twitching motility in Pseudomonas aeruginosa.

    PubMed

    Semmler, A B; Whitchurch, C B; Mattick, J S

    1999-10-01

    Twitching motility is a form of solid surface translocation which occurs in a wide range of bacteria and which is dependent on the presence of functional type IV fimbriae or pili. A detailed examination of twitching motility in Pseudomonas aeruginosa under optimal conditions in vitro was carried out. Under these conditions (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) twitching motility is extremely rapid, leading to an overall radial rate of colony expansion of 0.6 mm h(-1) or greater. The zones of colony expansion due to twitching motility are very thin and are best visualized by staining. These zones exhibit concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and consolidation/cell division. Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells which move away from the colony edge. Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells. The bacteria in the lattice network then appear to settle and divide to fill out the colonized space. Our observations redefine twitching motility as a rapid, highly organized mechanism of bacterial translocation by which P. aeruginosa can disperse itself over large areas to colonize new territories. It is also now clear, both morphologically and genetically, that twitching motility and social gliding motility, such as occurs in Myxococcus xanthus, are essentially the same process.

  10. Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine.

    PubMed

    Zhang, Yanyan; Hu, Zhiqiang

    2013-01-01

    Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10(7) PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10(7) PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre-existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre-existing biofilms. However, a combination of phages (3 × 10(7) PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one-time treatment at the concentration of 1.9 × 10(8) PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10(5) PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces.

  11. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

    PubMed

    Folders, J; Algra, J; Roelofs, M S; van Loon, L C; Tommassen, J; Bitter, W

    2001-12-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

  12. Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

    PubMed Central

    Borrero-de Acuña, José Manuel; Rohde, Manfred; Wissing, Josef; Jänsch, Lothar; Schobert, Max; Molinari, Gabriella; Timmis, Kenneth N.

    2016-01-01

    ABSTRACT Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO3− → NO2− → NO → N2O → N2. Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCE The processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes

  13. Liquid chromatography time of flight mass spectrometry based environmental metabolomics for the analysis of Pseudomonas putida Bacteria in potable water.

    PubMed

    Kouremenos, Konstantinos A; Beale, David J; Antti, Henrik; Palombo, Enzo A

    2014-09-01

    Water supply biofilms have the potential to harbour waterborne diseases, accelerate corrosion, and contribute to the formation of tuberculation in metallic pipes. One particular species of bacteria known to be found in the water supply networks is Pseudomonas sp., with the presence of Pseudomonas putida being isolated to iron pipe tubercles. Current methods for detecting and analysis pipe biofilms are time consuming and expensive. The application of metabolomics techniques could provide an alternative method for assessing biofilm risk more efficiently based on bacterial activity. As such, this paper investigates the application of metabolomic techniques and provides a proof-of-concept application using liquid chromatography coupled with time-of-flight mass spectrometry (LC-ToF-MS) to three biologically independent P. putida samples, across five different growth conditions exposed to solid and soluble iron (Fe). Analysis of the samples in +ESI and -ESI mode yielded 887 and 1789 metabolite features, respectively. Chemometric analysis of the +ESI and -ESI data identified 34 and 39 significant metabolite features, respectively, where features were considered significant if the fold change was greater than 2 and obtained a p-value less than 0.05. Metabolite features were subsequently identified according to the Metabolomics Standard Initiative (MSI) Chemical Analysis Workgroup using analytical standards and standard online LC-MS databases. Possible markers for P. putida growth, with and without being exposed to solid and soluble Fe, were identified from a diverse range of different chemical classes of metabolites including nucleobases, nucleosides, dipeptides, tripeptides, amino acids, fatty acids, sugars, and phospholipids.

  14. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

    PubMed

    van Beilen, J B; Panke, S; Lucchini, S; Franchini, A G; Röthlisberger, M; Witholt, B

    2001-06-01

    The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS: PMID:11390693

  15. Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

    PubMed Central

    Arif, Muhammad Irfan; Samin, Ghufrana; van Leeuwen, Jan G. E.; Oppentocht, Jantien

    2012-01-01

    A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a kcat of 17 s−1. 1H nuclear magnetic resonance experiments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferricyanide [K3Fe(CN)6] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid. PMID:22752160

  16. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid.

    PubMed

    Graf, Nadja; Altenbuchner, Josef

    2014-01-01

    Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficient to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86% within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

  17. Responses of unsaturated Pseudomonas putida CZ1 biofilms to environmental stresses in relation to the EPS composition and surface morphology.

    PubMed

    Lin, Huirong; Chen, Guangcun; Long, Dongyan; Chen, Xincai

    2014-12-01

    The extracellular polymeric substance (EPS) and surface properties of unsaturated biofilms of a heavy metal-resistant rhizobacterium Pseudomonas putida CZ1, in response to aging, pH, temperature and osmotic stress, were studied by quantitative analysis of EPS and atomic force microscope. It was found that EPS production increased approximately linearly with culture time, cells in the air-biofilm interface enhanced EPS production and decreased cell volume to cope with nutrient depletion during aging. Low pH, high temperature and certain osmotic stress (120 mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. In addition to the enhancement of EPS production in response to high osmotic (328 mM NaCl) stress, cells in the biofilm adhere tightly together to maintain a particular microenvironment. These results indicated the variation of EPS composition and the cooperation of cells in the biofilms is important for the survival of Pseudomonas putida CZ1 from environmental stresses in the unsaturated environments such as rhizosphere.

  18. 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

  19. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads.

    PubMed Central

    Timm, A; Steinbüchel, A

    1990-01-01

    Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent. Images PMID:2125185

  20. Organo-mineral interactions in Pseudomonas putida-birnessite assemblages: Impact on mineral reactivity

    NASA Astrophysics Data System (ADS)

    Simanova, Anna; Kroll, Alexandra; Pena, Jasquelin

    2016-04-01

    The ability of microorganisms to precipitate biogenic birnessite nanoparticles is widely spread in the bacterial and fungal trees of life, with this process accounting largely for the formation of birnessite in nature. Birnessite minerals occur typically as nanoparticles that exhibit significant chemical and structural disorder. Furthermore, the mineral is embedded within a biomass matrix composed of microbial cells and extracellular polymeric substances, where the biomass not only provides reactive surfaces but can mediate electron transfer reactions. The overarching question guiding our research is: How do nanoscale properties and admixing with microbial biomass modify the reactivity of Mn oxide minerals? In this study, we investigate the biomass-birnessite composites of Pseudomonas putida GB-1 biomass and δ-MnO2 nanoparticles. We characterized the structure and composition of the mineral fraction using X-ray diffraction, Mn K-edge X-ray absorption spectroscopy and wet-chemical methods. To characterize the biomass fraction, we employed FTIR spectroscopy and size-exclusion chromatography analysis of the extracellular polymeric substances. Finally, we measured Ni(II) sorption isotherms at pH 6 and Ni K-edge EXAFS spectra to determine the extent and mechanism of Ni sorption in the biomass-mineral composites and in biomass-only and mineral-only systems. This approach provided direct and indirect evidence for the extent of organo-mineral interactions in the composites, as well as a direct measure of sorption reactivity in the composites relative to biomass-only and mineral-only systems. We found that admixing of mineral nanoparticles with biomass reduced the reactivity of the edge sites of birnessite particles towards Ni(II) through the attachment of organic moieties to the mineral particles and/or modification of the assemblage surface charge properties. In addition, the interaction of biomass components with MnO2 particles leads to partial Mn(IV) reduction and

  1. Comprehensive analysis of the metabolome of Pseudomonas putida S12 grown on different carbon sources.

    PubMed

    van der Werf, Mariët J; Overkamp, Karin M; Muilwijk, Bas; Koek, Maud M; van der Werff-van der Vat, Bianca J C; Jellema, Renger H; Coulier, Leon; Hankemeier, Thomas

    2008-04-01

    Metabolomics is an emerging, powerful, functional genomics technology that involves the comparative non-targeted analysis of the complete set of metabolites in an organism. We have set-up a robust quantitative metabolomics platform that allows the analysis of 'snapshot' metabolomes. In this study, we have applied this platform for the comprehensive analysis of the metabolite composition of Pseudomonas putida S12 grown on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. This paper focuses on the microbial aspects of analyzing comprehensive metabolomes, and demonstrates that metabolomes can be analyzed reliably. The technical (i.e. sample work-up and analytical) reproducibility was on average 10%, while the biological reproducibility was approximately 40%. Moreover, the energy charge values of the microbial samples generated were determined, and indicated that no biotic or abiotic changes had occurred during sample work-up and analysis. In general, the metabolites present and their concentrations were very similar after growth on the different carbon sources. However, specific metabolites showed large differences in concentration, especially the intermediates involved in the degradation of the carbon sources studied. Principal component discriminant analysis was applied to identify metabolites that are specific for, i.e. not necessarily the metabolites that show those largest differences in concentration, cells grown on either of these four carbon sources. For selected enzymatic reactions, i.e. the glucose-6-phosphate isomerase, triosephosphate isomerase and phosphoglyceromutase reactions, the apparent equilibrium constants (K(app)) were calculated. In several instances a carbon source-dependent deviation between the apparent equilibrium constant (K(app)) and the thermodynamic equilibrium constant (K(eq)) was observed, hinting towards a potential point of metabolic regulation or towards bottlenecks in biosynthesis routes. For glucose-6

  2. Trace Metal Sequestration by and Structure of Mn Oxide Produced by Pseudomonas Putida

    NASA Astrophysics Data System (ADS)

    Manceau, A.; Toner, B.; Lanson, B.; Marcus, M. A.; Villalobos, M.; Sposito, G.

    2002-12-01

    The structure of the manganese oxide produced by Pseudomonas putida strain MnB1, and the sorption mechanism of zinc at the surface of the biogenic manganese oxide, were studied by X-ray diffraction and Mn- and Zn-K edge EXAFS spectroscopy. The X-ray diffraction pattern exhibits a broad basal reflection and asymmetrical hk0 bands characteristic of turbostratic birnessite. The average number of layers in birnessite particles and the lateral dimension of the MnO2 layers were estimated from the size of coherent scattering domains in the c* direction and in the ab plane. XRD simulations showed that the relative intensity and shape of the 200 and 020 reflections are sensitive to the amount of interlayer Mn and, therefore, can be used to constrain the composition and structure of the interlayer space. Both XRD and Mn K-edge EXAFS data are consistent with a structural model in which the interlayer space is devoid of manganese, all Mn atoms being located within the birnessite layer. Mn-Mn distances obtained by the two techniques are identical (2.84 \\x8F), and typical of pure Mn4+ manganese layers. The lack of Mn3+ within the manganese layer suggests that the deficit of structural charge from the biogenic birnessite arises from vacant octahedral sites (V). Zn-O and Zn-Mn EXAFS distances are consistent with the formation of tetrahedrally coordinated Zn complex on the face of vacant layer octahedral sites, i.e., with the formation of a tridentate corner-sharing interlayer complexes ([H2O]-IVZn-3Olayer-V-3Mnlayer). This [IV]TC complex was observed at low and high surface coverage, and this result contrasts with that obtained for abiotic hexagonal birnessite (HBi), in which Zn formed a [IV]TC complex at low surface coverage and a [VI]TC complex at higher coverage (Manceau et al., 2002). The structural reasons for this difference will be discussed, and it will be shown that the [IV]TC complex is the main binding form of Zn in natural phyllomanganates. Manceau, A., Lanson, B

  3. Draft Genome Sequence of Pseudomonas putida CBF10-2, a Soil Isolate with Bioremediation Potential in Agricultural and Industrial Environmental Settings

    PubMed Central

    Damania, Ashish

    2016-01-01

    Pseudomonas putida CBF10-2 is a microorganism isolated from farmland soil in Fairchild, TX, found to degrade high-impact xenobiotics, including organophosphate insecticides, petroleum hydrocarbons, and both monocyclic and polycyclic aromatics. The versatility of CBF10-2 makes it useful for multipurpose bioremediation of contaminated sites in agricultural and industrial environments. PMID:27417844

  4. Draft Genome Sequence of Hydrocarbon-Degrading Pseudomonas putida Strain KG-4, Isolated from Soil Samples Collected from Krishna-Godavari Basin in India

    PubMed Central

    Dawar, Chhavi

    2015-01-01

    We report here the 5.58-Mb draft genome of Pseudomonas putida strain KG-4 obtained from the oil fields of the Krishna-Godavari basin, Andhra Pradesh, India. The genome sequence is expected to facilitate identification and understanding of genes associated with hydrocarbon metabolism, which can help in developing strategies for managing oil spills and bioremediation. PMID:26044433

  5. Draft Genome Sequence of Pseudomonas putida CBF10-2, a Soil Isolate with Bioremediation Potential in Agricultural and Industrial Environmental Settings.

    PubMed

    Iyer, Rupa; Damania, Ashish

    2016-01-01

    Pseudomonas putida CBF10-2 is a microorganism isolated from farmland soil in Fairchild, TX, found to degrade high-impact xenobiotics, including organophosphate insecticides, petroleum hydrocarbons, and both monocyclic and polycyclic aromatics. The versatility of CBF10-2 makes it useful for multipurpose bioremediation of contaminated sites in agricultural and industrial environments. PMID:27417844

  6. Pseudomonas aeruginosa forms Biofilms in Acute InfectionIndependent of Cell-to-Cell Signaling

    SciTech Connect

    Schaber, J. Andy; Triffo, W.J.; Suh, Sang J.; Oliver, Jeffrey W.; Hastert, Mary C.; Griswold, John A.; Auer, Manfred; Hamood, Abdul N.; Rumbaugh, Kendra P.

    2006-09-20

    Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 hours of infection in thermally-injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections. P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild type and QS-deficient P. aeruginosa formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independent of QS.

  7. Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon.

    PubMed Central

    Wilson, S A; Wachira, S J; Norman, R A; Pearl, L H; Drew, R E

    1996-01-01

    In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon. A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination. Site-directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5' and 3' ends of the proposed AmiR binding site which are independently essential for antitermination. A T7 promoter/RNA polymerase-driven system shows AmiR-mediated antitermination, demonstrating a lack of promoter/polymerase specificity. Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC-AmiR complex has been identified and isolated. Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC. AmiC-AmiR-anti-inducer is a stable dimer-dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination. Images PMID:8918468

  8. Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa.

    PubMed

    Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas; Häussler, Susanne

    2016-08-01

    Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. PMID:27216077

  9. Electrical conductivity measurements of bacterial nanowires from Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Maruthupandy, Muthusamy; Anand, Muthusamy; Maduraiveeran, Govindhan; Sait Hameedha Beevi, Akbar; Jeeva Priya, Radhakrishnan

    2015-12-01

    The extracellular appendages of bacteria (flagella) that transfer electrons to electrodes are called bacterial nanowires. This study focuses on the isolation and separation of nanowires that are attached via Pseudomonas aeruginosa bacterial culture. The size and roughness of separated nanowires were measured using transmission electron microscopy (TEM) and atomic force microscopy (AFM), respectively. The obtained bacterial nanowires indicated a clear image of bacterial nanowires measuring 16 nm in diameter. The formation of bacterial nanowires was confirmed by microscopic studies (AFM and TEM) and the conductivity nature of bacterial nanowire was investigated by electrochemical techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), which are nondestructive voltammetry techniques, suggest that bacterial nanowires could be the source of electrons—which may be used in various applications, for example, microbial fuel cells, biosensors, organic solar cells, and bioelectronic devices. Routine analysis of electron transfer between bacterial nanowires and the electrode was performed, providing insight into the extracellular electron transfer (EET) to the electrode. CV revealed the catalytic electron transferability of bacterial nanowires and electrodes and showed excellent redox activities. CV and EIS studies showed that bacterial nanowires can charge the surface by producing and storing sufficient electrons, behave as a capacitor, and have features consistent with EET. Finally, electrochemical studies confirmed the development of bacterial nanowires with EET. This study suggests that bacterial nanowires can be used to fabricate biomolecular sensors and nanoelectronic devices.

  10. Functional analysis of the Pseudomonas aeruginosa autoinducer PAI.

    PubMed Central

    Passador, L; Tucker, K D; Guertin, K R; Journet, M P; Kende, A S; Iglewski, B H

    1996-01-01

    A series of structural analogs of the Pseudomonas aeruginosa autoinducer [PAI, N-3-oxo-dodecanoyl homoserine lactone] were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR. The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity. Replacement of the ring O by S in the homoserine lactone moiety can be tolerated. Tritium-labelled PAI ([3H]PAI) was synthesized and used to demonstrate the association of [3H]PAI with cells overexpressing LasR. The PAI analogs were also tested for their ability to compete with [3H]PAI for binding of LasR. Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity. The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of [3H]PAI. All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI. PMID:8830697

  11. 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

  12. Prevention of Pseudomonas aeruginosa adhesion by electric currents.

    PubMed

    Shim, Soojin; Hong, Seok Hoon; Tak, Yongsug; Yoon, Jeyong

    2011-02-01

    The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (∼81% at 15.0 μA cm(-2)). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm(-2)), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.

  13. 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

  14. Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa.

    PubMed

    Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas; Häussler, Susanne

    2016-08-01

    Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution.

  15. Uranyl Precipitation by Pseudomonas aeruginosa via Controlled Polyphosphate Metabolism

    PubMed Central

    Renninger, Neil; Knopp, Roger; Nitsche, Heino; Clark, Douglas S.; Keasling, Jay D.

    2004-01-01

    The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions. PMID:15574942

  16. 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

  17. Global transcriptional responses to triclosan exposure in Pseudomonas aeruginosa.

    PubMed

    Chuanchuen, Rungtip; Schweizer, Herbert P

    2012-08-01

    Global gene transcription was assessed by microarray experiments following treatment of a triclosan-susceptible Δ(mexAB-oprM) Pseudomonas aeruginosa strain with subinhibitory concentrations of triclosan. Expression patterns of selected genes were verified by quantitative real-time PCR analysis. The results showed that triclosan exposure had a profound effect on gene expression, affecting 44% of the genes present on the Affymetrix GeneChip(®), with 28% of genes being significantly upregulated and 16% being significantly downregulated in triclosan-treated cells. Genes encoding membrane proteins, transporters of small molecules, aspects of amino acid metabolism, and transcriptional regulators were significantly over-represented among the more strongly upregulated or downregulated genes in triclosan-treated cells. Quorum sensing-regulated genes were among the most strongly downregulated genes, presumably because of decreased acyl-acyl carrier protein pools and the resulting reduced acyl-homoserine lactone molecule synthesis. Surprisingly, iron homeostasis was completed perturbed in triclosan-exposed cells, with iron acquisition systems being strongly downregulated and iron storage systems significantly upregulated, thus mimicking conditions of excess iron. The profound perturbations of cellular metabolism via specific and global mechanisms may explain why triclosan is such a potent antimicrobial in susceptible bacteria.

  18. Heritability of Respiratory Infection with Pseudomonas aeruginosa in Cystic Fibrosis

    PubMed Central

    Green, Deanna M.; Collaco, J. Michael; McDougal, Kathryn E.; Naughton, Kathleen M.; Blackman, Scott M.; Cutting, Garry R.

    2013-01-01

    Objective To quantify the relative contribution of factors other than cystic fibrosis transmembrane conductance regulator genotype and environment on the acquisition of Pseudomonas aeruginosa (Pa) by patients with cystic fibrosis. Study design Lung infection with Pa and mucoid Pa was assessed using a co-twin study design of 44 monozygous (MZ) and 17 dizygous (DZ) twin pairs. Two definitions were used to establish infection: first positive culture and persistent positive culture. Genetic contribution to infection (ie, heritability) was estimated based on concordance analysis, logistic regression, and age at onset of infection through comparison of intraclass correlation coefficients. Results Concordance for persistent Pa infection was higher in MZ (0.83; 25 of 30 pairs) than DZ twins (0.45; 5 of 11 pairs), generating a heritability of 0.76. Logistic regression adjusted for age corroborated genetic control of persistent Pa infection. The correlation for age at persistent Pa infection was higher in MZ twins (0.589; 95% CI, 0.222-0.704) than in DZ twins (0.162; 95% CI, −0.352 to 0.607), generating a heritability of 0.85. Conclusion Genetic modifiers play a significant role in the establishment and timing of persistent Pa infection in individuals with cystic fibrosis. PMID:22364820

  19. Classification of Pseudomonas aeruginosa O antigens by immunoelectrophoresis.

    PubMed

    Lányi, B; Adám, M M; Szentmihályi, A

    1975-05-01

    Heated saline extracts of 89 strains, and (1) supernates of phenol-water extracts (L1 fractions), (2) purified lipopolysaccharide, (3) trichloracetic-acid (TCA) extracts, and (4) sodium-hydroxide extracts of 23 strains representing all Pseudomonas aeruginosa O antigens were subjected electrophoresis. Precipitation lines obtained with homologous and heterologous antisera were evaluated by electrodensitometric measurement. The characteristics of the immunoelectrophoretic groups established were as follows. Group I: two lines running at different rates towards the anode; three subgroups on the basis of the behaviour of alkali-treated antigens. Group II: triple line at the starting well, alkali sensitive. Group III: triple line at the starting well, alkali resistant; two subgroups according to reactivity or non-reactivity of L1 fractions. Group IV: triple line on the cathode side, alkali resistant, L1 fraction non-reactive. Group V: single line on the anode side, alkali sensitive, L1 fraction and TCA extract non-reactive. O antigens identified by agglutination corresponded closely with the immunoelectrophoretic pattern: strains with identical O antigens or sharing major somatic components fell, with one exception, into the same immunoelectrophoretic group. PMID:806687

  20. [Cervical lymphoadenopathy due to Pseudomonas aeruginosa following mesotherapy].

    PubMed

    Shaladi, Ali Muftah; Crestani, Francesco; Bocchi, Anna; Saltari, Maria Rita; Piva, Bruno; Tartari, Stefano

    2009-09-01

    Mesotherapy is a treatment method devised for controlling several diseases by means of subcutaneous microinjections given at or around the affected areas at short time intervals. It is used to treat a variety of medical conditions, amongst which all orthopaedic diseases and rheumatic pain. Mesotherapy is especially indicated for neck pain. The mechanism of action is twofold: a pharmacological effect due to the drug administered, and a reflexogenic effect, the skin containing many nerve endings that are sensitive to the mechanical action of the needle. Although this therapy is safe, like any other medical intervention it cannot be considered free of complications that may occur, such as allergies, haematomas, bruising, wheals, granulomas and telangiectasias. Infective complications are also possible, due to pathogenic bacteria that are inoculated through contamination of products, of the materials used for the procedure or even from germs on the skin. We present the case of a patient who had cervical lymphadenopathy due to Pseudomonas aeruginosa after mesotherapy treatment for neck pain. PMID:19838089

  1. Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis.

    PubMed

    Sheu, Der-Shyan; Lee, Chia-Yin

    2004-07-01

    The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Pp), class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA- as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB(-)4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB(-)4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB(-)4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum. PMID:15205419

  2. Pseudomonas aeruginosa facilitates Campylobacter jejuni growth in biofilms under oxic flow conditions.

    PubMed

    Culotti, Alessandro; Packman, Aaron I

    2015-12-01

    We investigated the growth of Campylobacter jejuni in biofilms with Pseudomonas aeruginosa under oxic flow conditions. We observed the growth of C. jejuni in mono-culture, deposited on pre-established P. aeruginosa biofilms, and co-inoculated with P. aeruginosa. In mono-culture, C. jejuni was unable to form biofilms. However, deposited C. jejuni continuously grew on pre-established P. aeruginosa biofilms for a period of 3 days. The growth of scattered C. jejuni clusters was strictly limited to the P. aeruginosa biofilm surface, and no intergrowth was observed. Co-culturing of C. jejuni and P. aeruginosa also enabled the growth of both organisms in biofilms, with C. jejuni clusters developing on the surface of the P. aeruginosa biofilm. Dissolved oxygen (DO) measurements in the medium showed that P. aeruginosa biofilms depleted the effluent DO from 9.0 to 0.5 mg L(-1) 24 hours after inoculation. The localized microaerophilic environment generated by P. aeruginosa promoted the persistence and growth of C. jejuni. Our findings show that P. aeruginosa not only prolongs the survival of C. jejuni under oxic conditions, but also enables the growth of C. jejuni on the surface of P. aeruginosa biofilms.

  3. Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida

    PubMed Central

    2014-01-01

    Background Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation. Results A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms. Conclusion Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis. PMID:24948031

  4. Contrasting colonization and plant growth promoting capacity between wild type and gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

    SciTech Connect

    Weyens N.; van der Lelie D.; Boulet, J.; Adriaensen, D.; Timmermans, J.-P.; Prinsen, E.; Van Oevelen, S.; D"Haen, J.; Smeets, K.; Taghavi, S.; Vangronsveld, J.

    2011-06-09

    This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

  5. Global Genome Comparative Analysis Reveals Insights of Resistome and Life-Style Adaptation of Pseudomonas putida Strain T2-2 in Oral Cavity

    PubMed Central

    How, Kah Yan; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Most Pseudomonas putida strains are environmental microorganisms exhibiting a wide range of metabolic capability but certain strains have been reported as rare opportunistic pathogens and some emerged as multidrug resistant P. putida. This study aimed to assess the drug resistance profile of, via whole genome analysis, P. putida strain T2-2 isolated from oral cavity. At the same time, we also compared the nonenvironmental strain with environmentally isolated P. putida. In silico comparative genome analysis with available reference strains of P. putida shows that T2-2 has lesser gene counts on carbohydrate and aromatic compounds metabolisms, which suggested its little versatility. The detection of its edd gene also suggested T2-2's catabolism of glucose via ED pathway instead of EMP pathway. On the other hand, its drug resistance profile was observed via in silico gene prediction and most of the genes found were in agreement with drug-susceptibility testing in laboratory by automated VITEK 2. In addition, the finding of putative genes of multidrug resistance efflux pump and ATP-binding cassette transporters in this strain suggests a multidrug resistant phenotype. In summary, it is believed that multiple metabolic characteristics and drug resistance in P. putida strain T2-2 helped in its survival in human oral cavity. PMID:25436236

  6. 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.

  7. A risk assessment of Pseudomonas aeruginosa in swimming pools: a review.

    PubMed

    Rice, Scott A; van den Akker, Ben; Pomati, Francesco; Roser, David

    2012-06-01

    Despite routine monitoring and disinfection, treated swimming pools are frequently contaminated with the opportunistic pathogen Pseudomonas aeruginosa, which can represent a significant public health threat. This review was undertaken to identify the current understanding of risk factors associated with pool operation with respect to P. aeruginosa. The ecology and factors that promote growth of P. aeruginosa in the pool environment are complex and dynamic and so we applied a systematic risk assessment approach to integrate existing data, with the aim to improve pool management and safety. Sources of P. aeruginosa, types of infections, dose responses, routes of transmission, as well as the efficacy of current disinfectant treatments were reviewed. This review also highlights the critical knowledge gaps that are required for a more robust, quantitative risk assessment of P. aeruginosa. Quantitative risk management strategies have been successfully applied to drinking water systems and should similarly be amenable to developing a better understanding of the risk posed by P. aeruginosa in swimming pools.

  8. High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply.

    PubMed

    Davis, Reeta; Duane, Gearoid; Kenny, Shane T; Cerrone, Federico; Guzik, Maciej W; Babu, Ramesh P; Casey, Eoin; O'Connor, Kevin E

    2015-04-01

    High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L(-1) h(-1) ) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L(-1) was achieved in 33 h with a biomass productivity of 3.1 g L(-1) h(-1) which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L(-1) h(-1) ) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA(-1) .

  9. Genome-Scale Reconstruction and Analysis of the Pseudomonas putida KT2440 Metabolic Network Facilitates Applications in Biotechnology

    PubMed Central

    Godinho, Miguel; Bielecka, Agata; Regenhardt, Daniela; Timmis, Kenneth N.

    2008-01-01

    A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, 13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype–phenotype relationships and provides a sound framework to explore this versatile bacterium and to

  10. Gibberellin secreting rhizobacterium, Pseudomonas putida H-2-3 modulates the hormonal and stress physiology of soybean to improve the plant growth under saline and drought conditions.

    PubMed

    Kang, Sang-Mo; Radhakrishnan, Ramalingam; Khan, Abdul Latif; Kim, Min-Ji; Park, Jae-Man; Kim, Bo-Ra; Shin, Dong-Hyun; Lee, In-Jung

    2014-11-01

    The physiological changes in tolerant soybean plants under salt and drought stress conditions with Pseudomonas putida H-2-3 were investigated. A bacterial isolate H-2-3 was isolated from soil and identified as Pseudomonas putida H-2-3 by 16S rDNA sequences. The treatment of P. putida H-2-3 significantly increased the length, fresh and dry weight of shoot and chlorophyll content in gibberellins (GAs) deficient mutant Waito-c rice seedlings over the control, it might be the presence of GA1, GA4, GA9 and GA20. The soybean plant growth was retarded in salt (120 mM sodium chloride) and drought (15% polyethylene glycol) stress conditions at 10 days treatments, while P. putida H-2-3 effectively enhanced the shoot length and fresh weight of plants suffered at salt and drought stress. The chlorophyll content was lower in abiotic stress conditions and bacterial inoculant P. putida H-2-3 mitigated the stress effects by an evidence of higher quantity of chlorophyll content in plants exposed to salt and drought. The stress hormonal analysis revealed that individual treatment of P. putida H-2-3, salt and drought significantly enhanced the abscisic acid and salicylic acid content than their control. P. putida H-2-3 applied to salt and drought stressed plants showed a lower level of abscisic acid and salicylic acid and a higher level of jasmonic acid content. Under stress condition induced by salt and drought in plants expressed higher level of total polyphenol, superoxide dismutase and radical scavenging activity and no significant changes in flavonoids. The bio-inoculant, P. putida H-2-3 modulated those antioxidants by declining superoxide dismutase, flavonoids and radical scavenging activity. P. putida H-2-3 induced tolerance against abiotic stress was confirmed by a reduction of Na content in abiotic stressed plants. The results suggest that P. putida H-2-3 application reprograms the chlorophyll, stress hormones and antioxidants expression in abiotic stress affected

  11. Nanoscale Adhesion Forces of Pseudomonas aeruginosa Type IV Pili

    PubMed Central

    2015-01-01

    A variety of bacterial pathogens use nanoscale protein fibers called type IV pili to mediate cell adhesion, a primary step leading to infection. Currently, how these nanofibers respond to mechanical stimuli and how this response is used to control adhesion is poorly understood. Here, we use atomic force microscopy techniques to quantify the forces guiding the adhesion of Pseudomonas aeruginosa type IV pili to surfaces. Using chemical force microscopy and single-cell force spectroscopy, we show that pili strongly bind to hydrophobic surfaces in a time-dependent manner, while they weakly bind to hydrophilic surfaces. Individual nanofibers are capable of withstanding forces up to 250 pN, thereby explaining how they can resist mechanical stress. Pulling on individual pili yields constant force plateaus, presumably reflecting conformational changes, as well as nanospring properties that may help bacteria to withstand physiological shear forces. Analysis of mutant strains demonstrates that these mechanical responses originate solely from type IV pili, while flagella and the cell surface localized and proposed pili-associated adhesin PilY1 play no direct role. We also demonstrate that bacterial–host interactions involve constant force plateaus, the extension of bacterial pili, and the formation of membrane tethers from host cells. We postulate that the unique mechanical responses of type IV pili unravelled here enable the bacteria to firmly attach to biotic and abiotic surfaces and thus maintain attachment when subjected to high shear forces under physiological conditions, helping to explain why pili play a critical role in colonization of the host. PMID:25286300

  12. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    PubMed

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga.

  13. The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa

    PubMed Central

    Nadal Jimenez, Pol; Koch, Gudrun; Thompson, Jessica A.; Xavier, Karina B.; Cool, Robbert H.

    2012-01-01

    Summary: Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce