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P S Phale - One of the best experts on this subject based on the ideXlab platform.
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bypassing isophthalate inhibition by modulating glutamate dehydrogenase gdh purification and kinetic characterization of nadp gdhs from isophthalate degrading pseudomonas aeruginosa strain pp4 and Acinetobacter Lwoffii strain isp4
Journal of Bacteriology, 2010Co-Authors: C Vamseekrishna, P S PhaleAbstract:Pseudomonas aeruginosa strain PP4 and Acinetobacter Lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDHI was produced when cells were grown on isophthalate, while GDHII was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDHP, when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDHI and GDHII were found to be homotetramers, while GDHP was found to be a homohexamer. GDHII was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDHI. Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDHI and GDHII, in strain PP4. In strain ISP4, irrespective of the carbon source, the GDHP produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDHP from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDHP from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDHP in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
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carbon source dependent modulation of nadp glutamate dehydrogenases in isophthalate degrading pseudomonas aeruginosa strain pp4 pseudomonas strain ppd and Acinetobacter Lwoffii strain isp4
Microbiology, 2008Co-Authors: C Vamseekrishna, P S PhaleAbstract:Acinetobacter Lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or α-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADP-dependent GDH (NADP-GDH), while cells grown on glucose, 2× yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon source-dependent presence of two (GDHI and GDHII), three (GDHA, GDHB and GDHC) and one (GDHP) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.
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biodegradation of phthalate isomers by pseudomonas aeruginosa pp4 pseudomonas sp ppd and Acinetobacter Lwoffii isp4
Applied Microbiology and Biotechnology, 2006Co-Authors: C Vamseekrishna, Y Mohan, P S PhaleAbstract:Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter Lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.
David L Gutnick - One of the best experts on this subject based on the ideXlab platform.
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involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil degrading strain Acinetobacter Lwoffii rag 1
Journal of Bacteriology, 2003Co-Authors: David Nakar, David L GutnickAbstract:The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter Lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.
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analysis of the wee gene cluster responsible for the biosynthesis of the polymeric bioemulsifier from the oil degrading strain Acinetobacter Lwoffii rag 1
Microbiology, 2001Co-Authors: David Nakar, David L GutnickAbstract:A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading Acinetobacter Lwoffii RAG-1 was isolated and characterized. The complete sequence of this cluster, termed wee, consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5–14% of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or ops sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.
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esterase from the oil degrading Acinetobacter Lwoffii rag 1 sequence analysis and over expression in escherichia coli
Fems Microbiology Letters, 1993Co-Authors: R Alon, David L GutnickAbstract:The est gene encoding an esterase from Acinetobacter Lwoffii RAG-1 was cloned into E. coli under the control of the PL promoter of the phage λ. The N-terminal sequence of the first 20 amino acids of the heterologous expressed esterase corresponded to that obtained from the nucleotide sequence. Antibodies prepared against the over-expressed recombinant esterase in E.coli were used to locate the enzyme primarily in the membrane fractions of A. LwoffiiRAG-1. Comparison with homologous proteins from both eukaryotic and prokaryotic organisms suggest that the RAG-1 esterase exhibits sequence motifs characteristic of both serine proteases and of lipases.
Seung Il Kim - One of the best experts on this subject based on the ideXlab platform.
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Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24
2016Co-Authors: Sang-yeop Lee, Sung Ho Yun, Young Ho Chung, Gun-hwa Kim, Chi-won Choi, Jonghyun Kim, Edmond Changkyun Park, Seung Il KimAbstract:Burkholderia sp. K24, formerly known as Acinetobacter Lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.
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characterization of a new catechol branch of the β ketoadipate pathway induced for benzoate degradation in Acinetobacter Lwoffii k24
Biochemical and Biophysical Research Communications, 2007Co-Authors: Youngho Yoon, Sun Hee Leem, Sung Ho Yun, Soonho Park, Soyoung Seol, Seung Il KimAbstract:Abstract Acinetobacter Lwoffii K24 is a known aniline-degrading bacterium. In previous studies, two catechol branches of the β-ketoadipate pathway were reported to be induced for aniline degradation, and related enzymes (CatA 1 and CatA 2 ) were identified from the aniline-induced proteome of A. Lwoffii K24 [S.I. Kim, S.H. Leem, J.S. Choi, Y.H. Chung, S. Kim, Y.M. Park, Y.K. Park, Y.N. Lee, K.S. Ha, Cloning and characterization of two catA genes in Acinetobacter Lwoffii K24, J. Bacteriol. 179 (1997) 5226–5231; and E.A. Kim, J.Y. Kim, S.J. Kim, K.R. Park, H.J. Chung, S.H. Leem, S.I. Kim, Proteomic analysis of Acinetobacter Lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry, J. Microbiol. Methods 57 (2004) 337-349]. A. Lwoffii K24 has also been found to utilize other aromatic compounds such as p -hydroxybenzoate, salicylate, and benzoate. In this study, we performed a comparative 2-DE/MS analysis of a benzoate-induced proteome and found that a new catechol 1,2-dioxygenase (CatA 3 ) and benzoate 1,2-dioxygenase were up-regulated as the primary dioxygenases responsible for benzoate degradation in A. Lwoffii K24. However, CatA 1 and CatA 2 were not detected on the same 2D gel as CatA 3 . Transcription analysis of three catA genes from A. Lwoffii K24 showed that these cat genes were specifically expressed under certain growth conditions using different aromatic compounds as the carbon source. While catA 1 and catA 2 were expressed under the aniline culture condition, catA 3 was expressed under the benzoate culture condition. A new cat gene cluster ( catB 3 C 3 A 3 F 3 ) was cloned and found to share sequence homology and a similar gene structure with the cat genes of Acinetobacter radioresistens . This result suggests that the third catechol branch ( cat 3 ) of the β-ketoadipate pathway was selectively induced for the degradation of benzoate in A. Lwoffii K24. It also provides evidence of multiple catechol branches in the β-ketoadipate pathway and the independent regulation of monocyclic aromatic compound degradation in A. Lwoffii K24.
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proteomic analysis of Acinetobacter Lwoffii k24 by 2 d gel electrophoresis and electrospray ionization quadrupole time of flight mass spectrometry
Journal of Microbiological Methods, 2004Co-Authors: Euna Kim, Soo Jung Kim, Sun Hee Leem, Kyeong Ryang Park, Jinyoung Kim, Haejin Chung, Seung Il KimAbstract:The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter Lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. Lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. Lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. Lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. Lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria.
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enhanced detection and characterization of protocatechuate 3 4 dioxygenase in Acinetobacter Lwoffii k24 by proteomics using a column separation
Biochemical and Biophysical Research Communications, 2002Co-Authors: Hyung Yeel Kahng, Kun Cho, Seung Yual Song, Soo Jung Kim, Sun Hee Leem, Seung Il KimAbstract:Acinetobacter Lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. Lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. Lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.
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proteome analysis of aniline induced proteins in Acinetobacter Lwoffii k24
Current Microbiology, 2002Co-Authors: Seung Il Kim, Soo Jung Kim, Myung Hee Nam, Soo Hyun Kim, Jongshin Yoo, Young Mok ParkAbstract:Acinetobacter Lwoffii K24 is a soil bacterium that can use aniline as a sole carbon and nitrogen source (by β-ketoadipate pathway genes (cat genes)) and has two copies of catABC gene separately located on the chromosome. In order to identify aniline-induced proteins, two-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of A. Lwoffii K24 cultured in aniline and succinate media. In the range of pH3–10, more than 370 spots were detected on the silver stained gels. Interestingly, more than 20 spots were selectively induced on aniline-cultured bacteria. Twenty-three protein spots of A. Lwoffii K24 were analyzed by N-terminal microsequencing and internal microsequencing with in-gel digestion. Of 20 aniline induced protein spots, we identified six β-ketoadipate pathway genes, one subunit of amino group transfer (putative subunit of aniline oxygenase), malate dehydrogenase, putative ABC transporter, putative hydrolase, HHDD isomerase, and five unknown proteins. Especially in case of two catechol 1,2-dioxygenases (CDI1 and CDI2), more than three isotypes were detected on the 2D gel. This study showed that the proteome analysis of A. Lwoffii K24 may be helpful for identification of genes induced by aniline and understanding of their function in the cell.
Otto Holst - One of the best experts on this subject based on the ideXlab platform.
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the allergy protective properties of Acinetobacter Lwoffii f78 are imparted by its lipopolysaccharide
Allergy, 2009Co-Authors: Jennifer Debarry, Anna Hanuszkiewicz, Otto Holst, Karina Stein, Holger HeineAbstract:To cite this article: Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter Lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010; 65: 690–697. Abstract Background: An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life (‘hygiene hypothesis’). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter Lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection. Methods: The impact of A. Lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their THelper cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments. Results: It was shown that A. Lwoffii F78 induced a TH1-polarizing program in human dendritic cells which led to TH1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. Lwoffii F78 was the responsible molecule promoting these effects. Conclusion: We found evidence that the allergy-protecting effects of A. Lwoffii F78 are because of the activation of a TH1-polarizing program in human dendritic cells, and that the LPS of A. Lwoffii F78 is responsible for these beneficial effects.
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structural analysis of the capsular polysaccharide from Acinetobacter Lwoffii f78
European Journal of Organic Chemistry, 2008Co-Authors: Anna Hanuszkiewicz, Zbigniew Kaczynski, Buko Lindner, Torsten Goldmann, Ekkehard Vollmer, Jennifer Debarry, Holger Heine, Otto HolstAbstract:The capsular polysaccharide from Acinetobacter Lwoffii F78 was isolated and purified, and its structure was elucidated by chemical analyses, NMR spectroscopy, and mass spectrometry. The presence of a capsule on this bacterium was confirmed by transmission electron microscopy experiments, and the utilization of the antilipid A monoclonal antibody proved the nonendotoxin origin of the isolated material. The structure represents a novel nonbranched aminopolysaccharide with high heterogeneity of amino groups substituents: →3)-α-L-FucNAc-(1→3)-β-D-QuiNR14NR2-(1→4)-β-L-GlcNR33NR4A-(1→, where R1 = 3-hydroxybutyric acid (3-HBA) or N-acetylated alanine (AlaNAc), R2 = AlaNAc or 3-HBA, R3 = acetyl or 3-HBA, and R4 = 3-HBA or acetyl.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
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structural and immunochemical analysis of the lipopolysaccharide from Acinetobacter Lwoffii f78 located outside chlamydiaceae with a chlamydia specific lipopolysaccharide epitope
Chemistry: A European Journal, 2008Co-Authors: Anna Hanuszkiewicz, Buko Lindner, Jennifer Debarry, Holger Heine, Goran Hubner, Evgeny Vinogradov, Lore Brade, Helmut Brade, Otto HolstAbstract:Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter Lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. Lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.
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Acinetobacter Lwoffii and lactococcus lactis strains isolated from farm cowsheds possess strong allergy protective properties
The Journal of Allergy and Clinical Immunology, 2007Co-Authors: Jennifer Debarry, Nicole Blümer, Anna Hanuszkiewicz, Harald Renz, Holger Garn, Nina Dickgreber, Erika Von Mutius, Albrecht Bufe, Soren Gatermann, Otto HolstAbstract:Background Children who grow up in a farming environment show lower levels of atopic sensitization, hay fever, and asthma than children of the same age not living in such an environment. A number of investigations provided good evidence that this is due to an early-life contact with cowsheds, farm animals, and/or consumption of products like raw milk. Also, it had been indicated that microorganisms might have an important effect on the development of allergies, and thus the question arose of which farm microbial organisms, their products, or both might induce or influence allergy-protective mechanisms. Objective We sought to gain further insight into the potential allergy-protective properties of microbes isolated from the farming environment. Methods Of a number of bacterial species identified in cowsheds of farms, 2 were selected, isolated, and characterized, namely Acinetobacter Lwoffii F78 and Lactococcus lactis G121. The isolates were investigated with regard to their activation of pattern-recognition receptors, the maturation of human monocyte-derived dendritic cells, the upregulation of inflammatory cytokines, the T H 1-polarizing Notch ligand expression, and their influence on the allergic phenotype. Results It is shown that both bacterial isolates were able to reduce allergic reactions in mice, to activate mammalian cells in vitro , and to induce a T H 1-polarizing program in dendritic cells. Conclusion Our data strongly support the hygiene hypothesis, which states that an environment rich in microbiologic structures, such as a farming environment, might protect against the development of allergies. Clinical implications This work provides the first data on a potential application of cowshed bacteria in allergy protection.
C Vamseekrishna - One of the best experts on this subject based on the ideXlab platform.
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bypassing isophthalate inhibition by modulating glutamate dehydrogenase gdh purification and kinetic characterization of nadp gdhs from isophthalate degrading pseudomonas aeruginosa strain pp4 and Acinetobacter Lwoffii strain isp4
Journal of Bacteriology, 2010Co-Authors: C Vamseekrishna, P S PhaleAbstract:Pseudomonas aeruginosa strain PP4 and Acinetobacter Lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDHI was produced when cells were grown on isophthalate, while GDHII was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDHP, when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDHI and GDHII were found to be homotetramers, while GDHP was found to be a homohexamer. GDHII was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDHI. Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDHI and GDHII, in strain PP4. In strain ISP4, irrespective of the carbon source, the GDHP produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDHP from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDHP from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDHP in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.
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carbon source dependent modulation of nadp glutamate dehydrogenases in isophthalate degrading pseudomonas aeruginosa strain pp4 pseudomonas strain ppd and Acinetobacter Lwoffii strain isp4
Microbiology, 2008Co-Authors: C Vamseekrishna, P S PhaleAbstract:Acinetobacter Lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or α-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADP-dependent GDH (NADP-GDH), while cells grown on glucose, 2× yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon source-dependent presence of two (GDHI and GDHII), three (GDHA, GDHB and GDHC) and one (GDHP) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.
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biodegradation of phthalate isomers by pseudomonas aeruginosa pp4 pseudomonas sp ppd and Acinetobacter Lwoffii isp4
Applied Microbiology and Biotechnology, 2006Co-Authors: C Vamseekrishna, Y Mohan, P S PhaleAbstract:Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter Lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.
