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  • 1
    Language: English
    In: The Journal of biological chemistry, 18 February 2011, Vol.286(7), pp.5151-6
    Description: Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.
    Keywords: Ubiquitination ; Unfolded Protein Response ; Endoplasmic Reticulum -- Metabolism ; Membrane Proteins -- Metabolism ; Ubiquitin-Protein Ligases -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 2
    Language: English
    In: Journal of Applied Physics, 01 March 2012, Vol.111(5)
    Description: Starting from a random or ordered distribution of 0.8%, 1.6%, 3.7%, and 12.5% dopants over the lattice sites of a simple cubic grid, we estimate the fraction of unclustered dopants after pulsed laser processing of different host:dopant systems. Initial clustering events are simulated with a greedy algorithm implemented in a Monte Carlo study. The greedy algorithm gives adequate results for dopants with low diffusivity and low solubility. The absolute initial dopant concentration and declustering strongly influence the kinetics of clustering. Particularly, we consider transition metal doped Si and GaAs after pulsed laser annealing, which are of interest for spintronics applications. An uncritical integral diffusion of Mn in GaAs:Mn and a tendency of Mn to form silicides in Si:Mn are simulated. These results are in good agreement with experimental observations.
    Keywords: Articles
    ISSN: 0021-8979
    E-ISSN: 1089-7550
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  • 3
    Language: English
    In: PLoS ONE, 2011, Vol.6(3), p.e17583
    Description: Genome characterization of the model PCB-degrading bacterium Burkholderia xenovorans LB400 revealed the presence of eleven central pathways for aromatic compounds degradation, among them, the homogentisate and the homoprotocatechuate pathways. However, the functionality of these central pathways in strain LB400 has not been assessed and related peripheral pathways has not been described. ; The aims of this study were to determine the functionality of the homogentisate and homoprotocatechuate central pathways in LB400 and to establish their role in 3-hydroxyphenylacetate (3-HPA) and 4-hydroxyphenylacetate (4-HPA) catabolism. Strain LB400 was able to grow using 3-HPA and 4-HPA as sole carbon source. A genomic search in LB400 suggested the presence of and genes clusters encoding proteins of the 3-hydroxyphenylacetate and 4-hydroxyphenylacetate peripheral pathways. LB400 cells grown with 3-HPA and 4-HPA degraded homogentisate and homoprotocatechuate and showed homogentisate 1,2-dioxygenase and homoprotocatechuate 2,3-dioxygenase activities. Transcriptional analyses by RT-PCR showed the expression of two chromosomally-encoded homogentisate dioxygenases (BxeA2725 and BxeA3900) and the gene encoding the homoprotocatechuate 2,3-dioxygenase during 3-HPA and 4-HPA degradation. The proteome analyses by two-dimensional polyacrilamide gel electrophoresis of LB400 grown in 3-HPA and 4-HPA showed the induction of fumarylacetoacetate hydrolase HmgB (BxeA3899). ; This study revealed that strain LB400 used both homogentisate and homoprotocatechuate ring-cleavage pathways for 3- hydroxyphenylacetate and 4-hydroxyphenylacetate catabolism and that these four catabolic routes are functional, confirming the metabolic versatility of LB400.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology ; Biotechnology ; Biochemistry
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: Applied and Environmental Microbiology, Feb 1, 2016, Vol.82(3), pp.888-896
    Description: The article describes the characterization of the short-lapse dynamics of soil microbial communities in response to hydrocarbon pollution and different bioremediation treatments. To this end, replicate diesel-spiked soil microcosms were inoculated with either a defined bacterial consortium or a hydrocarbonoclastic bacterial enrichment and incubated for 12 weeks, and Illumina 16S rRNA gene sequencing was used to follow the microbial community dynamics. Both the bacterial consortium and enrichment were found to enhance hydrocarbon degradation in diesel-polluted soils; moreover, all diesel-polluted soils showed a pronounced and rapid bloom of a native gammaproteobacterium.
    Keywords: Soil Microbiology – Research ; Bioremediation – Usage ; Hydrocarbons – Contamination
    ISSN: 0099-2240
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: PLoS ONE, 2011, Vol.6(3), p.e17555
    Description: Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds. ; To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel , genes and additional other genes was introduced into the bacterium by biparental mating. The transconjugant strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg, 0.12 and CHHg, 0.08. The addition of Hg (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h. ; A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.
    Keywords: Research Article ; Biology ; Microbiology ; Biotechnology
    E-ISSN: 1932-6203
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  • 6
    In: PLoS ONE, 2013, Vol.8(2)
    Description: In this study, the gentisate and protocatechuate pathways in Burkholderia xenovorans LB400 were analyzed by genomic and functional approaches, and their role in 3-hydroxybenzoate (3-HBA) and 4-hydroxybenzoate (4-HBA) degradation was proposed. The LB400 genome possesses two identical mhbRTDHI gene clusters encoding the gentisate pathway and one mhbM gene encoding a 3-HBA 6-hydroxylase that converts 3-HBA into gentisate. The pca genes encoding the protocatechuate pathway and the pobA gene encoding the 4-HBA 3-monooxygenase that oxidizes 4-HBA into protocatechuate are arranged in gene clusters and single genes mainly at the minor chromosome, but also at the major chromosome and the megaplasmid. Strain LB400 was able to grow on gentisate, protocatechuate, 3-HBA and 4-HBA. Transcriptional analyses showed that the mhbD gene encoding the gentisate 1,2-dioxygenase was expressed during growth on 3-HBA, 4-HBA and gentisate, whereas the pcaG gene encoding the protocatechuate 3,4-dioxygenase was expressed only during growth on 4-HBA and protocatechuate. The mhbM gene encoding the 3-HBA 6-hydroxylase was transcribed in strain LB400 during growth on HBAs, gentisate, protocatechuate and glucose. The pobA gene encoding the 4-HBA 3-monooxygenase was expressed during growth on HBAs and glucose. 3-HBA- and 4-HBA-grown LB400 cells showed gentisate 1,2-dioxygenase activity, whereas protocatechuate 3,4-dioxygenase activity was observed only in 4-HBA-grown cells. The mhbR gene encoding a MarR-type transcriptional regulator that probably regulates the expression of the MhbT transporter, and the pcaQ and pcaR genes encoding LysR-type transcriptional regulators that regulate pcaHG and pcaIJBDC genes, respectively, were transcribed during growth on both HBAs, gentisate, protocatechuate and glucose, suggesting a basal constitutive expression. The results indicate active gentisate, protocatechuate, 3-HBA and 4-HBA catabolic pathways in B. xenovorans LB400 and suggest that 3-HBA is channeled exclusively through the gentisate route, whereas 4-HBA is funneled into the protocatechuate central pathway and potentially into the gentisate pathway.
    Keywords: Research Article ; Biology
    E-ISSN: 1932-6203
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  • 7
    In: PLoS ONE, 2017, Vol.12(1)
    Description: p -Cymene is an aromatic terpene that is present in diverse plant species. The aims of this study were to study the p -cymene metabolism in the model aromatic-degrading bacterium Burkholderia xenovorans LB400, and its response to p -cymene. The catabolic p -cymene ( cym ) and p -cumate (cmt) genes are clustered on the LB400 major chromosome. B . xenovorans LB400 was able to grow on p -cymene as well as on p -cumate as a sole carbon and energy sources. LB400 growth attained higher cell concentration at stationary phase on p -cumate than on p -cymene. The transcription of the key cymAb and cmtAb genes, and p -cumate dioxygenase activity were observed in LB400 cells grown on p -cymene and on p -cumate, but not in glucose-grown cells. Diverse changes on LB400 proteome were observed in p -cymene-grown cells compared to glucose-grown cells. An increase of the molecular chaperones DnaK, GroEL and ClpB, the organic hydroperoxide resistance protein Ohr, the alkyl hydroperoxide reductase AhpC and the copper oxidase CopA during growth on p -cymene strongly suggests that the exposure to p -cymene constitutes a stress condition for strain LB400. Diverse proteins of the energy metabolism such as enolase, pyruvate kinase, aconitase AcnA, succinyl-CoA synthetase beta subunit and ATP synthase beta subunit were induced by p -cymene. Electron microscopy showed that p -cymene-grown cells exhibited fuzzy outer and inner membranes and an increased periplasm. p -Cymene induced diverse membrane and transport proteins including the p -cymene transporter CymD. Biofilm formation was reduced during growth in p -cymene in strain LB400 compared to glucose-grown cells that may be associated with a decrease of diguanylate cyclase protein levels. Overall, these results indicate active p -cymene and p -cumate catabolic pathways in B . xenovorans LB400. In addition, this study showed that p -cymene activated a stress response in strain LB400 and reduced its biofilm formation.
    Keywords: Research Article ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Physical Sciences ; Physical Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences
    E-ISSN: 1932-6203
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  • 8
    In: PLoS ONE, 2013, Vol.8(10)
    Description: 2-aminophenol (2-AP) is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB , amnBAC, amnACD , amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA − mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA), a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.
    Keywords: Research Article
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Applied microbiology and biotechnology, June 2014, Vol.98(11), pp.4781-94
    Description: Bioremediation is an environmental sustainable and cost-effective technology for the cleanup of hydrocarbon-polluted soils and coasts. In spite of that longer times are usually required compared with physicochemical strategies, complete degradation of the pollutant can be achieved, and no further confinement of polluted matrix is needed. Microbial aerobic degradation is achieved by the incorporation of molecular oxygen into the inert hydrocarbon molecule and funneling intermediates into central catabolic pathways. Several families of alkane monooxygenases and ring hydroxylating dioxygenases are distributed mainly among Proteobacteria, Actinobacteria, Firmicutes and Fungi strains. Catabolic routes, regulatory networks, and tolerance/resistance mechanisms have been characterized in model hydrocarbon-degrading bacteria to understand and optimize their metabolic capabilities, providing the basis to enhance microbial fitness in order to improve hydrocarbon removal. However, microbial communities taken as a whole play a key role in hydrocarbon pollution events. Microbial community dynamics during biodegradation is crucial for understanding how they respond and adapt to pollution and remediation. Several strategies have been applied worldwide for the recovery of sites contaminated with persistent organic pollutants, such as polycyclic aromatic hydrocarbons and petroleum derivatives. Common strategies include controlling environmental variables (e.g., oxygen availability, hydrocarbon solubility, nutrient balance) and managing hydrocarbon-degrading microorganisms, in order to overcome the rate-limiting factors that slow down hydrocarbon biodegradation.
    Keywords: Bacteria -- Metabolism ; Environmental Pollutants -- Metabolism ; Fungi -- Metabolism ; Hydrocarbons -- Metabolism ; Petroleum -- Metabolism
    ISSN: 01757598
    E-ISSN: 1432-0614
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  • 10
    Language: English
    In: Applied Microbiology and Biotechnology, 2010, Vol.87(4), pp.1543-1554
    Description: Polychlorobiphenyls (PCBs) are classified as “high-priority pollutants.” Diverse microorganisms are able to degrade PCBs. However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA, and degraded 3,5-CBA in the presence of m -toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4′-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of 2,4′-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the development of improved biocatalysts for remediation of PCB-contaminated environments.
    Keywords: PCBs ; Chlorobenzoate ; Cupriavidus necator ; Mineralization ; Genetically modified microorganism ; Bioremediation
    ISSN: 0175-7598
    E-ISSN: 1432-0614
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