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PloS One 2013The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a...
The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a heterohexameric (αβ)(3) Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356) with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356) and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.
Topics: Biphenyl Compounds; Burkholderiaceae; Catalytic Domain; Crystallography, X-Ray; Dioxygenases; Models, Molecular; Mutagenesis; Phylogeny; Polychlorinated Biphenyls; Protein Conformation; Protein Subunits; Substrate Specificity
PubMed: 23308114
DOI: 10.1371/journal.pone.0052550 -
Remarkable ability of Pandoraea pnomenusa B356 biphenyl dioxygenase to metabolize simple flavonoids.Applied and Environmental Microbiology May 2012Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to...
Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to degrade chlorobiphenyls in soil. However, the bases for this interaction are largely unknown. In this work, we found that BphAE(B356), the biphenyl/chlorobiphenyl dioxygenase from Pandoraea pnomenusa B356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAE(LB400) from Burkholderia xenovorans LB400. Unlike those of BphAE(LB400), the kinetic parameters of BphAE(B356) toward these flavonoids were in the same range as for biphenyl. In addition, remarkably, the biphenyl catabolic pathway of strain B356 was strongly induced by isoflavone, whereas none of the three flavonoids induced the catabolic pathway of strain LB400. Docking experiments that replaced biphenyl in the biphenyl-bound form of the enzymes with flavone, isoflavone, or flavanone showed that the superior ability of BphAE(B356) over BphAE(LB400) is principally attributable to the replacement of Phe336 of BphAE(LB400) by Ile334 and of Thr335 of BphAE(LB400) by Gly333 of BphAE(B356). However, biochemical and structural comparison of BphAE(B356) with BphAE(p4), a mutant of BphAE(LB400) which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAE(LB400), provided evidence that other residues or structural features of BphAE(B356) whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAE(B356). Together, these data provide supporting evidence that the biphenyl catabolic pathways have evolved divergently among proteobacteria, where some of them may serve ecological functions related to the metabolism of plant secondary metabolites in soil.
Topics: Amino Acid Substitution; Biphenyl Compounds; Burkholderiaceae; Dioxygenases; Flavonoids; Gene Expression Regulation, Bacterial; Kinetics; Metabolic Networks and Pathways; Protein Conformation
PubMed: 22427498
DOI: 10.1128/AEM.00225-12 -
Archives of Biochemistry and Biophysics Dec 2011In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356))...
In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity.
Topics: Amino Acid Sequence; Biphenyl Compounds; Burkholderia; Burkholderiaceae; Catalytic Domain; DDT; Dioxygenases; Insecticides; Models, Molecular; Molecular Sequence Data; Protein Binding; Sequence Alignment
PubMed: 22001737
DOI: 10.1016/j.abb.2011.09.016 -
The Journal of Biological Chemistry Oct 2011Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in...
Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.
Topics: Bacterial Proteins; Burkholderiaceae; Chlorophenols; Crystallography, X-Ray; Ligands; Oxidoreductases; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship; Substrate Specificity
PubMed: 21880718
DOI: 10.1074/jbc.M111.291013 -
Journal of Medical Microbiology Mar 2011Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species...
Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three showing a comparable or greater level of virulence in vivo relative to another CF pathogen, Burkholderia cenocepacia, whilst strains from two other species, Pandoraea apista and Pandoraea pnomenusa, were considerably less virulent. For all Pandoraea species, whole cells were required for larval killing, as cell-free supernatants had little effect on larval survival. Overall, invasive Pandoraea strains showed comparable invasion of two independent lung epithelial cell lines, irrespective of whether they had a CF phenotype. Pandoraea strains were also capable of translocation across polarized lung epithelial cell monolayers. Although protease secretion was a common characteristic across the genus, it is unlikely to be involved in pathogenesis. In conclusion, whilst multiple mechanisms of pathogenicity may exist across the genus Pandoraea, it appears that lung cell invasion and translocation contribute to the virulence of P. pulmonicola strains.
Topics: Adult; Animals; Bronchopneumonia; Burkholderia cenocepacia; Burkholderiaceae; Communicable Diseases, Emerging; Cystic Fibrosis; Epithelial Cells; Female; Humans; Larva; Lepidoptera; Lung; Survival Analysis; Virulence
PubMed: 21127160
DOI: 10.1099/jmm.0.022657-0 -
Acta Crystallographica. Section F,... Nov 2010cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa...
cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa strain B-356 was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method using polyethylene glycol 3350 and 0.2 M sodium malonate. A BphB crystal diffracted to 2.8 Å resolution and belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.2, c = 180.4 Å. Preliminary crystallographic analysis indicated the presence of two molecules in the asymmetric unit, giving a Matthews coefficient of 2.2 Å(3) Da(-1) and a solvent content of 44%.
Topics: Burkholderiaceae; Crystallization; Crystallography, X-Ray; Gene Expression; Oxidoreductases
PubMed: 21045310
DOI: 10.1107/S1744309110036894 -
International Journal of Systematic and... Sep 2011Five isolates, designated TA2, TA4, TA25(T), KOx(T) and NS15(T) were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole...
Five isolates, designated TA2, TA4, TA25(T), KOx(T) and NS15(T) were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole carbon source and were characterized using a polyphasic approach. The isolates were Gram-reaction-negative, aerobic, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA and DNA gyrase B subunit (gyrB) gene sequences confirmed that the isolates belonged to the genus Pandoraea and were most closely related to Pandoraea sputorum and Pandoraea pnomenusa (97.2-99.7 % 16S rRNA gene sequence similarity). The isolates could be differentiated from their closest relatives on the basis of several phenotypic characteristics. The major cellular fatty acid profiles of the isolates comprised C₁₆:₀, C₁₈:₁ω7c, C₁₇:₀ cyclo and summed feature 3 (C₁₆:₁ω7c and/or iso-C₁₅:₀ 2-OH). On the basis of DNA-DNA hybridization studies and phylogenetic analyses, the isolates represent three novel species within the genus Pandoraea, for which the names Pandoraea oxalativorans sp. nov. (TA25(T) = NBRC 106091(T) = CCM 7677(T) = DSM 23570(T)), Pandoraea faecigallinarum sp. nov. (KOx(T) = NBRC 106092(T) = CCM 2766(T) = DSM 23572(T)) and Pandoraea vervacti sp. nov. (NS15(T) = NBRC 106088(T) = CCM 7667(T) = DSM 23571(T)) are proposed.
Topics: Aerobiosis; Bacterial Typing Techniques; Burkholderiaceae; Cluster Analysis; Culture Media; DNA Gyrase; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Molecular Sequence Data; Nucleic Acid Hybridization; Oxalates; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 20952546
DOI: 10.1099/ijs.0.026138-0 -
Journal of Hazardous Materials Sep 2010Four oil component-degrading bacteria and one oil-tolerant microalgae, Scenedesmus obliquus GH2, were used to construct an artificial microalgal-bacterial consortium for...
Four oil component-degrading bacteria and one oil-tolerant microalgae, Scenedesmus obliquus GH2, were used to construct an artificial microalgal-bacterial consortium for crude-oil degradation. The bacterial strains included Sphingomonas GY2B and Burkholderia cepacia GS3C, along with a mixed culture, named GP3, containing Pseudomonas GP3A and Pandoraea pnomenusa GP3B. GY2B could only degrade polycyclic aromatic hydrocarbons, GS3C was able to degrade aliphatic chain hydrocarbons, and GP3 could utilize both saturated and aromatic hydrocarbons. In combination with unialgal or axenic algae, the bacteria showed different effects on oil degradation. Unialgal GH2 was not suitable for the consortium construction, as it could not cooperate well with GS3C and GP3. The axenic GH2 exhibited no oil-degrading ability; however, it significantly promoted the degradation ability of the oil component-degrading bacteria, especially for degrading biorefractory polycyclic aromatic hydrocarbons. Axenic S. obliquus GH2, combined with the four bacteria mentioned above, formed an optimal algal-bacterial consortium. The artificial consortium demonstrated an elevated efficiency in degrading both aliphatic and aromatic hydrocarbons of crude oil.
Topics: Alkanes; Bacteria; Biodegradation, Environmental; Eukaryota; Hydrocarbons, Aromatic; Petroleum
PubMed: 20638971
DOI: 10.1016/j.jhazmat.2010.05.033 -
Journal of Clinical Microbiology Nov 2009The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of...
The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of care for patients with cystic fibrosis (CF). Under the umbrella of EuroCareCF, two quality assurance trials of CF microbiology were performed in 2007 and 2008. Nine formulations with CF bacterial isolates were dispatched. A total of 31/37 laboratories from 18/21 European countries participated in the 2007 and 2008 trials. The common CF pathogens Pseudomonas aeruginosa and Staphylococcus aureus were correctly identified by almost all participants in both trials, even if the strains presented uncommon phenotypes. Burkholderia cenocepacia IIIB and Burkholderia vietnamensis CF isolates, however, were correctly assigned to the species level by only 26% and 27% of the laboratories, respectively. Emerging pathogens such as Achromobacter xylosoxidans, Inquilinus limosus, and Pandoraea pnomenusa were also not detected or were misclassified by many laboratories. One participant correctly identified all CF isolates in both trials. The percentages of correct classifications (susceptible, intermediate, resistant) by antimicrobial susceptibility testing ranged from 55 to 100% (median, 96%) per isolate and drug. The shortcomings in the diagnostics of rare and emerging pathogens point to the need for continuing education in CF microbiology and suggest the establishment of CF microbiology reference laboratories.
Topics: Bacteria; Bacterial Infections; Bacteriological Techniques; Bronchopneumonia; Cystic Fibrosis; Diagnostic Errors; Europe; Health Services Research; Humans; Microbial Sensitivity Tests; Quality Control
PubMed: 19741077
DOI: 10.1128/JCM.01182-09 -
International Journal of Systematic and... Jan 2010A facultatively chemolithoautotrophic, thiosulfate-oxidizing, Gram-negative, aerobic, motile, rod-shaped bacterial strain, designated ATSB16(T), was isolated from...
A facultatively chemolithoautotrophic, thiosulfate-oxidizing, Gram-negative, aerobic, motile, rod-shaped bacterial strain, designated ATSB16(T), was isolated from rhizosphere soils of sesame (Sesamum indicum L.). 16S rRNA gene sequence analysis demonstrated that this strain was closely related to Pandoraea pnomenusa LMG 18087(T) (96.7 % similarity), P. pulmonicola LMG 18016(T) (96.5 %), P. apista LMG 16407(T) (96.2 %), P. norimbergensis LMG 18379(T) (96.1 %) and P. sputorum LMG 18819(T) (96.0 %). Strain ATSB16(T) shared 96.0-96.4 % sequence similarity with four unnamed genomospecies of Pandoraea. The major cellular fatty acids of the strain ATSB16(T) were C(17 : 0) cyclo (33.0 %) and C(16 : 0) (30.6 %). Q-8 was the predominant respiratory quinone. The major polar lipids were phosphatidylmethylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine and two unidentified aminophospholipids. Hydroxyputrescine and putrescine were the predominant polyamines. The genomic DNA G+C content of the strain was 64.0 mol%. On the basis of the results obtained from this study, strain ATSB16(T) represents a novel species of the genus Pandoraea, for which the name Pandoraea thiooxydans sp. nov. is proposed. The type strain is ATSB16(T) (=KACC 12757(T) =LMG 24779(T)).
Topics: Burkholderiaceae; Chemoautotrophic Growth; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Molecular Sequence Data; Oxidation-Reduction; Phylogeny; RNA, Ribosomal, 16S; Sesamum; Soil Microbiology; Thiosulfates
PubMed: 19643869
DOI: 10.1099/ijs.0.012823-0