Burkholderia xenovorans LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the pha genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions

2018 ◽  
Vol 21 (1-2) ◽  
pp. 47-57 ◽  
Author(s):  
Viviana Urtuvia ◽  
Pamela Villegas ◽  
Sebastián Fuentes ◽  
Myriam González ◽  
Michael Seeger
Keyword(s):  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400 ◽  
Pha Genes ◽  
Anabolic Pathway

Genomic analysis of the phenylacetyl-CoA pathway in Burkholderia xenovorans LB400

2011 ◽  
Vol 193 (9) ◽  
pp. 641-650 ◽  
Author(s):  
Marianna A. Patrauchan ◽  
J. Jacob Parnell ◽  
Michael P. McLeod ◽  
Christine Florizone ◽  
James M. Tiedje ◽  
...  
Keyword(s):  
Genomic Analysis ◽  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400

scholarly journals Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

2006 ◽  
Vol 103 (42) ◽  
pp. 15280-15287 ◽  
Author(s):  
P. S. G. Chain ◽  
V. J. Denef ◽  
K. T. Konstantinidis ◽  
L. M. Vergez ◽  
L. Agullo ◽  
...  
Keyword(s):  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400

scholarly journals Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400

2006 ◽  
Vol 72 (1) ◽  
pp. 585-595 ◽  
Author(s):  
V. J. Denef ◽  
J. A. Klappenbach ◽  
M. A. Patrauchan ◽  
C. Florizone ◽  
J. L. M. Rodrigues ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Gene Clusters ◽  
Large Chromosome ◽  
Wild Type ◽  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400 ◽  
Cell Assays ◽  
Aromatic Degradation ◽  
Proteomic Analyses

ABSTRACT Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC ) and the megaplasmid (boxM ). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ΔbenABCD::kan mutant), the boxC pathway (the ΔboxABC ::kan mutant), and both pathways (the ΔbenABCDΔ boxABC ::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ΔbenABCD::kan and ΔbenABCD ΔboxABC ::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ΔbenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

scholarly journals Coping with Polychlorinated Biphenyl (PCB) Toxicity: Physiological and Genome-Wide Responses of Burkholderia xenovorans LB400 to PCB-Mediated Stress

2006 ◽  
Vol 72 (10) ◽  
pp. 6607-6614 ◽  
Author(s):  
J. Jacob Parnell ◽  
Joonhong Park ◽  
Vincent Denef ◽  
Tamara Tsoi ◽  
Syed Hashsham ◽  
...  
Keyword(s):  
Protein Function ◽  
Polychlorinated Biphenyl ◽  
Membrane Separation ◽  
Expression Patterns ◽  
Burkholderia Xenovorans ◽  
Inhibition Of Growth ◽  
Burkholderia Xenovorans Lb400 ◽  
Genome Wide ◽  
Differential Gene ◽  
Genome Wide Expression

ABSTRACT The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.

scholarly journals Family Shuffling of Soil DNA To Change the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase

2006 ◽  
Vol 189 (3) ◽  
pp. 779-788 ◽  
Author(s):  
Julie Vézina ◽  
Diane Barriault ◽  
Michel Sylvestre
Keyword(s):  
Amino Acid ◽  
Polychlorinated Biphenyl ◽  
Ethyl Benzene ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Degenerate Primers ◽  
Biphenyl Dioxygenase ◽  
Soil Dna ◽  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400

ABSTRACT Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2′-dichlorobiphenyl (2,2′-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2′-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2′-dichlorobiphenyl from 2,2′-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2′,3,3′-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2′,5,5′-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2′-CB at a rate of 16 ± 1 nmol min−1 per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2′-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M237S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.

scholarly journals Distinct Roles for Two CYP226 Family Cytochromes P450 in Abietane Diterpenoid Catabolism by Burkholderia xenovorans LB400

2007 ◽  
Vol 190 (5) ◽  
pp. 1575-1583 ◽  
Author(s):  
Daryl J. Smith ◽  
Marianna A. Patrauchan ◽  
Christine Florizone ◽  
Lindsay D. Eltis ◽  
William W. Mohn
Keyword(s):  
Cytochromes P450 ◽  
Degradation Pathway ◽  
Dehydroabietic Acid ◽  
Burkholderia Xenovorans ◽  
Substrate Removal ◽  
Burkholderia Xenovorans Lb400 ◽  
Abietane Diterpenoids ◽  
Insertion Mutants

ABSTRACT The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

scholarly journals Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400

2009 ◽  
Vol 284 (24) ◽  
pp. 16377-16385 ◽  
Author(s):  
Jasleen Bains ◽  
Rafael Leon ◽  
Martin J. Boulanger
Keyword(s):  
Active Site ◽  
Mechanistic Model ◽  
Titration Calorimetry ◽  
Biophysical Characterization ◽  
Docking Simulations ◽  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400 ◽  
C Terminus ◽  
Functionally Diverse

The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 Å resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu146 and Glu168) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys111 as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.

Sign in / Sign up

Export Citation Format

Share Document