Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas Wittichii RW1

Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF). A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3, and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1) were expected, whereas others (in a gene cluster for phenylacetate degradation) were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in parallel.

[1]  H. Heipieper,et al.  Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii , 2011, BMC Microbiology.

[2]  S. Salzberg,et al.  Genome Sequence of the Dioxin-Mineralizing Bacterium Sphingomonas wittichii RW1 , 2010, Journal of Bacteriology.

[3]  Leopold Parts,et al.  Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. , 2009, Genome research.

[4]  Z. Deng,et al.  New metabolites in dibenzofuran cometabolic degradation by a biphenyl-cultivated Pseudomonas putida strain B6-2. , 2009, Environmental Science and Technology.

[5]  W. Alkema,et al.  BioVenn – a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams , 2008, BMC Genomics.

[6]  S. Lemaire,et al.  The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii , 2008, Genetics.

[7]  V. Wray,et al.  Two Angular Dioxygenases Contribute to the Metabolic Versatility of Dibenzofuran-Degrading Rhodococcus sp. Strain HA01 , 2008, Applied and Environmental Microbiology.

[8]  Eoin L. Brodie,et al.  Temporal Transcriptomic Microarray Analysis of “Dehalococcoides ethenogenes” Strain 195 during the Transition into Stationary Phase , 2008, Applied and Environmental Microbiology.

[9]  J. Craig,et al.  Transcriptome Analysis Reveals that Multidrug Efflux Genes Are Upregulated To Protect Pseudomonas aeruginosa from Pentachlorophenol Stress , 2007, Applied and Environmental Microbiology.

[10]  S. Seah,et al.  Characterization of a C—C Bond Hydrolase from Sphingomonas wittichii RW1 with Novel Specificities towards Polychlorinated Biphenyl Metabolites , 2007, Journal of bacteriology.

[11]  Z. Deng,et al.  Cometabolic Degradation of Dibenzofuran and Dibenzothiophene by a Newly Isolated Carbazole-Degrading Sphingomonas sp. Strain , 2007, Applied and Environmental Microbiology.

[12]  V. de Lorenzo,et al.  Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene* , 2006, Journal of Biological Chemistry.

[13]  Jan Roelof van der Meer,et al.  A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes. , 2006, Environmental microbiology.

[14]  B. Mikami,et al.  Engineered membrane superchannel improves bioremediation potential of dioxin-degrading bacteria , 2006, Nature Biotechnology.

[15]  W. Verstraete,et al.  Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400 , 2006, Applied and Environmental Microbiology.

[16]  J. Ramos,et al.  Proteomic Analysis Reveals the Participation of Energy- and Stress-Related Proteins in the Response of Pseudomonas putida DOT-T1E to Toluene , 2005, Journal of bacteriology.

[17]  A. Hiraishi,et al.  Nocardioides aromaticivorans sp. nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments. , 2005, Systematic and applied microbiology.

[18]  T. Basta,et al.  Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains , 2004, Journal of bacteriology.

[19]  A. Guss,et al.  Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria , 2002, Archives of Microbiology.

[20]  S. Libby,et al.  The alternative sigma factor σE controls antioxidant defences required for Salmonella virulence and stationary‐phase survival , 2002, Molecular microbiology.

[21]  K. Timmis,et al.  A Second [2Fe-2S] Ferredoxin from Sphingomonas sp. Strain RW1 Can Function as an Electron Donor for the Dioxin Dioxygenase , 2000, Journal of bacteriology.

[22]  K. Timmis,et al.  Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans , 1999, Journal of Industrial Microbiology and Biotechnology.

[23]  J. Leveau,et al.  Dynamics of multigene expression during catabolic adaptation of Ralstonia eutropha JMP134 (pJP4) to the herbicide 2,4‐dichlorophenoxyacetate , 1999, Molecular microbiology.

[24]  K. Timmis,et al.  A Functional 4-Hydroxysalicylate/Hydroxyquinol Degradative Pathway Gene Cluster Is Linked to the Initial Dibenzo-p-Dioxin Pathway Genes inSphingomonas sp. Strain RW1 , 1999, Journal of bacteriology.

[25]  R. Halden,et al.  Removal of Dibenzofuran, Dibenzo-p-Dioxin, and 2-Chlorodibenzo-p-Dioxin from Soils Inoculated withSphingomonas sp. Strain RW1 , 1999, Applied and Environmental Microbiology.

[26]  K. Timmis,et al.  Genetic Analysis of Dioxin Dioxygenase ofSphingomonas sp. Strain RW1: Catabolic Genes Dispersed on the Genome , 1998, Journal of bacteriology.

[27]  K. Timmis,et al.  The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp. RW1 is related to class-I cytochrome P450-type reductases. , 1998, European journal of biochemistry.

[28]  C. Gross,et al.  SigmaE is an essential sigma factor in Escherichia coli , 1997, Journal of bacteriology.

[29]  K. Timmis,et al.  Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1. , 1997, European journal of biochemistry.

[30]  K. Timmis,et al.  Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1 , 1997, Applied Microbiology and Biotechnology.

[31]  Y. Ling,et al.  Reassessment of PCDD/DFs and Co-PCBs toxicity in contaminated rice-bran oil responsible for the disease "Yu-Cheng". , 1997, Chemosphere.

[32]  K. Bjerve,et al.  PCDDs, PCDFs, and PCBs in human blood in relation to consumption of crabs from a contaminated Fjord area in Norway. , 1996, Environmental health perspectives.

[33]  K. Timmis,et al.  Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Sphingomonas sp. Strain RW1 , 1996, Applied and environmental microbiology.

[34]  J. Ramos,et al.  Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control , 1994, Journal of bacteriology.

[35]  H. Beck,et al.  PCDD and PCDF exposure and levels in humans in Germany. , 1994, Environmental health perspectives.

[36]  T. Donohue,et al.  The activity of sigma E, an Escherichia coli heat-inducible sigma-factor, is modulated by expression of outer membrane proteins. , 1993, Genes & development.

[37]  K. Timmis,et al.  Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1 , 1993, Journal of bacteriology.

[38]  K. Olie,et al.  Effects of pre- and postnatal exposure to chlorinated dioxins and furans on human neonatal thyroid hormone concentrations. , 1993, Environmental health perspectives.

[39]  A. M. Cook,et al.  Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system , 1993, Journal of bacteriology.

[40]  T. Omori,et al.  Microbial degradation of dibenzofuran, fluorene, and dibenzo-p-dioxin by Staphylococcus auriculans DBF63 , 1993, Applied and environmental microbiology.

[41]  H. Wilkes,et al.  Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1 , 1992, Applied and environmental microbiology.

[42]  H. Harms,et al.  Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27 , 1990, Applied and environmental microbiology.

[43]  C. Rappe,et al.  Analysis of polychlorinated dibenzofurans, dioxins and related compounds in environmental samples. , 1985, Environmental health perspectives.

[44]  W. McNulty.  Toxicity and fetotoxicity of TCDD, TCDF and PCB isomers in rhesus macaques (Macaca mulatta). , 1985, Environmental health perspectives.

[45]  Y. Masuda,et al.  Inductive effect on hepatic enzymes and acute toxicity of individual polychlorinated dibenzofuran congeners in rats. , 1981, Toxicology and applied pharmacology.

[46]  J. C. Morgan,et al.  Bacterial and fungal oxidation of dibenzofuran. , 1979, The Biochemical journal.

[47]  A. Burlingame,et al.  The search for chlorinated dibenzofurans and chlorinated dibenzodioxins in wildlife populations showing elevated levels of embryonic death. , 1973, Environmental health perspectives.

[48]  O. Hutzinger,et al.  Acute and chronic oral toxicity of chlorinated dibenzofurans to salmonid fishes. , 1973, Environmental health perspectives.

[49]  R. Falchetto,et al.  Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran inSphingomonas sp. strain RW1 , 2004, Biodegradation.