Aquatic Environmental Microbiology and Chemistry

University of Wisconsin Milwaukee, Zilber School of Public Health    

 

                  

Genome sequence of Sphingomonas wittichii RW1

 

Following decades of intense international research efforts, Sphingomonas wittichii strain RW1 emerged as a unique bacterium unmatched in its ability to mineralize the organic backbone of toxic dioxin pollutants (the dibenzo-p-dioxin structure), and to co-oxidize a large number of mono-, di-, tri- and tetrachlorinated congeners of both dibenzo-p-dioxin (DD) and dibenzofuran (DF) (14, 16, 28). Environments throughout the globe contaminated with dioxin-like compounds represent a significant threat to human and ecological health due to the compounds’ unfavorable behavior, which includes environmental persistence, bioaccumulation, biomagnification in the food chain, acute and chronic animal and human toxicity, endocrine disrupting properties and immunosuppressive and tumor promoting effects (6, 10, 17-19, 24, 27).

Among the steadily growing list of microorganisms of potential usefulness for the bioremediation of dioxin-contaminated sites (12), S. wittichii strain RW1 is in a class by itself and stands out for multiple reasons: (i) RW1 can biotransform a larger number and greater diversity of chlorinated diaryl ethers than any other bacterium (14, 16, 28) (ii) despite prolonged cultivation in the laboratory, the strain remains a hardy and robust organism that survives reintroduction into contaminated soils (13); (iii) it biotransforms dioxins in situ (13), and (iv) it has an extensive arsenal of catabolic enzymes suitable for degradation of chlorinated phenolic intermediates that arise during dioxin transformation (3, 5). Due to these interesting characteristics, this bacterium is among the most promising species for use as a bioremediation agent.

S. wittichii is a member of the genus Sphingomonas, which was created recently by Yabuuchi et. al (31) and includes strictly aerobic gram negative, asporogenous rods that produce sphingoglycolipids (SGL) of the type glucuronosyl ceramide (1-1)(SGL-1) (29). They are found in a variety of habitats including subsurface soils (8), plant surfaces (26), freshwater lakes and rivers (21, 23, 25, 28), polar and oligotrophic ocean waters (7), coral reefs (22) and as nosocomial infections of humans (15). Many are involved in the degradation of large, complex aromatic compounds (8, 9, 28). Thus, Sphingomonas species play a profound role in the cycling of organic matter throughout the globe. Phylogenetically, Sphingomonas species form a tight grouping within the Alpha Proteobacteria subclass. While S. wittichii, RW1 is clearly a member of this genus, in neighbor-joining trees S. wittichii RW1 consistently clusters by itself indicating its distinctiveness within the group (29, 30). Its nearest relative is S. yanoikuyae (previously Beijerinckia sp.) with a 16S rRNA identity of 94% (1368 bp/1447 bp) and S. wittichii is one of just a few Sphingomonas species to produce the novel sphingoglycolipid galacturonosyl-(1®1) (SGL-1’) in addition to the common SGL-1 (30). Therefore, S. wittichii is a unique Sphingomonas species representing an unexplored branch of this important new genus.

The arrangement of genomic regions involved in dioxin degradation, are particularly important for understanding of the evolution of this degradative pathway and its ability to function within S. wittichii RW1. For example, genes for the dioxin dioxygenase, electron transport system and other ancillary proteins are thought to be dispersed throughout the entire genome (1). This is an unusual genetic arrangement compared to similar enzyme systems making it exceedingly difficult, if not impossible, to study by using traditional genetic approaches. In strain RW1, the dxnA1A2 genes coding for the dioxin dioxygenase are on the 240 Kb megaplasmid with a subset of other dioxin degradation related genes, whereas the reductase gene, redA2, and ferredoxin gene, fdx1, one of the two possible electron supply chains are dispersed on separate loci (1, 2). This genetic organization has led some to suggest that the elements constituting the entire catabolic pathway for dioxin degradation have been recruited from other bacteria and/or other genetic loci, and that chromosome and plasmid encoded pathways have converged in this bacterium, thereby leading to successful dioxin degradation capabilities that are still evolving (20). Indeed, the dioxygenase enzymes from S. wittichii strain RW1 are only distantly related to dioxygenases from other species (1). Further investigation of genomic regions involved in dioxin degradation are essential for obtaining clues as to how S. wittichii acquired its novel dioxin degradation potential and as to how gene transfer among sphingomonads occurs (4).

Regulatory proteins involved in dioxin degradation and their binding domains are currently unknown and are not linked to any of the known dioxin degradation genes. This is an important aspect of dioxin degradation since we and others have shown repression of the dioxin dioxygenase under certain physiological conditions (1, 11). The genome sequence in combination with a genetic and/or a proteomic approach using mass spectrometry will facilitate the identification of regulatory proteins. 

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References

1.         Armengaud, J., B. Happe, and K. N. Timmis. 1998. Genetic analysis of dioxin dioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersed on the genome. J. Bacteriol. 180:3954-3966.
2.         Armengaud, J., and K. N. Timmis. 1997. Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1. Eur. J. Biochem. 247:833-842.
3.         Armengaud, J., K. N. Timmis, and R.-M. Wittich. 1999. A functional 4-hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1. J. Bacteriol. 181:3452-3461.
4.         Basta, T., A. Keck, J. Klein, and A. Stolz. 2004. Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains. J. Bacteriol. 186:3862-3872.
5.         Bunz, P. V., and A. M. Cook. 1993. Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system. J. Bacteriol. 175:6467-6475.
6.         Cohen, M. D., R. R. Draxler, and R. Artz. 2002. Modeling the atmospheric transport and deposition of PCDD/F to the Great Lakes. Environ. Sci Tech. 36:4831-4845.
7.         Eguchi, M., M. Ostrowski, F. Fegatella, J. Bowman, D. Nichols, T. Nishino, and R. Cavicchioli. 2001. Sphingomonas alaskensis strain AFO1, an abundant oligotrophic ultramicrobacterium from the North Pacific. Appl. Environ. Microbiol. 67:4945-4954.
8.         Fredrickson, J. K., D. L. Balkwill, G. R. Drake, M. F. Romine, D. B. Ringelberg, and D. C. White. 1995. Aromatic-degrading Sphingomonas isolate from deep subsurface. Appl. Environ. Microbiol. 61:1917-1922.
9.         Gabriel, F. L. P., A. Heidlberger, D. Rentsch, W. Giger, K. Geunther, and H.-P. E. Kohler. 2005. A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonas xenophaga Bayram. Ipso-hydroxylation and intramolecular rearrangement. J. Biol. Chem. 280:M413446200.
10.       Gotz, R., B. Steiner, P. Friesel, K. Roch, F. Walkow, V. Maa[beta], H. Reincke, and B. Stachel. 1998. Dioxin (PCDD/F) in the River Elbe - investigations of their origin by multivariate statistical methods. Chemo. 37:1987-2002.
11.       Halden, R. U., D. R. Colquhoun, and E. S. Wisniewski. 2005. Identification and phenotypic characterization of Sphingomonas wittichii strain RW1 by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Appl. Environ. Microbiol. 71:2442-2451.
12.       Halden, R. U., and D. F. Dwyer. 1997. Biodegradation of dioxin-related compounds. Bioremed. J. 1:11-25.
13.       Halden, R. U., B. G. Halden, and D. F. Dwyer. 1999. Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin from soils inoculated with Sphingomonas sp. strain RW1. Appl. Environ. Microbiol. 65:2246-2249.
14.       Hong, H.-B., Y.-S. Chang, I.-H. Nam, P. Fortnagel, and S. Schmidt. 2002. Biotransformation of 2,7-dichloro- and 1,2,3,4-tetrachlorodibenzo-p-dioxin by Sphingomonas wittichii RW1. Appl. Environ. Microbiol. 68:2584-2588.
15.       Hsueh, P. R., L. J. Teng, P. C. Yang, Y. C. Chen, H. J. Pan, S. W. Ho, and K. T. Luh. 1998. Nosocomial infections caused by Sphingomonas paucimobilis: clinical features and microbiological characteristics. Clin. Infect. Dis. 26:676-681.
16.       Keim, T., W. Francke, S. Schmidt, and P. Fortnagel. 1999. Catabolism of 2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas sp strain RW1. J. Indust. Microb. Biotech. 23:359-363.
17.       Klamer, H. J. C., P. E. G. Leonards, M. H. Lamoree, L. A. Villerius, J. E. Akerman, and J. F. Bakker. 2005. A chemical and toxicological profile of Dutch North Sea surface sediments. Chemo. 58:1579-1587.
18.       Korhonen, M., M. Verta, J. Lehtoranta, H. Kiviranta, and T. Vartiainen. 2001. Concentrations of polychlorinated dibenzo-p-dioxins and furans in fish downstream from a Ky-5 manufacturing. Chemo. 43:587-593.
19.       McKinney, J. D., and C. L. Waller. 1994. Polychlorinated biphenyls as hormonally active structural analogues. Environ. Health. Perspect. 102:290-297.
20.       Nojiri, H., H. Habe, and T. Omori. 2001. Bacterial degradation of aromatic compounds via angular dioxygenation. J. Gen Appl. Microbiol. 47:279-305.
21.       Pinhassi, J., and T. Berman. 2003. Differential growth response of colony-forming Alpha and Gamma Proteobacteria in dilution culture and nutrient addition experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat. Appl. Environ. Microbiol. 69:199-211.
22.       Richardson, L. L., W. M. Goldberg, K. G. Kuta, R. B. Aronson, G. W. Smith, K. B. Ritchie, J. C. Halas, J. S. Feingold, and S. L. Miller. 1998. Florida's mystery coral-killer identified. Nature 392:557-558.
23.       Rickard, A. H., S. A. Leach, L. S. Hall, C. M. Buswell, N. J. High, and P. S. Handley. 2002. Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria. Appl. Environ. Microbiol. 68:3644-3650.
24.       Ross, P. S., S. J. Jeffries, M. B. Yunker, R. F. Addison, M. G. Ikonomou, and J. C. Calambokidis. 2004. Harbor seals (Phoca vitulina) in British Columbia, Canada, and Washington State, USA, reveal a combination of local and global polychlorinated biphenyl, dioxin, and furan signals. Environ. Toxicol. Chem. 23:157-65.
25.       Schweitzer, B., I. Huber, R. Amann, W. Ludwig, and M. Simon. 2001. Alpha and Beta Proteobacteria control the consumption and release of amino acids on lake snow aggregates. Appl. Environ. Microbiol. 67:632-645.
26.       Takeuchi, M., T. Sakane, M. Yanagi, K. Yamasato, K. Hamana, and A. Yokota. 1995. Taxonomic study of bacteria isolated from plants: proposal of Sphingomonas rosa sp. nov., Sphingomonas pruni sp. nov., Sphingomonas asaccharolytica sp. nov., and Sphingomonas mali sp. nov. Int. J. Syst. Bacteriol. 45:334-341.
27.       Vrzal, R., J. Ulrichova, and Z. Dvorak. 2004. Aromatic hydrocarbon receptor status in the metabolism of xenobiotics under normal and pathophysiological conditions. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 148:3-10.
28.       Wittich, R. M., H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel. 1992. Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1. Appl. Environ. Microbiol. 58:1005-1010.
29.       Yabuuchi, E., Y. Kosako, N. Fujiwara, T. Naka, I. Matsunaga, H. Ogura, and K. Kobayashi. 2002. Emendation of the genus Sphingomonas Yabuuchi et al. 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola. Intl. J. Evol. Syst. Microbiol. 52:1485-1496.
30.       Yabuuchi, E., H. Yamamoto, S. Terakubo, N. Okamura, T. Naka, N. Fujiwara, K. Kobayashi, Y. Kosako, and A. Hiraishi. 2001. Proposal of Sphingomonas wittichii sp. nov. for strain RW1(T), known as a dibenzo-p-dioxin metabolizer. Int. J. Sys. Evol. Microbiol. 51:281-292.
31.       Yabuuchi, E., I. Yano, H. Oyaizu, Y. Hashimoto, T. Ezaki, and H. Yamamoto. 1990. Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34:99-119.

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