Genome Sequence of the Dioxin-Mineralizing Bacterium Sphingomonas wittichii RW1

Abstract

Pollutants such as polychlorinated biphenyls and dioxins pose a serious threat to human and environmental health. Natural attenuation of these compounds by microorganisms provides one promising avenue for their removal from contaminated areas. Over the past 2 decades, studies of the bacterium Sphingomonas wittichii RW1 have provided a wealth of knowledge about how bacteria metabolize chlorinated aromatic hydrocarbons. Here we describe the finished genome sequence of S. wittichii RW1 and major findings from its annotation.

The alphaproteobacterium Sphingomonas wittichii strain RW1 is known as a potent degrader of toxic dioxin pollutants, as it completely mineralizes the organic backbone of the dibenzo-p-dioxin structure (9, 10). This organism was isolated 2 decades ago for its ability to grow on dioxin-like compounds as the sole carbon and energy source (10). Since that time, research has shown that S. wittichii RW1 can transform a larger number and greater diversity of chlorinated diaryl ethers than any other known bacterium (5, 7, 9).

The genome of S. wittichii RW1 was sequenced at the Joint Genome Institute (JGI) using a combination of 5-kb, 7-kb, and 36-kb DNA libraries. Draft assemblies were based on 88,105 total reads using the Phred/Phrap/Consed software package (3, 4). Assemblies were also constructed with Celera Assembler software (8) and checked for consistency. Possible misassemblies were corrected with Dupfinisher or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). A total of 453 primer walk reactions and one transposon bomb were necessary to close gaps. The finished genome assembly contains 36,718 reads, achieving an average of 11-fold sequence coverage with an error rate less than 1 in 100,000.

The completed 5,915,246-bp genome consists of a main chromosome (5,382,261 bp) and two megaplasmids, designated pSWIT01 (310,228 bp) and pSWIT02 (222,757 bp). The larger megaplasmid, pSWIT01, is similar to pNL1 from Novosphingobium aromaticivorans. Specifically, ∼17-kb regions from both plasmids code for similar proteins at a 50% similarity cutoff. These regions encode a reverse transcriptase and a type IV pilus. The smaller megaplasmid, pSWIT02, contains all of the genes previously shown to be required for dioxin degradation arranged in three disparate loci (1). The locus containing the dioxin dioxygenase coding sequence encodes all enzymes required to metabolize dioxin except for the reductase protein RedA2 and the 2,2′,3-trihydroxybiphenyl dioxygenase, DbfB. The start of the redA2 gene and that of the dbfB gene are approximately 6 and 21 kb from the dioxin dioxygenase locus, respectively. It appears that at least some of these genes may have been acquired horizontally, as the dioxin dioxygenase locus is flanked by multiple transposases; however, an intact transposon could not be identified.

The top three most abundant COG (clusters of orthologous groups) products encoded by the S. wittichii RW1 genome are TonB-dependent receptors (TBDRs; COG1629), dehydrogenases with different specificities (COG1028), and phenylpropionate dioxygenases and related ring-hydroxylating dioxygenases (COG4638). Based on a comparison of all bacterial genomes, S. wittichii RW1 appears to code for the most TBDRs. These receptors are known for binding iron-bound siderophores, facilitating their active transport into the periplasmic space. Recently TBDRs have been shown to bind a variety of other compounds, including sugars and alginate (2, 6). In S. wittichii RW1 the majority of TBDR genes reside within operons predicted to code for enzymes involved in aromatic hydrocarbon metabolism. This suggests yet another new role for TBDRs in S. wittichii RW1, specifically as transporters of aromatic hydrocarbons and/or their breakdown products. This may in part explain the unique ability of S. wittichii RW1 to consume a wide range of sparingly water-soluble aromatic hydrocarbons (6).

Nucleotide sequence accession numbers.

The nucleotide sequence of Sphingomonas wittichii RW1 has been deposited in GenBank under accession numbers CP000699 to CP000701.