Annotation of the Sphingomonas wittichii RW1 Genome

Figure 1. Electron micrograph of S. wittichii RW1. (D. Colquhoun, 2005)

Lead Investigators
Rolf U. Halden (Assistant Professor), Johns Hopkins Bloomberg School of Public Health
Todd R. Miller (Post doctoral fellow), Johns Hopkins Bloomberg School of Public Health

Collaborators
Steven Salzberg (Professor), University of Maryland Center for Bioinformatics and Computational Biology
Jonathan A. Eisen (Professor), University of California Davis Genome Center
Fernando Pineda (Associate Professor), Johns Hopkins Bloomberg School of Public Health
Robert N. Cole (Director), Johns Hopkins School of Medicine Mass Spectrometry Facility
David Colquhoun (PhD Candidate), Johns Hopkins Bloomberg School of Public Health

Paul Richardson, Joint Genome Institute Microbial Program
David Bruce, Joint Genome Institute Microbial Program

We are annotating the genome sequence and studying the proteomics of the dioxin degrading bacterium, Sphingomonas wittichii Strain RW1 (Figure 1). This unique bacterium completely mineralizes the organic backbone of dioxin pollutants, including many chlorinated congeners (8, 10, 17).  The genome sequence of S. wittichii Strain RW1 provides the means to study the evolution and ecology of dioxin degrading microorganisms, the organization of genetic loci required for dioxin degradation and the biochemistry of proteins and enzymes involved in the metabolism of halogenated aromatic compounds.  This research provides the foundation for applied projects such as the construction of recombinant strains more efficient at chemical degradation, or production of enzymes and biocatalysts for industrial waste cleanup.

Figure 3 shows an overview of the sequencing and annotation process as well as investigators participating in each step. Currently the genome is being used to identify expressed proteins via mass spectrometry (see RW1 Proteomics).

Figure 2. Structure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Figure 3. Sequencing and annotation of the S. wittichii RW1 genome. Total DNA was extracted and purified from S. wittichii RW1 cells grown in minimal medium containing dibenzofuran as the sole carbon source. The genome was sequenced by the Joint Genome Institute (JGI) using the whole-genome shotgun sequencing method via Sanger sequencing in which 3, 8 and 40 Kb sections of DNA are inserted into large BAC plasmids to create a library of S. wittichii RW1 DNA sections. Inserts are sequenced at random from both sides until the same sequences are encountered approximately 8-9 times (coverage or depth). The overlapping reads were then assembled by both JGI and S. Salzberg (UM-College Park) into the complete genome sequence. The sequence data was subjected to an automated annotation and is being studied by a team of scientists with expertise in a variety of disciplines. Currently the genome sequence is being used to identify expressed proteins (see RW1 Proteomics).

Taxonomy and Ecology

S. wittichii was isolated from the River Elbe, Germany and is considered an aquatic bacterium though similar bacteria have been detected in soils. It is a member of the genus Sphingomonas, which was created recently by Yabuuchi et. al (20) and includes strictly aerobic gram negative, asporogenous rods that produce sphingoglycolipids (SGL) of the type glucuronosyl ceramide (1 - 1)(SGL-1) (18). They are found in a variety of habitats including subsurface soils (5), plant surfaces (16), freshwater lakes and rivers (12, 14, 15, 17), polar and oligotrophic ocean waters (4), coral reefs (13) and as nosocomial infections of humans (9). Many are involved in the degradation of large, complex aromatic compounds (5, 6, 17). 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 (18, 19). 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 (19). Therefore, S. wittichii is a unique Sphingomonas species representing an unexplored branch of this important new genus.

Genetics

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 not contained within a single genetic locus (1). This is an unusual genetic arrangement compared to other bacterial enzyme systems making it exceedingly difficult 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 disparate pathways have converged in this bacterium, thereby leading to successful dioxin degradation capabilities that are still evolving (11). 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 (3).

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 (Figure 2) (1, 7). The genome sequence in combination with a genetic and/or a proteomic approach will facilitate the identification of regulatory proteins.    

Preliminary Results
One Chromosome:

• Waiting for final assembly

Two Megaplasmids:

• pSW1; 315,461 bp, GC% = 64.14, similar to pNL1 from Sphingomonas aromaticovorans F199
• pSW2; 225,258 bp, GC% = 61.24, codes for all proteins previously shown to be required for dioxin degradation and is similar to pCAR1 from Pseudomonas resinovorans CA10 and pCAR3 from Sphingomonas sp. KA1.

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