Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii Strain RW1 on Dibenzofuran and Dibenzo- p-Dioxin

Abstract

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-7320, 1993, https://doi.org/10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN, confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN, demonstrating that SWIT3046 is required for DXN degradation. A double-knockout strain does not grow on either DBF or DXN, demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. The replacement of dbfB by SWIT3046 results in a strain that grows normally (equal to the wild type) on both DBF and DXN, showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus, both dbfB- and SWIT3046-encoded enzymes are involved in DBF degradation, but only the SWIT3046-encoded enzyme is involved in DXN degradation.IMPORTANCES. wittichii RW1 has been the subject of numerous investigations, because it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis, our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid- and chromosomally encoded enzymes are necessary for DXN degradation, suggesting that the DXN pathway has only recently evolved.

Keywords: aromatic degradation; biodegradation; dibenzo-p-dioxin; dibenzofuran; dioxin; dioxygenase; ring cleavage.

FIG 1

Growth of RW1, RW1ΔdbfB, RW1Δ3046, and RW1ΔdbfBΔ3046 on dibenzofuran (DBF; left) and dibenzo-p-dioxin (DXN; right). Data are the averages from triplicates, and the error bars indicate their standard deviations. OD, optical density.

FIG 2

Diversity of RW1 meta ring cleavage enzymes with corresponding RNA-seq counts following growth on different substrates. In the amino acid sequence dendrogram, the ABQ number is the protein ID and the SWIT number is the gene ID in the GenBank database. The gene designation for SWIT4902 is dbfB. The RNA-seq numbers for each gene following growth on succinate (SUC), dibenzofuran (DBF), and dibenzo-p-dioxin (DXN) were extracted from Chai et al. (8) with GEO data set accession number GSE74831 and are the averages from multiple normalized replicates under each growth condition.

FIG 3

Complementation of the double dbfB and SWIT3046 knockout strain by either cloned dbfB or SWIT3046 on dibenzofuran (DBF; left) and complementation of the SWIT3046 knockout by cloned SWIT3046 on dibenzo-p-dioxin (DXN; right).

FIG 4

Growth of gene replacement constructs on dibenzofuran (DBF; left) and dibenzo-p-dioxin (DXN; right). Data are averages from triplicates, and the error bars indicate their standard deviations.

FIG 5

Metabolic map showing the role of DbfB and SWIT3046 in dibenzofuran and dibenzo-p-dioxin degradation by S. wittichii RW1. The redA2, fdx3, dxnA1, and dxnA2 genes code for the reductase, ferredoxin, and oxygenase alpha and beta subunits of the initial dioxygenase; dbfB codes for the THB ring cleavage dioxygenase; dxnB codes for a hydrolase; dxnC codes for a TonB-dependent receptor; and SWIT3048 to SWIT3043 encode (in order) a TonB-dependent receptor, a phytanoyl-CoA dioxygenase, a THB/THBE ring cleavage dioxygenase, an FAD-containing monooxygenase, a second TonB-dependent receptor, and a sulfatase.