Differential Roles of Three Different Upper Pathway meta Ring Cleavage Product Hydrolases in the Degradation of Dibenzo-p- Dioxin and Dibenzofuran by Sphingomonas wittichii Strain RW1

Abstract

Sphingomonas wittichii RW1 grows on the two related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (P. V. Bunz, R. Falchetto, and A. M. Cook, Biodegradation 4:171–178, 1993, https://doi/org/10.1007/BF00695119) identified two upper pathway meta cleavage product hydrolases (DxnB1 and DxnB2) active on the DBF upper pathway metabolite 2-hydroxy-6- oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate. We took a physiological approach to determine the role of these two enzymes in the degradation of DBF and DXN by RW1. Single knockouts of either plasmid-located dxnB1 or chromosome-located dxnB2 had no effect on RW1 growth on either DBF or DXN. However, a double-knockout strain lost the ability to grow on DBF but still grew normally on DXN, demonstrating that DxnB1 and DxnB2 are the only hydrolases involved in the DBF upper pathway. Using a transcriptomicsguided approach, we identified a constitutively expressed third hydrolase encoded by the chromosomally located SWIT0910 gene. Knockout of SWIT0910 resulted in a strain that no longer grows on DXN but still grows normally on DBF. Thus, the DxnB1 and DxnB2 hydrolases function in the DBF but not the DXN catabolic pathway, and the SWIT0190 hydrolase functions in the DXN but not the DBF catabolic pathway. IMPORTANCE S. wittichii RW1 is one of only a few strains known to grow on DXN as the sole source of carbon. Much of the work deciphering the related RW1 DXN and DBF catabolic pathways has involved genome gazing, transcriptomics, proteomics,heterologous expression, and enzyme purification and characterization. Very little research has utilized physiological techniques to precisely dissect the genes and enzymes involved in DBF and DXN degradation. Previous work by others identified and extensively characterized two RW1 upper pathway hydrolases. Our present work demonstrates that these two enzymes are involved in DBF but not DXN degradation. In addition, our work identified a third constitutively expressed hydrolase that is involved in DXN but not DBF degradation. Combined with our previous work (T. Y. Mutter and G. J. Zylstra, Appl Environ Microbiol 87:e02464-20, 2021, https://doi.org/ 10.1128/AEM.02464-20), this means that the RW1 DXN upper pathway involves genes from three very different locations in the genome, including an initial plasmid- encoded dioxygenase and a ring cleavage enzyme and hydrolase encoded on opposite sides of the chromosome.