Identification and characterization of three novel oxygenases from Sphingomonas wittichii RW1 and a Pseudomonas integrative and conjugative element (ICE)
Creators: name: Ivanovski, Igor
Publisher: name: No Publisher Supplied
Year: 2021

description: Aromatic hydrocarbons are pervasive in the environment with both natural and anthropogenic sources. Bicyclic compounds such as dibenzofuran (DF), dibenzo-p-dioxin (DD), biphenyl (BPH), and diphenylmethane (DPM) are of concern due to their ability to bioaccumulate, and when chlorinated, cause severe health effects. Sphingomonas wittichii RW1 is one of the few organisms which can grow on the related compounds DF and DD and Pseudomonas sp. strain AJR13 is one of the few strains that can grow on the related compounds BPH and DPM. In S. wittichii RW1 the DD and DF degradative pathway genes are scattered in the genome while in Pseudomonas sp. strain AJR13 the BPH and DPM degradative pathway genes are present on an integrative and conjugative element (ICE) in the chromosome. Aerobic degradation of bicyclic compounds such as DD, DF, BPH, and DPM typically proceeds through dioxygenase attack on one of the aromatic rings followed by ring cleavage and hydrolytic removal of the side chain. This results in a monoaromatic ring compound which then typically goes through another round of oxygenase attack, ring cleavage, and conversion to tricarboxylic acid cycle intermediates. This work examines three novel monocyclic aromatic oxygenases associated with bicyclic compound degradation in S. wittichii RW1 and Pseudomonas sp. strain AJR13.Examination of the genome sequence of Pseudomonas sp. strain AJR13 revealed a gene encoding a NahG family salicylate 1-monooxygenase associated with the ICE encoding BPH and DPM degradation even though AJR13 did not grow on salicylate. Transfer of the ICE to the well-studied P. putida KT2440 resulted in a KT2440 strain that could grow on salicylate. Knockout of the nahG type gene on the ICE in KT2440 eliminated the ability to grow on salicylate. Complementation of the knockout with the cloned nahG type gene restored growth on salicylate. Expression of the nahG gene in E. coli resulted in the production of catechol from salicylate, confirming that it is indeed a salicylate 1-monooxygenase. Interestingly, transfer of the cloned nahG type gene under control of the lac promoter to AJR13 resulted in a strain that could now grow on salicylate suggesting that the identical gene in the ICE is not expressed preventing the wild type AJR13 strain from growing on salicylate.Preliminary data from our laboratory suggested that the S. wittichii RW1 DD/DF initial dioxygenase shared electron transfer components with a tricomponent benzoate dioxygenase. To prove this hypothesis, the genes encoding the benzoate oxygenase component (benAB) were identified in the RW1 genome sequence through homology to known benzoate oxygenases. The RW1 benAB genes are upstream from a putative benD gene encoding a cis-benzoate dihydrodiol dehydrogenase. Knockout of the benA gene resulted in a strain unable to grow on benzoate. The knockout strain could not be complemented with the cloned benA gene but could be complemented with the cloned benABD genes, suggesting that the knockout insertion had a polar effect on the three gene operon. Expression of benAB in E. coli along with the fdx3 and redA2 genes encoding the ferredoxin and reductase components of the DD/DF dioxygenase produced a functional benzoate dioxygenase enzyme capable of converting benzoate to benzoate cis-dihydrodiol. This proves that the RW1 benzoate dioxygenase shares electron transfer components with the RW1 DD/DF dioxygenase.The ability to grow on salicylate was easily lost by S. wittichii RW1. Sequencing of the wild type strain and salicylate minus strains revealed a 14,116 base pair region missing from the published RW1 wild type genome sequence and missing from the spontaneous salicylate minus variants. This region contains genes encoding an oxygenase component of a NagGH family salicylate 5-oxygenase. Knockout of the nagGH-like genes resulted in a RW1 strain unable to grow on salicylate. Complementation with the cloned nagGH-like genes restored growth on salicylate. Heterologous expression of the nagGH genes along with the fdx3 and redA2 genes encoding the ferredoxin and reductase components of the DD/DF dioxygenase produced a functional salicylate 5-oxygenase that converts salicylate to gentisate. These data positively identify the genes encoding the oxygenase component of salicylate 5-oxygenase and demonstrates that they share electron transfer components with the RW1 DD/DF dioxygenase.