GENETIC-ANALYSIS OF DIOXIN DIOXYGENASE OF SPHINGOMONAS SP. STRAIN RW1- CATABOLIC GENES DISPERSED ON THE GENOME

The dioxin dioxygenase of Sphingomonas sp, strain RW1 activates dibenz o-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is inv olved in one of the bridges between the two aromatic rings, i.e., an a ngular dioxygenation. The dxnA1 and dxnA2 cistrons encoding this dioxy genase have been cloned and shown to be located just upstream of a hyd rolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electro n supply system of the dioxygenase are not clustered with the dioxygen ase gene but rather are located on two other distinct and separate gen ome segments. Moreover, whereas expression of dxnA1A2 is modulated acc ording to the available carbon source, expression of the dbfB gene enc oding the ring cleavage enzyme of the dibenzofuran pathway, which is l ocated in the neighborhood of dxnA1A2 but oriented in the opposite dir ection, is constitutive. The scattering of genes for the component pro teins of dioxin dioxygenase system around the genome of Sphingomonas s p, strain RW1, and the differential expression of dioxin pathway genes , is unusual and contrasts with the typical genetic organization of ca tabolic pathways where component cistrons tend to be clustered in mult icistronic transcriptional units, The sequences of the or and beta sub units of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) bindi ng site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxyge nase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron su pply system from RW1. Under these conditions, exclusively angular diox ygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxi n dioxygenase was not active in E. coli cells coexpressing a class IIB electron supply system, In the course of the isolation of the dxnA1 a nd dxnA2 cistrons, a number of other catabolic genes dispersed over di fferent genome segments were identified, which may indicate greater ca tabolic potential than was previously suspected, This finding is consi stent with the catabolic versatility of members of the genus Sphingomo nas, which is becoming increasingly evident, and may indicate a less w ell evolved and regulated but more dynamic genetic organization in thi s organism than is the case for better-studied pathways in organisms s uch as Pseudomonas species.