Evolution End Classification of tfd Gene Clusters Mediating Bacterial Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D)

The tfd (tfd(I) and tfd(II)) are gene clusters originally discovered in plasmid pJP4 which are involved in the bacterial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) via the ortho-cleavage pathway of chlorinated catechols. They share this activity, with respect to substituted catechols, with clusters tcb and clc. Although great effort has been devoted over nearly forty years to exploring the structural diversity of these clusters, their evolution has been poorly resolved to date, and their classification is clearly obsolete. Employing comparative genomic and phylogenetic approaches has revealed that all tfd clusters can be classified as one of four different types. The following four-type classification and new nomenclature are proposed: tfd(I), tfd(II), tfd(III) and tfd(IV(A,B,C)). Horizontal gene transfer between Burkholderiales and Sphingomonadales provides phenomenal linkage between tfd(I), tfd(II), tfd(III) and tfd(IV) type clusters and their mosaic nature. It is hypothesized that the evolution of tfd gene clusters proceeded within first (tcb, clc and tfd(I)), second (tfd(II) and tfd(III)) and third (tfd(IV(A,B,C))) evolutionary lineages, in each of which, the genes were clustered in specific combinations. Their clustering is discussed through the prism of hot spots and driving forces of various models, theories, and hypotheses of cluster and operon formation. Two hypotheses about series of gene deletions and displacements are also proposed to explain the structural variations across members of clusters tfd(II) and tfd(III), respectively. Taking everything into account, these findings reconstruct the phylogeny of tfd clusters, have delineated their evolutionary trajectories, and allow the contribution of various evolutionary processes to be assessed.