Operons, clusters of co-transcribed genes that often encode functionally linked proteins, are the principal form of gene organization and regulation in prokaryotes [1
]. Comparative analysis of bacterial and archaeal genomes has shown that only a few operons are conserved across large evolutionary distances. In general, gene order in prokaryotes is poorly conserved and prone to numerous rearrangements [3
]. A detailed analysis of gene order conservation has shown that only 5-25% of the genes in bacterial and archaeal genomes belongs to gene strings (probable operons) shared by at least two distantly related species [7
]. The presence of identical or similarly organized operons and suboperons in phylogenetically distant bacterial or archaeal lineages may result from three distinct evolutionary processes. Firstly, inheritance from the respective common ancestor - the core of the ribosomal protein superoperon is a case in point, but such conservation of operon organization is relatively rare; secondly, independent origin of identical operons or suboperons in different lineages; and thirdly, emergence of operons in a single lineage with subsequent dissemination by horizontal transfer. The potential central role of horizontal transfer in the evolution of operon organization of prokaryotic genomes is embodied in the 'selfish operon model' (SOM) [8
]. This model posits that "the physical proximity of genes in an operon provides no selective benefit to the individual organism but does enhance the fitness of the gene cluster itself, as clusters can be efficiently inherited horizontally as well as vertically" [11
]. Under SOM, operons are conceptually analogous to integrating viruses (phages), transposons and other mobile genetic elements, although coregulation of the genes in an operon could be an important selective factor that favors retention of operons during evolution.
Horizontal gene transfer (HGT) events have been classified into distinct categories of acquisition of new genes, acquisition of paralogs of existing genes and xenologous gene displacement whereby a gene is displaced by a horizontally transferred ortholog from another lineage (xenolog [12
]). Each of these types of horizontal transfer is common among prokaryotes, but their relative contributions differ in different lineages [13
]. Comparative-genomic analyses by many groups have suggested that, on the whole, horizontal gene transfer had substantial effects, albeit uneven in different lineages, on the gene content of bacterial and archaeal genomes [13
]. However, in spite of the considerable popularity of the selfish operon theory, we are unaware of systematic studies of horizontal gene transfer events at the level of operons. In part, this is likely to have been caused by the scarcity of experimental data on operon organization in any prokaryote other than Escherichia coli
Recent phylogenetic analyses of ribosomal proteins revealed several instances of apparent xenologous gene displacement within a conserved operon, in which other genes have not been horizontally transferred; in other words, these operons appear to represent an evolutionary mosaic [20
]. Another study demonstrated a complicated mosaic organization of the leukotoxin operon in bacteria of the genus Mannheimia
); the observed evolutionary pattern had to be explained through multiple gene transfer events, which led to the hypothesis that, in this case, frequent gene displacement conferred selective advantage onto the bacterium by maintaining antigenic variation [23
]. In earlier studies, evolution of operons from gene blocks with distinct evolutionary fates has been considered for rfb operons coding for lipopolysaccharide biosynthesis in enterobacteria [24
To assess the role of horizontal gene transfer in the evolution of operons systematically, we undertook phylogenetic analysis of members of highly conserved gene neighborhoods that are predicted to constitute operons [25
]. We focused primarily on mosaic operons in which one or more of the genes apparently have been transferred from distantly related species such that the phylogeny of the transferred genes is obviously incongruent with the phylogeny of the remaining genes in the respective operons.