Horizontal transfer beyond genes.

Horizontal gene transfer (HGT) is a major process in the evolution of bacteria and archaea (1–3) that is thought to be the principal source of functional innovation in microbial evolution (4, 5). The signal of vertical, tree-like evolution is detectable primarily among genes encoding components of information processing systems, whereas evolution of the operational genes (primarily, metabolic enzymes, transporters, and regulators of these processes) effectively shows network-type evolutionary dynamics (6, 7). Quantification of HGT across the diversity of bacteria reveals extremely high characteristic rates of gene transfer in most groups of bacteria, with multiple HGT events detectable per nucleotide substitution per gene, indicating that HGT is indeed the dominant mode of microbial evolution (8). Many HGT events involve individual genes, but transfer of larger portions of genomes, in particular operons, is common as well, leading to the selfish operon hypothesis under which operons represent self-contained and hence readily transferable units of evolution (9, 10). Accordingly, evolution of bacterial metabolic networks has been modeled as a process of acquisition of operons encoding distinct metabolic pathways, complete with the cognate regulatory elements (11). In PNAS, Oren et al. (12) add an unexpected, potentially important facet to the already extensive evidence of the dominance of horizontal transfer of genetic material in microbial evolution by describing a phenomenon they denote horizontal regulatory transfer (HRT). As implied by the name proposed for this distinct route of evolution, HRT involves horizontal transfer of noncoding regulatory regions of microbial DNA alone, without the adjacent regulated genes (Fig. 1). The HRT leads to a regulatory switch that modifies and in some cases rewires the regulatory network of the bacterial cell because nonhomologous regulatory regions are typically regulated by different transcription factors. The regulatory regions in microbial genomes that include promoters and transcription regulator-binding sites are much shorter than protein-coding genes and can change faster, because of which confident delineation of the evolutionary histories of these sequence is a nontrivial task. As in many other cases of evolutionary reconstruction, the key to the solution lies in the careful analysis of appropriately selected sets of closely related genomes, and Oren et al. take full advantage of this strategy. Analysis of the genomes of 46 sequenced isolates of Escherichia coli provides for a statistically supported comparison of the topologies of the phylogenetic trees for regulatory regions, their regulated genes, and the species tree. The results of this comparison strikingly show that evolution of the regulatory regions of over half of the core genes (i.e., genes shared by all isolates) was incongruent with the species tree. In the majority of these cases, the regulatory region appeared to have been transferred together with the downstream regulated gene(s), but about one third of these events seem to represent bona fide HRT (Fig. 1). Altogether, excluding cases that could be caused by artifacts of phylogenetic analysis, HRT appears to have affected more than 11% of the regulatory regions in E. coli, which is definitely a substantial contribution to the plasticity of the bacterial regulatory network. Using the RNA-seq technique, Oren et al. show that regulatory switching caused by HRT indeed leads to the divergence of transcription start sites and gene expression levels between E. coli strains, confirming the functional relevance of HRT. Furthermore, by experimentally reversing regulatory switching for a single gene, they demonstrate that HRT is adaptive for a uropathogenic strain of E. coli. Extension of this analysis to other groups of bacteria, for which multiple isolates are available, has shown that HRT is ubiquitous, but its extent significantly varies across bacterial taxa. At least some of this variation seems to make biological sense, with low levels of HRT detected in intracellular parasites compared with much higher levels in environmental bacteria. Notably, anecdotal cases of HRT over longer evolutionary distances, in particular from Enterobacter to E. coli, have been identified as well. The discovery of HRT substantially broadens the existing perspective on the HGT