Integration of horizontally transferred genes into regulatory interaction networks takes many million years.

Adaptation of bacteria to new or changing environments is often associated with the uptake of foreign genes through horizontal gene transfer. However, it has remained unclear how (and how fast) new genes are integrated into their host's cellular networks. Combining the regulatory and protein interaction networks of Escherichia coli with comparative genomics tools, we provide the first systematic analysis of this issue. Genes transferred recently have fewer interaction partners compared to nontransferred genes in both regulatory and protein interaction networks. Thus, horizontally transferred genes involved in complex regulatory and protein-protein interactions are rarely favored by selection. Only few protein-protein interactions are gained after the initial integration of genes following the transfer event. In contrast, transferred genes are gradually integrated into the regulatory network of their host over evolutionary time. During adaptation to the host cellular environment, horizontally transferred genes recruit existing transcription factors of the host, reflected in the fast evolutionary rates of the cis-regulatory regions of transferred genes. Further, genes resulting from increasingly ancient transfer events show increasing numbers of transcriptional regulators as well as improved coregulation with interacting proteins. Fine-tuned integration of horizontally transferred genes into the regulatory network spans more than 8-22 million years and encompasses accelerated evolution of regulatory regions, stabilization of protein-protein interactions, and changes in codon usage.