The complexity hypothesis posits that network connectivity and protein function are two important determinants of how a gene adapts to and functions in a foreign genome. Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the γ-proteobacterial family (extant and ancestral), supported growth. This demonstrates the limited functional interchangability of E. coli tuf with its homologs. Our data show a linear correlation between relative bacterial fitness and the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. Our data and analysis also suggest that the functional conservation of protein activity, and its network interactivity, act to constrain the successful transfer of this essential gene into foreign bacteria.