Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs

Abstract

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 billion years ago (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 gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Keywords: EF-Tu; ancient genes; horizontal gene transfer; proteobacteria; tuf.

FIG 1

Phylogenetic tree indicating the node and taxa of the ancestral and modern tuf (EF-Tu) homologs. Pink circles represent the ancestral EF-Tu nodes. E. coli was genetically engineered to carry ancestral or modern homologs of tuf, encoding translation elongation factor EF-Tu, replacing the native E. coli tufA gene. A shaded box indicates the area of viability of EF-Tu gene exchange. The scale bar expresses units of amino acid substitutions per site. The tree was created with data from references and .

FIG 2

Correlation between relative fitness and evolutionary distance for bacterial strains carrying a foreign tuf gene. Relative fitness is shown as a function of evolutionary divergence (see Materials and Methods) of EF-Tu homologs from E. coli (1 indicates greatest difference from E. coli). Each E. coli strain carried a foreign tuf gene at the tufA location and an intact native tufB gene. Fitness was measured as exponential growth rate, relative to the E. coli wild type carrying tufA and tufB. Species names and ancestor notations (AnEF, ancestral EF-Tu) refer to the source of the foreign tuf gene sequence in each strain. Strains shown in green carry a foreign tuf gene that can support viability even when the E. coli tufB gene has been deleted. Strains shown in black carry foreign tuf genes that do not support viability in the absence of the E. coli tufB gene. Empty symbols represent strains where the tufB region was amplified.

FIG 3

Fitness characteristics of strains carrying single tuf genes. (A) Relative fitness of strains carrying a single tuf gene, at the tufA locus, as a function of EF-Tu protein produced. EF-Tu concentration was normalized to the total protein concentration. Species names indicate the species origin of the only tuf gene present (linear regression involves only extant sequences; R² = 0.938). (B) Relative protein synthesis rate (amino acids/second) of the ribosome in each constructed bacterial strain (R² = 0.994), determined by the time that it takes to produce β-galactosidase activity. Relative fitness of strains carrying a single tuf gene at the tufA locus correlates with the rate of protein synthesis. Results are means for three biological replicates with error bars representing standard deviation values. (C) Relative fitness of strains expressing each of the five tuf genes at three different levels: no additional tuf (light blue), additional tuf expression under promoter J23105 (medium blue), and additional tuf expression under promoter J23100 (dark blue). Promoter J23105 expresses the gene at a medium level (represented by +), whereas promoter J23100 expresses the gene at a higher level (represented by ++). Results are means for three biological replicates with error bars representing standard deviation values.

FIG 4

Alignment of EF-Tu protein sequences encoded by foreign tuf variants. EF-Tu sequences were aligned using Clustal Omega. Sequences labeled in green indicate that the foreign tuf gene supports viability as the only tuf gene in the genome. Sequences labeled in black indicate that the strain requires E. coli tufB for viability.