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.

A beneficial synonymous substitution in EF-Tu is contingent on genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W=0.87, p < 0.01, ANOVA Tukey’s HSD) (Fig. 1C) (27–28), which upon 3000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (Evolved AnEF C45T, W= 1.6, p < 0.01, ANOVA Tukey’s HSD) (Fig. 3B ) .…”

A beneficial synonymous substitution in EF-Tu is contingent on genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W=0.87, p < 0.01, ANOVA Tukey’s HSD) (Fig. 1C) (27–28), which upon 3000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (Evolved AnEF C45T, W= 1.6, p < 0.01, ANOVA Tukey’s HSD) (Fig. 3B ) .…”

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W = 0.87, P < 0.01, ANOVA Tukey’s Honestly Significant Difference, HSD) ( Fig. 1C ) ( 27 , 28 ), which upon 3,000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (evolved AnEF C45T , W = 1.6, P < 0.01, ANOVA Tukey’s HSD) ( Fig. 3B ).…”

“…The transition of translation initiation to elongation is marked by the first translocation via EF-G ( 41 , 42 ), which is dependent on EF-Tu for bringing a cognate amino-acylated tRNA to form the first dipeptide bond ( 41 , 42 ). (ii) The increase in polysomes may be due to a decline in translation elongation rate in agreement with the previous studies showing that protein synthesis rate is reduced when translation is dependent on the EF-Tu ancestral variant ( 28 ). Furthermore, ancient EF-Tu variants were shown to exhibit significantly low K M values for dipeptide formation ( 48 ), indicating that ancestral EF-Tus are less efficient at sequestering cognate amino-acylated tRNAs to the ribosome.…”

Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor

“…In other words, EF-P homologs from closely related species, particularly other Gammaproteobacteria such as S. oneidensis and N. communis, tended to function more effectively in E. coli than those from more distantly related organisms like M. prima and H. aurantiacus. This pattern mirrors ndings from a similar study involving heterologous replacement of EF-Tu, where functionality in E. coli was typically limited to homologs from within the Gammaproteobacteria [39].…”

A beneficial synonymous substitution in EF-Tu is contingent on genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W=0.87, p < 0.01, ANOVA Tukey’s HSD) (Fig. 1C) (27–28), which upon 3000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (Evolved AnEF C45T, W= 1.6, p < 0.01, ANOVA Tukey’s HSD) (Fig. 3B ) .…”

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W = 0.87, P < 0.01, ANOVA Tukey’s Honestly Significant Difference, HSD) ( Fig. 1C ) ( 27 , 28 ), which upon 3,000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (evolved AnEF C45T , W = 1.6, P < 0.01, ANOVA Tukey’s HSD) ( Fig. 3B ).…”

“…The transition of translation initiation to elongation is marked by the first translocation via EF-G ( 41 , 42 ), which is dependent on EF-Tu for bringing a cognate amino-acylated tRNA to form the first dipeptide bond ( 41 , 42 ). (ii) The increase in polysomes may be due to a decline in translation elongation rate in agreement with the previous studies showing that protein synthesis rate is reduced when translation is dependent on the EF-Tu ancestral variant ( 28 ). Furthermore, ancient EF-Tu variants were shown to exhibit significantly low K M values for dipeptide formation ( 48 ), indicating that ancestral EF-Tus are less efficient at sequestering cognate amino-acylated tRNAs to the ribosome.…”

Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor

“…In other words, EF-P homologs from closely related species, particularly other Gammaproteobacteria such as S. oneidensis and N. communis, tended to function more effectively in E. coli than those from more distantly related organisms like M. prima and H. aurantiacus. This pattern mirrors ndings from a similar study involving heterologous replacement of EF-Tu, where functionality in E. coli was typically limited to homologs from within the Gammaproteobacteria [39].…”

A beneficial synonymous substitution in EF-Tu is contingent on genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W=0.87, p < 0.01, ANOVA Tukey’s HSD) (Fig. 1C) (27–28), which upon 3000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (Evolved AnEF C45T, W= 1.6, p < 0.01, ANOVA Tukey’s HSD) (Fig. 3B ) .…”

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background

“…In agreement with the previously published studies, the replacement of native E. coli EF-Tu with the ancestral EF-Tu AnEF causes a 13% decrease in fitness (W = 0.87, P < 0.01, ANOVA Tukey’s Honestly Significant Difference, HSD) ( Fig. 1C ) ( 27 , 28 ), which upon 3,000 generations of laboratory evolution is recovered and increased by 60% relative to the ancestor (evolved AnEF C45T , W = 1.6, P < 0.01, ANOVA Tukey’s HSD) ( Fig. 3B ).…”

“…The transition of translation initiation to elongation is marked by the first translocation via EF-G ( 41 , 42 ), which is dependent on EF-Tu for bringing a cognate amino-acylated tRNA to form the first dipeptide bond ( 41 , 42 ). (ii) The increase in polysomes may be due to a decline in translation elongation rate in agreement with the previous studies showing that protein synthesis rate is reduced when translation is dependent on the EF-Tu ancestral variant ( 28 ). Furthermore, ancient EF-Tu variants were shown to exhibit significantly low K M values for dipeptide formation ( 48 ), indicating that ancestral EF-Tus are less efficient at sequestering cognate amino-acylated tRNAs to the ribosome.…”

Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor

“…In other words, EF-P homologs from closely related species, particularly other Gammaproteobacteria such as S. oneidensis and N. communis, tended to function more effectively in E. coli than those from more distantly related organisms like M. prima and H. aurantiacus. This pattern mirrors ndings from a similar study involving heterologous replacement of EF-Tu, where functionality in E. coli was typically limited to homologs from within the Gammaproteobacteria [39].…”