Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria

Abstract

Horizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10–90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (k cat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.

Biophysical principles predict fitness landscapes of drug resistance

“…Fitting the data points with Eq. 1 resulted in an excellent quantitative prediction of fitness, in agreement with recent work (20). The parameter B resulting from the nonlinear fit of Eq.…”

“…A set of points obtained for untransformed parent E. coli strain MG1655, expressing solely its WT chromosomal DHFR, grown at different TMP concentrations shows a good superimposition as well [with slight deviation at higher fitness probably due to activation of the DHFR promoter through a feedback loop (23,24)]. Also included for comparison are the data points obtained in previous studies using chromosomal orthologous DHFR replacements (20) and an IPTG-inducible promoter-controlled chromosomal DHFR expression system (13). Fitting the data points with Eq.…”

“…1 with a = 1, and from which B was determined from nonlinear regression (1.3 ± 0.1 SE). Also shown for comparison are the fitness data obtained in previous works where orthologous DHFRs have been incorporated in E. coli chromosome (20) and where chromosomal DHFR was under IPTG-controlled expression (13) (red and orange points, respectively). (B) Comparison of experimental vs. predicted IC 50 for TMP in strains expressing DHFR mutants from pFLAG.…”

Identification by PCR signature-tagged mutagenesis of attenuated Salmonella Pullorum mutants and corresponding genes in a chicken embryo model

“…The folA gene is involved in encoding dihydrofolate reductase (DHFR), which catalyses an electron-transfer reaction to form tetrahydrofolate, a carrier of single-carbon functional groups utilized in central metabolism, including de novo purine biosynthesis, dTTP formation, and methionine and glycine production [21]; folA-lacking bacteria will use flavin-dependent thymidylate synthases for deoxythymidine-5'-monophosphate synthesis [22].…”

Experimental evolution of Escherichia coli harboring an ancient translation protein

“…It has been suggested that as the number of nodes connecting an extant protein within its protein-protein interaction network increases, the capacity to replace that protein with another homolog decreases despite the presumed functional equivalence between the endogenous gene and the homolog (Jain et al 1999). Several orthologous protein substitution experiments demonstrated successful transfer of an alien gene to a foreign genome (Bershtein et al 2015;LariosSanz and Travisano 2009;Lind et al 2010), resulting in a fitness decrease similar to our approach, which was referred to as the "weak link approach" (Counago et al 2006). After evolving these organisms under laboratory conditions, it was demonstrated that direct accumulation of convergent mutations on the extant alien gene exhibits adaptive behavior (Counago et al 2006;Miller et al 2010;Pena et al 2010).…”