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

doi:10.21203/rs.3.rs-7374903/v1

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

https://doi.org/10.21203/rs.3.rs-7374903/v1

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Horizontal gene transfer (HGT) is a major driver of microbial evolution, yet the influence of host cellular context on the integration and functionality of transferred genes remains underexplored. In this study, we investigate how host background affects the compatibility and consequences of acquiring post-translational modification (PTM) machinery through HGT using the heterologous expression of the highly conserved translational elongation factor P (EF-P) from diverse species in Escherichia coli as a model. EF-P and its PTM machinery have been horizontally transferred many times across the bacterial tree of life, and these experiments are meant to examine the consequences of these events. EF-P has a diverse and heterogenous relationship with PTMs; three characterized variants each undergo distinct PTM pathways, while others function effectively without any modification. In this study, we demonstrate that EF-P from Deinococcus radiodurans, Geoalkalibacter ferrihydriticus, and Nitrosomonas communis can complement an EF-P knockout in E. coli without requiring modification, suggesting they represent new examples of unmodified EF-Ps. We also found that the EF-P from the Thermotogota Mesotoga prima is post-translationally modified in an off-target reaction by the rhamnosylation enzyme EarP, thus interfering with its functionality. Conversely, we saw that rhamnosylation by EarP is fully compatible with the EF-P-like protein EfpL from Escherichia coli, thus presenting a promising opportunity to develop novel, catalytically active PTMs. These findings highlight that PTM systems introduced via HGT can have unintended effects on host proteins, emphasizing the complexity of gene integration and functional compatibility in foreign genomic contexts.

Evolution

bacterial diversity

horizontal gene transfer

Successful horizontal gene transfer (HGT) involves a complex series of events—from the initial entry of a gene into a new cellular environment, to its stable integration into the genome or other replicating element, to its eventual spread throughout a population [1]. Along this path, numerous barriers can hinder the integration of new genes, including high phylogenetic divergence between the donor and recipient and incompatible restriction-modification or CRISPR systems [1]. Yet one important aspect of this process remains relatively overlooked: the influence of the host’s physiological background on the successful ‘domestication’ of the incoming gene. In this study, we explore this critical gap through an experimental system inspired by patterns observed in genomic data—namely, the interference between horizontally acquired variants of elongation factor P (EF-P), their associated post-translational modification systems, and the host bacterium’s existing translational machinery.

EF-P is an essential protein in bacteria which tackles a problem shared by all forms of life: the translation of sequences composed of one or multiple prolines [2–5]. Proline is a uniquely rigid amino acid and causes the ribosome to stall during translation, especially during the formation of peptide bonds between multiple prolines (polyproline sequences) [6]. To overcome this, EF-P, along with its homolog IF-5A in the Archaea and Eukaryotes, interacts with the peptidyl-transferase center to reposition and stabilize the P-site tRNA, dramatically accelerating the rate of peptide bond formation between prolines [2–4]. Without EF-P, bacteria suffer species specific phenotypes that include reduced growth rate [7–11], reduced antibiotic resistance [11, 12], loss of mobility [13, 14], loss of virulence [7, 9, 11, 14], and cell death [14–18].

In many species, EF-P must be post-translationally modified to reach full activity [19, 20]. Although the specific type of modification varies, all known modifications target a single amino acid at the tip of a conserved loop in domain I (β3Ωβ4) of the protein, extending the “reach” of EF-P further towards the peptidyl-transferase center [2]. This amino acid is typically either a lysine—modified by β-(R)-lysylation (and hydroxylation) in many Gammaproteobacteria and others [21, 22] or by the addition of 5-aminopentanol in the Firmicutes [10, 23, 24]—or an arginine, which is modified through α-rhamnosylation by the glycosyltransferase EarP [7, 25, 26]. However, increasing evidence shows that some EF-P variants function effectively without any modification at the β3Ωβ4 tip [5, 27, 28]. These include the EF-P subtypes EfpL from the Gammaproteobacteria [5], and those from the PGKGP family, primarily found in the Actinobacteria and the Bacteroidetes [27, 28].

EF-P has been horizontally transferred many times [7, 29]—an unusual event for a protein important for fundamental cellular processes like translation [30–32]. In cases of horizontal gene transfer, post-translationally modified EF-P subtypes are typically transferred along with their cognate PTM enzymes as a single operon to the new host [29]. This co-transfer supports the idea that EF-P proteins and their PTM systems have co-evolved, and that switching between different PTM pathways may not be straightforward. Bioinformatics analyses have linked the horizontal transfer of EF-P and its PTM systems to broader signs of EF-P dysfunction, namely selective pressure against highly conserved polyproline motifs and the proteins which contain them [29]. However, genomic data offers only static snapshots—glimpses of genomes long after the original HGT event. As a result, genomics can at best suggest what might have occurred during the immediate aftermath of gene transfer, such as a potential ‘domestication’ phase where overall EF-P function is compromised. This dysfunction may reflect barriers to successful HGT, possibly influenced by the recipient cell’s physiology. In particular, pre-existing, ‘native’ EF-P may be sensitive to interference with the sudden appearance of a foreign EF-P PTM system.

In this study, we experimentally test the hypothesis that EF-P are vulnerable to off-target modifications following interaction with non-coevolved PTM machinery (Visual summary in Fig. 1). By examining 1) whether off-target modifications are possible, and 2) whether these modifications have an impact on the function of diverse EF-P, we aim to uncover molecular complications involved in horizontal gene transfer, gain insight into the domestication of foreign genes, and explore the broader evolutionary implications of these events.

Rhamnosylation by EarP as a model system for studying molecular complications in horizontal gene transfer

In order to investigate if any interaction occurs between EF-P PTM systems and EF-Ps that are not their natural targets, we first had to pick one of the three known PTMs of EF-P to focus on ((R)-β-lysylation, 5-aminopentanolylation, or α-rhamnosylation). We selected α-rhamnosylation for three reasons. One, the rhamnosylation pathway is comparatively straightforward and particularly amenable to HGT, as it is carried out by a single enzyme, EarP. Additionally, the enzymes required to synthesize the substrate of EarP, dTDP-β-L-rhamnose, are well distributed throughout the bacterial tree of life [7] as rhamnose is a common component of bacterial cell walls [33]. Two, EarP appears to be relatively promiscuous with regard to its EF-P substrates; EF-P which co-occur with EarP exhibit a relatively high degree of heterogeneity in the sequence of the conserved loop [20,34]. EarP can tolerate several substitutions in this region and still successfully produce rhamnosylated EF-P [20]. Three, there are genomic clues that the transfer of EarP into a new species may lead to off-target effects. Specifically, the horizontal transfer of EarP into the phylum Thermotogota was associated with loss of well-conserved polyproline motifs, a potential sign of global EF-P dysfunction [29].

Once we decided to focus on EarP, we next made a phylogenetic tree of EF-P sequences across the bacterial tree of life in order to find EF-P that were not natively rhamnosylated yet potentially could be. These EF-Ps should encode an arginine at the tip of the conserved loop in domain I of EF-P but should not co-occur with EarP. These analyses are complicated by the fact that while unmodified EF-P have been verified experimentally [5,27,28], an unmodified EF-P and an EF-P modified by an unknown pathway are impossible to differentiate with purely genomic data. In our dataset of 3588 EF-P from across 30 phyla, we identified 741 EF-P which encoded an arginine at the conserved position (Figure 2). We eliminated 535 of these EF-P as potential targets as they either co-occurred with EarP (291 proteins) or were identified as the paralogous EF-P like protein EfpL, which we did not initially consider as a valid target (244 proteins) [5]. This left a pool of 206 arginine-type EF-Ps across 14 distinct phyla that had no known post-translational modification system. From this pool, we selected 8 EF-P for further study, prioritizing varied sequence composition in the loop region and phylogenetic diversity (Table 1, and annotated on the tree in Figure 2). Two of these EF-P, those from G. ferrihydriticus and N. communis, co-occurred with another EF-P while all others were the sole encoded EF-P of the genome.

Table 1) Table with further details on each species and the exact EF-P loop sequence of each EF-P. The first two entries are two examples of the known arginine EF-P variants for comparison. The conserved arginine residue that is modified by rhamnosylation in EarP type EF-Ps is in bold.

New arginine-type EF-P variants bypass the need for post-translational activation

To analyze our target EF-P, we first needed to ensure that they could be expressed and were functionally active without an accompanying modification system in our model organism E. coli. We confirmed successful expression through mass spectrometry (Figure 5 & Supplemental Figure 3) for 6 of the 8 EF-P. Notably, despite several tactics, we could not express the EF-P from the Verrucomicrobia A. muciniphila, nor the Firmicute C. inoculum. Next, we screened the remaining EF-P for activity in E. coli using two different methods.

First, we tested the ability of these EF-P to restore a normal growth phenotype to an E. coli mutant strain deficient in polyproline synthesis. Specifically, we used the E. coli double knockout strain DefpDuup, which is missing the primary EF-P of E. coli, as well as the backup polyproline synthesis system Uup [35–37]. This strain has a more pronounced phenotype compared to the E. coli single knockout strain Defp (doubling time of 48.9 minutes for DefpDuup compared to 27.3 minutes in Defp) [5]. We trans-complemented this strain with our six remaining EF-P and measured colony size of all combinations after 18 hours of growth at 37° C (Figure 3). Strikingly, the EF-P from Deinococcus radiodurans (EF-P_Dera) and Nitrosomonas communis (EF-P_Nico) replaced EF-P_Eco nearly to wildtype levels, restoring colony size on average to 80.1% and 71.4% of the EF-P_Eco trans-complementation, respectively.

Next, we screened our heterologously expressed EF-Ps specifically for activity in polyproline synthesis in a Δefp E. coli BW25113 background using our well described in vivo luminescent reporter (Figure 4) [5,38]. With this assay, we can test the “strength” of specific amino acid motifs by linking translational pausing to luminescence (further details in Methods). In practice, this means the stronger the detected luminescence, the stronger the translational pause caused by a particular combination of amino acids. We tested the ability of each EF‑P to rescue ribosomal pausing at a diverse set of motifs: a strong stalling motif featuring three consecutive prolines (LPPP), a weak stalling motif featuring two consecutive prolines (TPPH), and a control motif featuring a single proline which does not cause any stalling (RPDG) [5]. The results of these in vivo assays echoed our findings in the colony size complementation assay; three EF-P were hardly functional (those from H. aurantiacus, M. prima, and D. acetiphilus) and three could rescue polyproline synthesis in E. coli (those from D. radiodurans, N. communis, and G. ferrihydriticus). Interestingly, for the TPPH stalling motif, EF-P_Dera and EF-P_Nicoperformed significantly better than EF-P_Eco (P < 0.05, T test). However, these same EF-Ps performed poorly when tested against the LPPP motif (Figure 4). Variance in EF-P performance across different amino acid motifs has been observed in other EF-P subtypes [5,27]. For example, while the primary EF-P of E. coli rescues ribosomal stalling at most motifs, a subset of motifs are rescued more efficiently by the EF-P paralog, EfpL [5]. Similarly, unmodified lysine EF-Ps from the PGKGP subgroup can restore most, but not all polyproline synthesis when expressed heterologously in E. coli [27]. These observations suggest that differences in the loop region among EF-P subtypes leads to functional variability, particularly in their capacity to reposition specific tRNA combinations within the peptidyl transferase center.

Generally speaking, we found that three EF-P functioned well in E. coli (EF-P_Dera, EF-P_Nico, and EF-P_Gefe), and three struggled to function (those from H. aurantiacus, M. prima, and D. acetiphilus; EF-P_Heau, EF-P_Mepr, and EF-P_Deac). In some cases, these latter EF-Ps even had a detrimental impact on polyproline synthesis (see Figure 4, LPPP). These findings imply that either these EF-Ps 1) need an unknown modification to function properly, similar to the detrimental effect of un-rhamnosylated EF-P from P. putida and S. oneidensis on E. coli [20], or that 2) these EF-P are simply too distantly related to function effectively in E. coli. We found stronger support for the second possibility: the functional performance of our foreign EF-P in E. coli was generally inversely correlated with their phylogenetic distance from E. coli (Supplemental Figure 1). 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 findings 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].

However, there were two clear exceptions to this general trend. In the first, we found that despite the substantial phylogenetic distance between D. radiodurans and E. coli (Supplemental Figure 1), EF-P_Dera consistently performed well in our assays. One possible explanation for these results is that EF-P_Dera may have been acquired via horizontal gene transfer from a lineage more closely related to E. coli, which could explain its higher level of function in our host background and its relatively high amino acid percent identity to EF-P_Eco (44.9%, Supplemental Figure 2). In the second, we found that the link between phylogenetic distance and EF-P function broke down in the specific context of our TPPH motif rescue experiments (Supplemental Figure 1). Here, we found no correlation between EF-P function and phylogenetic distance (P = 0.915, cor = -0.046, Pearson’s correlation). This divergence from the broader pattern was largely driven by the strong performance of EF-P_Dera and the relatively good performance of distantly related EF-P (EF-P_Heau and EF-P_Mepr). These findings suggest that, although phylogenetic distance can be a useful predictor of heterologous EF-P functionality in E. coli, specific tRNA within the peptidyl transferase center can override these general trends and produce more nuanced, context-dependent outcomes.

Promiscuous EarP activity targets the non-cognate EF-P from M. prima

Next, we wanted to know whether our six target EF-P could be post-translationally modified by EarP. Here, we aimed to simulate complications resulting from the horizontal transfer of a PTM system into a new physiological background. Therefore, we purified each EF-P after co-expression with EarP (from Shewanella oneidensis, EarP_Shon) in a wildtype LMG194 E. coli background and measured the molecular weight of these EF-P with mass spectrometry. We excluded the EF-P from D. acetiphilus from these experiments, as it consistently failed to rescue polyproline synthesis in E. coli (Figure 4). As a control, we also included the EarP-type EF-P from Pseudomonas putida (EF-P_Ppu) to test the efficiency of cross-species post-translational modification with EarP_Shon.

We found that the molecular weight of four of our target EF-P matched the figure calculated from their sequence and did not vary upon co-expression with EarP_Shon (Supplemental Figure 3), indicating that no modification occurred. These EF-P included those from D. radiodurans, H. aurantiacus, N. communis, and G. ferrihydriticus. We also found no change in molecular weight upon co-expression of EarP_Shon for our negative control, EF-P_Eco. This EF-P encodes a lysine at the position of post-translational modification and therefore cannot be modified by EarP. These results verified that EF-P_Dera and EF-P_Nico function well in E. coli without the need for any post-translational modification and imply that these EF-P may also be unmodified in their native host species.

Interestingly, one of our heterologous EF-P showed an altered mass upon co-expression of EarP_Shon; that of M. prima (EF-P_Mepr, Figure 5). While the His6-tagged EF-P_Mepr has an expected monoisotopic mass of 21935.07 Da, upon co-expression of EarP_Shon a peak appeared at a mass of 22081.15 Da. This corresponds to an increase in mass of 146.08 Da, equivalent to the attachment of a rhamnose moiety (146.06 Da) [7]. We found a similar pattern in our positive control for rhamnosylation—EF-P_Ppu also showed an increase in mass of roughly 146 Da upon co-expression of EarP_Shon (Figure 5). However, while EF-P_Ppu was fully rhamnosylated—as shown by the disappearance of the molecular weight corresponding to its unmodified form (Figure 5)—EF-P_Mepr was partially rhamnosylated, with a relative abundance of 20.06% compared to that of the unmodified mass.

These results raise the question: why were so few EF-P variants successfully modified by EarP_Shon, despite prior evidence that EarP can tolerate variation in the EF-P loop region [20,26]? One hypothesis is that evolutionary optimization for rhamnosylation and for unmodified EF-P function impose conflicting selective pressures. Mutations that enhance the efficiency of rhamnosylation or improve the function of the modified EF-P may simultaneously impair the activity of its unmodified counterpart. This tradeoff is supported by the poor performance of unrhamnosylated EF-P_Shon and EF-P_Ppuin E. coli [7,20,26], suggesting that these EF-P variants have evolved a dependency on their modification for proper function. In the absence of rhamnosylation, their activity drops dramatically. Together, these findings suggest that EF-P subtypes become functionally locked into their respective post-translational modification (PTM) systems, and that switching between modification pathways or from a natively unmodified to a modified form may incur fitness costs.

Off-target rhamnosylation has diverging impacts on the function of unmodified arginine-type EF-Ps

To assess the impact of the non-native rhamnosylation of EF-P_Mepr in more detail, we repeated previous experiments with co-expression of EarP_Shon or an empty control plasmid for EF-P_Mepr, EF-P_Eco (negative control), EF-P_Shon and EF-P_Ppu (positive controls). For the complementation assay, co-expression of EarP_Shon and EF-P_Eco had no impact on colony size, as expected (Figure 6). On the other hand, co-expression of EarP_Shon greatly enhanced colony size in the EF-P_Shon and EF-P_Ppu controls (Figure 6). This is also to be expected, as these EF-P are natively rhamnosylated and can be detrimental to E. coli in their un-rhamnosylated forms [7,20]. Lastly, co-expression with EarP_Shon significantly affected the EF-P_Mepr trans-complementation but resulted in only a very slight increase in average colony size (Figure 6).

Next, we repeated the in vivo stalling‐rescue assay with our luminescent reporter. Co‐expression of EarP_Shon had no significant impact on EF-P_Eco (Figure 7), but dramatically improved EF-P_Ppu rescue: unmodified EF-P_Ppu had a detrimental impact on polyproline synthesis (LPPP = 0.60, TPPH = 0.77), whereas rhamnosylated EF-P_Ppu performed well (LPPP = 2.48, TPPH = 1.57), consistent with Lassak et al. (2015) [7]. EF-P_Mepr also gained a modest benefit from EarP_Shon in the assay with LPPP (from 0.38 → 1.18), but only to levels equivalent to those seen in the Δefp strain (Figure 7). In other words, rhamnosylation of EF-P_Mepr merely mitigated its deleterious effect and did not enhance activity. These data suggest that adding a non-native rhamnosyl modification disrupts the interaction between EF-P_Mepr and the E. coli ribosome, possibly due to incompatibility at the peptidyl-transferase center.

Lastly, we sought to explore our proposed explanation for how rhamnosylation affects EF-P_Mepr on the molecular level, specifically in its interaction with the tRNA at the P-site of the peptidyl transferase center. Due to the lack of experimental structures of EF-P_Mepr and in particular rhamnosylations, we used structural modeling to suggest and compare the positioning of β3Ωβ4 loop tip regions in the following three variants: unmodified EF-P_Mepr, rhamnosylated EF-P_Mepr, and the naturally rhamnosylated EF-P from Pseudomonas aeruginosa (EF-P_Paer), in complex with tRNA^Pro (Figure 8). Our models suggest that while the unmodified EF-P_Meprcan form several favorable contacts with tRNA^Pro, these contacts are abolished upon the addition of the rhamnose modification, which introduces a steric clash (Figure 8). By contrast, based on the available high-resolution crystal structure [40] the β3Ωβ4 loop of EF-P_Paer is oriented in such a way as to take full advantage of the addition of the rhamnose moiety, leading to additional favorable contacts (Figure 8). While a full proof for these models can only come from respective experimental structures, these observations suggest that rhamnosylation supports EF-P function when co-evolved with the protein structure, as is the case with EF-P_Paer. In contrast, the rhamnosylation of EF-P_Mepr is predicted to disrupt the protein’s function despite a high overall structural conservation among EF-P across species. These results are in line with our experimental data: while EF-P_Meprshows some limited activity in polyproline synthesis (Figure 7 TPPH) and provides minor trans-complementation benefits in E. coli (Figure 6), rhamnosylation did not improve its function, but merely reduced detrimental effects (Figure 7 LPPP, RPDG). Altogether, these results support our hypothesis that the beneficial application of rhamnosylation requires adaptation of the critical EF-P loop region, and that simply adding a non-native modification is unlikely to immediately enhance function.

It is only natural that EF-P_Mepr is adapted to support translation in M. prima, rather than in E. coli. Given the evolutionary distance between these species, it’s likely that the ribosome of M. prima differs structurally from that of E. coli. We have shown that upon interaction with EarP_Shon, a substantial fraction of EF-P_Mepr is rhamnosylated and that this modification has a significant impact on the function of EF-P_Mepr, inasmuch as it can be measured in E. coli. This set of experiments, measuring the impact of EarP_Shon on EF-P_Mepr, mimics historical horizontal gene transfer events that actually occurred in the phylum Thermotogota. Long ago, members of the genera Geotoga and Oceanotoga acquired an EarP and an EarP-type EF-P which has since replaced their ancestral form of EF-P [29]. The ancestral EF-P in these genera was likely similar to the EF-P in their sister family, the Kosmotogaceae, to which M. prima belongs. It is therefore reasonable to infer that the initial introduction of an “invading” EarP into the Geotoga and Oceanotoga caused off-target rhamnosylation of their native EF-P, likely with negative functional consequences. These molecular mismatches would help explain the genomic signatures which coincide with this horizontal transfer event [29].

A contrasting illustration of this “invading-gene” scenario comes from the EF-P paralog EfpL, which we initially excluded as a target as it is a “secondary EF-P”, and always paired with an EF-P with a wider motif spectrum [5]. Although it is not natively post-translationally modified in E. coli, we surprisingly found that exposure to heterologously expressed EarP also leads to partial rhamnosylation of EfpL (Supplemental Figure 4). Importantly, in our in silico model, this modification had no detectable effect on EfpL activity in E. coli, presumably as a result of its unique β3Ωβ4 architecture [5] (Supplemental Figure 5). On the one hand, this indicates that, unlike EF-P_Mepr, the rhamnosylation of EfpL does not compromise its function. On the other hand, EfpL does not gain a benefit from the rhamnose moiety, as it is predicted to be positioned too far from the tRNA for any meaningful interaction (Supplemental Figure 5). To test whether our model could be supported experimentally, we investigated the effect of rhamnosylation of EfpL in our trans-complementation experiment. Once again, we measured colony size of our ΔefpΔuup strain in the presence of earP alone, efpL alone or upon co-expression of both. Expression of efpL increased colony size significantly. In line with our in silico model, we also found that rhamnosylation did not affect EfpL’s complementation efficiency (Supplemental Figure 6). This observation also aligns with the phylogenetic placement of EfpL as a sister clade to the EarP type EF-P (Figure 2) and evolutionary observations inferred from former analyses [20], which suggest that an EfpL-like ancestor was initially tolerant to rhamnosylation. Only later, a shortening of the extended EfpL loop—presumably by a single proline—created new structural contacts and, likely, a new EF-P functionality. This evolutionary change may have enabled the subsequent fixation of EarP-dependent EF-Ps in some lineages. Thus, the fate of an “invading” PTM system can depend not only on potential incompatibility, but also on whether it creates an opportunity for novel function.

Taken together, our findings indicate that the horizontal transfer of PTM machinery can produce complex, host-dependent outcomes: initial interference with host proteins followed by successful integration after a period of adaption in some lineages, such as the Thermotogota, and evolutionary opportunity in others, such as EfpL. This duality adds a rich new dimension to our understanding of HGT and cellular adaptation.

In conclusion, our study highlights contrasting evolutionary paths that can follow horizontal gene transfer, using horizontally transferred EF-P variants and their associated PTM machinery in foreign bacterial hosts as a study system. We demonstrated that certain EF-P proteins, including those from D. radiodurans,G. ferrihydriticus and N. communis, function efficiently in Escherichia coli without any post-translational modifications, further expanding known classes of unmodified EF-P. Importantly, we showed that the promiscuous rhamnosylation enzyme EarP can modify non-cognate EF-P proteins, as exemplified by M. prima (EF-P_Mepr). This off-target modification impaired rather than enhanced the functionality of EF-P_Mepr, underscoring the potential for PTM machinery introduced through horizontal gene transfer to disrupt existing cellular processes. However, we also identified a contrasting scenario involving the EF-P paralog EfpL from E. coli. Intriguingly, despite its native state as an unmodified protein, partial rhamnosylation of EfpL by EarP was also possible, and did not interfere with its functionality, possibly opening a path for evolutionary adaption. Thus, our work emphasizes the complex interplay between evolutionary constraints and functional innovation during gene integration.

Selection of EF-P candidates & phylogenetic trees

To identify EF-P candidates to test experimentally, we first extracted all efp genes found within a pre-validated set of 3273 bacterial genomes across 30 phyla [41]. Specifically, we used the hmmsearch command from HMMER v. 3.4 to extract genes that contained all three EF-P PFAM domains: PF01132 (EF-P OB domain), PF09285 (EF-P C-terminal), and PF08207 (EF-P KOW-like domain). We next aligned these protein sequences using default parameters in MUSCLE v. 5.1 and extracted the sequence of the conserved loop in domain I of the protein, using validated EF-P sequences as guides.

We next searched for genes related to the post-translational modification of EF-P, with the goal of identifying EF-P that do not co-occur with genes tied to a known modification. To do this, we followed parameters used in previous work [29]. To visualize the distribution of our target EF-P, we trimmed the MUSCLE aligned EF-P amino acid sequences using trimAl with a gap threshold of 0.1 [42] and used iqtree2 v. 2.3.6 [43] to build a phylogenetic tree, using the evolutionary model recommended by the ModelFinder feature [44]. We plotted this tree in R using the package ggtree v. 3.14.0 [45].

We created a second phylogenetic tree to obtain evolutionary distances between the different species used in this study (used in Supplemental Figure 1). This tree was created using Markerfinder to extract and concatenate 40 marker genes [46] from the genome of each species, MUSCLE to align these sequences, trimAl to trim the alignment, and finally iqtree2 to build the tree. We extracted phylogenetic distances between each species on this tree using the cophenetic function in base R.

Plasmid construction

All strains, plasmids, and oligonucleotides used in this study are listed and described in Supplementary Data 1. We isolated plasmid DNA using the Zyppy® Plasmid Miniprep Kit (Zymo Research) and purified PCR reactions using the DNA Clean & Concentrator®-5 DNA kit (Zymo Research) or the Zymoclean® Gel DNA Recovery Kit (Zymo Research). We purchased all polymerases for PCR amplification from New England BioLabs (NEB), and we used all kits and enzymes according to the manufacturer’s instructions.

We constructed plasmids for the expression of C-terminally His6-tagged efp genes under the control of an arabinose inducible promoter by first amplifying the corresponding genes from genomic DNA sourced from the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. We used primers specified in Supplementary Data 1. We then purified and cloned these DNA fragments into the pBAD24 vector [47] using the NEBuilder® HiFi DNA Assembly Master Mix (NEB).

Growth conditions

We routinely grew E. coli cells in Miller-modified lysogeny broth (LB) [48,49] or super optimal broth (SOB) [50] at 37 °C aerobically under agitation unless otherwise indicated. We measured growth using optical density at a wavelength of 600 nm (OD600). When required, we added 1.5% (w/v) agar to solidify media, and if needed, we added antibiotics at the following concentrations: 100 µg/ml carbenicillin sodium salt (pBAD24) or 25 µg/ml chloramphenicol (pBAD33). We induced plasmids carrying the P_BAD [47]promoter with L(+)-arabinose at a final concentration of 0.2% (w/v).

Measurement of colony size

To measure the ability of our foreign EF-Ps to restore polyproline synthesis in a deficient strain we first transformed the pBAD24 vectors carrying each efp into the E. coli double deletion strain DefpDuup. This strain was constructed in previous work [5], and has a strong growth deficit. We plated our transformants onto LB agar containing 100 µg/ml carbenicillin sodium salt and 0.2% L(+)-arabinose to induce expression and incubated them for up to 24h at 37 °C. We used ImageJ version 1.54g [51] to measure the diameter of each colony. We excluded colonies that were outside the 5^th and 95^th percentile in size and only considered up to the 300 largest colonies within this range. For the version of the experiment involving co-production of EarP, we added the pBAD33 vector carrying an arabinose inducible copy of earP and added 25 µg/ml chloramphenicol to our plates [26]. Alternatively, earP was expressed under control of its native constitutive promoter from an pBBR1MCS2 backbone [26].

Measurement of pausing strength in vivo

We measured the pausing strength of specific amino acid motifs using a previously developed luminescent reporter based on the E. coli histidine operon attenuation mechanism [38]. In this system, high histidine levels allow uninterrupted translation of the leader peptide HisL, forming a transcription-terminating stem loop that prevents expression of the downstream operon hisGDCBHAF. Low histidine causes ribosomal pausing at HisL, enabling transcription of the downstream biosynthesis operon. Our reporter fuses HisL and the 5′ UTR of hisGDCBHAF with the luxCDABE operon, integrated into the E. coli genome via single homologous recombination [52,53]. Test motifs are inserted into HisL and ribosome pausing at these motifs increases luminescence by promoting lux expression.

The strains used in this study, representing a strong stalling motif (featuring three consecutive prolines, LPPP), a weak stalling motif (featuring two consecutive prolines, TPPH) [54] , and a proline containing motif that does not cause stalling (RPDG) [5] were constructed in previous work using the aforementioned methods [5,38]. We measured the pausing strength of these motifs by detecting luminescence with a Tecan Infinity® plate reader using the following parameters: absorption at 600 nm (number of flashes: 10; settle time: 50 ms) and luminescence emission (attenuation: none; settle time: 50 ms; integration time: 200 ms) in between 10-min cycles of agitation (orbital: 180 rpm; amplitude: 3 mm) for around 16 h.

Protein overproduction and purification

To prepare proteins for mass spectrometry analysis, we overproduced C-terminally His6-tagged EF-Ps using the pBAD24 vector in E. coli LMG194 grown in SOB. We added 0.2% (w/v) L(+)-arabinose to induce gene expression during exponential growth and grew cells overnight at 18 °C. On the next day, we harvested these cells by centrifugation and resuspended the cell pellet in in 100 mM sodium phosphate buffer at pH 7.6. We then lysed cells using a continuous-flow cabinet (Constant Systems Ltd.) at 1.35 kbar and clarified our lysates by centrifugation at 4 °C at 234 998 × g for 1 h. Next, we purified our His₆-tagged proteins using Ni-NTA beads (Qiagen). We washed the beads and attached proteins using the sodium phosphate buffer plus 20mM imidazole and used 250mM imidazole for elution. Finally, we dialyzed the purified proteins overnight in the 100 mM sodium phosphate buffer (with one buffer change) to remove any lingering imidazole. We used SDS-PAGE to check the size and purity of our overproduced C-terminally His₆-tagged EF-Ps. For these analyses, we used 12.5% (w/v) SDS and stained with InstantBlue Coomassie Protein stain (Abcam).

Mass spectrometry for identification of modification status

For top-down EF-P measurements we desalted our proteins on the ZipTip with C4 resin (Millipore, ZTC04S096) and eluted with 50 % (v/v) acetonitrile 0.1 % (v/v) formic acid (FA) buffer resulting in ~10 μM final protein concentration in 200–400 μl total volume. We performed MS measurements on an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Fisher Scientific) via direct injection, a HESI-Spray source (Thermo Fisher Scientific) and FAIMS interface (Thermo Fisher Scientific) in a positive, peptide mode. Typically, we searched the FAIMS compensation voltage (CV) by a continuous scan. The most intense signal was usually obtained at -25 CV. We acquired the MS spectra with at least 120,000 FWHM, AGC target 100 and 2-5 microscans and deconvoluted the spectra in Freestyle (Thermo) using the Xtract Deconvolution algorithm.

To investigate the effect of EarP overproduction on the E. coli proteome, we heterologously produced EarP from Pseudomonas putida KT2440 (locus tag PP_1857) from an arabinose inducible promoter (P_BAD) using pBAD33 as vector backbone [47]; pBAD33PP1857-His6 [26]. We induced expression of earP and clarified lysates as described above. We performed MS analysis on these lysates following tryptic digestion and desalting on SDB-RPS StageTips [55]. We performed measurements on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled to an EasynLC 1200 nanoflow liquid chromatography system (Thermo Fisher Scientific). Peptides were separated within 120 min on a 75 µm x 50 cm in-house C₁₈ column at a flow rate of 300 nL/min with an active gradient from 5-30% B (80% acetonitrile/0.1% formic acid). Data were acquired in a data-dependent mode (top15) with the Orbitrap resolution set to 60,000 for MS1, and 15,000 for MS2 scans. Suitable precursor ion with charge states 2-5 were isolated in a 1.4 Th window and the maximum ion injection time was 28 ms to reach an AGC target of 100%. We processed the MS raw files with FragPipe v18.0, Philosopher v4.4.0 and MSFragger v3.5 [56–58]. We searched the resulting spectra against an E. coli reference proteome (UP000000625) with cysteine carbamidomethylation as a fixed modification; and N-terminal acetylation, methionine oxidation, and arginine modified with C₆H₁₀O₄ (146.0579 Da, rhamnose-H₂O) as variable modifications. We set the maximum absolute precursor mass tolerance to 20 ppm and filtered our results at 1% false discovery rate at the peptide spectrum match and protein level.

Modeling rhamnosylation of EF-P_Mepr, EF-P_Paer, and EfpL_Eco

We carried out all modeling and in silico modification in Pymol using release 2.5.5 (Schrödinger). We created structural models displaying the effects of rhamnosylation on interactions between EF-P_Mepr, EF-P_Paer or E. coli EfpL and the tRNA CCA by structurally aligning the respective EF-P proteins with PDB entry 6enj showing the full polyproline stalled E. coli ribosome [59]. For the E. coli EfpL we used the previously determined crystal structure (PDB entry 8s8u [5]), while for EF-P_Paer we used PDB entry 3oyy [40]. We used an AF3 model [60] of the full-length proteinas input for EF-P_Mepr, which showed a very high overall pLDDT confidence across the structured protein parts, and a good confidence in the relevant loop region. To account for possible slight deviations in the EF-P/EfpL tip/tRNA CCA region of different bacterial ribosomes, we confined alignments to the respective EF-P loops and the CCA trinucleotide, keeping the position of CCA. We inserted rhamnose modifications manually in a conformation according to https://www.ebi.ac.uk/chebi/searchId.do?chebiId=167445 based on the arginine sidechain conformation as given in the three structures. We derived polar contacts between the modified or unmodified loop tip residues automatically from the software.

Funding

This research was supported by the Swiss National Science Foundation (Grant number 210991 to TB). JL is grateful to the DFG (LA 3658/1-3). Protein mass spectrometry was supported by DFG grant SFB1309 – 325871075 to PK.

Acknowledgements

We thank Ralph Krafczyk for early work on rhamnosylation and Kirsten Jung, Alina Sieber, and Urte Tomasiunaite for helpful discussions. We particularly thank Giovanni Gallo for valuable suggestions throughout the project.

Authors’ contributions

TEB conceived the project together with JL. TEB and JL designed the experiments. TEB performed all experiments and computational analyses unless otherwise noted. PK was responsible for all mass spectrometry unless otherwise noted. AS performed modelling. JS performed in vivo studies with EfpL and FMR performed EfpL specific mass spectrometry. TEB and JL wrote the paper, which was edited by all authors.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All genomes used in this study are publicly available from JGI's IMG database [61]. R scripts and all files needed to reproduce these analyses, and most figures are available at: https://github.com/tessbrewer/arginine_ptms.

Good BH, Bhatt AS, McDonald MJ. Unraveling the tempo and mode of horizontal gene transfer in bacteria. Trends Microbiol. 2025;
Lassak J, Wilson DN, Jung K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A. Mol Microbiol. 2016;99:219–35.
Ude S, Lassak J, Starosta AL, Kraxenberger T, Wilson DN, Jung K. Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches. Science (1979). 2013;339:82–5.
Doerfel LK, Wohlgemuth I, Kothe C, Peske F, Urlaub H, Rodnina M V. EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues. Science (1979). 2013;339:85–8.
Sieber A, Parr M, von Ehr J, Dhamotharan K, Kielkowski P, Brewer T, et al. EF-P and its paralog EfpL (YeiP) differentially control translation of proline-containing sequences. Nat Commun. 2024;15:10465.
Tanner DR, Cariello DA, Woolstenhulme CJ, Broadbent MA, Buskirk AR. Genetic Identification of Nascent Peptides That Induce Ribosome Stalling. Journal of Biological Chemistry. 2009;284:34809–18.
Lassak J, Keilhauer EC, Fürst M, Wuichet K, Gödeke J, Starosta AL, et al. Arginine-rhamnosylation as new strategy to activate translation elongation factor P. Nat Chem Biol. 2015;11:266–70.
Tollerson R, Witzky A, Ibba M. Elongation factor P is required to maintain proteome homeostasis at high growth rate. Proc Natl Acad Sci U S A. 2018;115:11072–7.
Peng WT, Banta LM, Charles TC, Nester EW. The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis. J Bacteriol. 2001;183:36–45.
Rajkovic A, Hummels KR, Witzky A, Erickson S, Gafken PR, Whitelegge JP, et al. Translation control of swarming proficiency in Bacillus subtilis by 5-amino-pentanolylated elongation factor P. Journal of Biological Chemistry. 2016;291:10976–85.
Navarre WW, Zou SB, Roy H, Xie JL, Savchenko A, Singer A, et al. PoxA, YjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. Mol Cell. 2010;39:209–21.
Rajkovic A, Erickson S, Witzky A, Branson OE, Seo J, Gafken PR, et al. Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa. mBio. 2015;6.
Hummels KR, Kearns DB. Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P. PLoS Genet. 2019;15:e1008179.
Guo Q, Cui B, Wang M, Li X, Tan H, Song S, et al. Elongation factor P modulates Acinetobacter baumannii physiology and virulence as a cyclic dimeric guanosine monophosphate effector. Proceedings of the National Academy of Sciences. 2022;119.
Yanagisawa T, Takahashi H, Suzuki T, Masuda A, Dohmae N, Yokoyama S. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability. PLoS One. 2016;11:e0147907.
Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003;48:77–84.
Rubin BE, Wetmore KM, Price MN, Diamond S, Shultzaberger RK, Lowe LC, et al. The essential gene set of a photosynthetic organism. Proceedings of the National Academy of Sciences. 2015;112.
Rivas-Marin E, Moyano-Palazuelo D, Henriques V, Merino E, Devos DP. Essential gene complement of Planctopirus limnophila from the bacterial phylum Planctomycetes. Nat Commun. 2023;14:7224.
Hummels KR, Kearns DB. Translation elongation factor P (EF-P). FEMS Microbiol Rev. 2020;44:208–18.
Volkwein W, Krafczyk R, Jagtap PKA, Parr M, Mankina E, Macošek J, et al. Switching the post-translational modification of translation elongation factor EF-P. Front Microbiol. 2019;10.
Peil L, Starosta AL, Virumäe K, Atkinson GC, Tenson T, Remme J, et al. Lys34 of translation elongation factor EF-P is hydroxylated by YfcM. Nat Chem Biol. 2012;8:695–7.
Roy H, Zou SB, Bullwinkle TJ, Wolfe BS, Gilreath MS, Forsyth CJ, et al. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-β-lysine. Nat Chem Biol. 2011;7:667–9.
Witzky A, Hummels KR, Tollerson R, Rajkovic A, Jones LA, Kearns DB, et al. EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis. mBio. 2018;9.
Hummels KR, Witzky A, Rajkovic A, Tollerson R, Jones LA, Ibba M, et al. Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF-P modification state. Mol Microbiol. 2017;106:236–51.
Li X, Krafczyk R, Macošek J, Li Y-L, Zou Y, Simon B, et al. Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first Arg Rha specific antibody. Chem Sci. 2016;7:6995–7001.
Krafczyk R, Macošek J, Jagtap PKA, Gast D, Wunder S, Mitra P, et al. Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P. mBio. 2017;8.
Tomasiunaite U, Kielkowski P, Krafczyk R, Forné I, Imhof A, Jung K. Decrypting the functional design of unmodified translation elongation factor P. Cell Rep. 2024;43:114063.
Pinheiro B, Scheidler CM, Kielkowski P, Schmid M, Forné I, Ye S, et al. Structure and Function of an Elongation Factor P Subfamily in Actinobacteria. Cell Rep. 2020;30:4332-4342.e5.
Brewer TE, Wagner A. Horizontal Gene Transfer of a key Translation Factor and its Role in Polyproline Proteome Evolution. Mol Biol Evol. 2024;41.
Brockhurst MA, Harrison E, Hall JPJ, Richards T, McNally A, MacLean C. The Ecology and Evolution of Pangenomes. Current Biology. Cell Press; 2019. p. R1094–103.
Kanhere A, Vingron M. Horizontal gene transfers in prokaryotes show differential preferences for metabolic and translational genes. BMC Evol Biol. 2009;9.
Sorek R, Zhu Y, Creevey CJ, Francino MP, Bork P, Rubin EM. Genome-Wide Experimental Determination of Barriers to Horizontal Gene Transfer. Science (1979). 2007;318:1449–52.
Maki M. Biosynthesis of 6-deoxyhexose glycans in bacteria. Glycobiology. 2003;14:1R – 15.
Yakovlieva L, Wood TM, Kemmink J, Kotsogianni I, Koller F, Lassak J, et al. A β-hairpin epitope as novel structural requirement for protein arginine rhamnosylation. Chem Sci. 2021;12:1560–7.
Takada H, Fujiwara K, Atkinson GC, Chiba S, Hauryliuk V. Resolution of ribosomal stalling by EF-P and ABCF ATPases YfmR and YkpA/YbiT. Nucleic Acids Res. 2024;52:9854–66.
Hong H-R, Prince CR, Tetreault DD, Wu L, Feaga HA. YfmR is a translation factor that prevents ribosome stalling and cell death in the absence of EF-P. Proceedings of the National Academy of Sciences. 2024;121.
Chadani Y, Yamanouchi S, Uemura E, Yamasaki K, Niwa T, Ikeda T, et al. The ABCF proteins in Escherichia coli individually cope with ‘hard-to-translate’ nascent peptide sequences. Nucleic Acids Res. 2024;52:5825–40.
Krafczyk R, Qi F, Sieber A, Mehler J, Jung K, Frishman D, et al. Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria. Commun Biol. 2021;4:589.
Kacar B, Garmendia E, Tuncbag N, Andersson DI, Hughes D. Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio. 2017;8.
Choi S, Choe J. Crystal structure of elongation factor P from Pseudomonas aeruginosa at 1.75 Å resolution. Proteins. 2011;79:1688–93.
Brewer TE, Wagner A. Translation stalling proline motifs are enriched in slow-growing, thermophilic, and multicellular bacteria. ISME Journal. 2022;16:1065–73.
Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–3.
Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37:1530–4.
Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14:587–9.
Yu G, Lam TT-Y, Zhu H, Guan Y. Two Methods for Mapping and Visualizing Associated Data on Phylogeny Using Ggtree. Mol Biol Evol. 2018;35:3041–3.
Sunagawa S, Mende DR, Zeller G, Izquierdo-Carrasco F, Berger SA, Kultima JR, et al. Metagenomic species profiling using universal phylogenetic marker genes. Nat Methods. 2013;10:1196–9.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177:4121–30.
Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951;62:293–300.
Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory; 1972.
Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983;166:557–80.
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.
Lassak J, Henche A-L, Binnenkade L, Thormann KM. ArcS, the Cognate Sensor Kinase in an Atypical Arc System of Shewanella oneidensis MR-1. Appl Environ Microbiol. 2010;76:3263–74.
Fried L, Lassak J, Jung K. A comprehensive toolbox for the rapid construction of lacZ fusion reporters. J Microbiol Methods. 2012;91:537–43.
Peil L, Starosta AL, Lassak J, Atkinson GC, Virumäe K, Spitzer M, et al. Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P. Proc Natl Acad Sci U S A. 2013;110:15265–70.
Kulak NA, Pichler G, Paron I, Nagaraj N, Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 2014;11:319–24.
Kong AT, Leprevost F V, Avtonomov DM, Mellacheruvu D, Nesvizhskii AI. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nat Methods. 2017;14:513–20.
Teo GC, Polasky DA, Yu F, Nesvizhskii AI. Fast Deisotoping Algorithm and Its Implementation in the MSFragger Search Engine. J Proteome Res. 2021;20:498–505.
da Veiga Leprevost F, Haynes SE, Avtonomov DM, Chang H-Y, Shanmugam AK, Mellacheruvu D, et al. Philosopher: a versatile toolkit for shotgun proteomics data analysis. Nat Methods. 2020;17:869–70.
Huter P, Arenz S, Bock L V, Graf M, Frister JO, Heuer A, et al. Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P. Mol Cell. 2017;68:515-527.e6.
Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:493–500.
Chen I-MA, Chu K, Palaniappan K, Ratner A, Huang J, Huntemann M, et al. The IMG/M data management and analysis system v.6.0: new tools and advanced capabilities. Nucleic Acids Res. 2021;49:D751–63.

Table 1 is available in the Supplementary Files section.

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

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