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

Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor Tess E Brewer, Pavel Kielkowski, Jingzhi Stritzel, Florian Meier-Rosar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7374903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2026 Read the published version in BMC Biology → Version 1 posted 11 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background 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. Results and Discussion 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 D efp D uup , 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 D efp (doubling time of 48.9 minutes for D efp D uup compared to 27.3 minutes in D efp ) [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 Δ efp 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 Nico performed 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 Ppu in 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 Δ efp 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 Mepr can 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 Mepr shows 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 Δ efp Δ uup 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. Conclusions 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. Methods 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 D efp D uup . 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 His 6 -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 His 6 -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 C 18 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 C 6 H 10 O 4 (146.0579 Da, rhamnose-H 2 O) 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 protein as 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. Declarations 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. References 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. 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Additional Declarations No competing interests reported. Supplementary Files argsupplemental23Jul.pdf Table1.docx Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2026 Read the published version in BMC Biology → Version 1 posted Editorial decision: Revision requested 07 Oct, 2025 Reviews received at journal 06 Sep, 2025 Reviews received at journal 05 Sep, 2025 Reviewers agreed at journal 30 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviewers invited by journal 28 Aug, 2025 Editor assigned by journal 15 Aug, 2025 Submission checks completed at journal 15 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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14:09:00","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44290,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/63bf49cbbf9f143586f07de6.png"},{"id":93502919,"identity":"b0ccea03-1462-4c6f-80cc-45158ff6cf76","added_by":"auto","created_at":"2025-10-14 14:16:59","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52021,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/20370888e22084f0d562790c.png"},{"id":93501224,"identity":"45928eb3-940c-4c71-835a-96555da742f5","added_by":"auto","created_at":"2025-10-14 14:01:00","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151906,"visible":true,"origin":"","legend":"","description":"","filename":"411b2b4013e9485890b2018f049790ad1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/efcd002dff86c46fcaf1d0e0.xml"},{"id":93501223,"identity":"70d2c1a1-43c8-4f40-aac9-e8448a1cf0be","added_by":"auto","created_at":"2025-10-14 14:01:00","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166257,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/b578efceef07b5a30da6f066.html"},{"id":93501190,"identity":"4ba7c006-eb85-4402-9b3d-945c84b2c313","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296907,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic outlining the scenario investigated in this study. This study aims to simulate the impact of an HGT event that can introduce off-target complications to the recipient cell, using EF-P and one of its post-translational modification systems, EarP, as a study system. \u003cstrong\u003eLeft) \u003c/strong\u003eIllustration showing an idealized scenario of conjugation-based HGT of an \u003cem\u003eefp\u003c/em\u003e and \u003cem\u003eearP\u003c/em\u003e gene. \u003cstrong\u003eRight) \u003c/strong\u003eIllustration showing a hypothetical complication stemming from the lefthand panel, namely the off-target post-translational modification of the non-cognate, ‘native’ EF-P which existed before any HGT event.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/ae9f5b56dbb9bde2890ec4a4.png"},{"id":93502306,"identity":"49d01c81-5150-4811-8f0c-327e096108ce","added_by":"auto","created_at":"2025-10-14 14:08:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":526736,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of EF-P amino acid sequences with the EF-P chosen for further study\u003cem\u003e \u003c/em\u003elabelled. Color of the circles represents the phylum the species encoding the EF-P originates from (according to the Genome Taxonomy Database; GTDB), the inner ring surrounding the tree indicates which EF-P type each EF-P belongs to, and the outer ring specifically highlights EF-P which encode an arginine at the post-translationally modified position at the tip of the conserved loop in domain I (β3Ωβ4).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/a77bd4d2e420aedcd88812ca.png"},{"id":93501192,"identity":"2d86cb1a-fb72-4dc3-adc3-651e4589c1a4","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183039,"visible":true,"origin":"","legend":"\u003cp\u003eComplementation assay of \u003cem\u003eE. coli\u003c/em\u003e BW23113 mutant strain lacking \u003cem\u003eefp\u003c/em\u003e and \u003cem\u003euup\u003c/em\u003e(Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e). In strains overproducing EF-P (+EF-P). Protein production was confirmed by mass spectrometry (\u003cstrong\u003eFigure 5 \u0026amp; Supplemental Figure 3\u003c/strong\u003e). We used ImageJ to quantify the diameter of colonies on LB agar + 0.2% (w/v) arabinose plates after 18 h of cultivation at 37 °C. We excluded colonies that were outside the 5th and 95th percentile in size and only considered up to the 300 largest colonies within this range. The leftmost red dashed vertical line corresponds to the average colony size in the non-complemented Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e control, the rightmost line corresponds to the average colony size in the Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e + \u003cem\u003eE. coli\u003c/em\u003e EF-P control. All complemented strains were significantly different from both the Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e(P \u0026lt; 0.0001, Wilcoxon test) and Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e + \u003cem\u003eE. coli\u003c/em\u003e EF-P controls (P \u0026lt; 0.005, Wilcoxon test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/fd64685c2f5edd2f47963d8f.png"},{"id":93501197,"identity":"4af56b4c-9dc6-481b-9c5d-c24a8aa6494a","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e comparison of rescue efficiency of two stalling motifs (LPPP and TPPH) and the control motif RPDG. Given is the quotient of relative light units measured in Δ\u003cem\u003eefp\u003c/em\u003e and corresponding trans-complementations by different heterologous EF-P (+EF-P). Each condition contains at least 3 biological replicates. Red dashed line represents a rescue efficiency equal to that of the Δ\u003cem\u003eefp\u003c/em\u003e control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/8d19c7cdf1c1dc18e95e6e39.png"},{"id":93501198,"identity":"3df6fdb6-c92a-42d1-8458-bb5a22645141","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121231,"visible":true,"origin":"","legend":"\u003cp\u003eDeconvoluted intact protein mass spectra of the indicated His-tagged EF-P overproduced with or without co-expression of EarP from \u003cem\u003eShewanella oneidensis\u003c/em\u003e. MW\u003csub\u003efound\u003c/sub\u003e refers to the molecular weight (Da) of the dominant peak, while MW\u003csub\u003eEF-P \u003c/sub\u003erefers to the calculated monoisotopic mass of each EF-P based on the sequence of the expressed gene. Only dominant peaks and peaks of interest are labelled. Arrow represents expected increase in mass with successful rhamnosylation by EarP (+ 146.06 Da).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/886b06db652a5bc64a05d448.png"},{"id":93502915,"identity":"da3560bf-050f-408e-aca8-e1227adf803e","added_by":"auto","created_at":"2025-10-14 14:16:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":184642,"visible":true,"origin":"","legend":"\u003cp\u003eComplementation assay of \u003cem\u003eE. coli\u003c/em\u003e BW23113 mutant strain lacking \u003cem\u003eefp\u003c/em\u003e and \u003cem\u003euup\u003c/em\u003e (Δ\u003cem\u003eefp\u003c/em\u003eΔ\u003cem\u003euup\u003c/em\u003e). Same experimental set-up as in \u003cstrong\u003eFigure 3\u003c/strong\u003e, but heterologous EF-P are co-expressed with either EarP or an empty control plasmid. The \u003cem\u003eE. coli\u003c/em\u003eEF-P control should not show differences with EarP co-expression, while the \u003cem\u003eS. oneidensis\u003c/em\u003e and \u003cem\u003eP. putida\u003c/em\u003e EF-P should benefit from EarP co-expression, as these EF-P are naturally rhamnosylated. P-values are from Wilcoxon Rank Sum tests corrected for multiple comparisons with the Bonferroni method.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/c2f9ae5ab81c9176546d3161.png"},{"id":93501200,"identity":"e38283f0-7379-48a0-9eff-92f3d12a633d","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":147489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e comparison of rescue efficiency of two stalling motifs (LPPP and TPPH) and the control motif RPDG. Same general experimental set-up as in \u003cstrong\u003eFigure 4\u003c/strong\u003e, but here heterologous EF-P are co-expressed with either an empty control plasmid or EarP\u003csub\u003eShon\u003c/sub\u003e. Each condition was normalized to Δ\u003cem\u003eefp \u003c/em\u003ecarrying a control plasmid or EarP, as appropriate. The \u003cem\u003eE. coli\u003c/em\u003e EF-P control does not show differences with EarP co-expression, while the \u003cem\u003eP. putida\u003c/em\u003e EF-P benefits from EarP co-expression. Each condition contains at least 3 biological replicates. P-values are from t-tests and were corrected for multiple comparison using the Bonferroni method. Red dashed line represents a rescue efficiency equal to that of the Δ\u003cem\u003eefp \u003c/em\u003econtrol.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/af2cb2df894b7dabee805a95.png"},{"id":93501202,"identity":"969fe5ad-8bbc-4c9a-b9a6-5919b74a38a1","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":214197,"visible":true,"origin":"","legend":"\u003cp\u003eThree models showing possible alignments of EF-P with the tRNA CCA supporting the functional observations. The \u003cem\u003eMesotoga prima\u003c/em\u003e EF-P is shown in either an unrhamnosylated state (left) or rhamnosylated state (middle) while the \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e EF-P is rhamnosylated (right). The tRNA CCA is colored light blue. Dashed lines highlight polar contacts between protein and RNA with involved atoms colored by element. Structures were created with Pymol.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/12c96ad2c1d019398cd93eea.png"},{"id":101874473,"identity":"e14947d0-bb1b-4b63-af13-9c06d636f3a8","added_by":"auto","created_at":"2026-02-04 13:58:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2752997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/2dd1a8a3-0368-48db-8679-5f3f374eaba4.pdf"},{"id":93501196,"identity":"3694ee9f-fb2e-416a-b1ba-7c202b96f605","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1268226,"visible":true,"origin":"","legend":"","description":"","filename":"argsupplemental23Jul.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/702ac36c5bd70375891c9df3.pdf"},{"id":93501193,"identity":"2d354c22-8d56-40f5-91e2-e3cf4bbea401","added_by":"auto","created_at":"2025-10-14 14:00:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":92451,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7374903/v1/89cfa46611c862bc2bdcadb1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor","fulltext":[{"header":"Background","content":"\u003cp\u003eSuccessful horizontal gene transfer (HGT) involves a complex series of events\u0026mdash;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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Yet one important aspect of this process remains relatively overlooked: the influence of the host\u0026rsquo;s physiological background on the successful \u0026lsquo;domestication\u0026rsquo; of the incoming gene. In this study, we explore this critical gap through an experimental system inspired by patterns observed in genomic data\u0026mdash;namely, the interference between horizontally acquired variants of elongation factor P (EF-P), their associated post-translational modification systems, and the host bacterium\u0026rsquo;s existing translational machinery.\u003c/p\u003e\u003cp\u003eEF-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 [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Without EF-P, bacteria suffer species specific phenotypes that include reduced growth rate [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], reduced antibiotic resistance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], loss of mobility [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], loss of virulence [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and cell death [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn many species, EF-P must be post-translationally modified to reach full activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. 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 \u0026ldquo;reach\u0026rdquo; of EF-P further towards the peptidyl-transferase center [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This amino acid is typically either a lysine\u0026mdash;modified by β-(\u003cem\u003eR\u003c/em\u003e)-lysylation (and hydroxylation) in many Gammaproteobacteria and others [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] or by the addition of 5-aminopentanol in the Firmicutes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u0026mdash;or an arginine, which is modified through α-rhamnosylation by the glycosyltransferase EarP [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, increasing evidence shows that some EF-P variants function effectively without any modification at the β3Ωβ4 tip [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These include the EF-P subtypes EfpL from the Gammaproteobacteria [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and those from the PGKGP family, primarily found in the Actinobacteria and the Bacteroidetes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEF-P has been horizontally transferred many times [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u0026mdash;an unusual event for a protein important for fundamental cellular processes like translation [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, genomic data offers only static snapshots\u0026mdash;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 \u0026lsquo;domestication\u0026rsquo; phase where overall EF-P function is compromised. This dysfunction may reflect barriers to successful HGT, possibly influenced by the recipient cell\u0026rsquo;s physiology. In particular, pre-existing, \u0026lsquo;native\u0026rsquo; EF-P may be sensitive to interference with the sudden appearance of a foreign EF-P PTM system.\u003c/p\u003e\u003cp\u003eIn this study, we experimentally test the hypothesis that EF-P are vulnerable to off-target modifications following interaction with non-coevolved PTM machinery (\u003cb\u003eVisual summary in\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eRhamnosylation by EarP as a model system for studying molecular complications in horizontal gene transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 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 ((\u003cem\u003eR\u003c/em\u003e)-\u0026beta;-lysylation, 5-aminopentanolylation, or \u0026alpha;-rhamnosylation). We selected \u0026alpha;-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-\u0026beta;-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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOnce 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 (\u003cstrong\u003eFigure 2\u003c/strong\u003e). 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].\u0026nbsp;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 (\u003cstrong\u003eTable 1, and annotated on the tree in Figure 2\u003c/strong\u003e). Two of these EF-P, those from \u003cem\u003eG. ferrihydriticus\u003c/em\u003e and \u003cem\u003eN. communis\u003c/em\u003e, co-occurred with another EF-P while all others were the sole encoded EF-P of the genome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Table 1)\u0026nbsp;\u003c/strong\u003eTable 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNew arginine-type EF-P variants bypass the need for post-translational activation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u003cem\u003eE. coli\u003c/em\u003e. We confirmed successful expression through mass spectrometry (\u003cstrong\u003eFigure 5 \u0026amp; Supplemental Figure 3\u003c/strong\u003e) for 6 of the 8 EF-P. Notably, despite several tactics, we could not express the EF-P from the Verrucomicrobia \u003cem\u003eA. muciniphila\u003c/em\u003e, nor the Firmicute \u003cem\u003eC. inoculum\u003c/em\u003e. Next, we screened the remaining EF-P for activity in \u003cem\u003eE. coli\u003c/em\u003e using two different methods. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirst, we tested the ability of these EF-P to restore a normal growth phenotype to an \u003cem\u003eE. coli\u003c/em\u003e mutant strain deficient in polyproline synthesis. Specifically, we used the \u003cem\u003eE. coli\u003c/em\u003e double knockout strain D\u003cem\u003eefp\u003c/em\u003eD\u003cem\u003euup\u003c/em\u003e, which is missing the primary EF-P of \u003cem\u003eE. coli\u003c/em\u003e, as well as the backup polyproline synthesis system Uup [35\u0026ndash;37]. This strain has a more pronounced phenotype compared to the \u003cem\u003eE. coli\u003c/em\u003e single knockout strain D\u003cem\u003eefp\u003c/em\u003e (doubling time of 48.9 minutes for D\u003cem\u003eefp\u003c/em\u003eD\u003cem\u003euup\u003c/em\u003e compared to 27.3 minutes in D\u003cem\u003eefp\u003c/em\u003e) [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\u0026deg;\u0026nbsp;C (\u003cstrong\u003eFigure 3\u003c/strong\u003e). Strikingly, the EF-P from \u003cem\u003eDeinococcus radiodurans\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e) and \u003cem\u003eNitrosomonas communis\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003eNico\u003c/sub\u003e\u003c/em\u003e) replaced EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e nearly to wildtype levels, restoring colony size on average to 80.1% and 71.4% of the EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e trans-complementation, respectively.\u003c/p\u003e\n\u003cp\u003eNext, we screened our heterologously expressed EF-Ps specifically for activity in polyproline synthesis in a \u0026Delta;\u003cem\u003eefp E. coli\u003c/em\u003e BW25113 background using our well described \u003cem\u003ein vivo\u003c/em\u003e luminescent reporter (\u003cstrong\u003eFigure 4\u003c/strong\u003e) [5,38]. With this assay, we can test the \u0026ldquo;strength\u0026rdquo; of specific amino acid motifs by linking translational pausing to luminescence (further details in \u003cem\u003eMethods\u003c/em\u003e). 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 \u003cem\u003ein vivo\u003c/em\u003e assays echoed our findings in the colony size complementation assay; three EF-P were hardly functional (those from \u003cem\u003eH. aurantiacus\u003c/em\u003e, \u003cem\u003eM. prima\u003c/em\u003e, and \u003cem\u003eD. acetiphilus\u003c/em\u003e) and three could rescue polyproline synthesis in \u003cem\u003eE. coli\u003c/em\u003e (those from \u003cem\u003eD. radiodurans\u003c/em\u003e, \u003cem\u003eN. communis\u003c/em\u003e, and \u003cem\u003eG. ferrihydriticus\u003c/em\u003e). Interestingly, for the TPPH stalling motif, EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003eNico\u0026nbsp;\u003c/sub\u003e\u003c/em\u003eperformed significantly better than EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e (P \u0026lt; 0.05, T test). However, these same EF-Ps performed poorly when tested against the LPPP motif (\u003cstrong\u003eFigure 4\u003c/strong\u003e). 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\u003csub\u003e\u0026shy;\u003c/sub\u003e-P of \u003cem\u003eE. coli\u003c/em\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e [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.\u003c/p\u003e\n\u003cp\u003eGenerally speaking, we found that three EF-P functioned well in \u003cem\u003eE. coli\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e, EF-P\u003cem\u003e\u003csub\u003eNico\u003c/sub\u003e\u003c/em\u003e, and EF-P\u003cem\u003e\u003csub\u003eGefe\u003c/sub\u003e\u003c/em\u003e), and three struggled to function (those from \u003cem\u003eH. aurantiacus\u003c/em\u003e, \u003cem\u003eM. prima\u003c/em\u003e, and \u003cem\u003eD. acetiphilus\u003c/em\u003e; EF-P\u003cem\u003e\u003csub\u003eHeau\u003c/sub\u003e\u003c/em\u003e, EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, and EF-P\u003cem\u003e\u003csub\u003eDeac\u003c/sub\u003e\u003c/em\u003e). In some cases, these latter EF-Ps even had a detrimental impact on polyproline synthesis (see \u003cstrong\u003eFigure 4\u003c/strong\u003e, 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 \u003cem\u003eP. putida\u003c/em\u003e and \u003cem\u003eS. oneidensis\u003c/em\u003e on \u003cem\u003eE. coli\u0026nbsp;[20]\u003c/em\u003e, or that 2) these EF-P are simply too distantly related to function effectively in \u003cem\u003eE. coli\u003c/em\u003e. We found stronger support for the second possibility: the functional performance of our foreign EF-P in \u003cem\u003eE. coli\u003c/em\u003e was generally inversely correlated with their phylogenetic distance from \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e). In other words, EF-P homologs from closely related species, particularly other Gammaproteobacteria such as \u003cem\u003eS. oneidensis\u003c/em\u003e and \u003cem\u003eN. communis\u003c/em\u003e, tended to function more effectively in \u003cem\u003eE. coli\u003c/em\u003e than those from more distantly related organisms like \u003cem\u003eM. prima\u003c/em\u003e and \u003cem\u003eH. aurantiacus\u003c/em\u003e. This pattern mirrors findings from a similar study involving heterologous replacement of EF-Tu, where functionality in \u003cem\u003eE. coli\u003c/em\u003e was typically limited to homologs from within the Gammaproteobacteria [39]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, there were two clear exceptions to this general trend. In the first, we found that despite the substantial phylogenetic distance between \u003cem\u003eD. radiodurans\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e), EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e consistently performed well in our assays. One possible explanation for these results is that EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e may have been acquired via horizontal gene transfer from a lineage more closely related to \u003cem\u003eE. coli\u003c/em\u003e, which could explain its higher level of function in our host background and its relatively high amino acid percent identity to EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e (44.9%, \u003cstrong\u003eSupplemental Figure 2\u003c/strong\u003e). 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 (\u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e). Here, we found no correlation between EF-P function and phylogenetic distance (P = 0.915, cor = -0.046, Pearson\u0026rsquo;s correlation). This divergence from the broader pattern was largely driven by the strong performance of EF-P\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e and the relatively good performance of distantly related EF-P (EF-P\u003cem\u003e\u003csub\u003eHeau\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e). These findings suggest that, although phylogenetic distance can be a useful predictor of heterologous EF-P functionality in \u003cem\u003eE. coli\u003c/em\u003e, specific tRNA within the peptidyl transferase center can override these general trends and produce more nuanced, context-dependent outcomes.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePromiscuous EarP activity targets the non-cognate EF-P from \u003cem\u003eM. prima\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, 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 \u003cem\u003eShewanella oneidensis\u003c/em\u003e, EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e) in a wildtype LMG194 \u003cem\u003eE. coli\u003c/em\u003e background and measured the molecular weight of these EF-P with mass spectrometry. We excluded the EF-P from \u003cem\u003eD. acetiphilus\u003c/em\u003e from these experiments, as it consistently failed to rescue polyproline synthesis in \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003eFigure 4\u003c/strong\u003e). As a control, we also included the EarP-type EF-P from \u003cem\u003ePseudomonas putida\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e) to test the efficiency of cross-species post-translational modification with EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe 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\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e (\u003cstrong\u003eSupplemental Figure 3\u003c/strong\u003e), indicating that no modification occurred. These EF-P included those from \u003cem\u003eD. radiodurans\u003c/em\u003e, \u003cem\u003eH. aurantiacus\u003c/em\u003e, \u003cem\u003eN. communis\u003c/em\u003e, and \u003cem\u003eG. ferrihydriticus\u003c/em\u003e. We also found no change in molecular weight upon co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e for our negative control, EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e. 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\u003cem\u003e\u003csub\u003eDera\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003eNico\u003c/sub\u003e\u003c/em\u003e function well in \u003cem\u003eE. coli\u003c/em\u003e without the need for any post-translational modification and imply that these EF-P may also be unmodified in their native host species. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, one of our heterologous EF-P showed an altered mass upon co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e; that of \u003cem\u003eM. prima\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, \u003cstrong\u003eFigure 5\u003c/strong\u003e). While the His6-tagged EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e has an expected monoisotopic mass of 21935.07 Da, upon co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e 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\u0026mdash;EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e also showed an increase in mass of roughly 146 Da upon co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e (\u003cstrong\u003eFigure 5\u003c/strong\u003e). However, while EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e was fully rhamnosylated\u0026mdash;as shown by the disappearance of the molecular weight corresponding to its unmodified form (\u003cstrong\u003eFigure 5\u003c/strong\u003e)\u0026mdash;EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e was partially rhamnosylated, with a relative abundance of 20.06% compared to that of the unmodified mass.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results raise the question: why were so few EF-P variants successfully modified by EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e, 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\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003ein \u003cem\u003eE. coli\u003c/em\u003e [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.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOff-target rhamnosylation has diverging impacts on the function of unmodified arginine-type EF-Ps\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of the non-native rhamnosylation of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e in more detail, we repeated previous experiments with co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e or an empty control plasmid for EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e (negative control), EF-P\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u0026nbsp;\u003c/em\u003e(positive controls). For the complementation assay, co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e had no impact on colony size, as expected (\u003cstrong\u003eFigure 6\u003c/strong\u003e). On the other hand, co-expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e greatly enhanced colony size in the EF-P\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e and EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e controls (\u003cstrong\u003eFigure 6\u003c/strong\u003e). This is also to be expected, as these EF-P are natively rhamnosylated and can be detrimental to \u003cem\u003eE. coli\u003c/em\u003e in their un-rhamnosylated forms \u0026nbsp;[7,20]. \u0026nbsp;Lastly, co-expression with EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e significantly affected the EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e trans-complementation but resulted in only a very slight increase in average colony size (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we repeated the \u003cem\u003ein vivo\u003c/em\u003e stalling‐rescue assay with our luminescent reporter. Co‐expression of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u0026nbsp;\u003c/em\u003ehad no significant impact on EF-P\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e (\u003cstrong\u003eFigure 7\u003c/strong\u003e), but dramatically improved EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e rescue: unmodified EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e had a detrimental impact on polyproline synthesis (LPPP = 0.60, TPPH = 0.77), whereas rhamnosylated EF-P\u003cem\u003e\u003csub\u003ePpu\u003c/sub\u003e\u003c/em\u003e performed well (LPPP = 2.48, TPPH = 1.57), consistent with Lassak et al. (2015) [7]. EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e also gained a modest benefit from EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e in the assay with LPPP (from 0.38 \u0026rarr; 1.18), but only to levels equivalent to those seen in the \u0026Delta;\u003cem\u003eefp\u003c/em\u003e strain (\u003cstrong\u003eFigure 7\u003c/strong\u003e). In other words, rhamnosylation of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e 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\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e and the \u003cem\u003eE. coli\u003c/em\u003e ribosome, possibly due to incompatibility at the peptidyl-transferase center.\u003c/p\u003e\n\u003cp\u003eLastly, we sought to explore our proposed explanation for how rhamnosylation affects EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e 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\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e and in particular rhamnosylations, we used structural modeling to suggest and compare the positioning of \u0026beta;3\u0026Omega;\u0026beta;4 loop tip regions in the following three variants: unmodified EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, rhamnosylated EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, and the naturally rhamnosylated EF-P from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e), in complex with tRNA\u003csup\u003ePro\u003c/sup\u003e (\u003cstrong\u003eFigure 8)\u003c/strong\u003e. Our models suggest that while the unmodified EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003ecan form several favorable contacts with tRNA\u003csup\u003ePro\u003c/sup\u003e, these contacts are abolished upon the addition of the rhamnose modification, which introduces a steric clash (\u003cstrong\u003eFigure 8)\u003c/strong\u003e. By contrast, based on the available high-resolution crystal structure\u0026nbsp;[40] the \u0026beta;3\u0026Omega;\u0026beta;4 loop of EF-P\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e is oriented in such a way as to take full advantage of the addition of the rhamnose moiety, leading to additional favorable contacts (\u003cstrong\u003eFigure 8)\u003c/strong\u003e. 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\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e. In contrast, the rhamnosylation of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e is predicted to disrupt the protein\u0026rsquo;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\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003eshows some limited activity in polyproline synthesis (\u003cstrong\u003eFigure 7\u003c/strong\u003e TPPH) and provides minor trans-complementation benefits in \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003eFigure 6\u003c/strong\u003e), rhamnosylation did not improve its function, but merely reduced detrimental effects (\u003cstrong\u003eFigure 7\u003c/strong\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is only natural that EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e is adapted to support translation in \u003cem\u003eM. prima\u003c/em\u003e, rather than in \u003cem\u003eE. coli\u003c/em\u003e. Given the evolutionary distance between these species, it\u0026rsquo;s likely that the ribosome of \u003cem\u003eM. prima\u003c/em\u003e differs structurally from that of \u003cem\u003eE. coli\u003c/em\u003e. We have shown that upon interaction with\u0026nbsp;EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e, a substantial fraction of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e is rhamnosylated and that this modification has a significant impact on the function of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, inasmuch as it can be measured in \u003cem\u003eE. coli\u003c/em\u003e. This set of experiments, measuring the impact of EarP\u003cem\u003e\u003csub\u003eShon\u003c/sub\u003e\u003c/em\u003e on EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, mimics historical horizontal gene transfer events that actually occurred in the phylum Thermotogota. Long ago, members of the genera \u003cem\u003eGeotoga\u003c/em\u003e and \u003cem\u003eOceanotoga\u003c/em\u003e 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 \u003cem\u003eM. prima\u003c/em\u003e belongs. It is therefore reasonable to infer that the initial introduction of an \u0026ldquo;invading\u0026rdquo; EarP into the \u003cem\u003eGeotoga\u003c/em\u003e and \u003cem\u003eOceanotoga\u003c/em\u003e 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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA contrasting illustration of this \u0026ldquo;invading-gene\u0026rdquo; scenario comes from the EF-P paralog EfpL, which we initially excluded as a target as it is a \u0026ldquo;secondary EF-P\u0026rdquo;, and always paired with an EF-P with a wider motif spectrum [5]. Although it is not natively post-translationally modified in \u003cem\u003eE. coli\u003c/em\u003e, we surprisingly found that exposure to heterologously expressed EarP also leads to partial rhamnosylation of EfpL (\u003cstrong\u003eSupplemental Figure 4\u003c/strong\u003e). Importantly, in our \u003cem\u003ein silico\u003c/em\u003e model, this modification had no detectable effect on EfpL activity in \u003cem\u003eE. coli\u003c/em\u003e, presumably as a result of its unique \u0026beta;3\u0026Omega;\u0026beta;4 architecture\u0026nbsp;[5] (\u003cstrong\u003eSupplemental Figure 5\u003c/strong\u003e). On the one hand, this indicates that, unlike EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, 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\u0026nbsp;interaction (\u003cstrong\u003eSupplemental Figure 5\u003c/strong\u003e). 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 \u0026Delta;\u003cem\u003eefp\u003c/em\u003e\u0026Delta;\u003cem\u003euup\u003c/em\u003e strain in the presence of \u003cem\u003eearP\u003c/em\u003e alone, \u003cem\u003eefpL\u003c/em\u003e alone or upon co-expression of both. Expression of \u003cem\u003eefpL\u003c/em\u003e increased colony size significantly. In line with our \u003cem\u003ein silico\u003c/em\u003e model, we also found that rhamnosylation did not affect EfpL\u0026rsquo;s complementation efficiency (\u003cstrong\u003eSupplemental Figure 6\u003c/strong\u003e). This observation also aligns with the phylogenetic placement of EfpL as a sister clade to the EarP type EF-P (\u003cstrong\u003eFigure 2\u003c/strong\u003e) 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\u0026mdash;presumably by a single proline\u0026mdash;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 \u0026ldquo;invading\u0026rdquo; PTM system can depend not only on potential incompatibility, but also on whether it creates an opportunity for novel function. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken 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.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn 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 \u003cem\u003eD. radiodurans,\u003c/em\u003e\u003cem\u003eG. ferrihydriticus\u003c/em\u003e and \u003cem\u003eN. communis\u003c/em\u003e, function efficiently in \u003cem\u003eEscherichia coli\u003c/em\u003e 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 \u003cem\u003eM. prima\u003c/em\u003e (EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e). This off-target modification impaired rather than enhanced the functionality of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, 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 \u003cem\u003eE. coli\u003c/em\u003e. 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.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSelection of EF-P candidates \u0026amp; phylogenetic trees\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify EF-P candidates to test experimentally, we first extracted all \u003cem\u003eefp\u003c/em\u003e 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. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe 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]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe created a second phylogenetic tree to obtain evolutionary distances between the different species used in this study (used in \u003cstrong\u003eSupplemental Figure 1\u003c/strong\u003e). 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll strains, plasmids, and oligonucleotides used in this study are listed and described in \u003cstrong\u003eSupplementary Data 1\u003c/strong\u003e. We isolated plasmid DNA using the Zyppy\u0026reg; Plasmid Miniprep Kit (Zymo Research) and purified PCR reactions using the DNA Clean \u0026amp; Concentrator\u0026reg;-5 DNA kit (Zymo Research) or the Zymoclean\u0026reg; 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\u0026rsquo;s instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe constructed plasmids for the expression of C-terminally His6-tagged \u003cem\u003eefp\u003c/em\u003e 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 \u003cstrong\u003eSupplementary Data 1\u003c/strong\u003e. We then purified and cloned these DNA fragments into the pBAD24 vector [47] using the NEBuilder\u0026reg; HiFi DNA Assembly Master Mix (NEB). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe routinely grew \u003cem\u003eE. coli\u003c/em\u003e cells in Miller-modified lysogeny broth (LB) [48,49] or super optimal broth (SOB) [50] at 37\u0026thinsp;\u0026deg;C aerobically under agitation unless otherwise indicated. We measured growth using optical density at a wavelength of 600\u0026thinsp;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\u0026thinsp;\u0026micro;g/ml carbenicillin sodium salt (pBAD24) or 25\u0026thinsp;\u0026micro;g/ml chloramphenicol (pBAD33). We induced plasmids carrying the P\u003csub\u003eBAD\u003c/sub\u003e [47]\u003csup\u003e\u0026nbsp;\u003c/sup\u003epromoter with L(+)-arabinose at a final concentration of 0.2% (w/v).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of colony size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo measure the ability of our foreign EF-Ps to restore polyproline synthesis in a deficient strain we first transformed the pBAD24 vectors carrying each \u003cem\u003eefp\u003c/em\u003e into the \u003cem\u003eE. coli\u003c/em\u003e double deletion strain D\u003cem\u003eefp\u003c/em\u003eD\u003cem\u003euup\u003c/em\u003e. This strain was constructed in previous work [5], and has a strong growth deficit. We plated our transformants onto LB agar containing 100\u0026thinsp;\u0026micro;g/ml carbenicillin sodium salt and 0.2% L(+)-arabinose to induce expression and incubated them for up to 24h at 37\u0026thinsp;\u0026deg;C. We used ImageJ version 1.54g [51] to measure the diameter of each colony. We excluded colonies that were outside the 5\u003csup\u003eth\u003c/sup\u003e and 95\u003csup\u003eth\u003c/sup\u003e 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 \u003cem\u003eearP\u003c/em\u003e and added 25\u0026thinsp;\u0026micro;g/ml chloramphenicol to our plates [26]. Alternatively, \u003cem\u003eearP\u0026nbsp;\u003c/em\u003ewas expressed under control of its native constitutive promoter from an pBBR1MCS2 backbone [26]. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of pausing strength \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe measured the pausing strength of specific amino acid motifs using a previously developed luminescent reporter based on the \u003cem\u003eE. coli\u003c/em\u003e 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 \u003cem\u003ehisGDCBHAF\u003c/em\u003e. Low histidine causes ribosomal pausing at HisL, enabling transcription of the downstream biosynthesis operon. Our reporter fuses HisL and the 5\u0026prime; UTR of \u003cem\u003ehisGDCBHAF\u003c/em\u003e with the \u003cem\u003eluxCDABE\u003c/em\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 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\u0026reg; plate reader using the following parameters: absorption at 600\u0026thinsp;nm (number of flashes: 10; settle time: 50\u0026thinsp;ms) and luminescence emission (attenuation: none; settle time: 50\u0026thinsp;ms; integration time: 200\u0026thinsp;ms) in between 10-min cycles of agitation (orbital: 180\u0026thinsp;rpm; amplitude: 3\u0026thinsp;mm) for around 16\u0026thinsp;h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein overproduction and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare proteins for mass spectrometry analysis, we overproduced C-terminally His6-tagged EF-Ps using the pBAD24 vector in \u003cem\u003eE. coli\u003c/em\u003e 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\u0026thinsp;\u0026deg;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\u0026thinsp;kbar and clarified our lysates by centrifugation at 4\u0026thinsp;\u0026deg;C at 234 998\u0026thinsp;\u0026times;\u0026thinsp;g for 1\u0026thinsp;h. Next, we purified our His\u003csub\u003e6\u003c/sub\u003e-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 His\u003csub\u003e6\u003c/sub\u003e-tagged EF-Ps. For these analyses, we used 12.5% (w/v) SDS and stained with InstantBlue Coomassie Protein stain (Abcam).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometry for identification of modification status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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 \u0026mu;M final protein concentration in 200\u0026ndash;400 \u0026mu;l 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of EarP overproduction on the \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eproteome, we heterologously produced EarP from \u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440 (locus tag PP_1857) from an arabinose inducible promoter (P\u003csub\u003eBAD\u003c/sub\u003e) using pBAD33 as vector backbone [47]; pBAD33PP1857-His6 [26]. We induced expression of \u003cem\u003eearP\u0026nbsp;\u003c/em\u003eand 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 \u0026micro;m x 50 cm in-house C\u003csub\u003e18\u003c/sub\u003e 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\u0026ndash;58]. We searched the resulting spectra against an \u003cem\u003eE. coli\u003c/em\u003e reference proteome (UP000000625) with cysteine carbamidomethylation as a fixed modification; and N-terminal acetylation, methionine oxidation, and arginine modified with C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (146.0579 Da, rhamnose-H\u003csub\u003e2\u003c/sub\u003eO) 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModeling rhamnosylation of EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, EF-P\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e, and \u003cstrong\u003eEfpL\u003cem\u003e\u003csub\u003eEco\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe carried out all modeling and in silico modification in Pymol using release 2.5.5 (Schr\u0026ouml;dinger). We created structural models displaying the effects of rhamnosylation on interactions between EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, EF-P\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e EfpL and the tRNA CCA by structurally aligning the respective EF-P proteins with PDB entry 6enj showing the full polyproline stalled \u003cem\u003eE. coli\u003c/em\u003e ribosome [59]. For the \u003cem\u003eE. coli\u003c/em\u003e EfpL we used the previously determined crystal structure (PDB entry 8s8u [5]), while for EF-P\u003cem\u003e\u003csub\u003ePaer\u003c/sub\u003e\u003c/em\u003e we used PDB entry 3oyy [40]. We used an AF3 model [60] of the full-length protein\u003csub\u003e\u0026nbsp;\u003c/sub\u003eas input for EF-P\u003cem\u003e\u003csub\u003eMepr\u003c/sub\u003e\u003c/em\u003e, 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.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis 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 \u0026ndash; 325871075 to PK. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEB 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 \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003estudies with EfpL and FMR performed EfpL specific mass spectrometry. TEB and JL wrote the paper, which was edited by all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll genomes used in this study are publicly available from JGI\u0026apos;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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGood BH, Bhatt AS, McDonald MJ. Unraveling the tempo and mode of horizontal gene transfer in bacteria. Trends Microbiol. 2025;\u003c/li\u003e\n \u003cli\u003eLassak J, Wilson DN, Jung K. 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Nucleic Acids Res. 2021;49:D751\u0026ndash;63.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Evolution, bacterial diversity, horizontal gene transfer","lastPublishedDoi":"10.21203/rs.3.rs-7374903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7374903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHorizontal 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 \u003cem\u003eEscherichia coli\u003c/em\u003e 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 \u003cem\u003eDeinococcus radiodurans\u003c/em\u003e, \u003cem\u003eGeoalkalibacter ferrihydriticus\u003c/em\u003e, and \u003cem\u003eNitrosomonas communis\u003c/em\u003e can complement an EF-P knockout in \u003cem\u003eE. coli\u003c/em\u003e without requiring modification, suggesting they represent new examples of unmodified EF-Ps. We also found that the EF-P from the Thermotogota \u003cem\u003eMesotoga prima\u003c/em\u003e 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 \u003cem\u003eEscherichia coli\u003c/em\u003e, 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.\u003c/p\u003e","manuscriptTitle":"Horizontal transfer of post-translational modifiers brings evolutionary opportunity and challenges to a conserved translation factor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 14:00:54","doi":"10.21203/rs.3.rs-7374903/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-07T15:39:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T17:13:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T16:09:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67174967020016876924339484076649844552","date":"2025-08-30T16:30:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108507440000977083248527818815872706314","date":"2025-08-30T00:13:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64348519767658436971868145119178137944","date":"2025-08-29T14:58:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277070971462409506318566220457980307362","date":"2025-08-28T16:05:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-28T15:47:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T15:57:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-15T08:30:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2025-08-14T14:26:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba7f1a63-1f79-4bce-930d-54c66d9b6074","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T13:58:43+00:00","versionOfRecord":{"articleIdentity":"rs-7374903","link":"https://doi.org/10.1186/s12915-026-02531-9","journal":{"identity":"bmc-biology","isVorOnly":false,"title":"BMC Biology"},"publishedOn":"2026-01-30 00:00:00","publishedOnDateReadable":"January 30th, 2026"},"versionCreatedAt":"2025-10-14 14:00:54","video":"","vorDoi":"10.1186/s12915-026-02531-9","vorDoiUrl":"https://doi.org/10.1186/s12915-026-02531-9","workflowStages":[]},"version":"v1","identity":"rs-7374903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7374903","identity":"rs-7374903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}