
==== Front
Mol Biol Evol
Mol Biol Evol
molbev
Molecular Biology and Evolution
0737-4038
1537-1719
Oxford University Press UK

39189989
10.1093/molbev/msae180
msae180
Discoveries
AcademicSubjects/SCI01130
AcademicSubjects/SCI01180
Horizontal Gene Transfer of a key Translation Factor and its Role in Polyproline Proteome Evolution
https://orcid.org/0000-0001-7855-5610
Brewer Tess E Faculty of Biology, Microbiology, Ludwig Maximilian University of Munich, Munich, Germany

https://orcid.org/0000-0003-4299-3840
Wagner Andreas Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland
Swiss Institute of Bioinformatics, Lausanne, Switzerland
Santa Fe Institute, Santa Fe, NM, USA

Barlow Miriam Associate Editor
Corresponding authors: E-mails: tess@tess-brewer.com; andreas.wagner@ieu.uzh.ch.
9 2024
27 8 2024
27 8 2024
41 9 msae18009 10 2023
05 8 2024
20 8 2024
11 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.
2024
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Abstract

Prolines cause ribosomes to stall during translation due to their rigid structure. This phenomenon occurs in all domains of life and is exacerbated at polyproline motifs. Such stalling can be eased by the elongation factor P (EF-P) in bacteria. We discovered a potential connection between the loss of ancestral EF-P, the appearance of horizontally transferred EF-P variants, and genomic signs of EF-P dysfunction. Horizontal transfer of the efp gene has occurred several times among bacteria and is associated with the loss of highly conserved polyproline motifs. In this study, we pinpoint cases of horizontal EF-P transfer among a diverse set of bacteria and examine genomic features associated with these events in the phyla Thermotogota and Planctomycetes. In these phyla, horizontal EF-P transfer is also associated with the loss of entire polyproline motif-containing proteins, whose expression is likely dependent on EF-P. In particular, three proteases (Lon, ClpC, and FtsH) and three tRNA synthetases (ValS, IleS1, and IleS2) appear highly sensitive to EF-P transfer. The conserved polyproline motifs within these proteins all reside within close proximity to ATP-binding-regions, some of which are crucial for their function. Our work shows that an ancient EF-P dysfunction has left genomic traces that persist to this day, although it remains unclear whether this dysfunction was strictly due to loss of ancestral EF-P or was related to the appearance of an exogenous variant. The latter possibility would imply that the process of “domesticating” a horizontally transferred efp gene can perturb the overall function of EF-P.

genome evolution
bacterial diversity
comparative genomics
horizontal gene transfer
translation factor
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pmcIntroduction

Proline-rich regions are important for protein function. They are over-represented in protein domains that are important for interactions with other proteins and with nucleic acids (Hersch et al. 2013). They are also associated with high cellular complexity (Mandal et al. 2014; Brewer and Wagner 2022). However, proline-rich regions cause a problem during mRNA translation that is universal to all domains of life. Due to its uniquely rigid structure, proline is the slowest amino acid to be incorporated into proteins during translation. Proline causes translating ribosomes to pause or “stall” on mRNA, which slows down protein synthesis. This problem is compounded when an amino acid sequence contains multiple adjacent prolines (polyproline motifs, ≥2P). The severity of this stalling is determined by several factors. They include the identity of the surrounding amino acids (particularly the two amino acids before and the one after the prolines e.g. XXPPX; Starosta et al. 2014a), the translation initiation rate (Hersch et al. 2014), the position of the polyproline motif within a protein (Woolstenhulme et al. 2015), and the proline codon used (Krafczyk et al. 2021). Some species seem to exploit ribosomal pausing at polyproline motifs and may encode these motifs to provide additional time for translational regulation, protein folding, or membrane insertion (Qi et al. 2018). Polyproline motifs can have a dramatic impact on translational rate in highly expressed proteins, where they cause ribosomal queuing, wreak havoc on translational efficiency, and unlink the coupling between transcription and translation (Hersch et al. 2014; Elgamal et al. 2016; Tollerson et al. 2018).

To mitigate this problem, species in all three domains of life encode proteins that minimize the impact of ribosomal stalling at polyproline motifs (Lassak et al. 2016). In bacteria, elongation factor P (EF-P) is such a protein. EF-P binds to the ribosome between the peptidyl-tRNA binding and tRNA-exiting sites (Lassak et al. 2016). From there, EF-P interacts with the peptidyl-transferase center, alleviating prolyl-dependent stalling (Lassak et al. 2016). Because EF-P is important for efficient translation, loss of the efp gene can have dramatic and varied phenotypic consequences, including reduced growth rate (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Rajkovic et al. 2016; Tollerson et al. 2018), loss of motility (Hummels and Kearns 2019; Guo et al. 2022), loss of virulence (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Guo et al. 2022), reduced antibiotic resistance (Navarre et al. 2010; Rajkovic et al. 2015), and in the case of Acinetobacter baumannii (Guo et al. 2022) and Neisseria meningitidis (Yanagisawa et al. 2016), death. Many of these phenotypes are caused by the under-expression of proteins containing polyproline motifs (Peng et al. 2001; Navarre et al. 2010; Hersch et al. 2013; Starosta et al. 2014a, 2014b; Rajkovic et al. 2015; Hummels and Kearns 2019). In some proteins, altering such motifs to reduce the severity of ribosomal stalling can ease EF-P loss-of-function phenotypes. For example, swarming motility can be restored to Bacillus subtilis efp mutants by modifying a ribosomal stalling polyproline motif in the flagellar C-ring component FliY (Hummels and Kearns 2019). In other proteins, polyproline motifs cannot be altered without substantial negative impacts on protein function. Examples include a polyproline motif within the glucose importer component EIIGlc in Corynebacterium glutamicum, which cannot be altered without inactivating the protein (Pinheiro et al. 2021), and a proline triplet in valine tRNA synthetase that is crucial for efficient and accurate tRNA charging in E. coli (Starosta et al. 2014b). In other words, while some proteins can be modified to reduce their reliance on EF-P, other proteins seem unavoidably dependent on EF-P for normal expression.

In some species of bacteria, EF-P must be post-translationally modified to function properly. While three different types of modification are presently known, these modifications all reside at the same position within EF-P—on the amino acid positioned at the tip of the conserved loop region in domain I of the protein (Lassak et al. 2016). In most Gammaproteobacteria, the modified amino acid is a lysine (K34). It is modified by (R)-β-lysylation through chemical reactions that are catalyzed by the proteins EpmA and EpmB (Park et al. 2012), and in some species by an additional hydroxylation step carried out by EpmC (Peil et al. 2012). As the modification added by EpmC does not have a significant impact on EF-P function (Peil et al. 2012), we will subsequently refer to EF-Ps with these modifications as the lysine-EpmAB/C type. In some Firmicutes the modified amino acid is also a lysine (K32, equivalent to K34 in E. coli), but it is modified by the attachment of a 5-aminopentanol group carried out in several steps by the proteins YmfI, YnbB, and GsaB (lysine-YmfI type) (Hummels et al. 2017; Witzky et al. 2018). In Betaproteobacteria, the modified amino acid is an arginine (R32, equivalent to K34 in E. coli), which is rhamnosylated by the protein EarP (arginine-EarP type) (Lassak et al. 2015). Some bacteria, such as the Actinobacteria, use an unmodified EF-P that encodes a lysine at the conserved position and can be identified by the presence of a distinctive proline loop (lysine-unmodified type) (Pinheiro et al. 2020; Tomasiunaite et al. 2024). Finally, some species contain an EF-P paralog named EfpL (previously known as YeiP), which encodes an arginine at the conserved position (arginine-EfpL type) (Sieber et al. 2024). EfpL is not post-translationally modified and plays a reduced role in resolving ribosomal stalling compared with EF-P in E. coli (Sieber et al. 2024).

The post-translational modification of many EF-Ps is unknown—many species do not contain any of the aforementioned post-translational modification systems, yet they do not have the conserved proline loop sequence that defines the unmodified EF-P of the Actinobacteria. These species may contain a yet to be identified post-translational modification system, or simply encode unmodified EF-Ps with a different loop region (Pinheiro et al. 2020; Tomasiunaite et al. 2024). Lastly, while all EF-P types are thought to function in the same general way, by binding to the ribosome and adjusting the position of the tRNA, they may differ in their exact method of action. For example, the (R)-β lysylation modification carried out by EpmAB is expected to reach within 2Å of the proline attached to the P-site tRNA, while the rhamnose modification carried out by EarP is shorter and likely functions indirectly through stabilization of the P-site peptidyl-Pro-tRNA (Lassak et al. 2015).

In this study, we investigate a genomic mystery. In the course of a previous analysis (Brewer and Wagner 2022), we discovered that some species within the bacterial phylum Planctomycetota do not encode a proline triplet in valine tRNA synthase that is crucial to the enzyme's function, and that was thought to be universally conserved across all domains of life (Starosta et al. 2014b). Where the valine tRNA synthases of all other known forms of life encode the amino acids “PPP” (Starosta et al. 2014b), these species instead encode “PLP”. Pulling at this thread, we found that the loss of this motif coincides with the loss of the ancestral EF-P and the appearance of a horizontally transferred EF-P variant in these Planctomycete genomes. Taken together, these observations suggest that species which now contain horizontally transferred EF-P suffered an episode of EF-P dysfunction at some point in their evolutionary history. EF-P transfer has been observed previously (Lassak et al. 2015; Volkwein et al. 2019), but never studied in detail. For example, the arginine-EarP type EF-P is believed to have originated within the Betaproteobacteria but is also sporadically present within some Gammaproteobacteria, Thermotogota, Planctomycetes, spirochetes, and Fusobacteria (Lassak et al. 2015). In the underlying transfer events, the proteins that post-translationally modify EF-P are generally transferred together with the EF-P-coding gene in a single operon. This supports previous speculations that different EF-P types co-evolve with their modification systems (Lassak et al. 2015; Volkwein et al. 2019).

In this study, we examined how frequently horizontal transfer of EF-P occurs within bacterial genomes. We find that horizontally transferred EF-P do not often coexist with the “native” ortholog, that is, the variant of EF-P that existed within the recipient genome before the transfer. We also examined in more detail members of two phyla that have lost their native EF-P and now encode horizontally transferred EF-P, the Planctomycetota and Thermotogota. In these species, loss of the ancestral EF-P and appearance of an exogenous EF-P variant is consistently linked with loss of otherwise highly conserved polyproline motifs, such as the nearly universally conserved proline triplet in valine tRNA synthetase. Our work shows that volatility in the evolutionary history of EF-P is associated with proteome remodeling to alter polyproline motifs. In some species, this leads to the loss of entire proteins that appear dependent on EF-P for proper expression. We show that these events leave behind telltale genomic signatures and may have affected these species’ evolution, as many of these conserved polyproline motifs appear to be important for ATP binding and hydrolysis.

Results

Genome Dataset Overview

Although EF-P is an important protein in bacteria, as evidenced by its near universal conservation and the severe, detrimental effects of its loss (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Guo et al. 2022), it has been studied in only a handful of species (Hersch et al. 2013; Lassak et al. 2015; Yanagisawa et al. 2016; Witzky et al. 2018; Pinheiro et al. 2020; Guo et al. 2022). The phylogenetic diversity of these species is heavily biased toward the Gammaproteobacteria (Hersch et al. 2013; Lassak et al. 2015; Guo et al. 2022). In order to investigate EF-P from a wider diversity of species than purely experimental methods allow, we first needed to characterize these proteins. Presently, there are five known types of EF-P. They differ in the amino acid residing at the tip of the conserved loop region in domain I, and the post-translational modifications of this amino acid. As detailed further in the Introduction and Methods sections, these comprise the lysine-EpmAB/C type EF-P, the lysine-YmfI type, the lysine-unmodified type, the arginine-EarP type, and the arginine-EfpL type. We bioinformatically annotated all known EF-Ps within a dataset of more than 3,000 bacterial species, comprising 35 phyla spread across 382 families (Methods). This procedure left us with many EF-Ps that did not fit into the five known types. Almost half of the genomes in our dataset (1,606 genomes) encode an EF-P of unknown type.

To further characterize these unknown type EF-P, we sorted all EF-P sequences (3,638 proteins) into “families” using similarity network-based sequence homology clustering (Methods). We assigned protein sequences with ≥49% sequence identity and ≥80% sequence length alignment to the same family. We chose this threshold as a compromise between sorting known modification types into unique families and minimizing the number of families with few members due to poor representation of some clades in the dataset. This procedure left us with 15 EF-P families (supplementary fig. S1, Supplementary Material online). The largest of these families contained 2,979 EF-Ps, including members of every lysine type EF-Ps—the lysine-EpmAB/C type (13.9%), the lysine-YmfI type (6%), the lysine-unmodified type (15.2%)—and many EF-Ps of unknown modification (51%). The next two largest families contained 299 EF-Ps primarily of the arginine-EarP type, and 265 EF-Ps of the arginine-EfpL type, respectively. The remaining families were unique to specific phylogenetic clades. For example, we found the phyla Spirochaetota, Acidobacteriota, and Verrucomicrobiota to have particularly rich EF-P diversity, encoding seven unique EF-P families between them (supplementary fig. S1, Supplementary Material online and supplementary results, Supplementary Material online).

We found that the vast majority of bacterial genomes (88.8% of our dataset) contain just one efp gene copy. A small proportion encoded two copies of the efp gene (11.0%), and just one species encoded three copies (the Gammaproteobacterium Marinobacterium rhizophilum). When two efp genes are present in a genome they usually encode different amino acids at the tip of the conserved loop region (86.5% of genomes with two EF-Ps have different amino acids at this position). Furthermore, the two EF-Ps are usually quite distinct in sequence composition (in 83% of genomes encoding two EF-Ps these EF-Ps fall into different sequence cluster families). In most species with two efp genes, one of the genes encodes a lysine-EpmAB/C type EF-P, and the other an arginine-EfpL type EF-P (70.7% of genomes encoding two EF-P). This pairing is most common among the Gammaproteobacteria (245 genomes), but also occurs in the phyla Desulfobacterota (5 genomes) and Planctomycetota (6 genomes). Notably, we did not find any genome where an arginine-EfpL type EF-P exists alone—EfpL is always paired with another EF-P type, consistent with its identity as a supporting EF-P subfamily (Lassak et al. 2015). We found several other combinations of EF-P pairs, which are detailed in the supplementary results, Supplementary Material online.

Horizontal Transfer of EF-P

As a first pass at identifying horizontally transferred EF-Ps, we superimposed the bioinformatically inferred EF-P types onto the phylogenetic tree of our bacterial species (Fig. 1). Because EF-P modification types evolved in distinct phylogenetic clusters (Hummels et al. 2017; Volkwein et al. 2019), this procedure can help identify EF-P types that appear outside of the cluster where they originated. For example, the EarP rhamnose modification system originated within the Betaproteobacteria (Volkwein et al. 2019). Any instances of the EarP type EF-P residing outside this phylogenetic group therefore indicates HGT of efp. We note that this approach biases our detection of HGT events for horizontally transferred EF-Ps that uses a post-translational modification.

Fig. 1. EF-P modification systems evolved in phylogenetically conserved clusters of bacteria, making horizontal transfer of different efp types clearly discernable. We created this phylogenetic tree using the amino acid sequences of concatenated conserved proteins from >3,000 bacterial genomes from 35 phyla. The rings surrounding the tree indicate which EF-P type is predicted to be encoded by each genome (Methods). Some genomes encode two EF-P, indicated by a second ring, and one genome encodes three. Although the arginine-EfpL EF-P type appears to have been horizontally transferred into the phyla Planctomycetota and Desulfobacterota, this EF-P type plays a supporting role in polyproline stalling and we do not discuss it further. The color of the circle at the leaves of the tree indicates which phylum each EF-P sequence came from according to the GTDB (Chaumeil et al. 2020). For clarity, only 11 phyla are colored, all others are termed “other”. (We note that Betaproteobacteriales is technically classified as an order within the Gammaproteobacteria in the GTDB). Likely cases of EF-P transfer are highlighted directly on the tree with shading and letters, and correspond to: a) Classes Leptospirae and Spirochaetia (lysine-EpmAB) and family Treponemataceae (arginine-EarP), b) Phyla Planctomycetota and Verrucomicrobiota (lysine-EpmAB) and genus Planctopirus (arginine-EarP), c) Family Fibrobacteraceae (lysine-EpmAB), d) Family Fusobacteriaceae (arginine-EarP), e) Family Petrotogaceae (arginine-EarP), f) Family Dehalococcoidaceae (lysine-EpmAB), g) Phyla Aquificota and Campylobacterota (lysine-EpmAB), h) Members of the phyla Desulfobacterota, Desulfuromonadota, and Myxococcota (lysine-EpmAB), i) Many members of the Alphaproteobacteria (lysine-EpmAB), j) Gammaproteobacteria orders Pseudomonadales, Enterobacterales, Thiomicrospirales, and Thiotrichales (arginine-EarP).

Using this approach, we identified 10 likely instances of EF-P transfer, which are labeled individually in Fig. 1. They include 6 transfers of lysine-EpmAB type EF-Ps and 4 transfers of arginine-EarP type EF-Ps. Next, we used phylogenetic distance comparisons between species trees and EF-P gene (protein) trees to qualitatively examine these 10 instances further (supplementary fig. S2, Supplementary Material online). We plotted the distance (cumulative branch length) between pairs of species on the species tree (Fig. 1) and a phylogenetic tree built from aligned EF-P protein sequences. If an efp gene has been horizontally transferred, the species tree and the EF-P tree will be discordant, because the transferred efp gene has not evolved within the clade it now resides in (see Methods for more details). These associations rendered some potential HGT events visually obvious (supplementary fig. S2, Supplementary Material online, especially b and e, the Planctomycetota & Verrucomicrobia and Thermotogota). We detail the results of these analyses further in the supplementary results, Supplementary Material online.

We decided to investigate two instances of EF-P transfer within the phyla Planctomycetota and Thermotogota further (Letters b and e in Fig. 1 and supplementary fig. S2, Supplementary Material online, respectively). We chose the Planctomycetota specifically because, as mentioned in the Introduction, they do not encode an otherwise universally conserved proline triplet in Valine tRNA synthetase (Starosta et al. 2014b). Otherwise, these phyla have sufficiently many genomes (>20) to reliably identify HGT, and they have highly discordant species and EF-P trees (supplementary fig. S2, Supplementary Material online). In addition, we found that these phyla do not show strong signatures of genome degradation, a confounding phenomenon that leads to the accumulation of many detrimental mutations and could complicate our analyses (supplementary fig. S3, Supplementary Material online and supplementary results, Supplementary Material online).

EF-P Transfer is Associated with Loss of Conserved Polyproline Motifs in the Thermotogota

The phylum Thermotogota consists of mostly thermophilic, fermentative anaerobes with a distinctive “toga”-shaped outer cell envelope (Pollo et al. 2015). Members of the phylum are commonly found within hydrothermal vents, petroleum reservoirs, and hot springs (Pollo et al. 2015). Our phylogenetic tree indicates that an efp gene may have been horizontally transferred into this phylum (Fig. 1 and supplementary fig. S2, Supplementary Material online, Letter e). Specifically, while most Thermotogota species encode an arginine EF-P of unknown modification (categorized as Family 1, supplementary fig. S1, Supplementary Material online), two species from the Petrotogaceae family (Geotoga petraeae and Oceanotoga teriensis) encode the arginine-EarP type EF-P and its modification system EarP (Fig. 1 and Family 3, supplementary fig. S1, Supplementary Material online). We used gene-species tree phylogenetic distance comparisons (Fig. 2, HGT 1) and gene synteny (Fig. 3, HGT 1) to verify the exogenous origin of this arginine-EarP type EF-P. These analyses showed that the gene synteny of this EF-P is inconsistent with other members of the phylum (Fig. 3, HGT 1). In addition this EF-P type shows greater sequence divergence from other Thermotogota EF-Ps than their shared phylogeny would predict (Fig. 2, HGT 1). For example, the arginine-EarP type EF-P of O. teriensis is more similar in sequence composition to EF-Ps from the Gammaproteobacteria family Burkholderiaceae (49.7% amino acid [aa] identity to the Parapusillimonas granuli EF-P) than to EF-Ps within the same phylogenetic family (39.5% aa identity to the intrafamily member M. hydrogenitolerans EF-P).

Fig. 2. Pairwise cophenetic distances between species’ positions on the species and EF-P protein sequence tree support horizontal transfer of EF-P in the Thermotogota. The vertical axis shows the cumulative branch length between two species on the species tree (Fig. 4), while the horizontal axis shows the cumulative branch length between the same two species on the EF-P protein sequence tree (Fig. 3). These phylogenetic distances correspond roughly to the number of amino acid substitutions per amino acid site. The dashed line indicates a 1:1 relationship between the species and EF-P trees. The color of the circles indicates from which phylogenetic families the pair of species being compared come from. For example, “Petrotogaceae × Thermotogaceae” indicates that a species from the family Petrotogaceae and a species from the family Thermotogaceae are being compared. We use this comparison as a visualization tool to identify potential horizontally transferred genes, as indicated by a phylogenetic distance that is not representative of the overall phylogenetic history of the clade. Two regions with anomalous evolutionary trajectories are outlined with solid lines. They indicate EF-P protein sequences that are more distantly related than the background phylogeny (HGT event 1, below the dashed 1:1 line), and EF-P protein sequences more closely related than the background phylogeny (HGT event 2, above the dashed 1:1 line). Within the Petrotogaceae family two separate gene transfer events have occurred, one from outside the phylum to the Oceanotoga and Geotoga genera (HGT Event 1, all colors) and one from within the family Thermotogaceae to the genera Petrotoga and Defluviitoga (HGT Event 2, Petrotogaceae × Thermotogaceae).

Fig. 3. Horizontal gene transfer of efp in the phylum Thermotogota. Left: Phylogenetic tree of EF-P amino acid sequences in the phylum Thermotogota (Methods). The phylogeny of EF-P in the family Petrotogaceae is not congruent with the overall phylogenetic tree of the family's species (Fig. 2). Within the Petrotogaceae family two separate gene transfer events of the efp gene have occurred (highlighted with shading across the figure), one arginine-unknown type from within the Thermotogaceae family (top shaded box) and one arginine-EarP type from outside the phylum (bottom shaded box). The color of the circle at the tree's tips represents the family these genomes belong to, according to the GTDB (Chaumeil et al. 2020). Level of ultrafast bootstrap support is indicated by the color of each node. Right: Gene synteny of each efp gene from the tree on the left. For clarity, only select genes of interest are indicated with color. The “native” copy of efp (centered) within the Thermotogota consistently co-occurs with yloU and nusB. This high conservation of synteny is absent for the efp genes highlighted with shading, which supports their horizontal transfer into the corresponding genomes. Additionally, the arginine-EarP type efp genes (bottom shaded box) co-occur with the genes for their modifying enzyme (earP), and with several genes in the rhamnose biosynthesis pathway (rfbB, rfbC, rfbD).

Unexpectedly, these analyses uncovered a second case of EF-P transfer within the Petrotogaceae family. That is, the efp genes within the genera Petrotoga and Defluviitoga have also lost the conserved gene synteny present in all other Thermotogota (Fig. 3, HGT 2). As opposed to the preceding example, they encode EF-Ps that is more similar in sequence to EF-P from the Thermotogaceae family than their shared phylogeny would predict (Fig. 2, HGT 2). For example, the EF-P of D. tunisiensis shares 75.1% aa identity to the EF-P of Thermotoga sp. RQ7 of the Thermotogaceae family, but only 61.1% aa identity to the EF-P of the intrafamily member M. hydrogenitolerans. It appears that species within the genera Petrotoga and Defluviitoga encode an arginine type EF-P of unknown modification that originated from a different family within Thermotogota, the Thermotogaceae. We further confirmed both these predicted horizontal transfer events with two independent computational methods (supplementary fig. S4, Supplementary Material online, supplementary results, Supplementary Material online).

Next, we investigated connections between the horizontal transfer of EF-P and polyproline motifs in the Thermotogota. We first clustered all proteins within the phylum into families based on amino acid sequence identity (Methods), then identified conserved polyproline motifs and polyproline motif-containing proteins within these families. Subsequently, we performed phylogenetic ANOVAs (Revell 2012) to test the null hypothesis that horizontal transfer of EF-P is not associated with the loss of conserved polyproline motifs, or of the proteins which contain them, while taking the phylogenetic relationships of our species into consideration. We found that the horizontal transfer of EF-P within the Thermotogota is significantly associated with the loss of 11 polyproline motifs or polyproline motif-containing proteins (Fig. 4). Loss of entire polyproline motif-containing proteins may indicate that the polyproline motif is crucial to their function, as is the case for the polyproline motif in the glucose importer component EIIGlc of C. glutamicum (Pinheiro et al. 2021). Interestingly, some of the polyproline motifs and polyproline containing-proteins linked to EF-P transfer were also lost among the genus Marinitoga within the Petrotogaceae family (Fig. 4). The synteny of the efp gene in the Marinitoga is similar to other families within the Thermotogota (Fig. 3). However, both tools we used to independently verify our HGT predictions indicated that this gene may be horizontally transferred (supplementary results, Supplementary Material online, supplementary fig. S4, Supplementary Material online). If true, this may explain why some cases of polyproline loss occur in the genus Marinitoga (Fig. 4).

Fig. 4. Eleven polyproline motifs and polyproline motif-containing proteins are significantly associated with horizontal transfer of EF-P into the Petrotogaceae family. We built this phylogenetic tree using IQ-TREE (Nguyen et al. 2015) with amino acid sequences of 43 concatenated and conserved marker genes generated by CheckM (Parks et al. 2015) (details in Methods). The color of the circle at the tree's tips represents the family these genomes belong to, according to the GTDB (Chaumeil et al. 2020). Level of ultrafast bootstrap support is indicated by the color of each node. Loss of “native” EF-P is indicated with shading across the figure, while gain of a horizontally transferred EF-P is indicated with the corresponding HGT Event as in Figs. 2 and 3. The exact timing and order of these events is unknown. The left-most heatmap shows, for eleven proteins, the presence of a conserved polyproline motif, the absence of the polyproline motif in the same position of the protein, or the complete absence of the protein. The eleven proteins are, from the left-most to the right-most column, as indicated by their acronyms: The putative ATPase YcaJ, the cell division protease FtsH, the ATP-dependent Clp protease ATP-binding subunit ClpC, the cation efflux protein FAM556, the polymerase primary sigma factor RpoD, the tryptophan synthase beta chain TrpB, the ATP-dependent Lon protease, the uncharacterized B12-binding/radical SAM-type protein FAM304, the uncharacterized metalloprotease FAM437, the type IV pilus assembly protein PilC, and the trehalose synthase TreT. Proteins with the prefix FAM- are not annotated by the KEGG database (Kanehisa et al. 2023), and this designation refers to their Silix (Miele et al. 2011) identifier (Methods). The right-most heatmap shows the EF-P type present in the corresponding species.

Next, we examined the polyproline motif-containing proteins that were significantly associated with the horizontal transfer of EF-P within certain members of the family Petrotogaceae. These proteins were functionally diverse. They include three ATP-dependent proteases (Lon, ClpC, and FtsH), two enzymes related to metabolism (the tryptophan synthase beta chain TrpB, the trehalose synthase TreT), four poorly characterized proteins (the putative ATPase YcaJ, a cation efflux protein annotated as FAM556 by our amino acid sequence clustering (Methods), an uncharacterized B12-binding/radical SAM-type protein FAM304, an uncharacterized metalloprotease FAM437), and two proteins of varied functions (type IV pilus assembly protein PilC, RNA polymerase primary sigma factor RpoD). Several of the polyproline motifs these proteins contain are highly conserved among our 3,000 bacterial genome dataset (supplementary table S1, Supplementary Material online). For example, the polyproline motifs located within the bacterial proteases are widely conserved among bacteria—85.5%, 97.9%, and 98.8% of FtsH, ClpC, and Lon proteins contain polyproline motifs at these exact positions, respectively. This strong conservation implies that these polyproline motifs are important to the proteins harboring them, and that these proteins likely depend on EF-P for expression.

EF-P Transfer is Associated with Loss of Conserved Polyproline Motifs in the Planctomycetota

Next, we examined the suspected horizontal transfer of EF-P to members of the phylum Planctomycetota. In a previous study (Brewer and Wagner 2022), we had discovered that the loss of a polyproline triplet in the protein Val tRNA synthetase, which is otherwise conserved across all three domains of life (Starosta et al. 2014b), coincided with a case of horizontally transferred EF-P in this phylum. We wanted to find out if additional patterns of polyproline motif loss coincided with this event. The phylum Planctomycetota includes cosmopolitan species that can be found in soil, aquatic, and wastewater habitats (Wiegand et al. 2018). These bacteria possess diverse cell structures and metabolisms. Some species divide by budding, some have cytoplasmic compartments, some perform anaerobic ammonium oxidation, and many have complex life cycles featuring transitions between sessile and swimming states (Wiegand et al. 2018). Our initial analyses indicated that Planctomycetota species have undergone at least three EF-P transfer events (Fig. 1, Letter b). As a result, they now encode four distinct types of EF-P, including EF-P types with unknown modifications (which cluster into three distinct EF-P families and likely include the “native” EF-P type, supplementary fig. S1, Supplementary Material online), lysine-EpmAB type EF-P, arginine-EarP type EF-P, and arginine-EfpL type EF-P.

As before, we verified HGT events using gene synteny (supplementary fig. S5, Supplementary Material online) and phylogenetic distance comparisons between gene and species trees (supplementary fig. S6, Supplementary Material online). We found that members of the class Planctomycetes, including the families Planctomycetaceae, Pirellulaceae, and Thermoguttaceae, no longer encode the native EF-P type of unknown modification that is present at the base of the Planctomycetes tree. Instead they encode a lysine-EpmAB type EF-P (HGT 1, supplementary fig. S5, Supplementary Material online). This EF-P is distinct in sequence to all other types in the phylum (supplementary fig. S6, Supplementary Material online), and in most species is flanked by the epmB gene (HGT 1, supplementary fig. S5, Supplementary Material online). A different, arginine-EarP type EF-P, is encoded by members of the genus Planctopirus, and is flanked by the earP gene (HGT 2, supplementary fig. S5, Supplementary Material online). These arginine-EarP type EF-Ps are more closely related to EF-P from the Gammaproteobacteria than to those from the Planctomycetota (supplementary fig. S2, Supplementary Material online, Letter b). The final horizontally transferred EF-P type, arginine-EfpL, is encoded somewhat sporadically within the class Planctomycetes, including within the Pirellulaceae, Isosphaeraceae, and Thermoguttaceae families (HGT 3, supplementary fig. S5, Supplementary Material online). In all species where it occurs, the arginine-EfpL type EF-P is paired with a nonEfpL type EF-P. As with the Thermotogota, we confirmed these horizontal transfer events with two independent methods (supplementary results, Supplementary Material online, supplementary fig. S7, Supplementary Material online).

Following the same procedure as for the phylum Thermotogota, we found that three polyproline motifs are significantly associated with the horizontal transfer of EF-P, but in two different subsets of the class Planctomycetes (Fig. 5). We found that all members of the class Planctomycetes that no longer encoded the native Planctomycetota EF-P also lost the polyproline motif containing Lon protease, similar to what we had observed in the Thermotogota (Fig. 4). Additionally, all members of the class Planctomycetes with HGT efp lost the polyproline-motif containing protein isoleucine tRNA synthetase type 1, instead encoding isoleucine tRNA synthetase type 2. Two distinct forms of isoleucine-tRNA synthase exist among bacteria—the typical form present in E. coli and most other species (type 1) and a second form (type 2) more closely related to eukaryotic type IleS, which lacks tRNA-dependent pretransfer editing activity (Cvetesic et al. 2016). In just the family Planctomycetaceae, we found that the loss of the conserved polyproline motif in valine tRNA synthetase was associated with EF-P transfer (Fig. 5).

Fig. 5. Three polyproline motifs and polyproline motif-containing proteins are significantly associated with horizontal transfer of EF-P in the class Planctomycetes. We built this phylogenetic tree using amino acid sequences of 43 concatenated and conserved marker genes generated by CheckM (Parks et al. 2015) (details in Methods). The color of the circle at the tree's tips represents the family these genomes belong to, according to the GTDB (Chaumeil et al. 2020). Level of ultrafast bootstrap support is indicated by the color of each node. Loss of “native” EF-P is indicated with shading across the figure, while gain of a horizontally transferred EF-P is indicated with the corresponding HGT event, as in supplementary figs. S5 and S6, Supplementary Material online. The exact timing and order of these events is unknown. The left-most heatmap reports the presence of a conserved polyproline motif, the absence of the polyproline motif in the same position in the protein, or the complete absence of the corresponding protein. The four proteins displayed are, from left to right: isoleucine tRNA synthetase type 2 (IleS2), valine tRNA synthetase (ValS), isoleucine tRNA synthetase type 1 (IleS1), and ATP-dependent Lon protease (Lon). Sequence homology clustering of Planctomycetes proteins revealed two distinct forms of isoleucine-tRNA synthase, the typical form present in E. coli and most other bacteria (type 1) and a second form more closely related to eukaryotic type IleS, which lacks tRNA-dependent pretransfer editing activity (type 2) (Cvetesic et al. 2016). While loss of the conserved polyproline motif in IleS2 was not significantly linked with horizontal transfer of EF-P after phylogenetic correction, its similar pattern of co-occurrence and conservation with ValS led us to include it here. The right-most heatmap shows the EF-P type present in the corresponding species; several species encode the EF-P paralog arginine-EfpL.

Characteristics of Proteins Sensitive to Horizontal Transfer of EF-P

From our investigations of the phyla Thermotogota and Planctomycetota, two major groups of proteins that appear to be consistently sensitive to EF-P transfer emerged. These are ATP dependent proteases (Lon, ClpC, and FtsH) and class I tRNA synthetases (ValS, IleS1, and IleS2). The polyproline motifs these proteins contain are well conserved throughout our wider dataset (supplementary table S1, Supplementary Material online), meaning that they may rely on EF-P for expression in most bacteria. We searched for similarities within these groups and tried to determine whether the polyproline motifs linked to EF-P transfer are important for the function of these proteins.

The polyproline motifs within the three proteases (Lon, ClpC, and FtsH) are well conserved among our wider dataset of over 3,000 bacterial species. Specifically, 98.8% of Lon, 97.9% of ClpC, and 85.5% of FtsH proteins have polyproline motifs in this position (supplementary table S1, Supplementary Material online). Upon further examination, we discovered that each of these well-conserved polyproline motifs is located within the same PFAM domain (PF00004) (Mistry et al. 2021). The same holds for some nonproteases. For example, we found that the well-conserved polyproline motif in the putative ATPase YcaJ (Fig. 4) is also located within domain PF00004. This PFAM domain characterizes a diverse ATPase family associated with a broad range of cellular activities (Neuwald et al. 1999). Within this domain, each polyproline motif is positioned within the ATP binding pocket (Fig. 6). Mutations of residues within this ATP binding pocket inactivate Lon protease (Fischer and Glockshuber 1994) and FtsH (Karata et al. 1999) in E. coli. Furthermore, the expression of Lon and Clp proteins is dependent on EF-P in E. coli (Peil et al. 2013; Woolstenhulme et al. 2015) and Salmonella enterica (Hersch et al. 2013). Expression of FtsH (known by the synonym HflB) is also EF-P-dependent in S. enterica (Hersch et al. 2013). Together, these observations suggest that these highly conserved polyproline motifs are important for ATP binding and hydrolysis in ATP-dependent proteases. Consequently, these proteins are prone to depend on EF-P for proper expression.

Fig. 6. Polyproline protein sequence motifs sensitive to the loss of native EF-P. Left: We found conserved polyproline (PP) motifs within three tRNA synthetases (ValS, IleS1, and IleS2) and three proteases (FtsH, Lon, and ClpC) to be linked to the loss of native EF-P. That is, in species that no longer encode their phylogenetically “native” EF-P, these motifs are often absent. Protein length is indicated by narrow horizontal bars, on top of which PFAM domains annotated by InterProScan (Jones et al. 2014) are displayed, colored by either their PFAM or PFAM clan (Mistry et al. 2021) description. Locations of conserved PP motifs are indicated with an X. Vertical bars correspond to regions predicted to interact with ATP. More specifically, they represent (i) the conserved tRNA class I His-Ile-Gly-His “HIGH” consensus motifs (Arnez and Moras 1997) InterproID (IPR001412) for the tRNA synthetases, and (ii) ATP binding regions (InterproID IPR001270 or PIRsitepredict ID PIRSR001174-2) for the proteases. For consistency, all proteins shown in the figure are from the genome of Kosmotoga arenicorallina (which encodes all indicated PP motifs and the native unknown-EF-P type of the phylum Thermotogota), except for IleS2, which is from Mesotoga prima. (K. arenicorallina encodes only IleS1, not IleS2.) M. prima also encodes the native unknown-EF-P type of the phylum Thermotogota). Right: Sequence logos of the well conserved PP motifs represented with an X in the left panel within our dataset of >3,000 bacterial genomes from 35 phyla. Specifically, 99.8% of ValS, 99.7% of IleS1, 99.3% of IleS2, 85.5% of FtsH, 98.8% of Lon, and 97.9% of ClpC proteins in our dataset have PP motifs in the indicated position. The consensus motifs of the species that do not encode the canonical PP motifs are, following the figure order from top to bottom: “MIPLP,” “DGPIY,” “DGPIT,” “VGSPG,” “VGAPG,” and “GSAPG”.

The polyproline motifs within the three class I tRNA synthetases (ValS, IleS1, and IleS2) are the most strongly conserved in our wider 3,000 species dataset. Specifically, 99.8% of ValS, 99.7% of IleS1, and 99.3% of IleS2 proteins have polyproline motifs in this position. Indeed, a previous study found that these polyproline motifs are invariant across all domains of life (Starosta et al. 2014b). The ValS polyproline motif is encoded by all nonPlanctomycetaceae species in our wider genome dataset, with only one exception, the Cyanobacterium Pseudanabaena sp. PCC 7367. However, all other available genomes from the family Pseudanabaenaceae encode the polyproline motif, meaning there are no additional genomes that can help validate the accuracy of this exception. Similar to the ATP-dependent proteases, the polyproline motif in ValS lies within the ATP binding region of this protein, near the His-Ile-Gly-His (“HIGH”) motif that is characteristic of this class of tRNA synthetases (Fig. 6) (Arnez and Moras 1997; Starosta et al. 2014b). Mutation of this polyproline motif in E. coli ValS results in a protein that nonproductively hydrolyzes ATP to ADP and mischarges tRNAVal with threonine (Starosta et al. 2014b). The polyproline motifs of IleS type 1 and 2 also lie next to the HIGH motif of these proteins (Fig. 6). While the function of this motif in IleS has not been characterized, it is also likely to be involved in ATP binding and hydrolyzation (Arnez and Moras 1997). While the loss of the highly conserved polyproline motif in ValS severely impairs its function in E. coli, we found no genomic evidence of a malfunctioning ValS or IleS in the Planctomycetes (supplementary results, Supplementary Material online, supplementary fig. S8, Supplementary Material online). Expression of ValS is strongly dependent on EF-P in E. coli (Starosta et al. 2014b; Woolstenhulme et al. 2015) and S. enterica (Hersch et al. 2013). Indeed, under-expression of ValS in the E. coli efp deletion mutant is partially responsible for its strong growth defect (Starosta et al. 2014b).

In summary, we find that the highly conserved polyproline motifs present in both the ATP-dependent proteases and class I tRNA synthetases are likely involved in ATP binding. Many of these proteins are dependent on EF-P for normal expression across multiple species (Starosta et al. 2014b; Woolstenhulme et al. 2015). The alteration of these highly conserved polyproline motifs, or the loss of the proteins containing them, coincides with EF-P transfer in two independent phyla. We conclude from these observations that loss of the ancestral EF-P type and appearance of a horizontally transferred EF-P variant is associated with a disruption in global EF-P function. In response to this disruption, selective pressure against polyproline motifs emerged, namely in proteins that are dependent on EF-P for proper expression.

Discussion

In a previous analysis (Brewer and Wagner 2022), we discovered hints of a connection between the horizontal transfer of efp and disruption of EF-P activity through the loss of highly conserved polyproline motifs. In this work, we thoroughly investigate this connection and study how disruption of EF-P activity may have affected modern-day bacterial species from a wider phylogenetic diversity than experimental methods allow. To this end, we first characterized EF-P from a set of over 3,000 bacterial genomes from 35 phyla. We found that horizontal transfer of the efp gene has occurred multiple times along the bacterial tree of life, with several cases of the arginine-EarP type EF-P and lysine-EpmAB type EF-P found outside their clades of phylogenetic origin (Fig. 1 and supplementary fig. S2, Supplementary Material online). We examined in more detail two phyla whose members contain horizontally transferred EF-P, the Thermotogota (Fig. 2 and supplementary fig. S3, Supplementary Material online) and the Planctomycetota (supplementary figs. S5 and S6, Supplementary Material online) and found that loss of the ancestral EF-P type and appearance of a horizontally transferred EF-P variant is consistently associated with the loss of polyproline motifs and polyproline motif-containing proteins (Figs. 4 and 5). In particular, we found two groups of proteins that seem to be particularly sensitive to disruptions in efp. These are the ATP-dependent proteases Lon, ClpC, and FtsH, as well as the class I tRNA synthetases IleS (type 1 and 2) and ValS (Fig. 6). We show that the position of the polyproline motifs within these proteins is highly conserved within the wider diversity of our 3,000 bacterial species (supplementary table S1, Supplementary Material online). Additionally, these motifs are all within or in close proximity to ATP-binding domains (Fig. 6). While the polyproline motif within ValS is known to be crucial for proper ATP hydrolyzation (Starosta et al. 2014b), the other polyproline motifs have not been studied in detail.

Our analyses suggest that species from the Thermotogota and Planctomycetota phyla which now encode horizontally transferred efp experienced a disruption in EF-P activity at some point in their evolutionary history. This disruption would have negatively impacted the expression of polyproline containing proteins, as has been shown experimentally in multiple species (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Guo et al. 2022). With only modern-day genomes to work from, we cannot conclusively determine the order of subsequent events. It may be that species that experienced this disruption in EF-P activity already encoded key proteins that had lost their characteristic polyproline motifs. A lower starting dependence on EF-P for protein expression may then have allowed these species to survive long enough to eventually acquire new EF-P via HGT. In this scenario, the original impetus for polyproline loss is unclear.

In our view, a more likely scenario is that a disruption of EF-P activity triggered selective pressure against polyproline motifs, which led some proteins to lose highly conserved polyproline motifs, thereby reducing their dependence on EF-P for expression. In other proteins, like Lon protease, these highly conserved polyproline motifs may be critical to the function of the protein, leading instead to the loss of the entire protein. Again, with only modern-day genomes to work from, we can merely hypothesize what the cause of the original disruption in EF-P activity was. One possibility is that species within the Planctomycetota and Thermotogota first lost their “native” form of EF-P, which would make EF-P transfer especially advantageous. The modern-day Thermotogota and Planctomycetota species, which encode horizontally transferred versions of EF-P, no longer encode the EF-P native to their phylum (Fig. 3 and supplementary fig. S5, Supplementary Material online), but we do find cases of horizontally transferred EF-P co-existing with native EF-P, albeit only rarely (supplementary results, Supplementary Material online). Furthermore, EF-P loss has not been observed in free-living organisms. EF-P is universally conserved in bacteria, with the exception of a few endosymbiotic species undergoing genome degradation (Sabree et al. 2012; Lassak et al. 2016). EF-P loss can have stark consequences. Experiments show that EF-P loss is lethal in two species (Yanagisawa et al. 2016; Guo et al. 2022), and causes severely deleterious phenotypes in other species (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Rajkovic et al. 2016; Tollerson et al. 2018). Furthermore, a recent study using transposon mutagenesis revealed that both the horizontally transferred arginine-EarP type EF-P and its modification system EarP are essential in the Planctomycete Planctopirus limnophilia. (Rivas-Marin et al. 2023).

Yet another possibility is that the transfer of an exogenous EF-P and its modification system into the Thermotogota and Planctomycetota happened before any loss of native EF-P. In this case, deleterious interactions between the native and horizontally transferred proteins may have perturbed the expression of EF-P-dependent proteins. It is tempting to speculate that these perturbations arose from deleterious interactions between the horizontally transferred modification system and the native EF-P. Different EF-P types and their corresponding modification systems co-evolved, such that switching just the conserved, post-translationally modified amino acid of an EF-P is not sufficient to switch its modification system (Lassak et al. 2015; Volkwein et al. 2019). For example, the function of an arginine-EarP type EF-PR32K from Shewanella oneidensis cannot be rescued by expression of the EpmAB system from E. coli, and neither can the function of a lysine-EpmAB type EF-PK34R from E. coli be rescued by the expression of EarP from S. oneidensis (Lassak et al. 2015). Indeed, subjecting an EF-P to a non-native post-translational modification can inhibit its function below that of the unmodified protein (Volkwein et al. 2019). If a non-native modification system can impair the function of the native EF-P type, overall EF-P function could be compromised, leading to the genome evolution patterns we observe. In support of this hypothesis, we note that the conserved positions within EF-P that bear post-translational modifications in both the Thermotogota and the Planctomycetes are theoretically compatible with their horizontally transferred modification systems. That is to say, the native EF-P within the Thermotogota is an arginine type EF-P of unknown modification, and the horizontally transferred EarP modifies arginine residues (supplementary fig. S9, Supplementary Material online). Likewise, one of the native EF-P within the Planctomycetota is a lysine type of unknown modification (supplementary fig. S10, Supplementary Material online), and the horizontally transferred EpmAB system modifies lysine residues.

Horizontal transfer of new EF-Ps and their post-translational modification systems have occurred multiple times in bacterial evolution (Fig. 1 and supplementary fig. S2, Supplementary Material online). This suggests that the horizontal transfer of post-translationally modified EF-P has benefits. This is plausible, as post-translational modifications permit additional interactions with the P-site tRNA, as exemplified by the (R)-β-lysylation of the E. coli EF-P (Huter et al. 2017). These interactions lead to improved functionality of the E. coli EF-P when compared with its shorter, unmodified form (Huter et al. 2017). Furthermore, post-translational modification systems of EF-P have evolved at least three independent times in bacteria, i.e. rhamnosylation by EarP (Lassak et al. 2015), lysylation by EpmAB (Park et al. 2012; Peil et al. 2012), and aminopentanolylation by YmfI, YnbB, and GsaB (Hummels et al. 2017; Witzky et al. 2018). The orthologs of EF-P in archaea and eukaryotes are also post-translationally modified (Lassak et al. 2016). These incidents of convergent evolution suggest the general utility of the post-translational modification of EF-P, and the benefit that extending the “reach” of EF-P further into the peptidyl-transferase center of the ribosome confers.

Lastly, we note that the horizontal transfer of EF-P is an anomaly when considered in the context of typical HGT. EF-P is a highly conserved protein involved in the fundamental cellular process of translation. The transfer of foreign genes into a new host almost always results in strong fitness costs (Acar Kirit et al. 2020), and genes related to transcription and translation are rarely transferred, possibly because their transfer is especially costly (Sorek et al. 2007; Kanhere and Vingron 2009; Brockhurst et al. 2019). It may be that horizontally transferred translation-related genes are more readily maintained in species with lifestyles capable of tolerating “hiccups” in translation due to decreased selection pressure on translational speed. In this regard, it is relevant that our two focal phyla in which the horizontal transfer of EF-P occurred have such lifestyles. The Thermotogota are mostly thermophiles with optimal growth temperatures between 45 and 60 °C (Pollo et al. 2015). The Planctomycetota are mostly mesophiles and notoriously slow-growing, with doubling times ranging from 6 h to 1 month (Jeske et al. 2016). In a previous study, we found that both slow-growing bacteria and thermophilic bacteria are enriched in polyproline motifs when compared with fast-growing mesophiles (Brewer and Wagner 2022). We hypothesized that this enrichment occurred for two reasons. The first is that slow-growing organisms experience lower selective pressure on translation speed and thus do not need to synthesize proteins rapidly. The second is that thermophiles derive a catalytic “boost” from high growth temperatures, which may lead to naturally higher rates of translation (Vieira-Silva and Rocha 2010). This catalytic boost may also accelerate the formation of proline-proline bonds (Brewer and Wagner 2022). If so, these two groups of organisms may be particularly well poised to endure disruptions in EF-P function, which may give them sufficient time to adapt evolutionarily by altering polyproline motifs to make key proteins EF-P independent. Tentative support for this hypothesis comes from studies of the efp deletion mutant phenotype in E. coli (Tollerson et al. 2018). The growth defect of these mutants is less severe when E. coli cells grow more slowly, indicating that dependence on EF-P is strongest when protein expression and demand for translational efficiency are high (Tollerson et al. 2018). However, these speculations require experimental validation, because all EF-P mutants thus far have been studied in relatively fast-growing, mesophilic organisms (Peng et al. 2001; Navarre et al. 2010; Lassak et al. 2015; Rajkovic et al. 2016; Tollerson et al. 2018).

The power of comparative genomics comes from the ability to leverage the evolutionary history of thousands of species and to make predictions based on their signatures of genomic change. It has allowed us to discover a recurrent connection between ancestral EF-P disruption and the loss of highly conserved polyproline motifs and polyproline-motif containing proteins. Ancient disruptions in EF-P activity have not only left clear traces in the genomes of modern-day species, but they may also have impacted the evolution of these species to the present day.

Materials and Methods

Selection of Study Phyla

We used a set of 3,265 phylogenetically diverse bacterial genomes that we characterized in a previous study (Brewer and Wagner 2022). These genomes span 35 phyla and were selected to maximize phylogenetic diversity—we included only one genome per Average Nucleotide Identity cluster, or in other words, only one genome per species present in the Integrated Microbial Genomes (IMG) database (Chen et al. 2021). We checked each genome for completeness and contamination with CheckM (Parks et al. 2015) (with cutoffs of ≥90% completeness and ≤5% contamination), and reassigned taxonomy using the Genome Taxonomy Database and GTDB-Tool kit version 0.2.2 (Chaumeil et al. 2020).

We used these genomes to identify taxonomic groups that may have lost their “native” EF-P and obtained exogenous EF-P through horizontal gene transfer, as identified through “non-native” types of post-translational EF-P modifications. The presence of such modifications can be computationally inferred by identifying the genes that encode the proteins which perform the modification, as well as by identifying the modified amino acid within EF-P. Because EF-P modification types originated in distinct phylogenetic clusters of bacteria, EF-P modification types that occur outside their cluster of origin indicate that an EF-P-coding gene and/or its associated modification genes have been transferred.

To detect such instances of HGT we first created a phylogenetic tree of all our genomes using 43 concatenated and conserved marker protein sequences generated by CheckM (v1.0.12) (Parks et al. 2015), then used IQ-TREE (v1.6.12) (Nguyen et al. 2015) to build the tree. We used the model finder feature (Kalyaanamoorthy et al. 2017) included in IQ-TREE to determine the best-fit substitution model for our tree (which was the “LG + F + R10” model). This tree is shown in Fig. 1 and is rooted with the genome of the Archaeon Haloquadratum walsbyi.

Next, we determined the post-translationally modified amino acid residue of each EF-P. We did this by first aligning all proteins annotated as EF-P by the KEGG functional database (Kegg Orthology term: K02356) using MUSCLE (v3.8.31) (Edgar 2004) with default settings. We then identified the post-translationally modified amino acid residue using the MUSCLE alignment with validated EF-P sequences as a guide. We assigned EF-P modification types by searching for a mix of annotations from the Clusters of Orthologous Groups (COG) (Galperin et al. 2021) and Pfam (Protein families) databases (Mistry et al. 2021). First, we considered a species to have an EF-P modified by β-(R)-lysylation if its genome encoded the genes for EpmA (COG2269) and EpmB (COG1509) (Park et al. 2012). To identify the optional hydroxylation of EpmAB-modified EF-P, we looked for the gene encoding EpmC (pfam04315) (Peil et al. 2012). Second, we considered an EF-P to be modified by rhamnosylation if its genome encoded EarP (pfam10093) (Lassak et al. 2015). Third, we considered an EF-P to be modified by the 5-aminopentanol moiety if its genome yielded BLASTP (v2.13.0+) (Camacho et al. 2009) hits in a search for the three proteins that carry out this attachment (YmfI, YnbB, and GsaB) (Hummels et al. 2017; Witzky et al. 2018), using B. subtilis orthologs of these proteins as our query sequences. The proteins responsible for the aminopentanolylation of EF-P are known to be homologous to many other broadly conserved proteins (Witzky et al. 2018) and do not have a consistent annotation in functional databases. Therefore we used different bit-score cutoffs for each gene after manual inspection of the results to reduce false positives from Gammaproteobacteria (YmfI: 145, YnbB: 200, and GsaB: 525). Fourth, we identified EfpL type EF-Ps using the TIGRfam (The Institute for Genomic Research Protein Families) database (Li et al. 2021). TIGRfam is a collection of manually curated protein families similar to Pfam, and it is the only database that distinguishes between canonical EF-P (TIGR00038) and the EfpL (TIGR02178) subtypes. We validated these four EF-P modification type assignments by confirming that the respective efp genes encoded lysine (EpmAB/C and YmfI/YnbB/GsaB) or arginine (EarP and EfpL) at the conserved modification position. Lastly, we identified EF-Ps that function without any modification by searching for the characteristic unmodified proline loop in the conserved positions 30 and 34 (P30GKGP34) within EF-P protein sequences (Pinheiro et al. 2020).

We used these assigned EF-P types to identify which bacterial phyla to target further for in-depth investigation. Our initial findings led us to focus on the Planctomycetota, and we chose the Thermotogota because they are a free-living clade unlikely to be undergoing genome degradation (supplementary fig. S3, Supplementary Material online). Also, the genomes of both phyla are well-represented in our data set (>20 genome sequences per phylum).

Verifying HGT of the efp Gene

To verify horizontal transfer of EF-P, we first constructed an EF-P specific phylogenetic tree using the EF-P amino acid sequences we aligned in previous steps. We built this phylogenetic tree with IQ-TREE (v1.6.12) (Nguyen et al. 2015) using the “LG + I + G4” model. We used comparisons between this EF-P tree and the species tree (created in previous steps with 43 concatenated and conserved marker protein sequences) to identify putative horizontally transferred EF-Ps. If an EF-P-coding gene has been transferred horizontally, the species tree and the EF-P tree will be discordant, because the transferred EF-P gene has not evolved within the clade it now resides in (supplementary fig. S2, Supplementary Material online).

We next used two methods to manually corroborate putative horizontal transfers of EF-P. First, we compared the species and EF-P trees by plotting the distance (cumulative branch length) between pairs of species on each tree. We found that generally, these cumulative branch lengths are highly correlated between the two trees. In other words, the rate at which amino acid substitutions occur in the native, translated efp gene is similar to the rate of amino acid substitutions within the 43 conserved marker protein sequences we used to build the species tree. This association renders likely EF-P horizontal transfer events visually obvious outliers (Fig. 2, supplementary figs. S2 and S6, Supplementary Material online). Second, we investigated the gene synteny between the efp gene and adjacent genes for each genome from our phyla of interest to verify that gene order differs between putatively transferred and native efp copies (Fig. 3 and supplementary fig. S5, Supplementary Material online). While the efp genes we classify as “native” do not show evidence of recent arrival at their corresponding genomes, this method would not necessarily detect a sufficiently ancient HGT event that preserves synteny or occurred phylum wide.

As a final step to verify the horizontal transfers we found, we checked whether independent computational tools designed to detect HGT agreed with our conclusions. We used two tools that use distinct methods to predict HGT. First, we used Alienness versus Predictor (AvP: Koutsovoulos et al. 2022) to calculate the HGT index metric for each efp gene in our target phyla using a composition-based, sequence similarity approach. The HGT index is based on the results of a sequence similarity search and represents the difference between the bit-score of the best outgroup match and the bit-score of the best ingroup match (Boschetti et al. 2012). For each efp gene, we used efp genes from within the same phylogenetic family as our ingroup, and efp genes outside the respective phylogenetic order as our outgroups. As a database we used every EF-P protein sequence in the UniProt database that contained all three canonical EF-P pFAM domains (PF01132, PF08207, PF09285: 46,112 sequences). The second tool we used is amalgamated likelihood estimation (ALE: Szöllősi et al. 2015), which detects HGT using a phylogeny-based approach. ALE predicts gene loss, duplication, and transfer events in order to reconcile species and gene trees and produce a summary gene tree that maximizes joint likelihood. We used the ALE_undated command which does not require dated input trees. As input trees, we used our main efp gene and species trees subset for our phyla of interest and related groups with 1,000 ultrafast bootstraps computed by IQ-TREE. We only considered HGT events predicted by ALE that had an inferred reconciliation frequency exceeding 0.35, similar to (Dharamshi et al. 2023). Detailed results of these analyses are presented in the supplementary results, Supplementary Material online.

Loss of Conserved Polyproline Motifs

To find polyproline motifs whose loss coincided with horizontal transfer of EF-P, we used a computational method that is independent of annotation databases. This is important because our phyla of interest are poorly studied, and thus poorly annotated. (For example, 45% of genes in the Planctomycete Planctopirus limnophila have no predicted function). For each phylum we were interested in, we combined all proteins from each genome within that phylum into one file (50 species for the Planctomycetes and 24 species for the Thermotogota), then performed an all-versus-all BLASTP search, using default parameters. With the resulting BLASTP output, we performed de novo clustering of the proteins using Silix (v1.3.0) (Miele et al. 2011). Silix is a software tool that clusters protein sequences into homologous families using similarity networks (Miele et al. 2011). Sequences with ≥35% sequence identity and ≥80% sequence alignment were assigned to the same family. This generated 2,379 and 74,409 protein families respectively, for the phyla Thermotogota and Planctomycetes. We aligned each family of proteins with MUSCLE and used custom python scripts to locate polyproline motifs within the proteins.

We tested the null hypothesis that horizontal transfer of the efp gene into a genome is not associated with (i) the loss of conserved polyproline motifs within particular proteins, or (ii) the loss of entire proteins containing well conserved polyproline motifs. We sub-selected the proteins we tested using the following criteria. First, we created two groups of genomes—those which encoded a putative horizontally transferred efp gene (HGT group) and those which still encoded their native efp gene (non-HGT group). To select which conserved polyproline motifs within particular proteins to test, each polyproline motif and the protein it resides within had to meet at least one of two conditions:

The protein and polyproline motif must be well conserved among the non-HGT group and poorly conserved among the HGT group (70% of genomes in the non-HGT group encode the protein, and the polyproline motif is present at a conserved position in at least 85% of these, while at most 20% of genomes in the HGT group encode the polyproline motif).

The protein and polyproline motif must be well conserved among the non-HGT group, but the protein itself is rare in the HGT group (same cutoffs as above for the non-HGT group, but the polyproline containing protein must be present in no more than 20% of genomes in the HGT group).

For polyproline motifs or polyproline containing proteins that passed these filters, we used the phylANOVA function from the R package phytools (v. 0.7-70) (Revell 2012) to test our null hypothesis that horizontal transfer is not associated with a loss of polyproline motifs or of proteins harboring them. PhylANOVA allowed us to control for the phylogenetic interdependence of protein sequences using the species trees described above. We corrected P-values for multiple testing using the Benjamini & Hochberg method at a false discovery rate of 0.1.

Characterizing Conservation of Polyproline Motifs

For each annotated protein and/or polyproline motif whose loss was significantly associated with the HGT of efp, we also determined how conserved the relevant polyproline motif is in our wider 3,000 species genome set. To this end, we first extracted the amino acid sequences of these proteins (YcaJ, FtsH, ClpC, RpoD, TrpB, Lon, PilC, TreT, ValS, and IleS) from each genome in our dataset using KEGG annotations (Kanehisa et al. 2023). For each set of proteins, we again used Silix to cluster homologs into families using the same parameters as described above. We clustered these protein sequences because we found some orthologous proteins to be annotated with the same KEGG Orthology term. For example, the Lon protease, the Lon-like protease BrxL, and the sporulation protease LonC are all annotated as ATP-dependent Lon proteases (K01338) by KEGG. We next aligned the Silix families bearing our protein of interest using MUSCLE, and then used our custom python scripts to locate conserved polyproline motifs. We then calculated how conserved each of these polyproline motifs are within each target protein across our 3,000 genomes dataset (supplementary table S1, Supplementary Material online).

Supplementary Material

msae180_Supplementary_Data

Acknowledgments

We acknowledge funding from the European Research Council under Grant Agreement No. 739874, as well as from Swiss National Science Foundation grant 31003A_172887, and from the University Priority Research Program in Evolutionary Biology at the University of Zurich. Additionally, we thank Michael Engel, Danielle Godreau, and Jürgen Lassak for helpful suggestions and discussions.

Supplementary Material

Supplementary material is available at Molecular Biology and Evolution online.

Data Availability

All genomes used in this study are publicly available from JGI's IMG database (Chen et al. 2021). R scripts and all files needed to reproduce these analyses and figures are available at: https://github.com/tessbrewer/pattern_project.
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