i) Ribosomal and other proteins that are involved in the translation process.

Ribosomes are essential cellular complexes composed of small proteins and RNA molecules. They play a crucial role across all three domains of life, Bacteria, Archaea, and Eukarya, in translating mRNA into proteins by catalyzing the assembly of amino acids into polypeptide chains. However, the complexity of ribosomes varies depending on the phylogenetic origin of the organism. Bacterial organisms show the lowest, followed by archaeal organisms, while eukaryotic organisms exhibit the highest level of complexity in their ribosomal composition [11, 12].

Despite the differences in composition, ribosomes of all three domains of life possess a common set of 34 proteins that are universally conserved and are the central core of their structure. This set of proteins is responsible for the ribosome´s main functions, such as peptide bond formation during protein synthesis [11, 12]. In addition, there is a set of ribosomal proteins common to archaeal and eukaryotic organisms, while some others are exclusively found in bacterial and eukaryotic organisms.

To investigate the phylogenetic distribution of proteins involved in the translation process, we utilized PhyloString to analyze the functional relationships within the COG0199, which clusters the S14 orthologous ribosomal proteins. The resulting analysis is presented in the heatmap shown in Fig 2. This figure reveals that COGs of modules (4,1), (4,2), and (4,3) (COG group 4, S3 Table) form part of the core ribosomal proteins shared by Bacteria, Archaea, and Eukarya (phylogenetic groups 1–3, S4 Table). The statistical values for the COG enrichment within this group can be found in the supplementary material (S5 Table). Furthermore, we observed that COGs of modules (3,2) and (3,3) mainly correspond to proteins shared by Archaea and Eukarya, while COGs of modules (2,1) and (2,3) are primarily associated with proteins shared by Bacteria and Eukarya. Lastly, module (1,1) corresponds to proteins mainly present in Bacteria.

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Fig 2. Heatmap of COG0199 involved in the translation of protein process.

Ribosomal protein S14 orthologous belongs to this COG0199. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 850, COG centers: 4, Phylogenetic centers: 3. Conserved COGs found across all three kingdoms of life, showing widespread distribution, are often crucial in the translation protein process. As a result, they typically appear in dark red in color, with STRING index values approaching the maximum of 1,000.

https://doi.org/10.1371/journal.pone.0297010.g002

ii) Amino acid biosynthesis. Tryptophan autotrophic and heterotrophic organisms identified by PhyloString.

L-tryptophan is one of the scarcest amino acids found in most proteins and the most costly to synthesize in terms of cell resources [13]. In many bacterial organisms, the genes coding for the enzymes involved in the tryptophan biosynthesis are transcribed in the same polycistronic unit to facilitate their coordinated expression. To identify the overall distribution of L-tryptophan autotrophic and heterotrophic organisms, we used the COG0147 that clustered the anthranilate synthases component I enzymes, TrpE. The result of this analysis is shown in Fig 3.

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Fig 3. Heatmap of COG0147 involved in tryptophan biosynthesis.

The COG0147 clusters the Anthranilate synthases component I enzymes, TrpE. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 800, COG centers: 3, Phylogenetic centers: 3. COGs that are crucial in the biosynthesis of the amino acid tryptophan are predominantly depicted with a dark red color, indicating STRING index values nearing the maximum of 1,000. The first phylogenetic group comprises phyla whose organisms are typically heterotrophic with respect to tryptophan.

https://doi.org/10.1371/journal.pone.0297010.g003

Module (2,1) of the heatmap of Fig 3 clusters all the autotrophic organisms having the enzymes involved in L-tryptophan biosynthesis, which are clustered COG group 2 (S6 Table), and include the following enzymes: Anthranilate synthase TrpE (COG0147), which forms a complex with TrpG (COG0512) to convert chorismate and glutamine into anthranilate [11]; TrpD (COG0547), known as anthranilate phosphoribosyl transferase, which transfers the phosphoribosyl group from 5-phosphorylribose-1-pyrophosphate to anthranilate, resulting in phosphoribosyl anthranilate; TrpF (COG0135), or phosphoribosyl anthranilate isomerase, which converts the phosphoribosyl anthranilate into carboxyphenylamino-deoxyribulse-5-phosphate. Additionally, TrpC (COG0134), referred to as indol-3-glycerol phosphate synthase, removes the carbon dioxide from carboxyphenylamino-deoxyribulse-5-phosphate resulting in the formation of indole-3-glycerol phosphate. Finally, the tryptophan synthase TrpA alpha chain (COG0159) cleaves the indoleglycerol phosphate to generate glyceraldehyde 3-phosphate and indole. The indole molecule generated in the previous step is used with L-serine by TrpB (COG0133), the beta chain of tryptophan synthase, to synthesize L-tryptophan.

Although the biosynthesis of L-tryptophan from chorismate commonly requires the presence of all the pathway’s enzymes, in exceptional cases, the absence of an enzyme could be complemented by enzymes with a related activity. Notably, this complementation has been documented in the case of Actinobacteria that have lost the phosphoribosyl anthranilate isomerase trpF gene (COG0135), which has been complemented by the enzyme PirA that provides the required isomerase activity [14]. Using PyloString, we identified that analogous complementation could also have occurred in some organisms that are members of the Thaumarchaeota phylum (see module (2,1)). Furthermore, the absence of certain enzymes within the modules (2,1) could indicate that certain organisms are in the process of losing the L-Trp pathway, as could be the case of the organisms belonging to the Annelida phylum. This phenomenon has been observed in various pathogens, including certain species within the genera Haemophilus, Coxiella, and Propionibacterium [15].

Organisms of the phylogenetic phyla of the module (2,1) of Fig 3 are considered prototrophs and are predominantly bacteria (Phyla group 1, S7 Table). On the other hand, organisms of modules (2,2), and (2,3) comprise auxotrophic organisms that require external sources of tryptophan for their growth and survival. This group of organisms primarily includes eukaryotic organisms (groups 2 and 3, S7 Table). The under or over-representation of the COGs within the different modules for the heatmap of COG0147 is presented in the S8 Table.

To further investigate the phylogenetic distribution of L-tryptophan auxotroph organisms, we repeated a similar analysis by selecting the "Class" option in the Phylogenetic level panel of the PhyloString web page. The heatmap and corresponding Tables are presented as Supplementary Material (S2 Fig and S9–S11 Tables). Interestingly, the COG groups obtained using both the "Phylum" and the "Class" options are almost the same (S6 and S9 Tables), indicating that the functional relationships of the proteins involved in L-tryptophan biosynthesis are well-conserved across different phylogenetic lineages. However, when dividing the Phyla into their corresponding phylogenetic classes, specific tendencies within smaller groups of organisms become apparent. For instance, the Tissierellia and Erysipelotrichia classes, despite belonging to the Firmicutes phylum, which commonly comprises prototrophic organisms, lack the main enzymes of the L-trp biosynthesis pathway. A similar scenario is observed in the Proteobacteria class of Oligoflexia (S2 Fig and S10 Table).

iii) Key proteins involved in the eukaryotic transcription initiation process.

Transcription is the process by which the RNA polymerases (RNAPs) copy the information contained in the DNA into molecules of RNA. Although RNAPs in all three domains of life share a common evolutionary origin, important differences have been documented in the molecular mechanisms and proteins related to the transcription initiation process. In the case of bacterial RNAPs, the recognition of specific promoter sequences in DNA is facilitated by sigma factors. However, in the case of archaeal and eukaryotic RNAPs, sigma factors are absent. Instead, their recognition of promoter elements relies on different generalized transcription factors (GTFs) [16, 17]. Furthermore, in eukaryotes, multiple types of nuclear RNAPs are responsible for transcribing distinct types of RNA. These transcription processes involve additional elements such as enhancers, silencers, and chromatin modifications (review in [18]).

One of the key components of the eukaryotic transcription initiation complex is the TATA-binding protein (TBP). Its primary function is to initiate transcription by RNA polymerase II at the promoters of protein-coding genes. TBP binds to a specific DNA sequence known as the TATA box, typically found in the promoter region of genes. The binding of the TBP to DNA initiates the recruitment of other transcription factors and RNA polymerase II. TBP It is an integral part of a larger protein complex known as the TFIID complex, which includes various TBP-associated factors.

To have a broad overview of the proteins related to the eukaryotic transcription initiation process, we used the PhyloString web server with KOG1932 as input. This KOG corresponds to a TATA-binding protein-associated factor. The heatmap resulting from this analysis is shown in Fig 4, while the corresponding lists of COG groups and phylogeny groups, along with the statistical values indicating COG enrichment within this group, are provided in S12–S14 Tables, respectively.

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Fig 4. Heatmap of KOG1932 involved in the transcription initiation process.

The KOG1932 corresponds to a TATA-binding protein-associated factor. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 800, COG centers: 2, Phylogenetic centers: 5. The first COG group includes proteins crucial to the process of transcription initiation in eukaryotic organisms. As anticipated, these proteins are absent in the fourth phylogenetic group, primarily composed of Bacteria and Archaea. This block also encompasses the phyla Euglenozoa and Fornicata, known for being among the more ancient phyla within Eukarya.

https://doi.org/10.1371/journal.pone.0297010.g004

Modules (1,1), (1,2), (1,3), and (1,5) in the heatmap shown in Fig 4 illustrate the presence of essential proteins involved in the RNA polymerase II transcription initiation. In contrast, modules (2,1), (2,2), and (2,3) cluster proteins with less significant relationships. These proteins are linked to various functions, including interactions with vitamin receptors, histone acetyltransferases, and proteins with annotations as “uncharacterized conserved protein”.

Remarkably, our PhyloString analysis, using KOG1932 as a seed, uncovered that some clades of eukaryotic organisms lack most of the proteins associated with the canonical eukaryotic transcription initiation process. These organisms are positioned near the root of the phylogenetic tree that divides Bacteria, Archaea, and Eukarya. The most striking instances are the Fornicata and Euglenozoa phyla, which represent the closest lineages to the Last Eukaryotic Common Ancestor (LECA, S3 Fig). In the heatmap of Fig 4, module (1,4), Fornicata and Euglenozoa cluster with the bacterial phyla (S11 Table) since they lack most of the KOGs related to transcription initiation that are present in eukaryotic organisms. Additional examples of primitive eukaryotic phyla that lack important KOGs associated with transcription initiation, as identified by PhyloString, include Apicomplexa, Ciliophora, Bacillariophyta, and Parabasalia. These phyla are clustered in the phylogenetic group 5 (S11 Table) and the module (1,5) of the Fig 4.

Examples of lesser striking instances where KOGs involved in transcription initiation are absent can be observed in the Microsporidia, Haptista, Heterolobosea, and Rhodophyta phyla, which lack certain subunits of the transcription initiation factors. These phyla cluster together in the second phylogenetic group of Fig 4 and S13 Table.

iv) Photosynthesis.

Photosynthesis is a vital biological process that many organisms employ to convert light energy into chemical energy and store it in organic compounds that can later be metabolized through cellular respiration to provide the energy required to perform different biological activities.

To have an overview of the functional network of proteins involved in this process, we used the COG5701, designated as Photosystem I reaction center subunit A1, PsaA, as input of our PhyloString web server. The corresponding heatmap is shown in Fig 5.

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Fig 5. Heatmap of COG5701 involved in the photosynthesis process.

The COG5701 corresponds to the photosystem I reaction center subunit A1, PsaA. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 700, COG centers: 2, Phylogenetic centers: 3. Cyanobacteria, found in phylogenetic group 1, and specific eukaryotic phyla commonly recognized as green plants, possess the capability to carry out photosynthesis. This shared ability is reflected in the heatmap by the presence of essential orthologous proteins, represented as dark red cells.

https://doi.org/10.1371/journal.pone.0297010.g005

Remarkably, phylogenetic group 1 of the heatmap of Fig 5 exclusively comprises the Cyanobacteria phylum. These are photosynthetic bacteria that utilize chlorophyll and various pigments for photosynthesis. Unlike eukaryotic algae, they lack chloroplasts and instead perform photosynthesis within specialized cellular membrane structures. Furthermore, many cyanobacteria have the remarkable ability to capture atmospheric nitrogen. Their distinct blue-green color results from the presence of pigments like phycocyanin and chlorophyll, which overshadow the green tint of chlorophyll, providing cyanobacteria with their distinctive appearance [19, 20].

Phylogenetic group 2 of the heatmap of Fig 5 includes various phyla within the monophyletic group known as Viridiplantae, commonly referred to as green plants. These organisms get their green color from pigments like chlorophyll. Viridiplantae is characterized by its members being photosynthetic and having chloroplasts, the cellular organelles responsible for photosynthesis. Viridiplantae is a highly diverse group, including green algae, land plants (Embryophyta), and their closest algal relatives (Charophyta). This diversity spans from unicellular green algae to complex, multicellular land plants, such as mosses, ferns, conifers, and flowering plants. Viridiplantae can be found in various habitats, including freshwater and marine environments, moist soil, and terrestrial ecosystems. Green algae in this group are typically aquatic, while land plants have adapted to life on land [21].

Interestingly, most of the COGs in the heatmap of Fig 5 are clustered in the second COGs group and are related to the different Photosystem reaction center proteins I and II, and cytochrome b6f complex subunit (S15 Table). The Photosystem II reaction center is a crucial component of the photosynthetic process in plants, algae, and cyanobacteria. Its role is to initiate the light-dependent reactions of photosynthesis. The heatmap of Fig 5 shows that a core of this set of enzymes is almost exclusively found in organisms of the Cyanobacteria phylum, which forms the first phylogenetic group. This core is also found in members of the second phylogenetic group, which includes various phyla of Viridiplantae, such as Chlorophyta, Streptophyta, Haptista, Bacillariophyta, and Rhodophyta (S16 Table). The values indicating COG enrichment within this group are detailed in S17 Table.

v) Endocytosis and intracellular vesicle trafficking.

Conventional clathrin-mediated endocytosis and intracellular vesicle trafficking are essential and highly conserved cellular mechanisms in eukaryotes. They are responsible for transporting proteins and lipids either from the plasma membrane or their site of biosynthesis to their respective functional locations within the cell. Through endocytosis, the cell takes solutes (cargo) and fluids from the external environment. It also plays a key role in regulating the signaling cascade mediated by plasma membrane receptors; it is the route of entry for some pathogens, among other functions [22, 23]. On the other hand, intracellular vesicle trafficking is responsible for the molecular transport between endomembrane-based organelles, such as endoplasmic reticulum, Golgi complex, endosome, and vacuole/lysosome. Additionally, it is involved in exocytosis and the recycling of endosomes back to the plasma membrane. These processes are vital for maintaining cellular organization and functionality [24, 25].

Vesicles are formed by the cytosolic-oriented bending of the donor plasma membrane, which is accompanied by the assembly of a cage-like structure on the cytosolic side of the nascent vesicle. This cage-like structure can be composed of either clathrin light and heavy chains in clathrin-coated vesicles (CCVs) or the coatmer protein complex I and II in COPI and COPII vesicles, respectively [26–29].

In addition to cargo molecules, the membrane bending site recruits various accessory proteins, including Adaptor proteins (AP), cargo-receptors, ADP-ribosylation factor (Arf) small GTPases, RabGTPases, their specific guanine-exchange factors (GEFs), and activators (GAPs), as well as VAMP (vesicle-associated membrane proteins), SNARE proteins, among others [30–33]. Following the vesicle scission from the donor membrane, the cage-like structure disassembles. Afterwards, vesicle membrane fuses to the acceptor membrane. The membrane fusion process is facilitated by accessory proteins, including tethering proteins and SNAREs [34–38].

According to Thattai M. [39], there are "budding specificity " and "fusion specificity" modules comprising coat proteins and the corresponding paralogous variants of accessory proteins acting at different locations, respectively. Recent genetic findings on the First and Last eukaryotic common ancestor (FECA and LECA) provide information on the origin and evolution of organelles and the molecular and cellular mechanisms involved in intracellular vesicle trafficking (reviewed by [33, 40]).

We employed the PhyloString web server to explore clathrin-mediated endocytosis and vesicular trafficking among different phylogenetic groups. In this case, we employed KOG0985 (Vesicle coat protein clathrin, heavy chain) as the query seed KOG. The result of our analysis is presented as the heatmap of Fig 6 and Table 1, while the corresponding lists of COG groups and phylogeny groups, along with the statistical values indicating COG enrichment within this group, are provided in S18–S20 Tables, respectively.

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Fig 6. Heatmap of KOG0985 involved in clathrin-mediated endocytosis.

The KOG0985 corresponds to the “Vesicle coat protein clathrin, heavy chain”. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 850, COG centers: 2, Phylogenetic centers: 3. The first phylogenetic group is mainly composed of bacterial and archaea phyla; it also includes the eukaryotic phyla Fornicata and Microsporidia, which exhibit a notable deficiency in proteins related to the clathrin-mediated endocytosis process. The second phylogenetic group encompasses phyla with organisms having structural components of endocytic and intracellular vesicles. It also includes components somewhat associated with specific functional or cellular processes related to endocytosis. The third phylogenetic group is predominantly composed of unicellular eukaryotic organisms such as algae and protozoa, along with land plants in the Streptophyta phylum. Organisms in this group generally possess a reduced set of structural components related to endocytic and intracellular vesicles.

https://doi.org/10.1371/journal.pone.0297010.g006

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Table 1. Phylogenetic distribution of the KOGs related to the Clathrin-mediated endocytosis.

The presence (checkmarks) and absence (crosses) of the main KOGs involved in Clathrin-mediated endocytosis are indicated for the most important phylogenetic groups, as could be inferred from our PhyloString analysis.

https://doi.org/10.1371/journal.pone.0297010.t001

As anticipated, structural, and accessory proteins related to clathrin-coated vesicles are exclusively grouped within the Eukarya phyla. These proteins include KOG0985 and KOG4031 (corresponding to vesicle coat protein clathrin’s heavy and light chains, respectively), KOG0934, KOG0935, and KOG0936 (AP/clathrin adaptor complex, small subunits), along with other components linked to the clathrin-mediated endocytosis machinery (see Fig 6 and Table 1). Remarkably, the first phylogenetic group in the heatmap of Fig 6 reveals that the eukaryotic phyla Fornicata and Microsporidia are grouped with bacterial and archaeal phyla that lack clathrin-mediated endocytosis and intracellular vesicle trafficking. Consequently, related KOGs are absent in this grouping. Fornicata and Microsporidia phyla exhibit a limited number of KOGs, including KOG0985 (Vesicle coat protein clathrin), KOG0934 (Clathrin adaptor complex), KOG0935 (Clathrin adaptor complex), as well as additional adaptor proteins such as those of KOG1061 (Vesicle coat complex AP-1/AP-2/AP-4, beta subunit), KOG0937 and KOG0938 (members of the Adaptor complexes medium subunit family). Surprisingly, KOG4031 (vesicle coat protein clathrin, light chain) is absent in the Fornicata and Microsporidia phyla, as observed in the Parabasalia and Euglena phyla of the third phylogenetic group. The clathrin light chain is crucial for clathrin-coated vesicle (CCVs) formation, playing a key role in polymerization and serving as a connecting link with the actin cytoskeleton [27, 41]. The absence of KOG4031 may suggest the formation of CCVs with different properties or efficiencies. The second phylogenetic group in the heatmap of Fig 6 outlines KOGs present in metazoan, fungi, and amebozoan. It includes clathrin-mediated endocytosis and intracellular vesicle trafficking, as well as components somewhat associated with specific functional or cellular processes related to endocytosis (Fig 6 and Table 1). Conversely, the third phylogenetic group mainly consists of unicellular and pluricellular algae, microalgae, land plants, and protozoa. This group clusters a reduced set of KOGs related to structural components of CCVs and intracellular vesicles.

As KOGs related to COPI (KOG0292, Vesicle coat complex COPI, alpha subunit) and COPII (KOG1986, Vesicle coat complex COPII, subunit SEC23, and KOG0307, Vesicle coat complex COPII, subunit SEC31) vesicles were also observed in the heatmap of Fig 6, we conducted a new PhyloString analysis, utilizing KOG1078 (Vesicle coat complex COPI, gamma subunit) as query seed to find the phylogenetic distribution of other KOGs of proteins that are part of the Vesicle coat complex. The outcome of this analysis is illustrated in the heatmap of Fig 7, while the corresponding lists of COG groups and phylogeny groups, along with the statistical values indicating COG enrichment within this group, are provided in S21–S23 Tables, respectively.

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Fig 7. Heatmap of KOG1078 involved in COPI-coated intracellular vesicles.

The KOG1078 corresponds to the “Vesicle coat complex COPI, gamma subunit”. The parameters used in the analysis were: Phylogenetic level: Phylum, STRING cut-off: 850, COG centers: 2, Phylogenetic centers: 3. The first phylogenetic group is composed of Bacterial and Archaea phyla, which lack COPI intracellular vesicles. The second phylogenetic group includes Eukarya phyla. The phyla of this group is composed of organisms that possesses the structural components of COPI- and COPII-coated vesicles, as well as components somewhat associated to specific functional or cellular processes related to intracellular trafficking mediated by these vesicles. The third phylogenetic group is formed by the Fornicata and Microsporida phyla. Organisms of these phyla have all the structural components of COPI- and COPII-coated vesicles, except for the epsilon, gamma and zeta subunits of the COPI complex (KOG3081, KOG1078 and KOG3343, respectively).

https://doi.org/10.1371/journal.pone.0297010.g007

Fig 7 reveals a set of KOGs involved in vesicle trafficking, including subunits of the Vesicle coat complex COPI (one alpha—KOG0292, two beta—KOG1058 and KOG0276, one epsilon—KOG3081, one gamma—KOG1078, and one zeta—KOG3343). These groups of orthologous proteins are consistently present across all Eukarya phyla. However, it is noteworthy that KOG1078 and KOG3343 are absent in the Fornicata phylum, as indicated in Fig 7 and Table 1.

Apart from the Vesicle coat complex COPI subunits highlighted in the Fig 7 heatmap, we have also identified the four structural subunits of the Vesicle coat complex COPII: Sec23 (KOG1986), Sec24 (KOG1985), Sec13 (KOG1332), and Sec31 (KOG0307), alongside the COPII-specific GTPase subunit SAR1 (KOG0077). The presence of these components spans across all Eukarya phyla, as depicted in the Fig 7 heatmap and succinctly outlined in Table 1.

In summary, the heatmaps of Figs 6 and 7 disclosed fascinating information concerning organisms of the primitive eukaryotic group Fornicata, as exemplified by the diarrheal pathogen Giardia intestinalis [42]. These findings suggest that these organisms might possess relatively rudimentary clathrin-coated vesicle (CCV) and COPI-like vesicle systems. While this hypothesis requires experimental validation, it is consistent with the observed reduced subcellular organization observed in Giardia trophozoites.

Giardia trophozoites exhibit a distinct endoplasmic reticulum network (ER) characterized by the presence of a unique vesicle system, known as peripheral vacuoles (PVs), positioned beneath the plasma membrane. This configuration may signify an evolutionary adaptation of the endocytic-exocytic pathways, potentially dependent on CCV- and COPI-like structures but lacking the clathrin light chain, as well as the COPI gamma and zeta subunits. Notably, PVs are contiguous with the ER but lack Golgi stacks [42, 43].

Giardia trophozoites exhibit a distinct endoplasmic reticulum network (ER) characterized by the presence of a unique vesicle system, known as peripheral vacuoles (PVs), positioned beneath the plasma membrane. This configuration may signify an evolutionary adaptation of the endocytic-exocytic pathways, potentially dependent on CCV- and COPI-like structures but lacking the clathrin light chain, as well as the COPI gamma and zeta subunits. Notably, PVs are contiguous with the ER but lack Golgi stacks [42, 43].

Typically, COPI vesicles play a crucial role in transporting cargo molecules between the Golgi apparatus and the ER, as well as within Golgi stacks [26]. The lack of certain components of the CCV and COPI machineries in other primitive Eukarya phyla, such as Parabasalia, Apicomplexa and Euglenozoa (Table 1) seem to be an indicative of the evolutionary events that contributed to the origin of multicellular organisms with a more complex cell biology, an avenue that would be interesting to explore.