==== Front PLoS GenetPLoS GenetplosplosgenPLoS Genetics1553-73901553-7404Public Library of Science San Francisco, USA 24809820PGENETICS-D-13-0258510.1371/journal.pgen.1004363Research ArticleBiology and life sciencesBiochemistryRNARNA synthesisNucleic AcidsProteinsCell BiologyMolecular Cell BiologyEvolutionary BiologyGeneticsGene ExpressionProtein TranslationMicrobiologyMedical MicrobiologyMicrobial PathogensBacterial PathogensBacillusBacillus SubtilisEscherichia ColiResearch and Analysis MethodsModel OrganismsProkaryotic ModelsPredicting the Minimal Translation Apparatus: Lessons from the Reductive Evolution of Mollicutes Minimal Translation Machinery in MollicutesGrosjean Henri 1 Breton Marc 2 3 Sirand-Pugnet Pascal 2 3 Tardy Florence 4 5 Thiaucourt François 6 Citti Christine 7 8 Barré Aurélien 9 Yoshizawa Satoko 1 Fourmy Dominique 1 de Crécy-Lagard Valérie 10 Blanchard Alain 2 3 * 1 Centre de Génétique Moléculaire, UPR 3404, CNRS, Université Paris-Sud, FRC 3115, Gif-sur-Yvette, France2 INRA, UMR 1332 de Biologie du Fruit et Pathologie, Villenave d'Ornon, France3 Univ. Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, Villenave d'Ornon, France4 Anses, Laboratoire de Lyon, UMR Mycoplasmoses des Ruminants, Lyon, France5 Université de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, Marcy L'Etoile, France6 Centre International de Recherche en Agronomie pour le Développement, UMR CMAEE, Montpellier, France7 INRA, UMR1225, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France8 Université de Toulouse, INP-ENVT, UMR1225, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France9 Univ. Bordeaux, Centre de bioinformatique et de génomique fonctionnelle, CBiB, Bordeaux, France10 Department of Microbiology and Cell Science, University Florida, Gainesville, Florida, United States of AmericaGojobori Takashi EditorNational Institute of Genetics, Japan* E-mail: pmcdaniel@example.com authors have declared that no competing interests exist. Conceived and designed the experiments: HG MB PSP VdCL ABl. Performed the experiments: HG MB PSP VdCL ABl FTa FTh CC ABa SY DF. Analyzed the data: HG MB PSP VdCL ABl SY DF. Contributed reagents/materials/analysis tools: FTh FTa ABa. Wrote the paper: HG VdCL ABl. Contributed to the design of the figures: HG MB PSP SY DF VdCL ABl. Provided some input to improve the paper and approved its final version: HG MB PSP VdCL ABl FTa FTh CC ABa SY DF. 5 2014 8 5 2014 10 5 e100436320 9 2013 24 3 2014 © 2014 Grosjean et al2014Grosjean et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches. Author Summary In all cells, proteins are synthesized from the message encoded by mRNA using complex machineries involving many proteins and RNAs. In this process, named translation, the ribosome plays a central role. The elements involved in both ribosome biogenesis and its function are extremely conserved in all organisms from the simplest bacteria to mammalian cells. Most of the 260 known proteins involved in translation have been identified and studied in the bacteria Escherichia coli and Bacillus subtilis, two common cellular models in biology. However, comparative genomics has shown that the translation protein set can be much smaller. This is true for bacteria belonging to the class Mollicutes that are characterized by reduced genomes and hence considered as models for minimal cells. Using homology inference approach and expert analyses, we identified the translation apparatus proteins for 39 of these organisms. Although striking variations were found from one group of species to another, some Mollicutes species require half as many proteins as E. coli or B. subtilis. This analysis allowed us to determine a set of proteins necessary for translation in Mollicutes and define the translation apparatus that would be required in a cellular chassis mimicking a minimal bacterial cell. This work was supported by grant EVOLMYCO project (ANR-07-GMGE-001) from the French national funding research agency (ANR), by INRA and Région Aquitaine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Mollicutes constitute a monophyletic class that share a common ancestor with Gram-positive bacteria of low G+C content or Firmicutes but have adopted a parasitic life style (Figure S1) [1]. During their coevolution with their eukaryotic hosts, mollicutes progressively lost the genes coding for cell-wall synthesis enzymes and for enzymes involved in the synthesis of small metabolites, such as amino acids, nucleotides and lipids that were available in the host. As a result, mollicute genomes are much smaller (580–1,840 Kbp; eg: about 482–2,050 CoDing Sequences or CDSs, Table S1) than those of model bacteria such as Escherichia coli or Bacillus subtilis (4,639–4,215 Kbp; eg: 4,320–4,176 CDSs respectively). These bacteria have nevertheless retained the full capacity to synthesize DNA, RNA and all the proteins required to sustain a parasitic life-style. In addition most of them are still able to grow in axenic conditions in rich media usually containing 20% serum (see [2] for review); only the hemoplasmas and the Candidatus phytoplasma species have yet to be cultured in vitro. Mollicutes are therefore considered as the smallest and simplest known bacteria capable of autonomous multiplication [3], [4]. 'Simple' does not mean 'simplistic'. One should not underestimate the elaborate solutions that mollicutes have used to solve problems related to their peculiar macromolecular organization and cellular compactness (discussed in [3], [5], [6] and references therein). From an evolutionary point of view, mollicutes should be considered as some of the most evolved prokaryotes that still have retained ability to perform the complex reactions that encompass DNA, RNA and protein synthesis, with possibly new tricks and inventions to make the most of their limited genetic capacities [7], [8]. For these reasons, specific Mollicutes strains have been used as a test bench to improve our understanding of the basic principles of a cell and for reconstructing a microbe that would function with a synthetic minimal genome (see [3], [4], [9], [10], [11] for examples). Identification of essential proteins is a long-standing problem that is directly linked to the concept of a minimal cell [12]. The approaches used in Mollicutes to identify the set of essential genes have been: i) comparative genomic analyses to create an overview of the protein content in model mycoplasmas (notably Mycoplasma genitalium and Mycoplasma pneumoniae) [5], [13], [14], [15], ii) identification of genes that cannot be individually inactivated [16], [17], [18], [19], iii) reconstruction of synthetic genomes and transplantation into a recipient cell [10]. Depending on the Mollicutes species considered and the method of analysis, the number of essential genes varies from 256 to 422. For M. genitalium, 256 were identified by in silico comparative genomics analysis [15] but over 382 were found by saturation transposon mutagenesis experiments [16], [19]. For Mycoplasma pulmonis and Mycoplasma arthritidis, saturation transposon mutagenesis identified 422 and 417 essential genes respectively [17], [20]. Messenger-RNA-dependent protein synthesis is one of the most complex cellular processes both in its biogenesis and its function. For a cell with a reduced genome such as M. genitalium, more than 25% of the genome encoding capacity is mobilized to build this complex machinery [2]. The bacterial ribosome is a giant multicomponent complex of several millions of daltons, composed of 3 RNA species (5S, 16S and 23S rRNA) and many structural proteins (60–70). Together with other RNAs (tRNAs, tmRNA and RNA-P) and a large repertoire of enzymes and protein factors, this protein synthesis machinery allows translation of mRNAs into polypeptides according to precise rules. Comparative analysis of bacterial genomes reveals that the majority of genes coding for the ribosomal proteins, aminoacyl-tRNA synthetases, translation factors and several ribosome biogenesis/maturation enzymes are universal [7], [21] and essential [22], [23], [24]. Genes coding for enzymes involved in rRNA and protein processing, RNA or protein modification, and ribosome maturation RNases appear less important, as deleting these does not lead to severe growth defects, and are the most easily lost genes during genomic erosion in Mollicutes species (see below). As the number of sequenced Mollicutes genomes has significantly increased, most of the phylogenetic sub-groups of this class of bacteria are now covered allowing for the analysis of the erosion of translation from an evolutionary perspective. This analysis defined the minimal set of proteins needed to sustain protein synthesis in various mollicutes. A major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in Mollicutes that are model organisms of choice for synthetic biology. Also, by careful analysis of the evolutionary pattern of gene losses and a few cases of gene gain in different individual Mollicutes species, light was shed on the progressive adaptation of an ancestral and complex cellular proteome towards a simpler, yet functional alternative one. Results and Discussion Prediction of proteins involved in translation machinery Selection of Mollicutes species Mollicutes have been subdivided by phylogenetic analysis into 5 main sub-groups: Spiroplasma, Pneumoniae, Hominis, Anaeroplasma and Asteroleplasma [1]. The sub-group Asteroleplasma, which includes the single species Asteroleplasma anaerobium, is marginal, and mixed with other Firmicutes species questioning its membership to the Mollicutes class [25]. With the exception of asteroleplasmas, which could not be included in this study because of the lack of genome sequences, Mollicutes represent a monophyletic class of bacteria. The Anaeroplasma group is most commonly referred as the AAP sub-group as it includes the Acholeplasma and Anaeroplasma genera together with the Candidatus phytoplasma species. A set of 39 genomes from distinct species that sample the diversity within Mollicutes were selected among the 60 sequenced genomes available at the time of this study. These include 9 species from the Spiroplasma sub-group, 16 from the Hominis sub-group, 10 from the Pneumoniae sub-group and 4 from the AAP sub-group. Among these 39 species, 27 have an animal host, including 7 a human host. Among the 5 species that are associated with plants, 4 are pathogens transmitted by sap-sucking insects. Culture as free living cells in axenic conditions has been achieved for 33 out of the 39 selected species: the uncultured ones are 3 hemoplasmas (Mycoplasma haemofelis, Mycoplasma haemocanis and Mycoplasma suis) and 3 Candidatus phytoplasma species (Ca. Phytoplasma mali, Ca. P. australiense and Ca. P. asteri) – they are boxed within a red dotted line in Figure S1. The 39 corresponding genomes have sizes ranging from 0.58 Mbp (482 predicted CDS) to 1.84 Mbp (2,050 predicted CDS) for M. genitalium and Spiroplasma citri, respectively (Table S1). Selection of bacterial protein queries Our work deals exclusively with the mechanistic aspect of RNA-to-Proteins machinery and not with the transcription of DNA-to-RNA. We first had to define the set of protein queries. The Gram-negative bacterium E. coli is the organism for which almost all components of the translation machinery have been identified and experimentally characterized and this set was used as a starting point [26]. Since Mollicutes species are phylogenetically closer to Gram-positive Firmicutes than to Gram-negative E. coli, additional proteins from B. subtilis were also used [27]. Although B. subtilis homologs exist for most of the E. coli proteins involved in translation, there are a few B. subtilis translation proteins for which no homologs are found in the E. coli genome and vice-versa (Table S2). Altogether, we selected 260 protein queries, of which 228 are encoded by genes found in E. coli, 210 by genes found in B. subtilis and 179 are common between the two bacteria ( Table 1 ). These proteins are involved in the biogenesis, maturation and proteosynthetic function of the ribosome and tRNAs. Not included were the proteins involved in RNA synthesis, in SRP/Sec-dependent membrane proteins translocation/secretion, in protein activation, in regulatory processes and in responses to stress or changes of environment and defense systems. The final 260 selected proteins were arbitrarily split into 7 categories according to their roles in the protein synthesis machinery ( Table 1 ). For each of these 7 categories, a color code was used throughout the paper to facilitate understanding of the data ( Figure 1 ). 10.1371/journal.pgen.1004363.g001Figure 1 Genes coding for proteins implicated in translation in Mollicutes. Using queries from E. coli (Ec) and from B. subtilis (Bs), the presence of homologous proteins was searched in 39 Mollicutes genomes (see list of selected species below part B of the figure). This figure corresponds to the raw data given in Table S3. The results were grouped into three panels: conserved core of genes involved in translation (A), genes lost in some species only (B) and genes absent in all Mollicutes species (C). In panels A and C, only data concerning Ec and Bs are shown. In part B, the selected species clustered according to the 4 phylogenetic groups; Spiroplasma, Hominis, Pneumoniae and AAP [25]. The queries, of which names of corresponding acronyms are given in Table S2, are ordered from top to bottom, first according to the highest number of occurences and second according to the 7 protein categories following this sequence: ribosomal proteins, tRNA aminoacylation, rRNA modifications, tRNA modifications, ribosome assembly, translation and RNA processing. The different categories are color coded as shown in Table 1 and below part C of the figure. The presence or absence of a given gene in a Mollicutes species is indicated by "1" in a grey background or by "0" in a white background, respectively. The 17 genes missing in some of the non-cultivated Mollicutes are indicated within a dashed-red box. The total number of genes in each category is indicated in panel D. 10.1371/journal.pgen.1004363.t001Table 1 Genes coding for proteins implicated in the biosynthesis and functions of translation machinery in model bacteria. Functional categories Subcategories Total of Escherichia coli queries Total of Bacillus subtilis queries Total of queries present in Ec and Bs Total of queries not present in Ec Total of queries not present in Bs Total Ribosomal proteins 55 59 54 5 1 60 Large subunit proteins 33 37 33 4 0 Small subunit proteins 22 22 21 1 1 tRNA aminoacylation 28 28 23 5 5 33 AA-tRNA synthetases 23 24 21 2 2 Related Proteins 5 4 2 3 3 rRNA modifications 30 23 20 3 10 33 Specific for 23S rRNA 19 11 10 0 9 Specific for 16S rRNA 11 10 10 1 1 unknown target 0 2 0 2 0 tRNA modifications 41 30 26 4 15 45 Enzymes 35 24 21 3 14 Protein cofactors 8 6 5 1 1 Ribosome assembly 33 30 26 5 7 38 GTPases and factors 25 25 20 5 4 r-protein modifications 8 5 5 0 3 Translation GTPases and factors 24 20 20 0 4 24 RNA processing RNases 17 20 10 10 7 27 TOTAL 228 210 179 32 49 260 Ec: Escherichia coli. Bs: Bacillus subtilis. Mollicutes share a core of ubiquitous genes encoding proteins involved in translation Inferring homology between each of the 260 protein queries and the predicted proteome of the 39 mollicutes was performed as described in Materials and Methods, using a combination of complementary approaches including sequence similarity searches, identification of conserved domains and phylogenetic analyses. The proteins involved in translation are known to be among the most conserved proteins in living organisms, which facilitated homolog predictions, especially in the monophyletic group of Mollicutes. The results of this data mining are summarized in the composite Figure 1 . In Figure 1A are listed the 104 genes that are present in the 39 genomes analyzed. The corresponding full names are given in Table S3. The presence of homologue genes in E. coli (Ec) and B. subtilis (Bs) are indicated in the small grey boxes adjacent to the acronyms. Only RpmGb, a duplicant of r-protein L31 of 50S subunit, is absent in E. coli (white small box). All these genes but three were shown to be essential in the model organisms (E. coli, B. subtilis) and/or in mycoplasmas (M. genitalium, M. pulmonis - Figure S2, part A). This core of ubiquitous genes represents 40% of the total queries (or 49% if only the genes present in B. subtilis are considered). Figure 1B displays the 88 additional genes that have been lost (white small boxes) in at least one Mollicutes species. This data clearly shows that some genes are more persistent than others (i.e. the genes are conserved in a majority of genomes; [28]). Also, the non-culturable species (species 33 to 38, comprised in doted red box) have lost the most translation genes (vertical white small boxes), with several being lost only in M. suis (species 35) or in M. suis plus the two M. haemofelis/canis (species 33, 34). Out of these 17 persistent genes identified in non-culturable species, 14 are essential by gene deletion analysis in M. genitalium and/or M. pneumoniae (indicated in Figure S2, B, in orange background). Since non-cultivability is associated with the loss of genes that are required for growth in axenic conditions [29], this set of 17 genes should be considered as essential elements of a minimal translation machinery (discussed below). In all other cases, the individual genes are often absent in Mollicutes from different sub-groups. A few of these were found to be essential when tested individually in M. genitalium and/or M. pulmonis (Figure S2, B). All other genes are dispensible or can easily be lost because of the presence of paralogous or analogous genes with redundant or overlapping functions (discussed below). Most genes were lost early during Mollicutes evolution and subsequent genome downsizing. In a few cases, a gene present in a single or in a limited set of Mollicutes species but absent in B. subtilis, may correspond to a lateral gene transfer event (discussed below). In Figure 1C are listed the 68 genes missing in all 39 mollicutes. Most are genes present in the Gram positive B. subtilis but absent in the Gram negative E. coli. Some of these could have emerged later during the evolution, after the separation of Firmicutes from other bacteria. Some genes are more easily lost than others As shown in Figure 1D , the genes that are the most easily lost in Mollicutes code for proteins involved in post-transcriptional modifications of t+rRNA (indicated in blue and green), in ribosome biogenesis and maturation – including post-translational modifications of ribosomal proteins (in pink), and ribonucleases involved in t+r+mRNA processing (in light blue). In contrast, genes coding for ribosomal proteins (in red), aminoacyl-tRNA synthetases (in yellow) and a few related proteins such as aspartyl/glutamyl-tRNA amidotransferases as well as a subset of translation factors are among the genes most resistant to loss (yellow and magenta in Figure 1D ). The minimal translation apparatus set of proteins depends on the Mollicutes sub-groups The total number of proteins involved in translation for each Mollicutes species was then tabulated ( Figure 2 ). It is clear that gene erosion is not uniform in each sub-group of the Mollicutes tree and that different sets of persistent genes exist in each Mollicutes sub-groups. In other words there are different ways to evolve towards a minimal and functionally coherent cell. The Spiroplasma sub-group retained the largest numbers of genes (from 158 to 167). At the other extreme, the species that shed the most genes lost are the three hemoplasmas (116, 121 and 121 genes). At variance, the three phytoplasmas, which share with the hemoplasmas the inability to grow in axenic conditions, have a larger set of genes (142, 143 and 144 genes), closer to that found in the other mollicutes. Among them, two different minimal sets are found in the Hominis group (138 genes for Mycoplasma ovipneumoniae, and for Mycoplasma hyopneumoniae) and in Pneumoniae group (144 genes for the closely related M. genitalium and M. pneumoniae). These data also indicate that there is no strict relationship between genome sizes, cell cultivability and the number of genes dedicated to translation (compare Figure 2 with genome sizes indicated in Table S1). Indeed, the hemoplasma M. haemofelis genome (1.1 Mbp) is larger than the phytoplasma genomes, and yet has over 26 less translation genes (see above). Similarly, the genome of M. ovipneumoniae, is almost twice the size of M. genitalium, and yet this species has a smaller number of translation genes (138 vs 144). This lack of correlation is not unexpected because genome downsizing during the Mollicutes evolution can be followed (or paralleled) by an expansion phase resulting from duplications [30], [31] and/or from acquisitions by lateral transfer [32], [33], [34], [35]. 10.1371/journal.pgen.1004363.g002Figure 2 Total number of proteins involved in translation for each Mollicutes species. The number of proteins involved in translation for each Mollicutes species was tabulated in reference to the number found for the two model bacteria E. coli (Ec) and B. subtilis (Bs). The numbering of species is the same as in Figure 1. The data corresponding to non-cultivated Mollicutes are framed with a red dashed line as in Figure 1. The horizontal blue dashed line indicates 104, which correspond to the core of translation proteins shared by all Mollicutes. Scenario for genome erosion during Mollicutes evolution Overview: loss and gain of genes Using our dataset of translation genes, we performed a reconstruction of gene gain and loss events in Mollicutes evolution. In this reconstruction, we hypothesized that the last ancestor common between the Mollicutes and B. subtilis was a virtual organism with 220 genes involved in translation (i.e. 208 B. subtilis query genes +12 genes found in Mollicutes but not in the modern B. subtilis). Ancestral gene content at each node of the phylogenetic tree was inferred using the posterior probabilities calculated from the birth-and death model implemented in the COUNT software package [36]. Taking into account that the genome downsizing was probably a major component in Mollicutes evolution, the scenario was built allowing no gene gain in B. subtilis ( Figure 3 ). This evolutionary scenario is supported by the similar results obtained using the Wagner parsimony method with a high penalty for gene acquisition [37]; only 17 out of the 220 genes were found to have a different history in this reconstruction. 10.1371/journal.pgen.1004363.g003Figure 3 Reconstruction of the evolution of translation-related gene set in mollicutes. Ancestral gene content at each node of the phylogenetic tree was inferred using the posterior probabilities calculated from the birth-and death model implemented in the COUNT program. Genes gained and lost are framed and highlighted with colors corresponding to gene categories, respectively. Very similar results were obtained using Wagner parsimony method with a gain penalty of 4. The phylogenetic tree was inferred using the maximum likelihood method from the concatenated multiple alignments of 79 proteins encoded by genes present at one copy in each genome. The phylogenetic groups are indicated: S for Spiroplasma, H for Hominis, P for Pneumoniae and AAP. The non-cultivated Mollicutes are framed by a red dashed line. Using this method, we found 26 gene gain events involving 20 different genes. The acronyms of the corresponding genes are indicated in open boxes with lines corresponding to the color code as defined in Table 1 . In contrast to these rare cases of gene gains, there were 255 gene losses with 3 major nodes totaling 99 loss events (39%): node 38 that represents the entrance in the Mollicutes class with 25 losses, node 2 that represents the separation between phytoplasmas and acholeplasmas with 38 gene losses and node 5 leading to the hemoplasmas with another 36 genes losses ( Figure 3 ). Once again these results emphasize the particular status of the non-cultivated Mollicutes surviving with a minimal set of proteins ( Figure 3 ). There are also other nodes showing major gene losses, including node 27 (15 losses) that corresponds to the separation of the Hominis group from the other mollicutes. This is quite remarkable because it involves a large cluster of species (16 altogether) that are characterized by a great diversity of animal hosts (Table S1). The events at node 38 are dependent of the arbitrary choice of B. subtilis as a model for last common ancestor for the Mollicutes The various gains and losses of genes for each category of proteins considered ( Table 1 ) in the 39 mollicutes are discussed below. The ones that have been lost in all Mollicutes ( Figure 1C ) are not systematically discussed. Ribosomal proteins The major function of ribosomal proteins (r-proteins) is stabilization of rRNA structure, although some of them are also involved in functional interactions with m+t+tmRNAs and translation factors. Of the 60 query genes coding for r-proteins present in ribosomes of E. coli and/or B. subtilis, 49 are present in the 39 mollicutes genomes examined ( Figure 1A , Table S3). Five genes encoding r-proteins (S14b/RpsNb, the ribosomal associated protein SRA or S22/RpsV, L31b/RpmEb, L7b/RplGb and L25/RplY) are missing in all Mollicutes ( Figure 1C ). These could have been lost very early in the genomic erosion (node 38, Figure 3 ) or could have emerged later in B. subtilis lineage, after the separation of the Mollicutes lineage. For the other r-proteins, the situation varies with the specific mollicute analyzed ( Figure 1B and Table S3). In contrast with the S14, L31, L7a (RplGb) cases discussed above, where one of the two encoding paralogous genes is absent at the emergence of Mollicutes class, most species tend to retain the two genes encoding L33a (RpmGa) and L33b (RpmGb), L33a being lost only in the 3 hemoplasmas (node 5). Protein L9 (RplI) with two globular domains (one being exposed out of the 50S subunit) normally interacts with tRNA in the P site and limits mRNA slippage (frameshift) [38]. It is also lost in the 3 hemoplasmas (node 5) and in the single Mycoplasma penetrans (node 10). S21 (RpsU) was lost only once at the root of the Hominis sub-group (node 27), whereas S1 (RpsA) was lost independently seven times (nodes 2, 5, 7, 10 13, 19 and 34), remaining in several species of the Hominis sub-group, and absent in most of the other mollicutes. S1 is important for translation initiation of Shine-Dalgano (SD)-containing mRNAs and becomes obsolete for reading leaderless-mRNAs [39]. Proteins S1 and S21, both playing a role in the initiation process, seem mutually exclusive. Finally, L30 (RpmD, lost at node 37) is found only in the AAP sub-group. Of note, S1, S21, S22 (SRA), L7a, L25 and L30 are absent in many other bacterial species [21]. Together with L33a mentioned above, they are known to be responsible for cellular ribosome heterogeneity, probably generating specialized ribosomes in response to stress conditions and environmental changes [40]. These r-proteins could have arisen during evolution to fufil specific non-essential innovations [41], and hence could be easily lost during the reductive evolution of Mollicutes ribosomes. Moreover, systematic chromosomal deletion studies of bacterial r-protein genes showed that many of these (24/55 in E. coli and 22/57 in B.subtilis) were not essential (Figure S2 and: [42], [43], [44] [45]). Translation factors In addition to the core ribosomal components, protein synthesis requires a series of translation factors. These factors ensure the speed and the fidelity of translation, as well as the functionality of the nascent polypeptide. Most of them are found in all Mollicutes illustrating again the conservation of the translation apparatus in the bacterial world. Translation factors present in all Mollicutes are the initiation factors IF1, IF2, IF3, the elongation factors EF-G, EF-P, EF-Ts and EF-Tu, the peptide chain release factor RF1, the recycling factor RRF, the back translocation elongation factor LepA (also designated EF4), the peptidyl hydrolase PTH, the methionine aminopeptidase (MAP) that releases non-formylated methionine from the N-terminal nascent peptide, and SmpB associated to tmRNA that rescues ribosomes stalled on truncated mRNAs. All of the above, except LepA, correspond to essential genes in bacteria including M. genitalium and M. pneumoniae (Figure S2). In E. coli, LepA becomes essential only under unfavourable growth conditions, such as low temperature or high ionic strength [46]. The ribosome-associated trigger factor TIG (also designated TF) is not essential in E. coli and is missing only in the non-culturable M. suis. In M. genitalium, TIG has two activities: the co-translational folding of nascent polypeptide and a peptidyl-prolyl isomerase activity [47], [48]. Together with DnaK/DnaJ/GrpE and GroEL/GroES, TIG belongs to the essential polypeptide chaperone networking system (see below and [49], [50]. A few translation factors are dispensable in several mollicutes. Methionyl-tRNA formyltransferase (FMT) that catalyzes the formylation of Met on initiator Met-tRNAMeti and peptide deformylase (DEF) that subsequently removes the formyl group from the N-terminal methionine of translated peptides, are both absent in the six non-culturable hemoplasmas/phytoplasmas (nodes 2 and 5) and the three species of the Hominis subgroup, Mycoplasma hyorhinis, M. ovipneumoniae and M. hyopneumoniae (node 20 in Figure 3 ). The concomitant loss of both these proteins, while the methionine aminopeptidase (MAP) remains ubiquitous ( Figure 1B ), agrees with the observation that in E. coli the def gene could be inactivated only if the fmt gene was also inactivated [51]. Of the two E. coli ribosome-associated bi-functional stringent factors RelA and SpoT, only one (designated RelA/SpoT) is present in Firmicutes [52]. These bi-functional enzymes carry a GDP/GTP-dependent (p)ppGpp synthetase and a phosphohydrolase activity that regulate the concentration of the alarmone (p)ppGpp in response to various environmental stresses, such as temperature change, transition to the stationary phase, or limitation of essential metabolites. In Mollicutes RelA/SpoT is lost in all the Hominis species (node 27) and in the 3 hemoplasmas (node 5). The GTPase TypA (or BipA), universally conserved in Bacteria, is another translation regulator that exhibits differential ribosome association in response to stress-related events [53]. Homolog of TypA is lost in all phytoplasmas, in all Hominis and Pneumoniae species (nodes 2 and 28), plus the single S. citri. The release factor 2 (RF2), required for reading the UGA termination stop codon, is missing in all mollicutes but the three phytoplasmas and A. laidlawii (node 37, Figure 3 ). The UGA codon is decoded as Trp in all mollicutes lacking RF2 [54] by an extra tRNATrp harboring a U*CA anticodon [55]. In the case of M. capricolum, the wobble base (U*34) is post-transcriptionally modified to cmnm5U [56]. In agreement with RF2 being absent, two other proteins (ArfA and YaeJ) are also absent, another example of concerted elimination of proteins belonging to the same biochemical process. ArfA rescues stalled-ribosomes from mRNA by recruiting RF2 to release tRNA, and YaeJ hydrolyzes peptidyl-tRNA (without RF2) on stalled ribosomes. The use of UGA codon as a Trp codon in most Mollicutes species also agrees with the lack of co-translational incorporation system (SelA, SelB, SelC and SelD) [57] for selenocystein in mollicutes as it uses the same UGA codon. Aminoacyl-tRNA synthetases and a few related proteins All Mollicutes genomes analyzed encoded the complete set of aminoacyl-tRNA synthetases (aaRS) and protein cofactors required to charge all 20 canonical amino acids. They need only 19 classical aaRSs as the gene coding for glutaminyl-tRNA synthetases (GlnS), found in many other bacteria (including E. coli), is missing in most mollicutes [58] [59]. Like their Firmicutes progenitor, mollicutes encode a non-discriminating type of glutamyl-tRNA synthetase (GltX) that charges both tRNAGlu and tRNAGln with Glu, and the heterotrimeric enzyme encompassing GatA, GatB and GatC (Gln-tRNA amidotransferase complex) that amidates Glu-tRNAGln to Gln-tRNAGln [60]. The loss of genes coding for the GatA/B/C enzymatic system and the gain of GlnS are concomitant and occurred at the root of the AAP sub-group (node 3 in Figure 3 , see also in Figure 1B ). This mutually exclusive process seemed to have occurred repeatedly in bacterial evolution [58]. Bacterial GlyRSs are of two types: a tetrameric form (α2β2) and a dimeric form (α2), the corresponding subunits being encoded by glyS (α subunit) and glyQ (β subunit) genes, respectively. B. subtilis str. 168 harbors a α2β2 type GlyRS, whereas other bacilli, such as Bacillus anthracis str. A2012 or Bacillus thuringiensis serovar konkukian str. 97-27, harbor an α2 type enzyme [61]. All mollicutes encode only GlyS and no GlyQ homologs (Table S3), suggesting that the homodimeric form of GlyRS was already present in the Mollicutes progenitor. PheRS is the only α2β2 heterodimeric aaRS found in all mollicutes, each subunit being encoded by the co-transcribed tandem pheS (for α subunit) and pheT (for β subunit) genes [62]. Interestingly in M. pneumoniae, the PheRS α2β2 was detected in vivo in a complex with four other synthetases (TyrS, MetG, ThrS, GltX) [11]. This multiprotein complex is reminiscent of the multi-synthetase complex found in Eukarya and Archaea, but elusive in Bacteria [63]. Lastly, a single gene is found for LysRS, TyrRS and ThrS, no duplicant for LysU, ThyZ and ThrR like in other bacteria. Many aaRSs are prone to mistakes and mischarge structurally similar amino acids. To minimize mistranslation, these enzymes harbor an editing activity to hydrolyze mischarged tRNAs. In Mollicutes, several aaRS carry mutations or even deletions in their editing domains that increase mistranslation frequency. Such genetic variants have been identified in LeuRS, PheRS and ThrRS editing domain of several Mycoplasma species [64], [65], [66]. In addition, the Mollicutes ProRSs are of the eukaryotic/archaeal type that lack the cis-editing domain [67]. Finally, no homologs are found in any Mollicutes species of the stand-alone bacterial editing proteins like the YbaK, ProX or AlaX families that hydrolyze misacylated Cys-tRNAPro, Ala-tRNAPro, Ser-tRNAAla and Gly-tRNAAla, respectively [68]. The systematic absence of aaRS editing functions in mollicutes suggested high misincorporation rate that were experimentally validated in a few cases [64], leading to a 'statistical proteome' that could be one of the reasons Mollicutes species are evolving faster than any other extant bacteria (discussed in: [69], [70], [71]). In E. coli, a D-aminoacyl-tRNA deacylase (dtd, yihZ gene in E. coli) allows recycling the D-containing misaminoacylated tRNA [72]. Orthologs of the yihZ gene occur in nearly all bacteria, including B. subtilis (yrvI) but in Mollicutes only A. laidlawii harbors a yihZ homolog. Finally, because the terminal 3′-CCA sequence of mature tRNAs in all Mollicutes species are generally encoded in the genome [73], the tRNA nucleotidyl transferase (CCAase) became obsolete and the pre-tRNA processing machinery exactly trims the tRNA at the CCA end with one RNase only [74], while in other bacteria several accessory RNases are needed (see below). The loss of the encoded CCAase gene occurred very early in Mollicutes evolution (node 37, Figure 3 ). As a consequence, one can expect the absence of 3′-CCA end turnover and of repair of tRNAs lacking the terminal amino acceptor adenosine. The systematic presence of CCA sequence at the end of all tRNA primary transcripts, instead of longer 3′-tail as in majority of bacteria, exemplifies again the genome economy strategies of mollicutes. Transfer RNA modification enzymes tRNA precursors are subject to enzymatic post-transcriptional modifications at many positions of the base or ribose moieties. These modifications stabilize the tRNA tertiary structure, introduce recognition determinants and antideterminants towards RNA-interacting macromolecules and fine-tune the decoding process at the level of both efficiency and fidelity. Genes coding for almost all E. coli tRNA modification enzymes have been identified and experimentally verified, and most of them have homologs in B. subtilis. A few additional B. subtilis genes coding for enzymes that are absent in E. coli have also been characterized (Table S3 and Figure S3). Of the 45 query genes coding for tRNA modification enzymes only a handful of homologs are predicted to resist genomic erosion in Mollicutes. These encode the two proteins TsaC and TsaD of the multienzymatic complex involved in t6A formation composed of 4 subunits in E. coli (TsaB, TsaC, TsaD, TsaE) [75], the site-specific methyltransferase TrmD catalyzing formation of m1G, and thiouridine synthetase MnmA catalyzing the thiolation of wobble uridine (s2U). All these modifications are located in the anticodon loop (position 34 or 37) of a subset of tRNAs (Figure S3). Of the other proteins of the t6A synthesis machinery, TsaB is missing in the 3 hemoplasmas (node 5, Figure 3 ), while TsaE is missing in all species of the Pneumoniae subgroup (node 12, Figure 3 ). In these latter organisms the t6A machinery is reminiscient of the recently elucidated mitochondrial pathway also composed of only two proteins [76]. In Mollicutes, the sulfur relay system working in conjunction with MnmA has yet to be characterized. Of the complex sulfur relay encompassing at least 7 components (IscU/IscS/TusA/TusB/TusC/TusD/TusE) identified in E. coli but not in B. subtilis [77], only IscU and IscS are present in all mollicutes. The most parsimonious explanation would be that the cysteine desulfurase IscS/IscS/NifS and/or the alternative SufU/SufU/NifU present also in B. subtilis suffice to provide the sulfur moiety by direct transfer of the sulfhydryl group to the wobble U34 [78], [79]. The next most persistent tRNA modification genes in Mollicutes are those coding for: i) MnmE and MnmG (formation of cmnm5U), both lost only in M. suis, ii) the two methyltransferases TrmL and TrmB catalyzing respectively the 2′O-ribose methylation of the wobble pyrimidine (C and cmnm5-containing U) and the formation of an m7G+ (carrying a positive charge) in the extra arm (variable loop) of a large subset of tRNAs (position 46), both missing only in 6 non-culturable mollicutes (nodes 2 and 5 and Figure 1B ), and iii) the site-specific TruB catalyzing the formation of ψ in all tRNAs, missing in the three hemoplasmas (node 5) and in M. genitalium and M. pneumoniae (node 6). Except for m7G+ at position 46 in the variable loop and ψ at position 55 of the Tψ-loop, these modifications are again located in the anticodon loop of tRNAs (wobble position 34, Figure S3). The MnmA/MnmE/MnmG and TrmL enzymes all play key roles by restricting the corresponding modified tRNAs in decoding only the 2 purine-ending codons of a 4-synonymous codon set, while m7G+46 and ψ55 allow stabilization of the L-shape 3D-conformation of all tRNAs [80]. The essential E. coli and B. subtilis tRNA-A34 deaminase (TadA, formation of the wobble inosine) is present only in species of the Spiroplasma and AAP groups (lost at node 28). The complete elimination of tadA was shown to be a stepwise process. It started with specific mutations in the active site of TadA, was followed by the lost of one tRNAArg (anticodon CCG) that became useless before the final loss of the tadA gene [81]. Similarly, the loss of the essential tRNA-lysidine synthetase TilS (k 2C34) in M. mobile and the three hemoplasmas (node 5) correlates with a compensatory C-to-U mutation at the wobble position 34 in the tRNAIle substrate. In the case of M. mobile, the mutant tRNAIle was shown to harbor an unmodified wobble U34 instead of the normal k2C34. Using M. mobile ribosome in a cell-free in vitro system, this mutant U34-containing tRNAIle was shown to decipher preferentially Ile-AUA codon but not when E. coli ribosome was used, suggesting changes in the mollicute ribosome. This decoding readjustment is also dependent on additional mutations in M. mobile IleRS, allowing the mutated enzyme to preferentially aminoacylate U34-containing tRNAIle [82]. In the case of M. penetrans, MetRS was shown to better discriminate between tRNAIle-CAU and tRNAMet-CAU than the canonical bacterial MetRS [83]. These examples demonstrate the high plasticity of the various components of translation machinery subsequent to the elimination of experimentally determined essential genes in E. coli or B. subtilis such as TilS and TadA, while preserving the accuracy of the decoding process. The less persistent tRNA modification enzymes are: the site-specific methyltransferase TrmK (m1A+22, also carrying a positive charge) missing only in the Hominis sub-group (node 27); the methyltransferase TrmN (alias TrmN6; m 6A37) missing in all species of the Pneumoniae sub-group (except Ureaplasma parvum and U. urealyticum) and in the six non-culturable mollicutes (nodes 2 and 5); the multi-site specific pseudouridine synthase TruA (Psi38–40) that is missing in all species of the Hominis sub-group (node 27) plus the three non-culturable hemoplasmas (node 5). For the remaining tRNA modification enzymes, a few are retained in a small subset of Mollicutes ( Figure 1B ). These are the G-to-Q transglycosylase (Tgt) acting at the wobble position 34 of a subset of tRNAs and its associated enzymes QueA, QueG, all lost very early (nodes 2 and 37 in Figure 3 ), leaving only A. laidlawii with tRNAs possibly containing Q34. Out of three dihydrouridine synthases characterized in E. coli, only one is present in B. subtilis and in the Spiroplasma group and A. laidlawii. Likewise, isopentenyl transferase MiaA responsible for i 6A37 formation, MiaB plus MtaB responsible for the subsequent methylthiolation of i6A37 (ms 2i6A) and t6A37 (ms 2t6A) respectively, are lost several times independently in the majority of mollicutes. With the exception of an E. coli TtcA homolog (s2C32 formation), possibly acquired by lateral gene transfer in M. penetrans, all modification enzymes present in E. coli and absent in B. subtilis are also absent in Mollicutes. Examples include MnmC (mnm5U34 from cmnm5U34), CmoA/CmoB (cmo5U34), SelU (seU34 and ges2U34 from s2U34), TmcA (ac4C34), TsaA (m6t6A37 from t6A37), TrmH (Gm18), TrmA (m5U54), TruC (ψ65) and TruD (ψ13). These modification enzymes obviously emerged in other phyla than the Firmicutes. An interesting case concerns TrmFO catalyzing the folate-dependent methylation of the conserved uridine at position 54 (m5U54) in the Tψ-loop of tRNAs of Gram-positive bacteria [84]. Sequencing of tRNAs from M. capricolum and M. mycoides revealed the absence of m5U54 in tRNAs, while two and even three TrmFO homologs were found in the Spiroplasma sub-group (Table S3). Only one of the three isoforms is present in a few species of the Hominis sub-group and was probably inherited by lateral gene transfer (node 14 in Figure 3 ), possibly from another ruminant mycoplasma from the Spiroplasma sub-group [34]. The target specificities of the two TrmFO homologs in M. capricolum and M. mycoides while still to be determined, are obviously distinct from the B. subtilis tRNA-specific TrmFO, which illustrates again the evolutionary malleability of modification enzymes. The special case of tmRNA In addition to tRNAs, a transfer-messenger RNA (tmRNA) and its associated protein SmpB have also been identified in all mollicutes. Their function is to rescue stalled ribosomes during translation. This tmRNA folds into a tRNA-like domain (TLD), that shares many structural and functional similarities with tRNAs. In particular, the UUC sequence of the T-arm loop of E. coli tmRNA is post-transcriptionally modified into m5UψC. The m5U residue is introduced by the S-Adenosyl-L-Methionine-dependent TrmA and the ψ probably by TruB [84]. As mentioned above, TrmA is missing in all Mollicutes and the function of TrmFO in these organisms is still unclear. The pseudouridine synthase TruB, present in many mollicutes (see above), could therefore also catalyze ψ formation in the Mollicutes tmRNAs. Ribosomal RNA modification enzymes Many bases and riboses of rRNAs are post-transcriptionally modified like in tRNAs (Figure S3). Most modifications are introduced during pre-rRNA maturation and ribosome assembly, and just a few are formed at the level of the 30S and 50S subparticles or of the entire 70S ribosome. The conservation and clustering of modifications in the decoding center of the 30S subunit and in the peptidyl-transferase center of the 50S subunit, attests their important roles in the translation process. Out of the total 33 genes coding for rRNA modification enzymes in both E. coli and B. subtilis, only 19 remain in Mollicutes, and only four are ubiquitous ( Figure 1A ). These are: i) the region-specific RsmA, catalyzing the dimethylation of two adenines at positions 1518 and 1519 (m6,6A, E. coli numbering) of helix 45 located close to the decoding site in 16S rRNA, ii) the site-specific RsmH catalyzing, the formation of m4C1402 of helix 44 at the P-site of the 30S subunit, iii) the multi-site specific RluD, catalyzing the isomerization of uridine into pseudouridine at three neighboring positions (1911, 1915 and 1917, E. coli specificity) of helix 69 in 23S rRNA, and iv) the site-specific RlmB, catalyzing the methylation of 2′-hydroxyl group of G2251 (Gm) in the P-loop (helix 80) of 23S rRNA (Figure S3). The ubiquitous m4C1402 of helix 44 of 16S rRNA can be further methylated on the ribose into m4Cm1402 by RsmI, an enzyme found in all Mollicutes except the three hemoplasmas (node 5 in Figure 3 ), whereas the ubiquitous ψ1915 of helix 69 in 23S rRNA can be further hypermodified into m3ψ by RlmH only after the 70S ribosome is formed, thus at very late stage of ribosome assembly. RlmH is lost in the hemoplasmas (node 5), the three phytoplasmas (node 2), and three of the six members of the Pneumoniae group (node 7). In the 3D-architecture of the ribosome, this hypermodified helix 69 extrudes from the 50S subunit toward the decoding center of the 30S subunit, close to helices 44 and 45, where the other universally conserved multi-modified rRNA sequences are located. Among other fairly persistent genes are those encoding RluC catalyzing the isomerization of U955, U2504 and U2580 into pseudouridines, two of which belong to the peptidyl transferase center (PTC)-loop of 50S subunit, and RsmG catalyzing the formation of m7G+527 (carrying a positive charged on methylated N7) in helix 18 of the decoding center of 30S subunit. RluC is absent only in the three phytoplasmas (node 2) and RsmG is absent in Ca. Phytoplasma mali and in the hemoplasmas (node 5, Figure 3 ). Many rRNA modification enzymes are lost in a large group of Mollicutes but with different patterns ( Figure 1B ). RluB catalyzing the formation of ψ2605 in helix 93 of the peptidyl-transferase center and RsmD catalyzing the formation of m2G966 in helix 31 of the decoding center are both absent in the group Pneumoniae (lost at node 12). Whereas, RsmB, catalyzing the formation of m5C967 located next to m2G966 mentioned above, is present in all species of the Spiroplasma sub-group and absent in all species of the Pneumoniae, Hominis and AAP sub-groups (loss at nodes 3 and 28). RsmE, catalyzing the formation of m3U1498 nearby the conserved m4Cm1402 in helix 44 of the decoding center of 16S rRNA, is present in all species of the Hominis sub-group and a few species of the Spiroplasma and Pneumoniae sub-groups. The case of RlmCD is special. It catalyzes the formation of m5U at two positions (747 in helix 35 and 1939 in helix 71) in 23S rRNA of B. subitilis, while in E. coli two paralogous enzymes (RlmC and RlmD) are needed to catalyze m5U747 and m5U1939 formation respectively [85]. RlmC/RlmCD is present in a few species of the Hominis sub-group only, while RlmD is absent in all mollicutes. Finally, RsuA, catalyzing formation of ψ516 in helix 18 of rRNA 16S, is present in A. laidlawii and few species of the Hominis sub-group only, whereas the dual t+rRNA specific RlmN (m2A2503 in 23S rRNA + m2A37 in tRNA, E. coli specificity) remains in only four mollicutes: S. citri, Ureaplasma spp., M. penetrans and A. laidlawii ( Figure 3 ). Two orphan RNA methylase genes are found in B. subtilis but absent in E.coli: YsgA, encoding a putative TrmH/SPOUT-like 2′-O-ribose RNA methyltransferase (COG0566C) and renamed rlmB2 because of its close relationship with rlmB catalyzing the formation of Gm2251 (see above) and yqxC, encoding an another similar FtsJ/Spb1/SPOUT-like 2′-O-ribose RNA methyltransferase. Because B. subtilis harbors a modified Gm2553 in the P-loop (helix 92, see Figure S3), for which the corresponding gene is unknown [86], we speculate that one of these two orphan genes correspond to the missing but important G2553-2′-O-ribose-rRNA methyltransferase, while the second one probably catalyzes 2′-O-ribose methylation at a yet unidentified nucleotide of RNA. Both YsgA/RlmB2 and YqxC are present in about half the Mollicutes analyzed but always present together ( Figure 1B ). The A. laidlawi species seems to have conserved more rRNA modifications genes than other mollicutes. For example, RsmC (m2G1207) is found in A. laidlawii only (early loss at nodes 2 and 37 in Figure 3 ). Moreover, A. laidlawii harbors four RlmCD copies instead of only one in other Mollicutes. These enzymes should catalyze the formation of the six m5U identified in A. laidlawii 23S rRNA, their exact locations remaining to be determined [87]. The case of E. coli RluF (Psi2604) is special as no homolog is present in B. subtilis but
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