Evolutionary distance between clusters.

<p>Amino acid sequences of genes encoding α-like and β-like proteins of the Type 2 and Type 3 clusters (panels A, C and E) and α- and β-subunits of F₁F₀ Type 1 clusters (panels B, D and F) were concatenated and multiple alignments were generated. Multiple sequence alignments were curated with GBLOCK to remove unreliable sites and a final round of manual editing was performed with Jalview. Evolutionary distances were calculated with Type 3 pairs thought to have been exchanged through HGT as references. These distances are shown as a function of the evolutionary distance between species inferred from 16S rDNA data. The Type 3 pairs concerned were MAG2930/2940 (<i>M. agalactiae</i>), MHO_3130/3120 (<i>M. hominis</i>) and MGA_1321d (<i>M. gallisepticum</i>). In the last case, the analysis was based exclusively on the truncated <i>atpD</i>-like gene. Homologs from a phylogenetic group are circled: H, Hominis; P, Pneumoniae; S, Spiroplasma.</p

Evolution of <i>atpA</i> and <i>atpA</i>-like genes in bacteria.

<p>The phylogenetic tree was inferred from the amino acid sequences of ATPase alpha subunits encoded by <i>atpA</i> and <i>atpA</i>-like genes. Multiple alignment was generated with MUSCLE. The phylogenetic tree was generated by the ML method. Branches corresponding to Type 1, Type 1′, Type 2 and Type 3 proteins were supported by 96–100% bootstrap values. The ML, NJ, MP and ME methods generated trees with similar topologies, except alternative branching of N-ATPases using NJ or ME (indicated by a star). Main bacterial groups are indicated. Proteins from mollicutes are named by their mnemonics, others by the species name. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038793#pone.0038793.s002" target="_blank">Table S2</a></b> for details.</p

Model of an F₁-likeX₀ ATPase encoded by the seven-gene clusters of Types 2 and 3 specific to mycoplasmas.

<p><b>A</b>. The F₁-like complex model of <i>Mmm</i> was drawn by similarity with the crystal structure of the <i>E. coli</i> F₁-ATPase (Pdb id: 3oaa) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038793#pone.0038793-Cingolani1" target="_blank">[27]</a> with the help of the Pymol software (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038793#pone.0038793-DeLano1" target="_blank">[28]</a>. The X₀ complex proteins of <i>Mmm</i> were schematized on the basis of 2D structure predictions. Proteins 1 and 5 are depicted associated with the membrane, in accordance with the predicted transmembrane segments. Based on <i>in silico</i> and experimental results, the F₁-like complex, Protein 2 and the main part of Protein 5 were predicted to be cytoplasmic. Within this model, the F₁-like and the X₀ sectors are represented, but the way they could interact remains largely unclear. <b>B</b>. The genes of the clusters were arbitrarily numbered from 1 to 7. Gene names are indicated above the boxes representing the genes. TM, transmembrane segments. The proteins encoded by genes 3, 4, 6 and 7 were found to be related to the subunits γ, ε, α and β of the F₁F₀ ATPase, respectively.</p

Growth and ATPase activity of <i>Mmm</i> T1/44 and the Δ619 mutant.

<p><b>A</b>. Growth of <i>Mmm</i> T1/44 (▪) and Δ619 (□) in Hayflick medium at 37°C. <b>B</b>. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□). The figure shows representative results of five independent experiments. <b>C</b>. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□) transformed with the control plasmid pMYSO1, and Δ619 complemented with the MSC_0619 (α-like) and MSC_0618 (β-like) proteins, generated from the plasmid pCC1 (▴).</p

Operon structure and expression of the genes of the Type 3 cluster in <i>Mmm</i>.

<p><b>A</b>. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the <i>MSC_0618</i> to <i>MSC_0627</i> genes. The region of the genome region surrounding the Type 3 cluster in <i>Mmm</i> is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the <i>MSC_0619</i> disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). <b>B</b>. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). <b>C</b>. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. <b>D</b>. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of <i>Mmm</i> T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).</p

ATPase F₁F₀ in mycoplasmas.

<p><b>A</b>. Bacterial ATPase F₁F₀. <b>B</b>. Organization of the operon encoding the ATPase F₁F₀ in mycoplasmas. In <i>E. coli</i> and mycoplasma species, the F₁F₀ ATPase operon and likely the 3D structure are similar.</p

Genomic contexts of Type 2 and Type 3 clusters in mycoplasmas.

<p>Homologous genes are indicated by boxes of the same colour, connected by dashed lines. The genomic regions containing the Type 2 (A) and Type 3 (B) clusters are framed in red. The schematic diagram was generated from screenshots obtained from the MBGD database. Mnemonics and gene names are indicated; genes from the clusters are numbered arbitrarily from 1 to 7. The genome structures of <i>M. mycoides</i> subsp. <i>capri</i> and <i>M. capricolum</i> subsp. <i>capricolum</i> were identical to that of <i>Mmm</i>.</p

TAG species analysis and uptake of labeled oleate.

<p>(A) Dominant TAG species in procyclic <i>T. brucei</i> cells identified by ESI/MS/MS after oleate feeding for three days (black columns) or in the control (white columns). For a complete list of TAG species detected see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s001" target="_blank">S1 Figure</a>. The nomenclature 54:X indicates the total carbon number of all three acyl chains and the sum of all unsaturated double bonds within the acyl chains. (B) Uptake kinetics upon growth in the presence of radiolabeled oleate for up to 8 h. The incorporation of ¹⁴C oleate into lipid species was quantified by HPTLC and a Storm 860 phosphorimager. PPL, phospholipids; TAG, triacylglycerol; SE, Steryl-esters; DAG, diacylglycerol.</p

NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.

1<p>WCE, whole cell exctract.</p>2<p>glyco, partially purified glycosome fraction.</p>3<p>Mean ± SEM of n experiments (mU/mg of protein).</p>4<p>+gluc: cells cultured in SDM79 containing 10 mM glucose.</p>5<p>−gluc: cells cultured in glucose-depleted SDM79GluFree.</p><p>NADPH-dependent 3-hydroxyacyl-CoA dehydrogenase activity in WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cells.</p

Phenotypic analysis of Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell.

<p>(A) growth curve of WT and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells in glucose-rich (SDM79 with 10 mM glucose) or glucose-free (SDM79GluFree) conditions. (B) Global protein abundance in the partially purified glycosome fraction of WT (x-axis) and Δ<i>tfeα1</i>/Δ<i>tfeα1</i> cell knock cells (y-axis). Each protein identification is presented by a point at log₁₀ of normalized peptide count values taken from the proteome data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114628#pone.0114628.s004" target="_blank">S4 Figure</a>. Proteins on the dashed grey line have identical normalized peptide counts in both samples; the grey lines represent a 2-fold abundance in one condition.</p