Clustering analysis of the metabolic and cellular process proteins of 27 Mycoplasma species/strains and Phytoplasma OY.

<p>The order of the <i>Mycoplasma</i> species is according to their phylogenetic relationship (23S rRNA tree). Each column represents the proteins of one single species/strain and each row (horizontal line) represents the copy number (grey color scale on top) of one gene. The sizes of the genomes in Mbp are indicated below the 3-letter abbreviated names of the organisms. Functional clustering and comparison of the proteins among species were based on KO (KEGG Orthology) assignments. The conservation of each gene among the <i>Mycoplasma</i> species (i.e. the number of <i>Mycoplasma</i> species possessing the gene) is shown on the right side of clustering result. <i>Abbreviations</i> – mha: <i>M. haemofelis</i>; mss: <i>M. suis</i> Illinois; msk: <i>M. suis</i> KI3806; mpe: <i>M. penetrans</i>; mga: <i>M. gallisepticum</i>; mge: <i>M. genitalium</i>; mpn: <i>M. pneumoniae</i>; mmy: <i>M. mycoides</i>; mcp: <i>M. capricolum</i>; mlc: <i>M. leachii</i>; mat: <i>M. arthritidis</i>; mho: <i>M. hominis</i>; mmo: <i>M. mobile</i>; mhr: <i>M. hyorhinis</i>; mhy: <i>M. hyopneumoniae</i> 232; mhj: <i>M. hyopneumoniae</i> J; mhp: <i>M. hyopneumoniae</i> 7448; mco: <i>M. conjunctivae</i>; mpu: <i>M. pulmonis</i>; mfm: <i>M. fermentans</i> M64; mfr: <i>M. fermentans</i> JER; maa: <i>M. agalactiae</i> PG2; mal: <i>M. agalactiae</i> 5632; mbv: <i>M. bovis</i> PG45; msy: <i>M. synoviae</i>; mcd: <i>M. crocodyli</i>; and poy: <i>Phytoplasma</i> OY.</p

Representative 2-DE gel of <i>Mycoplasma fermentans</i> M64.

<p>A 100 µg of total proteins were separated by a 24 cm pH 3–10 linear IPG strip for the first dimension IEF and a 24×20 cm 11–13% gradient gel for the second dimension SDS-PAGE. After silver staining, an average of 700 spots can be detected on a 2-DE gel. Spots are numbered according to the matched ORFs.</p

A representative scheme of <i>M. fermentans</i> proteins involved in the glycolysis pathway.

<p>The identified enzymes were labeled with asterisk (*) and the predicted enzymes but not identified in this study were labeled with hash (#). Further details were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035304#pone.0035304.s007" target="_blank">Table S5</a>.</p

A representative phylogenetic tree for the selected Mollicutes species.

<p>The tree was constructed by neighbor-joining algorithm using 16 S ribosomal gene sequences from selected species of Mollicutes. The bootstrap values indicate the percentages of 1000 bootstrap replicates that support the branch. Most of the mycoplasmas can be categorized into four phylogenetic groups, namely pneumoniae, hominis, spiroplasma and haemoplasma. The 16 S sequence of <i>M. fermentans</i> M64, JER and PG18 are identical. All human pathogens were underlined. <i>Acholeplasma laidlawii</i>, AYWB phytoplasma and OY phytoplasma were used as the outgroup species in this analysis.</p

Proportion of <i>M. fermentans</i> M64 homologs in 26 mycoplasmal genomes.

<p>The three-letter KEGG organism codes were used in the x-axis. The full species names and the values used to plot the bar chart can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035304#pone.0035304.s005" target="_blank">Table S3</a>.</p

Phylogenetic conservation analysis of nucleotide (A) and carbohydrate (B) metabolic networks of M. fermentans M64 and 20 other Mycoplasma species.

<p><i>M. fermentans</i> M64 metabolic networks were served as the references in the comparisons. The small open squares represent the compounds and the colored circles represent the proteins (enzymes) participating in the metabolisms. Circles with thick and light blue circumference denote the potential essential genes inferred from the comparison with <i>M. pulmonis</i>, <i>M. arthritidis</i>, and <i>M. genitalium</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032940#pone-0032940-g005" target="_blank">Figure 5</a>). The arrows and lines linking the enzymes and substrates indicate the direction of the reactions. Enzymes involved in more than one metabolism are annotated with a single letter code of the other metabolisms (A: Amino acids; C: Carbohydrate; N: Nucleotide; V: cofactors and Vitamins) next to the circles. The color scale (bottom) indicates the phylogenetic conservation of proteins in the analyzed <i>Mycoplasma</i> species.</p

Terminal and junction sequences of the IS elements, ICEFs, and ΦMFV1 prophage DNAs in the M. fermentans M64 genome.

<p>The terminal inverted repeats and flanking sequences of 15 complete and 4 partial copies of three families of IS elements including the IS<i>1550</i>, IS<i>1630</i>, and ISMf<i>1</i>, 7 copies of three families of ICEFs, and 2 prophage genomes are shown. Bold uppercase letters with arrows on top indicate the terminal inverted repeats. The target site duplications are shown in underlined bold lowercase. The elements interrupted by other transposable elements are indicated.</p

The number of transmembrane helices predicted using TMHMM.

<p>The prediction was performed on all <i>M. fermentans</i> M64 predicted and identified proteins. Of the 1050 total predicted ORFs, 791 (75%) have no predicted TMD. Whereas 161 of 181 (89%) identified ORFs have no predicted TMD.</p

An adaption of the schematic representation of the nucleoside salvage pathway by Wang, et al., 2001 [<b>50</b>].

<p>Substrates/products: dN/N (deoxy)nucleoside; dNMP/NMP (deoxy)nucleoside monophosphate; dNDP/NDP (deoxy)nucleoside diphosphate; dNTP/NTP (deoxy)nucleoside triphosphate; PRPP 5-phosphoribosyl diphosphate; ATP adenosine 5′-triphosphate; PPi pyrophosphate. Identified enzymes were labeled with asterisk (*) and predicted enzymes but not identified were labeled with hash (#), details can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035304#pone.0035304.s006" target="_blank">Table S4</a>.</p

Dotplot of the genomes of M. fermentans strains M64 versus JER (A) and PG18 (B).

<p>The graphs were generated with the <i>Gepard</i> 1.21 with word length of 20 and window size of 0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032940#pone.0032940-Krumsiek1" target="_blank">[78]</a>. The long repetitive sequences are indicated by horizontal arrows: ICEF-I and -II (unfilled arrows), ICEF-III (black arrows), and ΦMFV1 (gray arrows). The locations of the IS elements of the ISMf<i>1</i>, IS<i>1550</i> and IS<i>1630</i> families are indicated by small black and gray (elements interrupted by ICEFs) triangles. The two types of non-IS small duplications are indicated by small filled (16–23S rRNA operon) and open triangles (uncharacterized repetitive sequence containing a hypothetical protein-coding gene).</p