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

General features of M. fermentans M64 genome.

*<p>Repetitive sequences included the transposable elements and the duplicated 16 and 23S rRNA.</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

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

Sequence analysis of KPX plasmids.

<p>Two circular sequences are shown for the organization of pKPX-1 (<b>A</b>) and pKPX-2 (<b>B</b>). Mapping shotgun sequencing reads of pECX-1 to the pKPX-1 is indicated by the red half-circle. A large part of the plasmid, corresponding to the nucleotide positions 23,125 to 145,377 of pKPX-1, was not found in pECX-1. Only the part on the left side, totaling 128,191-bp, is retained. Two genes encoding chloramphenicol and amikacin resistance were identified by functional library screening. Their positions in the deleted region are indicated. Nucleotides are numbered according to the replication origin. Genes are color coded: yellow, β-lactamase; red, antimicrobial resistance associated; blue, plasmid replication and partitioning; black, transposases or IS elements; and white, other coding sequences of miscellaneous features. The arrows on the open reading frames (ORFs) indicate the gene orientation. Gene clusters involved in gene transfer or mobility are marked in green. <i>Xba</i>I and <i>Avr</i>II restriction sites are shown inside the circle.</p

Tandem duplication of the <i>bla</i>_NDM-1 gene in pKPX-1 and pECX-1.

<p>(<b>A</b>) Diagrammatic representation of the analysis of <i>bla</i>_NDM-1 copy number by Southern blot. The probe is shown with a red arrow, and the tandem duplication of the 8588-bp repeat is indicated by the bracket. The asterisks indicate the methylated <i>Nru</i>I sites. The sizes of <i>Bam</i>HI or <i>Hind</i>III digested fragments depend on the copy number of the repeat. The pound sign indicates 79.1 kb and 71.8 kb for <i>Bam</i>HI and <i>Hind</i>III restrictions, respectively, when there are 8 copies of the tandem repeat, as in the case of pECX-1. (<b>B</b>) Sequencing read distribution and Southern analysis of the <i>bla</i>_NDM-1 region for pECX-1. The upper panel shows the relative coverage depth of the repeat region and its flanking sequences. The average coverage of <i>bla</i>_NDM-1 is 7–8 fold of those sequences of the immediately adjacent regions, suggesting that there are eight copies of the repeat. As shown in the lower panel, Southern analysis confirms this model of tandem duplication. (<b>C</b>) <i>Bla</i>_NDM-1 copy number variation detected by the Southern analysis. Sequence depth of the region revealed an average of 3–4 copies of the repeat sequence in pKPX. <i>Bam</i>HI and <i>Hind</i>III digestion gave a series of ladder bands, corresponding to different copy numbers of the repeat. By contrast, <i>Avr</i>II and <i>Nru</i>I both deliberated a single major band of 8.6 kb, representing the unit length of the tandem repeats.</p

Restriction analysis of the KPX plasmids by PFGE.

<p>Plasmid DNA isolated from the KPX isolate was analyzed using pulsed field gel electrophoresis (PFGE). The restriction patterns of the plasmid DNA were compared to those of pECX-1 and pECX-2 in <i>Escherichia coli</i>. The size of the DNA markers is shown in kilobases (kb) on the left side.</p

Antimicrobial resistance determinants in pKPX-1 and pKPX-2.

*<p>Nonfunctional; ^† Deleted in pECX-1.</p><p>Abbreviations: AAC, aminoglycoside acetyltransferase; APH, aminoglycoside phosphotransferase; ANT, aminoglycoside nucleotdyltransferase; Rmt, 16S rRNA methyltransferase; Qnr, quinolone resistance protein; TetA, tetracycline efflux protein; Cat, chloramphenicol acetyltransferase; ARR-2, rifampin ADP-ribosyltransferase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; Mph, macrolide phosphotransferase.</p

Minimal inhibitory concentrations (MICs)^* for different antimicrobial agents of <i>K. pneumoniae</i> KPX, <i>E. coli</i> DH10B, and <i>E. coli</i> transformants of DNA derived from <i>K. pneumoniae</i> KPX.

*<p>MIC interpretations are based on Clinical and Laboratory Standards Institute (CLSI) breakpoints (CLSI M100-S21, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062774#pone.0062774-Clinical1" target="_blank">[28]</a>), except for polymyxin E and tigecycline, which are based on EUCAST (<a href="http://www.eucast.org/clinical_breakpoints/" target="_blank">http://www.eucast.org/clinical_breakpoints/</a>) breakpoints.</p