Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

Abstract

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.

Fig. 1

Genetic map of the 24-kb repeat locus (A), the 13-kb repeat locus (B), the 12-kb repeat locus (C) and the 8-kb repeat locus (D) in type strain PG1 of M. mycoides subsp. mycoides SC. Horizontal black arrowheads indicate position of the various oligonucleotide primers and the gaps between two paired arrowheads show the amplified PCR fragments A–D. The fragments A–D relate to the PCR products shown in Fig. 2, Fig. 3. Restriction sites for SacI, ClaI, MluNI, SspBI, EcoRI, SpeI, HindIII, and NruI are indicated by vertical arrows. Double-headed arrows with integrated fragment lengths indicate the generated digestion fragments that react positively with DNA probes on Southern blots. The solid vertical bars show the borders of the repeated segments and the corresponding figures give the exact position in nucleotides (nt) referring to the genomic sequence NC_005364. The exact number of basepairs of the individual repeated segments is indicated on each brace. Genes are represented by pentagons (arrowed boxes). Negatively arrowed boxes or chevrons indicate truncated genes. In each panel, the same shading was used for the same genes. IS elements are indicated by white boxes and their inverted repeats by gray triangles. Gene abbreviations: rpoC, DNA-directed RNA polymerase beta' chain; asnA, aspartate-ammonia ligase; gidA, glucose inhibited division protein A; nox, NADH oxidase; pncA, pyrazinamidase/nicotinamidase; lppQ, prolipoprotein Q; lpp, putative variable lipoprotein; ptsG, PTS system, glucose-specific IIBC component; glk, glucokinase; arc, carbamate kinase; mgtA, Mg²⁺-transport ATPase; phnB, alkylphosphonate ABC transporter, permease component; phnC, alkylphosphonate ABC transporter, ATP-binding component; phnD, alkylphosphonate ABC transporter, substrate binding component; patB, aminotransferase (PLP-dependent); abc, ABC transporter, permease component; had, hydrolase of the HAD family; oppF, oligopeptide ABC-transporter; glf, UDP-galactopyranose mutase; galE, UDP-glucose 4-epimerase; epsG, glycosyltransferase; cps, glycosyltransferase. Asterisks (*) indicate truncated or nonfunctional genes or genetic elements.

Fig. 2

PCR and Southern blot results of a selection of eight strains of M. mycoides subsp. mycoides SC for investigation of the 24-kb repeats. PCR was performed with 50 ng of genomic DNA using oligonucleotide primers: (A) MmmSC_23760fwd and MmmSC_1rev to obtain fragment A of Fig. 1A; (B) MmmSC_rpoC_fwd and MmmSC_MSC1011rev to obtain fragment B of Fig. 1A; and (C) MmmSC_23760fwd and MmmSC_MSC1061rev to obtain fragment C of Fig. 1A. (D) Southern blot hybridization of ClaI-, SacI-, and MluNI-digested DNA hybridized to the asnA probe. Std, molecular mass standards: 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb.

Fig. 3

PCR and Southern blot results of a selection of eight strains of M. mycoides subsp. mycoides SC for investigation of the 13-kb repeats. (A) PCR performed with 50 ng of genomic DNA using oligonucleotide primers MmmSC_mgtA_fwd and MmmSC_ptsG_rev to obtain fragment D of Fig. 1B. (B) Southern blot hybridization of EcoRI- and SspBI-digested DNA hybridized to the ptsG probe. Std, molecular mass standards.

Fig. 4

Southern blot hybridization of SpeI-digested DNA of a selection of eight M. mycoides subsp. mycoides SC strains hybridized to the patB probe for investigation of the 12-kb repeats. Std, molecular mass standards.

Fig. 5

Southern blot hybridization of HindIII-, ClaI-, and NruI-digested DNA of a selection of eight M. mycoides subsp. mycoides SC strains hybridized to the oppF probe for investigation of the 8-kb repeats. Std, molecular mass standards.

Fig. 6

Plasticity of the 13-kb repeats. PCR performed using oligonucleotide primers MmmSC_mgtA_fwd and MmmSC_ptsG_rev to obtain fragment D of Fig. 1B with lysates of passaged (up to 200 generations) M. mycoides subsp. mycoides SC strains: (A) Afadé and (B) 91130. (C) Southern blot hybridization of EcoRI- and SspBI-digested DNA of strains Afadé, 91130, and L2 after passages 0 or 30 (corresponding to 200 generations, 200G) hybridized to the ptsG probe. Std, molecular mass standards.