Mycoplasma pneumoniae large DNA repetitive elements RepMP1 show type specific organization among strains

Abstract

Mycoplasma pneumoniae is the smallest self-replicating bacterium with a streamlined genome of 0.81 Mb. Complete genome analysis revealed the presence of multiple copies of four large repetitive elements (designated RepMP1, RepMP2/3, RepMP4 and RepMP5) that are implicated in creating sequence variations among individual strains. Recently, we described RepMP1-associated sequence variations between reference strain M129 and clinical isolate S1 that involved three RepMP1-genes (i.e. mpn130, mpn137 and mpn138). Using PCR and sequencing we analyze 28 additional M. pneumoniae strains and demonstrate the existence of S1-like sequence variants in nine strains and M129-like variants in the remaining nineteen strains. We propose a series of recombination steps that facilitates transition from M129- to S1-like sequence variants. Next we examined the remaining RepMP1-genes and observed no other rearrangements related to the repeat element. The only other detected difference was varying numbers of the 21-nucleotide tandem repeats within mpn127, mpn137, mpn501 and mpn524. Furthermore, typing of strains through analysis of large RepMPs localized within the adhesin P1 operon revealed that sequence divergence involving RepMP1-genes mpn130, mpn137 and mpn138 is strictly type-specific. Once more our analysis confirmed existence of two highly conserved groups of M. pneumoniae strains.

Figure 1. Sequence variations involving MPN137 and MPN138.

A. PCR amplification of MPN138/7 regions. Chromosomal DNA of four type 1 and eight type 2 strains was used, and generated products were electrophoresed on 1% agarose. Amplicon size was estimated using 1-kb DNA ladder. B. Comparison of MPN137 and MPN138 regions in different clinical strains. The analyzed region contains loci MPN139 (open arrow), MPN138 (red arrow) and MPN137 (blue arrow) in M129 strain and all tested type 1 isolates. The fused reading frame (MPN138/7, red and blue arrow) was found in all tested type 2 strains. The location of RepMP1-core elements and short repeats B and C within analyzed regions is indicated. The position of amplified genomic regions (line) and primers (▸,◂) is shown for both type 1 and type 2 strains. The region deleted in type 2 strains is presented (striped bar) and the 49 nt-region originally not found in either MPN137 or MPN138 is indicated (orange stripe, **).

Figure 2. Type-specific deletion of MPN130.

A. PCR amplification of MPN129 to MPN131 regions. Chromosomal DNA of four type 1 and eight type 2 strains was used and generated products were visualized on 1% agarose for analysis. Amplicon size was estimated using 1-kb DNA ladder. B. Comparison of MPN129-MPN131 regions among M. pneumoniae clinical strains. In reference strain M129 and other type 1 isolates, the RepMP1-containing gene (MPN130, orange arrow) is located between coding regions MPN129 and MPN131. In all tested type 2 strains, MPN130 is missing. The location of RepMP1-core element and sRepB within analyzed regions is indicated. The position of amplified genomic regions (line) and primers (▸,◂) is shown for both type 1 and type 2 strains, and the region deleted in type 2 strains is presented (striped bar).

Figure 3. Model of two proposed recombination events.

A and B. Chromosomal region MPN129-MPN140 in M. pneumoniae M129. Position, length and orientation of all genes are presented and color-coded as above (arrows). C. Identification of sReps within MPN130, MPN137 and MPN138. Short repeats B (green arrows labeled B) were identified within both intergenic regions surrounding MPN130. Analyses revealed copies of sRepD and sRepE (red and blue arrows labeled D and E, respectively). Corresponding sReps involved in homologous recombination are connected by dotted lines. A diagram illustrates exchange of chromosomal regions during homologous recombination. D. Chromosomal regions after homologous recombination. Coding regions of three RepMP1-genes with rearranged domains are shown. sRepBs presumably involved in sequence deletion are indicated (asterisks). E. Deletion of two RepMP1-genes. Deleted region containing two RepMP1-genes (dotted loop) and implicated sRepB (green arrow) are represented. F. Chromosomal region MPN129-MPN140 in S1. Detailed depiction of MPN129-MPN131 and MPN138/7 regions and organization of the chromosomal region are presented.

Figure 4. Predicted secondary structure of modified RepMP1-proteins.

A. Type-specific modification of Mpn130, Mpn137 and Mpn138 proteins. Three proteins (within grey box) are predicted in type 1 isolates. In type 2 strains, fused protein Mpn138/7 consists of Mpn138-N-terminal and Mpn137-C-terminal region that is shorter by one heptad repeat (*). Only the indicated 16-aa region of MPN130 (**) is retained in type 2 strains. Regions of coiled coils are shown (numbers represent start and end of region). Locations of leucine zipper (LZ) in Mpn138 and Mpn130 and of leucine repeats (LR) in Mpn138 and Mpn138/7 are indicated. B. Type-specific modification of Mpn524. The position of direct tandem heptad repeats (DR) and coiled coil region is presented for type 1 Mpn524. In all type 2 strains one heptad repeat is deleted (*). C. Mpn501 modification. The positions of direct tandem heptad repeats (DR) and coiled coil domains are indicated. The insertion of an additional heptad repeat (+1) is the only not type-specific modification identified among strains. Transmembrane domains (TM) were predicted by DAS analysis.