Evidence for the Rapid and Divergent Evolution of Mycoplasmas: Structural and Phylogenetic Analysis of Enolases

Abstract

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae. Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor.

Keywords: crstal structure; divergent evolution; enolase; mollicutes; mycoplasma.

FIGURE 1

Sequence similarity matrix between two enolases from different species. Heat map of the sequence identity matrix between two enolases from different species. The species names for every row and column are noted. The block shows the sequence identity between the enolases of the two intersecting species. The degree of sequence similarity is colored from dark orange to light blue.

FIGURE 2

Different sequence patterns among different enolases. Partial sequence alignment of enolases from different species. The full sequence identities are shown at the end of each sequence. The secondary structure of Mhp Eno is shown above the alignment. Different sequence patterns are highlighted with orange and brown rectangles for Mollicutes and non-Mollicutes, respectively. For sequence accession numbers, please refer to supplementary materials.

FIGURE 3

Mp Eno and Mb Eno oligomeric conformations. (A,B) Gel filtration of Mp Eno (A) and Mb Eno (B). Mp Eno and Mb Eno purities were checked by SDS-PAGE and are shown in the inset images. (C) Native PAGE analysis of Mhp Eno, Mb Eno and Mp Eno. The protein marker sizes are indicated to the right of the picture. (D) Negative staining of Mb Eno; the scale is indicated.

FIGURE 4

The overall structures of Mp Eno and Mb Eno. (A,B) Structures of one molecule each of Mp Eno (A) and Mb Eno (B). β-Strands and α-helices are sequentially numbered; “S” indicates a β-strand, and “H” indicates an α-helix. The β-strands are shown in orange. The α-helices of the N-terminal domains are shown in blue and green, in Mp Eno and Mb Eno, respectively. (C,D) Overall structures of Mp Eno (C) and Mb Eno (D). (C) Mp Eno is a dimer. (D) Mb Eno is an octamer.

FIGURE 5

Structural comparisons of mycoplasma enolases and other enolases. All the enolases are shown in cartoon mode and in the color of blue-white-red spectrum according to crystallographic B factors. The color bars show the B-factor scales for the corresponding forms. Species names are below corresponding enolase structure modes. Helix 7, H13, H6/S6 loop, S6/H7 loop regions are marked by rectangles. The accession numbers for enolases are the same as used in Supplementary Table S2.

FIGURE 6

Helix 7 motifs and structures. (A) Sequence alignment of the H7/H8 regions of different enolases. For sequence accession numbers, please refer to supplementary materials. The secondary structure of Mhp Eno is indicated above the alignments. The conserved sites in the mycoplasma enolase H7 region are highlighted with orange rectangles. The absence of H7 and H8 in enolase structures is highlighted with brown rectangles. (B) Structures of helix 7 from three mycoplasma enolases. Blue, green and orange indicate Mp Eno, Mb Eno and Mhp Eno, respectively. Conserved sites are indicated and shown stick models. The H7 motifs in Mp enolase, Mb enolase and Mhp enolase are shown under the H7 structures. (C) Structural overlap of the S6/H7 loops (corresponding to Helix 7 of Mycoplasma enolases) of enolases from non-Mycoplasma species. Enolases from H. sapiens, S. cerevisiae, E. coli and E. hirae are shown in light green, light blue, brown and pink, respectively, and other enolases are shown in white.

FIGURE 7

Mycoplasmataceae evolutionary trees generated from enolase, EF-TU and 16S rRNA sequences. (A–C) Evolutionary trees generated with enolase, 16S rRNA and EF-TU sequences. Bars indicate distances under the corresponding tree. Mycoplasma bovis, M. pneumoniae and M. hyopneumoniae are shown in green, blue and orange, respectively. The branches for M. pneumoniae, M. genitalium, M. hyopneumoniae and M. ovipneumoniae are red in all three trees to clarify the distance between neighboring species.

FIGURE 8

Phylogenetic trees generated from the S3/H1 loop, H4/S4 loop and H6/H8 regions of enolases from Mycoplasmataceae.