Comparative genome analysis of Mycoplasma pneumoniae

Abstract

Background: Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40% of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England.

Results: Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99% identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes.

Conclusions: These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Fig. 1

Overall sequence identity of the 15 sequenced strains with the reference M129 genome. BLAST-based similarity of a given strain versus the M129 reference is represented as a colored ring. Colors by strain are indicated to the right. Solid coloration indicates >99 % identity and transparent grey indicates approximately 95 % identity. Location in the reference genome is indicated by numeration on the inside of the ring. GC content in the reference genome is indicated by the black bar graphs between the genomic coordinates and the colored rings (bars pointing toward the outside of the circle indicate high GC content). Note that genomic structural alterations are not visible using this method

Fig. 2

Whole genome alignment of the 15 sequenced strains using MAUVE. Regions colored in mauve are conserved across all strains. Differently colored blocks are conserved in some strains. Blocks that are lower are inverted relative to the other strains. Open boxes indicate the location of genes. tRNA genes are shaded in green and rRNA genes are shaded in red. Genes affected by the indicated variants are labeled. Numbers above intervals indicate locations relative to the M129 strain. a Alignment showing all 15 strains. b Close up of the type 2-specific insertion. M129 and FH are shown and are typical of the other type 1 and 2 strains, respectively. Lines indicate relative point of insertion. c Close up of the type 1-specific insertion. M129 and FH are shown and are typical of the other type 1 and 2 strains, respectively. Lines indicate relative point of insertion

Fig. 3

Phylogenetic tree based on whole genome alignment of the 15 sequenced strains. The 15 sequenced M. pneumoniae strains and M. hominis (included as an outgroup) were aligned, and a tree was generated using the alignment. Confidence values, represented as percent of supporting bootstrapping iterations are shown for each node. Scale, in differences per site, is indicated at the bottom. The branches between M. hominis and the M. pneumoniae strains have been truncated (indicated by double slashes), and the branch length (in differences per site) is indicated above the branch. The inset shows the same tree (rescaled, note the new scale bar) without any branches truncated

Fig. 4

Multiple protein sequence alignments showing the differences in P1 and ORF6 between type 1 and type 2 strains. a The large region of variation in P1. Type1 is representative sequence for all type 1 strains and type2 is representative of all type 2 strains. b The large region of variation in ORF6. Type1 is representative of all type 1 strains, except M129, which is also shown (differences in M129 highlighted in red). Type2 is representative of all type 2 strains except for MAC, which is also shown (differences in MAC highlighted in red)

Fig. 5

Multiple protein sequence alignments showing strain-specific differences in P1 and ORF6. Type1 and 2 strain names are highlighted in blue and green, respectively. a A poly-serine repeat in P1 with varying lengths in various strains. 19294 has a uniquely long allele of the poly-serine repeat, and the repeat-length in the other strains does not strictly correspond to strain type. b A stop-loss mutation in MAC results in an additional 7 amino acids added to the protein sequence. c A pair of frameshifts results in the truncation of the beginning of ORF6 in MAC. Type1 and Type2 are representative sequences for type 1 and other type 2 strains, respectively. The new protein is predicted to use an alternate start codon by RAST: the starting methionine in MAC is the same codon as that which codes for the leucine in other type 1 and type 2 strains

Fig. 6

Phylogenetic tree of ArcA. The protein sequences of the two ArcA protein sequences from M. pneumoniae (MPN304 and MPN560), ArcA sequences from other Mycoplasma species, and the protein sequence of arginine deiminase from Streptococcus pneumoniae strain R6 (spr0822_Spn_R6) were aligned and a tree was generated from the alignment. Confidence values, represented as percent of supporting bootstrapping iterations are shown for each node. Scale, in differences per site, is indicated at the bottom

Fig. 7

Multiple protein sequence of the variable regions in the hsdS genes. Both copies of the hsdS gene had a repetitive region of varying length consisting of TELS and AELS units (highlighted in orange and yellow, respectively). Note that in both copies, the length and composition of the repeat does not correspond to strain subtype. Strain names are to the right and highlighted in blue for type 1 and green for type 2. a Repeat region in the MPN089 copy of the hsdS gene. This is part of the variation in the 108000–126000 region shown in Fig. 2c. b Repeat region in the MPN343 copy of the hsdS gene. This is the variation in the 409700–410900 region shown in Fig. 2c