Complete Genome Sequences of the p1 Gene Type 2b and 2c Strains Mycoplasma pneumoniae KCH-402 and KCH-405.

Here, we present the complete genome sequences of Mycoplasma pneumoniae KCH-402 and KCH-405, which are p1 gene type 2b and 2c strains, respectively. These strains harbor variations in the orf6 gene, which encodes the cytadherence-related proteins P40 and P90.

GENOME ANNOUNCEMENT

Mycoplasma pneumoniae, a common cause of pneumonia and bronchitis in humans (1–3), is classified into several types according to the sequence polymorphism of the p1 gene, which encodes a major adhesin P1 protein. KCH-402 and KCH-405, two M. pneumoniae strains isolated in Japan, are p1 gene type 2b and 2c strains, respectively. The p1 gene type 2b sequence, originally detected in Germany (4, 5), is also referred to as type 2v (6). The sequence of p1 gene type 2c was identified in Dutch and Chinese strains (7, 8). Currently, type 2c is among the most prevalent clinical isolates in Japan; type 2b strains are rare.

We extracted genomic DNA from KCH-402 and KCH-405 cultured in PPLO medium (Becton Dickinson, Sparks, MD, USA) using the QIAamp DNA mini kit (Qiagen, Hilden, Germany). DNA libraries with insert sizes of 300 to 500 bp were prepared using the Nextera XT sample preparation kit (Illumina, San Diego, CA, USA). Genome sequencing was performed using the Illumina HiSeq 4000 platform. Paired-end reads (2 × 150 bp) were assembled de novo using CLC Genomics Workbench version 8.0.2 (Qiagen). Assembled contigs were mapped on the type 2a reference genome of strain 309 (GenBank accession no. AP012303.1) to identify gap regions. PCR amplicons containing gap regions were amplified and sequenced by Sanger sequencing and used to assemble the complete genome sequences. The complete genomes of KCH-402 and KCH-405 were 817,074 and 817,099 bp, respectively. Annotation was performed manually based on the gene nomenclature of reference strains M129 (GenBank accession no. U00089.2) and 309.

The KCH-402 and KCH-405 genomes harbor characteristic variations in orf6, which encodes the cytadherence-related proteins P40 and P90 (9). Compared to the type 2 reference strain FH (GenBank accession no. CP010546.1), the variation in KCH-402 was in the P40 part of orf6, spanning an approximately 680-bp region. Meanwhile, the variation in KCH-405 was in a 35-bp region of the P90 part of orf6. These variations were probably generated by DNA recombination events between the orf6 locus and RepMP5 repetitive sequences in the M. pneumoniae genome (10). Next, we inspected our M. pneumoniae strain collection and confirmed that three other type 2b strains carried orf6 sequences identical to that of KCH-402. The KCH-405 orf6 sequence was commonly found in type 2c strains but was also shared by several type 2a strains of our collection. A KCH-405-like orf6 gene was also present in the draft genome sequence of the type 2d strain 3896 (GenBank accession no. LHPS00000000).

Comparative analysis of the KCH-402 and KCH-405 genomes with the reported M. pneumoniae genomes by whole-genome, single-nucleotide variation analysis revealed that the type 2b strain KCH-402 clustered within the type 2 strain clade and the type 2c strain KCH-405 was within a distinct clade comprising type 2a and 2c strains.

The polymorphism of both orf6 and p1 might be useful for strain classification and discrimination. The complete genome sequences of KCH-402 and KCH-405 may be used as reference sequences for type 2b and 2c strains and for detailed phylogenetic and epidemiological characterization of M. pneumoniae clinical strains.

Accession number(s).

The complete genome sequences of strains KCH-402 and KCH-405 have been deposited in DDBJ/ENA/GenBank under the accession numbers AP017318 and AP017319, respectively.