Complete Genome Sequence of Mycoplasma flocculare Strain Ms42T (ATCC 27399T).

Mycoplasma flocculare is a commensal or low-virulence pathogen of swine. The complete 778,866-bp genome sequence of M. flocculare strain Ms42(T) has been determined, enabling further comparison to genomes of the closely related pathogen Mycoplasma hyopneumoniae. The absence of the p97 and glpD genes may contribute to the attenuated virulence of M. flocculare.

GENOME ANNOUNCEMENT

Mycoplasma flocculare is generally considered to be a commensal colonizer of the porcine nasopharynx but an opportunist pneumonic pathogen in coinfections with Mycoplasma hyopneumoniae (1). M. hyopneumoniae is the agent of swine enzootic pneumonia, a disease with high morbidity that significantly impacts swine production (2). Based on 16S rRNA phylogeny, M. flocculare and M. hyopneumoniae are each other’s closest relatives (3), prompting comparative analysis of representative genomes of these taxa (4). A draft genome sequence of M. flocculare ATCC 27716 (15 contigs; 763,948 bp) has been determined and extensively analyzed by comparative genomics and transcriptional profiling (4, 5). Presented herein is the completely assembled genome of the M. flocculare type strain Ms42T (ATCC 27399T), the parental strain from which isolate ATCC 27716 was derived by three additional filter cloning steps.

Genomic DNA was prepared from strain Ms42T (obtained from the American Type Culture Collection as ATCC 27399T) and sequenced at the National Center for Genomics Research (Santa Fe, NM, USA) using the Pacific Biosciences platform. Reads from two SMRT cells were assembled into a single contig using HGAP version 2 (6) with 574× coverage. An approximately 10.9-kb duplication was verified by PCR and Sanger sequencing. The 778,866-bp genome was auto-annotated using the PGAP pipeline at NCBI, followed by manual curation. The complete genome comprises 629 genes: 563 open reading frames (ORFs), 31 pseudogenes (22 independently confirmed with alternate sequencing chemistries), 30 tRNAs, and 3 rRNA genes with the 5S rRNA gene separated from the 16S-23S rRNA operon. The G+C content is 28.94%.

A comprehensive comparative analysis of the genomes of several swine mycoplasmas has been documented (4). In that study, it was noted that although multiple adherence-related genes of the M. hyopneumoniae P97/P102 families were present in M. flocculare, orthologs encoding the primary cilium adhesin P97 (7, 8) and downstream ORF P102 were absent. The same holds true for parental strain Ms42T, indicating that the absence of the P97-P102 locus was not due to inadvertent selection of a deletion during subcloning.

Another feature that may contribute to the differential virulence of M. flocculare and M. hyopneumoniae is the absence of glpD (encoding glycerol 3-phosphate oxidase) in M. flocculare. Orthologs of glpD have been found in multiple Mycoplasma species, including the six M. hyopneumoniae isolates for which genome sequences are available. The product, GlpD, is responsible for the glycerol-dependent production of H2O2. This activity has been shown to be cytotoxic to eukaryotic cells when infected by Mycoplasma mycoides subspecies mycoides SC biotype (9), Mycoplasma pneumoniae (10), and Mycoplasma gallisepticum (11), although in the latter study, glpD was found to be dispensable for virulence in an infection model. The recent development of genetic tools for M. hyopneumoniae (12) should enable the potential roles of glpD and H2O2 production to be ascertained.

The first completely assembled M. flocculare genome will enable more detailed analyses of genome structure and plasticity among the neurolyticum cluster of Mycoplasma species and provides a complete reference for further postgenomic applications.

Nucleotide sequence accession number.

This complete genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession number CP007585.