Genome Sequence for “Candidatus Mycoplasma haemominutum,” a Low-Pathogenicity Hemoplasma Species

Abstract

We present the genome sequence of “Candidatus Mycoplasma haemominutum” strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.

GENOME ANNOUNCEMENT

Hemoplasmas are uncultivatable hemotropic bacteria within the genus Mycoplasma (8). “Candidatus Mycoplasma haemominutum” was identified as being distinct from Mycoplasma haemofelis on the basis of 16S rRNA gene sequence data comparison (12). “Ca. Mycoplasma haemominutum” is the most prevalent of the feline-infecting hemoplasmas (10), and infection doesn't usually result in clinical disease in immunocompetent cats, but decreases in hematocrit have been reported (11, 17).

Genomic DNA from “Ca. Mycoplasma haemominutum” strain Birmingham 1 was purified from blood taken from an experimentally infected specific-pathogen-free cat at high parasitemia, as described previously (2). This low-passage strain, derived from a naturally infected cat (15), has been used in previous infection kinetics studies (9, 14, 16, 17). Whole shotgun pyrosequencing was performed by generating a standard DNA library with a 3-kb insert size using the 454 library preparation kit (Roche Applied Sciences, Indianapolis, IN) and sequenced on a GS-FLX using Titanium chemistry (454 Life Sciences, Roche Applied Sciences). The 454 reads were assembled with Newbler (August 2010 R&D version; Roche Applied Sciences). The final assembly was performed with gap4 (http://staden.sourceforge.net). Open reading frames were identified with GLIMMER (3) and GENEMARK (7), and tRNA genes were identified by tRNAscan-SE (6). Putative functions were inferred using BLAST against the National Center for Biotechnology Information databases (1) and InterProScan (4). Artemis v12 was used to organize data and facilitate annotation (13). Orthologs/paralogs were defined using ORTHOMCL (5).

Final assembly (>50-fold coverage) of the “Ca. Mycoplasma haemominutum” genome gave a single linear contig of 513,880 bp with 35.5% GC content. The GC skew and gene organization, compared to those of other hemoplasma genomes, suggested that a region of ∼3,000 bp, comprising the origin of replication and genes dnaA and dnaN, was missing. Despite many attempts, this region could not be amplified by PCR from “Ca. Mycoplasma haemominutum” genomic DNA, and, hence, a circular chromosome could not be confirmed.

Of the 547 putative proteins identified, 219 (40%) matched functionally characterized proteins, 149 (27.2%) matched hypothetical proteins of other bacterial species, and 179 (32.7%) had no matches. Of the matched hypothetical proteins, 38 had predicted biochemical functions. In common with other sequenced hemoplasmas, the number of known functional proteins in the genome is small, with energy metabolism appearing to be restricted to the glycolytic pathway and ATP synthesis. Series of paralogs containing a total of 159 hypothetical protein repeats were present; these included 47 hypothetical proteins that had homologs in Mycoplasma suis but not in other hemoplasma or bacterial species, suggesting a recent common ancestor in M. suis. Unlike other hemoplasmas, “Ca. Mycoplasma haemominutum” lacks an intact GMP synthase pathway, which could impair its ability to synthesize purine nucleotides de novo, and lacks a type 1 restriction modification system and DNA methylases, found in M. suis and M. haemofelis, which could explain why it has markedly fewer paralog repeats (159 versus 229 to 240 in M. suis and 1,013 to 1,115 in M. haemofelis).

We report the nearly complete annotated “Ca. Mycoplasma haemominutum” genome. Feline hemoplasma genomes can now be compared to identify pathogenic determinants.

Nucleotide sequence accession number.

The genome sequence of “Ca. Mycoplasma haemominutum” strain Birmingham 1 was deposited in the EMBL database under accession number HE613254.