Comparison of metabolic capacities and inference of gene content evolution in mosquito-associated Spiroplasma diminutum and S. taiwanense

Abstract

Mosquitoes are hosts of several Spiroplasma species that belong to different serogroups. To investigate the genetic mechanisms that may be involved in the utilization of similar hosts in these phylogenetically distinct bacteria, we determined the complete genome sequences of Spiroplasma diminutum and S. taiwanense for comparative analysis. The genome alignment indicates that their chromosomal organization is highly conserved, which is in sharp contrast to the elevated genome instabilities observed in other Spiroplasma lineages. Examination of the substrate utilization strategies revealed that S. diminutum can use a wide range of carbohydrates, suggesting that it is well suited to living in the gut (and possibly the circulatory system) of its mosquito hosts. In comparison, S. taiwanense has lost several carbohydrate utilization genes and acquired additional sets of oligopeptide transporter genes through tandem duplications, suggesting that proteins from digested blood meal or lysed host cells may be an important nutrient source. Moreover, one glycerol-3-phosphate oxidase gene (glpO) was found in S. taiwanense but not S. diminutum. This gene is linked to the production of reactive oxygen species and has been shown to be a major virulence factor in Mycoplasma mycoides. This finding may explain the pathogenicity of S. taiwanense observed in previous artificial infection experiments, while no apparent effect was found for S. diminutum. To infer the gene content evolution at deeper divergence levels, we incorporated other Mollicutes genomes for comparative analyses. The results suggest that the losses of biosynthetic pathways are a recurrent theme in these host-associated bacteria.

Keywords: Mollicutes; Spiroplasma diminutum; Spiroplasma taiwanense; genome; mosquito; virulence factor.

Fig. 1.—

Molecular phylogeny of spiroplasmas. The maximum likelihood tree was inferred based on the concatenated alignment of the 16S ribosomal RNA gene and RNA polymerase subunit beta (rpoB). The numbers on the internal branches indicate the percentage of bootstrap support based on 1,000 replicates (only values >70% are shown). The sequences from Bacillus subtilis are included as the outgroup. The two species with genome sequences reported in this study (i.e., Spiroplasma diminutum and S. taiwanense) are highlighted in bold. The hosts of the Spiroplasma species in the Apis clade are labeled, with the host genus name inside the parentheses.

Fig. 2.—

Genome maps of Spiroplasma diminutum and S. taiwanense. Rings from the outside in: (1) scale marks; (2) protein-coding genes on the forward strand; (3) protein-coding genes on the reverse strand (color-coded by the functional categories); (4) rRNA (purple) and tRNA genes (green); (5) pseudogenes (orange) and intergenic regions >300 bp (black); (6) species-specific regions identified in the pairwise comparison between S. diminutum (blue) and S. taiwanense (red); (7) GC skew; and (8) GC content.

Fig. 3.—

Pairwise genome alignments. The color blocks represent regions of homologous backbone sequences without rearrangement. The average nucleotide sequence identities were calculated based on single-copy genes that are conserved between the two genomes compared. (A) Between S. diminutum and S. taiwanense. One inversion was found, which corresponds to the ∼459–581 kb region of the S. diminutum genome and the ∼503–649 kb region of the S. taiwanense genome. (B) Between S. citri and S. melliferum. These two genome sequences are incomplete draft assemblies; the vertical red bars indicate the boundaries of individual contigs.

Fig. 4.—

Sugar uptake and utilization. Comparison of the phosphotransferase system (PTS) transporters and enzymes involved in sugar uptake and utilization between S. diminutum and S. taiwanense. Gene names are color-coded according to their patterns of presence/absence (gray: shared; blue: S. diminutum-specific; red: S. taiwanense-specific). DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; ROS, reactive oxygen species.

Fig. 5.—

Comparative analysis of gene content among Spiroplasma species. The numbers of shared and species-specific homologous gene clusters from a three-species comparison are shown in the Venn diagram. Gene names in the metabolic map are color-coded based on their patterns of presence/absence among the three species compared. DMAPP, dimethylallyl pyrophosphate; GlcNAc, N-acetylglucosamine; IPP, isopentenyl pyrophosphate; MurNAc, N-acetylmuramic acid; PRPP, phosphoribosyl pyrophosphate; PTS, phosphotransferase system.

Fig. 6.—

Phylogenetic distribution pattern of homologous gene clusters. The organismal phylogeny is inferred from the concatenated protein alignment of 259 single-copy genes shared by all species. All internal nodes received 100% bootstrap support based on 1,000 replicates and maximum likelihood inference. The numbers in parentheses below species names indicate the number of homologous gene clusters found in each species. The numbers above a branch and preceded by a “+” sign indicate the number of homologous gene clusters that are uniquely present in all daughter lineages; the numbers below a branch and preceded by a “−” sign indicate the number of homologous gene clusters that are uniquely absent. For example, 127 gene clusters are shared by S. diminutum and S. taiwanense and do not contain a homolog from all four other species compared; similarly, three gene clusters are missing in these two Spiroplasma species but are present in all four other species.