Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium

Abstract

Background: The genus Spiroplasma contains a group of helical, motile, and wall-less bacteria in the class Mollicutes. Similar to other members of this class, such as the animal-pathogenic Mycoplasma and the plant-pathogenic 'Candidatus Phytoplasma', all characterized Spiroplasma species were found to be associated with eukaryotic hosts. While most of the Spiroplasma species appeared to be harmless commensals of insects, a small number of species have evolved pathogenicity toward various arthropods and plants. In this study, we isolated a novel strain of honeybee-associated S. melliferum and investigated its genetic composition and evolutionary history by whole-genome shotgun sequencing and comparative analysis with other Mollicutes genomes.

Results: The whole-genome shotgun sequencing of S. melliferum IPMB4A produced a draft assembly that was ~1.1 Mb in size and covered ~80% of the chromosome. Similar to other Spiroplasma genomes that have been studied to date, we found that this genome contains abundant repetitive sequences that originated from plectrovirus insertions. These phage fragments represented a major obstacle in obtaining a complete genome sequence of Spiroplasma with the current sequencing technology. Comparative analysis of S. melliferum IPMB4A with other Spiroplasma genomes revealed that these phages may have facilitated extensive genome rearrangements in these bacteria and contributed to horizontal gene transfers that led to species-specific adaptation to different eukaryotic hosts. In addition, comparison of gene content with other Mollicutes suggested that the common ancestor of the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) clade may have had a relatively large genome and flexible metabolic capacity; the extremely reduced genomes of present day Mycoplasma and 'Candidatus Phytoplasma' species are likely to be the result of independent gene losses in these lineages.

Conclusions: The findings in this study highlighted the significance of phage insertions and horizontal gene transfer in the evolution of bacterial genomes and acquisition of pathogenicity. Furthermore, the inclusion of Spiroplasma in comparative analysis has improved our understanding of genome evolution in Mollicutes. Future improvements in the taxon sampling of available genome sequences in this group are required to provide further insights into the evolution of these important pathogens of humans, animals, and plants.

Figure 1

Phylogenetic placement of Spiroplasma melliferum IPMB4A. The maximum likelihood phylogenetic tree was inferred using the 16S ribosomal RNA gene, GenBank accession numbers were listed in square brackets following the species names. The numbers on the internal branches indicated the percentage of bootstrap support based on 1,000 re-samplings.

Figure 2

Functional classification of annotated protein-coding genes. The functional categorization of each protein-coding gene was classified according to the COG assignments, genes that did not have any inferred COG annotation were assigned to a custom category X. The number of protein-coding genes in each set was labeled in the center of each pie chart. (A) All 932 annotated protein-coding genes in the S. melliferum IPMB4A genome. (B) The 392 protein-coding genes that have specific functional category assignments.

Figure 3

Extensive level of genome rearrangement between closely related Spiroplasma spp. The color blocks represent regions of homologous backbone sequences without rearrangement among the genomes compared. The vertical red bars indicate the boundaries of individual contigs. The average nucleotide sequence identities were calculated based on single-copy genes that were conserved among the three genomes compared in each group. (A) Comparison among Spiroplasma melliferum IPMB4A, S. melliferum KC3, and S. citri GII3-3X. (B) Comparison among Mycoplasma hyopneumoniae strains 7448, J, and 232. (C) Comparison among Bacillus anthracis str. Ames, B. cereus ATCC 10987, and B. thuringiensis serovar konkukian str. 97-27.

Figure 4

Numbers of shared and genome-specific homologous gene clusters. The Venn diagrams show the number of shared and genome-specific homologous gene clusters among the genomes compared. (A) Comparison among Spiroplasma melliferum IPMB4A, S. melliferum KC3, and S. citri GII3-3X. (B) Comparison among S. melliferum IPMB4A, Mycoplasma genitalium G37, and ‘Candidatus Phytoplasma asteris’ OY-M.

Figure 5

Sugar uptake and utilization in Spiroplasma spp. Comparison of the phosphotransferase system (PTS) transporters and enzymes involved in sugar uptake and utilization between Spiroplasma melliferum and S. citri. (A) Genes shared between S. melliferum and S. citri (B) S. melliferum-specific systems, genes that were present in S. melliferum and absent in S. citri were highlighted in red. Abbreviations: N-acetylglucosamine (GlcNAc); N-acetylmuramic acid (MurNAc).

Figure 6

Highlights of selected metabolic pathways in Mollicutes. The analysis was based on a three-way comparison among Spiroplasma melliferum IPMB4A, Mycoplasma genitalium G37, and ‘Candidatus Phytoplasma asteris’ OY-M. The color-coded circles indicated the presence (filled) or the absence (unfilled) of a gene in each genome. Genes that were present in S. melliferum IPMB4A and absent in the other two genomes were highlighted in red.