Genome degeneration and adaptation in a nascent stage of symbiosis

Abstract

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Keywords: IS elements; comparative genomics; degenerative genome evolution; pseudogenes; recent symbiont.

Fig. 1.—

Microscopic images of SOPE. The main panel shows cells from a 5th instar bacteriome of SOPE stained with FM4-64 (red) and DAPI (blue). Rod-shaped bacteria are densely packed into the cytoplasm of the insect cells, whose nuclei display extensive DAPI staining. The inset panel shows isolated SOPE cells stained with DAPI, illustrating their filamentous morphology.

Fig. 2.—

Whole-genome sequence alignment of strain HS and SOPE. Bezier curves highlight regions of synteny shared between strain HS and SOPE. Uninterrupted blocks of synteny are rendered in the same hue. Matches occurring on the same DNA strand are rendered in the purple spectrum, whereas those occurring on different strands are rendered in the yellow spectrum. Thus, to maintain replicational symmetry, matches highlighted in purple should remain on the same replichore, whereas those highlighted in yellow should switch replichore. Sequences shared between the strain HS megaplasmid and SOPE chromosome are rendered in gray. The outer plots represent GC skew with positive skew depicted in red and negative skew depicted in blue. The strand-specific locations of KOPS (FtsK orienting polar sequences) sites are shown as tickmarks overlaid on the plot of GC skew. Inside the plots, the pink and blue tick marks correspond to the positions of phage and IS element sequences, respectively. These and any other forms of repetitive DNA were masked in the generation of the alignment. The location of the dif (terminus) sequence in strain HS is highlighted and intersects with the switch in GC skew, as expected.

Fig. 3.—

IS element dense regions in SOPE. Scalar illustration of two IS element dense regions in the SOPE chromosome. The top row corresponds to the region encompassing SOPEG_ps0144-SOPEG_0160, and the bottom row corresponds to SOPEG_2674-SOPEG_2683. IS element ORFs are colored according to their family (see key). ORFs are shaded in accordance with their positional synteny in comparison with a full length HS ortholog. The full spectrum of shading (5′–3′) is depicted in the key. ORFs disrupted by an IS element insertion are connected by curved lines. All non-IS element ORFs are labeled with their SOPE locus tags.

Fig. 4.—

Plot of dN versus dS of orthologous genes in strain HS and SOPE. (A) Plot of dN versus dS of 1,601 orthologous genes in strain HS and SOPE. Each point depicted on the plot represents a single gene with the radius of each point proportional to gene size. Mean dN/dS ratio is plotted as a gray dotted line. Genes with a dN/dS ratio greater than 0.4 are annotated with their product. Genes with a dN/dS ratio greater than 0.3 are depicted in red and those genes with dN/dS ratios less than 0.3 are depicted in green. Mean ORF size was calculated for genes with dN/dS greater than 0.3 (red) and dN/dS less than 0.3 (green). (B) Plot of SOPE dN/dS versus Sodalis glossinidius dN/dS for 1,229 orthologous genes in strain HS, SOPE, and So. glossinidius. Each point on the plot represents a single orthologous gene.

Fig. 5.—

Overview of SOPE metabolism. The names in the yellow boxes indicate the genes predicted to be responsible for a given reaction. The generation of ATP is indicated. Abbreviations (besides the accepted symbols): CMP-KDO, CMP-3-deoxy-d-manno-octulosonate; DHF, dihydrofolate; ECA, enterobacterial common antigen; GlcNac-6P, N-acetyl glucosamine-6-phosphate; H/dH, nucleoside not G; DHAP, dihydroxyacetonephosphate, hydroxymethylpyrimidine; PEP, phospoenolpyruvate; PG, peptidoglycan; PRPP, phosphoribosyl pyrophosphate; SAM, S-adenosylmethionine; THF, tetrahydrofolate; UQ: ubiquinone.

Fig. 6.—

Comparison of TTSS gene clusters. Scalar illustration of the chromosomal regions encoding SSRs (SSR1–3) in Sodalis glossinidius (top), strain HS (middle), and SOPE (bottom). Genes are colored according to their predicted functions as secretion apparatus (tan), secreted effectors (green), chaperones (purple), transcriptional regulators (pink), hypothetical (white), or IS elements (brown). Pseudogenes are colored in red. Frameshifting indels and premature stops are shown as blue and red tick marks at their respective positions within the ORF. Pseudogenes without tick marks are inactivated by an IS element insertion within an ORF or by truncations of greater than 10% compared with the intact strain HS ortholog. Gene names are shown based on the strain HS annotation.