Polyester synthesis genes associated with stress resistance are involved in an insect–bacterium symbiosis

Abstract

Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the β-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ΔphaB, ΔphaC, and ΔphaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ΔphaB and ΔphaC, but only moderately in ΔphaP. In symbiosis, the host insects infected with ΔphaB and ΔphaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ΔphaB and ΔphaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.