Significance This study reports a previously unrecognized involvement of polyhydroxyalkanoate (PHA), known as a bacterial endocellular storage polymer, in an insect–bacterium symbiosis. Many bacteria in the environment accumulate PHA granules within their cells, which provide resistance to nutritional depletion and other environmental stresses. Here we demonstrate that synthesis and accumulation of PHA in the symbiont cells are required for normal symbiotic association with, and, consequently, positive fitness effects for the host insect. The requirement of PHA for symbiosis suggests that, contrary to the general expectation, the within-host environment may be, at least in some aspects, stressful for the symbiotic bacteria.

The Riptortus–Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect–bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host’s symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host’s symbiotic organ at early instar stages and reduced the host’s fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host’s symbiotic organ and enhancing the host’s fitness values.

cThe Riptortus-Burkholderia symbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut of Riptortus pedestris was investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizing Burkholderia symbionts were occupied by an extracellular matrix consisting of polysaccharides. This observation prompted us to search for symbiont genes involved in the induction of biofilm formation and to examine whether the biofilms are necessary for the symbiont to establish a successful symbiotic association with the host. To answer these questions, we focused on purN and purT, which independently catalyze the same step of bacterial purine biosynthesis. When we disrupted purN and purT in the Burkholderia symbiont, the ⌬purN and ⌬purT mutants grew normally, and only the ⌬purT mutant failed to form biofilms. Notably, the ⌬purT mutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the ⌬purN mutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the ⌬purT mutant, which might operate via impaired purine biosynthesis. The host insects infected with the ⌬purT mutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the ⌬purN mutant. These results show that the function of purT of the gut symbiont is important for the persistence of the insect gut symbiont, suggesting the intricate biological relevance of purine biosynthesis, biofilm formation, and symbiosis.