Bacterial Cell Wall Synthesis Gene uppP Is Required for Burkholderia Colonization of the Stinkbug Gut

To establish a host-bacterium symbiotic association, a number of factors involved in symbiosis must operate in a coordinated manner. In insects, bacterial factors for symbiosis have been poorly characterized at the molecular and biochemical levels, since many symbionts have not yet been cultured or are as yet genetically intractable. Recently, the symbiotic association between a stinkbug, Riptortus pedestris, and its beneficial gut bacterium, Burkholderia sp., has emerged as a promising experimental model system, providing opportunities to study insect symbiosis using genetically manipulated symbiotic bacteria. Here, in search of bacterial symbiotic factors, we targeted cell wall components of the Burkholderia symbiont by disruption of uppP gene, which encodes undecaprenyl pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall components. Under culture conditions, the Delta uppP mutant showed higher susceptibility to lysozyme than the wild-type strain, indicating impaired integrity of peptidoglycan of the mutant. When administered to the host insect, the Delta uppP mutant failed to establish normal symbiotic association: the bacterial cells reached to the symbiotic midgut but neither proliferated nor persisted there. Transformation of the Delta uppP mutant with uppP-encoding plasmid complemented these phenotypic defects: lysozyme susceptibility in vitro was restored, and normal infection and proliferation in the midgut symbiotic organ were observed in vivo. The Delta uppP mutant also exhibited susceptibility to hypotonic, hypertonic, and centrifugal stresses. These results suggest that peptidoglycan cell wall integrity is a stress resistance factor relevant to the successful colonization of the stinkbug midgut by Burkholderia symbiont.