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.

Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins ␣ 1 and ␣ 2 (␣ 1 I and ␣ 2 I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by ␣ 1 I and ␣ 2 I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both ␣ 1 I and ␣ 2 I with high affinity and effectively inhibit the binding of ␣ 1 I and ␣ 2 I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.Collagen is a major component of the extracellular matrix (ECM). 2 At least 27 genetically different collagen types have been identified, each containing at least one dominant collagenous domain (1). These collagenous domains have a characteristic triple helical structure formed by repeating Gly-X-Y sequences in each participating polypeptide, where X often is proline and Y hydroxyproline. The collagen monomers often assemble into more complex structures of varying organizations, such as fibrils (types I-III, V, and XI), networks (types IV, VIII, and X), and beaded filaments (type VI) (2). The fibrillar collagen types I and III are the major structural components of the ECM of skin, cardiac, and vascular tissues, whereas type II collagen is a major component of cartilage. In addition to contributing to the structural integrity of the tissues, collagens also affect cell behavior through interactions with other matrix proteins and cellular receptors (3-6).The integrins are a family of heterodimeric cell surface receptors involved in cell-cell and cell-substrate adhesion. They act as bridging molecules that link intracellular signaling molecules to the ECM, controlling cell behavior and tissue architecture through bi-directional signaling (7). Four integrins, ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 , have been shown to bind collagens (8 -10). Of these, the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins have been studied in more detail compared with the others. Collagenintegrin interactions play a role in normal and pathological physiology; these interactions directly affect cell adhesion, migration, proliferation, and differentiation, as well as angiogenesis, platelet aggregation, and ECM assembly (11). The precise molecular events that lead to these activi...

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.

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.

dTo 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 ⌬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 ⌬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 ⌬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 ⌬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. Many insects are in intimate symbiotic associations with bacteria. Such symbiotic bacteria exist in the gut lumen, body cavity, or inside cells. To establish a successful host-symbiont association, a number of molecular factors from the symbiont side, and also from the host side, must work in a coordinated manner. To understand the mechanisms of these intricate host-symbiont interactions, several model symbiotic systems have been used to identify novel symbiotic factors and to determine their molecular functions (1). For example, the legume-Rhizobium nitrogen-fixing symbiosis and the squid-Vibrio luminescent symbiosis have been studied in depth. In both systems, the symbiotic bacteria are easily cultivable and genetically manipulatable and are thus suitable for elucidating the molecular properties of their symbiotic factors (2-8).However, among insect-microbe symbiotic systems, molecular factors relevant to symbiosis have been poorly characterized except for inferences from genomic information (9-11). The paucity of molecular and biochemical studies is attributed to the difficulty in isolating and culturing symbiotic bacteria outside insect hosts. Consequently, powerful mutant-based molecular genetic approaches have not been effectively applied to insect-microbe symbiotic ...