Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug

Abstract

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.

Purine Biosynthesis, Biofilm Formation, and Persistence of an Insect-Microbe Gut Symbiosis

“…While we tried to find other candidates for the biofilm study, we have studied the purine biosynthesis-deficient mutants, the purL and purM strains (see Fig. S2 in the supplemental material), and found that these strains exhibit defects in biofilm formation that are independent of their auxotrophic growth (13). In other previous studies, purine nucleotide biosynthesis was shown to affect biofilm formation through the secondary messenger c-di-GMP, which plays a central role in the transition from a motile lifestyle to a biofilm lifestyle in Gram-negative bacteria (15,18,(27)(28)(29).…”

“…When the uppP gene of the Burkholderia symbiont, which is known to be involved in the biosynthesis of bacterial cell wall components, is mutated, the peptidoglycan integrity of the bacteria is weakened and the uppP mutant is unable to colonize the host midgut (12). When the purine nucleotide biosynthesis genes purL and purM are mutated, the mutants can colonize the host midgut but fail to reach a normal population level (13). When the bacterial polyhydroxyalkanoate (PHA) synthesis genes phaB and phaC are mutated, the PHA-deficient mutant strains are able to colonize and reach a normal population, but their population decreases in the later stages of symbiotic association, indicating that PHA is important for bacterial persistence in the host midgut (14).…”

“…Recently, the genome of the Burkholderia symbiont strain RPE64 has been sequenced (11), and using genetically manipulated Burkholderia symbionts, several novel bacterial factors necessary for establishing symbiosis with the host have been identified (12)(13)(14). When the uppP gene of the Burkholderia symbiont, which is known to be involved in the biosynthesis of bacterial cell wall components, is mutated, the peptidoglycan integrity of the bacteria is weakened and the uppP mutant is unable to colonize the host midgut (12).…”

Glycolysis and pyrimidine biosynthesis are required for replication of adherent–invasive Escherichia coli in macrophages

“…The purM gene encodes an enzyme that catalyses the fifth step in the de novo biosynthesis of purines. Whilst there are no reports of any requirement for purine biosynthesis during bacterial intramacrophagic replication, it has been shown that purine biosynthesis-deficient mutants of Burkholderia, Sinorhizobium and Photorhabdus are defective in their ability to form symbiotic associations with insects, plants and nematodes, respectively (Ruisheng & Grewal, 2011;Buendía-Clavería et al, 2003;Kim et al, 2014). Moreover Pseudomonas aeruginosa must synthesize purines during both chronic and acute infections of animals (Turner et al, 2014).…”

Culturing and Characterization of Gut Symbiont Burkholderia spp. from the Southern Chinch Bug, Blissus insularis (Hemiptera: Blissidae)

“…Biofilms produced by crypt-associated exocellular Burkholderia spp. are detected in the crypt lumens of R. pedestris as an exocellular matrix with polysaccharides (52), and they are associated with host insect fitness (56). However, the nature of the biofilm, and the potential role it plays, in the Burkholderia-Blissus interaction is unknown.…”