PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris

Abstract

Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (ΔphaP1, ΔphaP2, ΔphaP3, and ΔphaP4 mutants), one phaR gene-depleted mutant, and a phaR-complemented mutant (ΔphaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the secondinstar nymphs, and colonization ability and fitness parameters were examined. In vitro, the ΔphaP3 and ΔphaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the ΔphaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia-infected insects. However, other phaP family genedepleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the ΔphaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut.IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA granules during host-gut symbiont interactions are not fully understood. Here, we report the effects on colonization ability in the host midguts and the fitness of host insects after feeding Burkholderia mutant cells (four phaP-depleted mutants and one phaR-depleted mutant) to the host insects. Analyses of both synthesized PHA granule amounts and CFU numbers suggest that the phaR gene is closely related to synthesis of the PHA granule and the colonization of the Burkholderia gut symbiont in the host insect's midgut. Like our previous report, this study also supports the idea that the environment of the host midgut may not be favorable to symbiotic Burkholderia cells and that PHA granules may be required to adapt in the host midgut.

Genomic Comparison of Insect Gut Symbionts from Divergent Burkholderia Subclades

“…We first searched whether the five symbiont genomes carry the representative virulence or symbiosis factors previously reported in pathogenic species of BCC&P, such as B. pseudomallei ; plant growth-promoting rhizobacteria (PGPR) of PBE, such as B. phytofirmans ; and bean bug-associated SBE, such as B. insecticola [ 17 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ] ( Table 3 ).…”

“…We subsequently determined the complete genomes of two isolated symbionts of P. gutta and then compared them with the whole genomes of other three SBE symbionts. Studies on an SBE symbiont, B. insecticola RPE64, of the bean bug have revealed that several symbiosis factors, such as genes related to motility and cell wall synthesis, are important for colonization in the midgut crypts [17,[57][58][59][60][61][62][63][64][65][66]. The presence of all these symbiosis factors Figure 5B shows the comparison of COG functional categories between the above four classes of orthogroups, i.e., conserved in the seven species, conserved in only the five insect symbionts, conserved in only the two iPBE symbionts, and conserved in only the three SBE symbionts.…”

“…The COG functional categories of the orthogroups were assigned based on the functional categories of genes of PGU19 and B. insecticola RPE64, which were included in the orthogroup. The genes involved in the virulence in B. pseudomallei K96243 [53,54], in symbiotic association with plants in B. phytofirmans PsJN [55,56], and in symbiosis with stink bugs in B. insecticola RPE64 [17,[57][58][59][60][61][62][63][64][65][66] were searched in the resultant orthogroups and their orthologs were identified.…”

“…We first searched whether the five symbiont genomes carry the representative virulence or symbiosis factors previously reported in pathogenic species of BCC&P, such as B. pseudomallei; plant growth-promoting rhizobacteria (PGPR) of PBE, such as B. phytofirmans; and bean bug-associated SBE, such as B. insecticola [17,[53][54][55][56][57][58][59][60][61][62][63][64][65][66] (Table 3). The five symbionts consistently possessed the following factors that are commonly important in both pathogenesis and symbiosis: flagella, chemotaxis, lipopolysaccharide (LPS) biosynthetic cluster, phospholipase C, MucD Ser protease, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and all the symbiosis factors reported in B. insecticola.…”

Host–symbiont specificity determined by microbe–microbe competition in an insect gut

“…Recent studies have revealed part of the symbiont's mechanisms involved in these processes. The Burkholderia symbiont employs a unique corkscrew flagellar motility to pass through the constricted region (16); it undergoes cytological and metabolic alterations, such as flagella loss and polyhydroxyalkanoate accumulation, to cope with the crypt environment (17)(18)(19); and it rapidly proliferates to occupy entirely the available space in the lumen of the symbiotic M4 midgut region, notably by recycling host metabolic wastes into essential amino acids and B vitamins in the M4 crypts, which are thought to be part of the symbiont services provided to the host (17).…”