A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes the in vitro gene expression with bacterial ribosomes

Microbes utilize secondary metabolites to mediate interactions with neighboring organisms. Such molecules exhibit diverse biological activities, some of which facilitate symbiotic relationships between the microbes and their animal hosts (1, 2).

Diaphorin is a polyketide produced by “Candidatus Profftella armatura” (Gammaproteobacteria: Burkholderiales), an intracellular symbiont harbored alongside the primary symbiont “Candidatus Carsonella ruddii” (Gammaproteobacteria: Oceanospirillales) (3, 4) in the bacteriome organ (5–7) of the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) (8–11). D. citri is a serious agricultural pest that transmits “Candidatus Liberibacter” spp. (Alphaproteobacteria: Rhizobiales), the pathogens of the most destructive and incurable citrus disease, huanglongbing (12, 13). Conserved presence of Profftella and its diaphorin-synthesizing gene clusters in Diaphorina spp. underlines the physiological and ecological significance of diaphorin for the host psyllids (14, 15). Diaphorin, which D. citri contains at a concentration as high as 2–20 mM in the body (16), exerts inhibitory effects on various eukaryotes (8, 17, 18) and Bacillus subtilis (Firmicutes: Bacilli) (19) but promotes the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales) (19), implying that this secondary metabolite serves as a defensive agent of the holobiont (host-symbiont assemblage) against eukaryotes and some bacterial lineages but is beneficial for other bacteria (8, 17, 19). Besides “Ca. Liberibacter” spp. and the bacteriome-associated mutualists, D. citri may harbor various secondary symbionts of a facultative nature, including Wolbachia (Alphaproteobacteria: Rickettsiales) and Arsenophonus (Gammaproteobacteria: Enterobacterales) (14). Recent studies are revealing that interactions among these bacterial populations are important for psyllid biology and host plant pathology (10, 14, 20–22). In this context, the unique property of diaphorin may affect the microbiota of D. citri, potentially influencing the transmission of “Ca. Liberibacter” spp. Moreover, this distinct activity of diaphorin may be exploited to improve the efficacy of industrial production by E. coli, which is frequently used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels (19).

Diaphorin belongs to the family of pederin-type compounds (8, 19), which exhibit toxicity and antitumor activity by suppressing eukaryotic protein synthesis through binding to the E-site of the 60S subunit of eukaryotic ribosomes (23). However, little is known about the effects of these compounds on bacterial gene expression (24). To explore the possibility that diaphorin exerts its unique activity on bacteria by directly targeting bacterial gene expression, our previous study analyzed the effects of diaphorin on the in vitro gene expression using ribosomes isolated from B. subtilis and E. coli, quantifying production of the super folder green fluorescent protein (sfGFP) (25). Five-millimolar diaphorin was used for the analysis because this concentration exhibited significant inhibitory and promoting effects on B. subtilis and E. coli, respectively, in culture experiments (19). The result showed that 5-mM diaphorin inhibits gene expression involving ribosomes from both B. subtilis and E. coli, suggesting that the adverse effects of diaphorin on B. subtilis are attributed to, at least partly, its inhibitory effects on gene expression (25). On the other hand, the result did not explain the promoting effects of diaphorin on E. coli. Moreover, the concentration of diaphorin in the intracellular environment, where the inherent gene expression machinery works, may be lower than in the culture medium. Therefore, in the present study, we analyzed the effect of 50 and 500 µM of diaphorin on bacterial gene expression using the same assay system.

Cell-free translation of sfGFP with diaphorin at final concentrations of 50 and 500 µM demonstrated that this concentration range of diaphorin promotes the in vitro gene expression involving ribosomes of both E. coli and B. subtilis (Fig. 1). Namely, the relative activity of gene expression using the E. coli ribosome treated with 50-µM diaphorin was 1.079 ± 0.012 (mean ± standard error, n = 48), which was moderately (7.9%) but significantly (P < 0.001, Steel test) higher than that of the control (1.000 ± 0.008, n = 96, Fig. 1A). Furthermore, the relative gene expression activity using the E. coli ribosome treated with 500-µM diaphorin was 1.089 ± 0.017 (n = 48), which was again moderately (8.9%) but significantly (P < 0.001, Steel test) higher than that of the control (Fig. 1A). These results imply that the positive effects of diaphorin on the growth and metabolic activity of E. coli (19) can be attributed to its direct effects on the core gene expression machinery. When cultured in media containing 5-mM diaphorin (19), E. coli may be able to keep the intracellular diaphorin concentration within this range, positively affecting their vital activities. Regarding B. subtilis, although the relative gene expression activity using the B. subtilis ribosome along with 50-µM diaphorin (0.992 ± 0.023, n = 48) was not significantly different (P > 0.05, Steel test, Fig. 1B) from the control (1.000 ± 0.011, n = 96), the gene expression using the B. subtilis ribosome with 500-µM diaphorin (1.084 ± 0.034, n = 48) was moderately (8.4%) but significantly (P < 0.001, Steel test) higher than the control (Fig. 1B). This result appears inconsistent with previously observed adverse effects of the same concentration of diaphorin on the cultured B. subtilis (19). However, transmission electron microscopy showed that diaphorin also damages the B. subtilis cell envelope (19), which may negate the positive effects of the appropriate concentration of diaphorin on the gene expression machinery of B. subtilis.

This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property of diaphorin to pest management and industrial use. Moreover, this study demonstrated for the first time that a pederin-type compound promotes the gene expression of organisms.

Diaphorin was extracted and purified as described previously (8, 17, 19, 25). Adult D. citri was ground in methanol, and the extracts were purified using an LC10 high-performance liquid chromatography system (Shimadzu) with an Inertsil ODS-3 C18 reverse-phase preparative column (GL Science).

The B. subtilis ribosomes were purified as described previously (25). B. subtilis cells were passed through a French press cell (Ohtake) at approximately 110 MPa (16,000 psi), and ribosomes were captured using HiTrap Butyl FF columns (Cytiva). The eluent was ultracentrifuged (100,000 × g, 4°C, 16 h) using Optima L-100 XP Ultracentrifuge (Beckman Coulter) to sediment ribosomes.

The in vitro gene expression activities involving ribosomes of E. coli and B. subtilis were evaluated utilizing a PUREfrex 2.0 kit (GeneFrontier) as previously described (25). With distinct concentrations of diaphorin included in the reaction solution, sfGFP was synthesized at 37°C for 4 h, which was then separated by SDS-PAGE. After renaturation, the fluorescence of sfGFP was elicited at 488 nm, passed through a 520-nm band pass filter, and recorded using a Typhoon 9400 image analyzer (GE Healthcare). The fluorescence intensity of sfGFP was quantified using the ImageQuant TL software (version 8.1, GE Healthcare).

All statistical analyses were conducted using R version 4.1.3. Multiple comparisons were conducted using the Kruskal-Wallis test, followed by the Steel test.

This work was supported by the Japan Society for the Promotion of Science (https://www.jsps.go.jp) KAKENHI (grant number - 20H02998) to A.N. The funder had no role in the study design, data collection and analysis, the decision to publish, or manuscript preparation.