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

ABSTRACT Diaphorin is a polyketide produced by “Candidatus Profftella armatura” (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2–20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications. IMPORTANCE This study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. 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 to pest management and industrial use.

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, respec tively, 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 organ isms.

Preparation of diaphorin
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).

Quantification of cell-free synthesis of sfGFP
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 fluores cence intensity of sfGFP was quantified using the ImageQuant TL software (version 8.1, GE Healthcare).

Statistical analysis
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.

FIG 1
FIG 1 Cell-free gene expression with bacterial ribosomes is promoted by a limited concentration range of diaphorin.(A) Relative gene expression with the E. coli ribosome.The signal intensity of synthesized sfGFP in each sample is normalized to the mean signal intensity of control samples.Jitter plots of all data points (control, n = 96; others, n = 48) and box plots (gray, control; orange, 50-µm and 500-µm diaphorin) showing their distributions (median, quartiles, minimum, and maximum) are indicated.Blue dots represent the mean.Asterisks indicate a statistically significant difference (***, P < 0.001, Steel test).For reference, previously published data of 5-mM diaphorin treatment (19) are shown in purple dots (n = 48) with a box plot.(B) Relative gene expression with the B. subtilis ribosome.The signal intensity of synthesized sfGFP in each sample is normalized to the mean signal intensity of control samples.Jitter plots of all data points (control, n = 96; others, n = 48) and box plots (gray, control; green, 50-µm and 500-µm diaphorin) showing their distributions (median, quartiles, minimum, and maximum) are indicated.Blue dots represent the mean.Asterisks indicate a statistically significant difference (***, P < 0.001, Steel test).Previously published data of 5-mM diaphorin treatment (19) are shown in purple dots (n = 48) and a box plot.