Phenotype Overlap in Xylella fastidiosa Is Controlled by the Cyclic Di-GMP Phosphodiesterase Eal in Response to Antibiotic Exposure and Diffusible Signal Factor-Mediated Cell-Cell Signaling

Abstract

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3=-5=)-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

Cyclic‐di‐GMP levels affect Pseudomonas aeruginosa fitness in the presence of imipenem

“…One report shows that P. aeruginosa cells with low c-di-GMP levels are more resistant to the antimicrobial peptide colistin than cells with high c-di-GMP levels (Chua et al, 2013). In the phytopathogenic bacterium Xylella fastidiosa, subinhibitory concentrations of tobramycin increased the expression of the eal gene coding for a phosphodiesterase, and an eal mutant was more susceptible to this antibiotic (de Souza et al, 2013). In this work, we investigated whether there is a correlation between c-di-GMP levels in planktonic cultures and adaptation in the presence of an antibiotic.…”

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces

“…fastidiosa uses diffusible signaling factors (XfDSF), a family of related unsaturated fatty acids, to regulate its behavior in a cell density-dependent manner (10,11). XfDSF-mediated signaling suppresses motility and stimulates the production of cell-surface adhesins, thus increasing cell aggregation, surface attachment, and biofilm formation (10,(12)(13)(14). A ΔrpfF mutant of X. fastidiosa, blocked in the production of XfDSF, is hypervirulent to grapevine but is impaired in insect colonization and transmission (11,12,15).…”

New insights on molecular regulation of biofilm formation in plant‐associated bacteria

“…Moreover, mutation of csgA led to a dramatic reduction in virulence in grape and insect transmission rate. Similar regulation of biofilm formation was exhibited when high levels of c-di-GMP were induced by mutation of eal, a PDE harboring an EAL domain (de Souza et al 2013). These results are evidence of a particular signaling network where at high cell-density levels biosynthesis of c-di-GMP is repressed, promoting EPS biosynthesis and cellular aggregation.…”