Abstract
In
Xanthomonas
, RpfF is a bifunctional enzyme having both enoyl-ACP dehydratase and acyl-ACP thioesterase activities catalyzed by a single active site. While the hydratase activity is essential for producing quorum-sensing signals of the diffusible signal factor (DSF) family, the physiological role of the thioesterase activity remained unclear. Singh and coworkers have reported that deletion of the gene encoding RpfF (a ∆
rpfF
strain) of the rice pathogen
X
.
oryzae
pv.
oryzae
(
Xoo
) resulted in loss of membrane integrity (Singh
et al
, 2022). We report that the loss in membrane integrity is due to lack of the RpfF thioesterase activity. This was demonstrated by restoration of membrane integrity in the Δ
rpfF
strain by expression of a foreign thioesterase. Thioesterases originating in either
E. coli
or a plant enzyme fully restored virulence in rice, bacterial growth, and tolerance of membrane stress. The thioesterase overcame the defects in membrane lipid synthesis reported previously and restored the lipid components to wild-type levels. Radiolabeling revealed that fatty acid synthesis in the Δ
rpfF
strain proceeded at only one-third of the wild-type rate, which was rescued by complementation with thioesterase encoding genes. Thus, the acyl-ACP thioesterase activity of RpfF is a key regulator of
de novo
fatty acid synthesis and membrane integrity. We propose a dual-function model wherein RpfF maintains membrane lipid homeostasis via its thioesterase activity while concurrently producing DSF signals via its hydratase activity to orchestrate quorum sensing.