RpfF is a bifunctional enzyme. Using a single active site, it performs two chemically distinct reactions. As an enoyl-acyl carrier protein (ACP) dehydratase, it helps convert intermediates of the bacterial fatty acid synthesis pathway, known as FASII, into the DSF family of signaling molecules, which include DSF, BDSF, IDSF, and CDSF. As an acyl-ACP thioesterase, it cleaves the thioester bond linking fatty acid chains to ACP, releasing free fatty acids. While the dehydratase role in quorum sensing has been studied extensively, the physiological purpose of the thioesterase activity remained a puzzle for more than a decade.

The clue that something more was going on came from earlier work showing that deleting the rpfF gene from Xoo caused a striking loss of membrane integrity. The mutant became hypersensitive to detergents, leaked proteins, and lost much of its ability to cause disease. But because rpfF deletion removes both enzymatic activities at once, it was unclear which function was responsible. The research team, led by Lizhen Luo, Zhe Hu, and Haihong Wang of South China Agricultural University together with John E. Cronan of the University of Illinois, resolved this ambiguity with an elegant trick: they replaced the missing thioesterase activity with unrelated enzymes borrowed from other organisms.

The team inserted into the chromosome of the rpfF deletion mutant two foreign thioesterase genes: tesA from the gut bacterium Escherichia coli and fatB1 from the camphor tree, Cinnamomum camphorum. Because these enzymes share no evolutionary history with RpfF and cannot make DSF signals, any rescue of the mutant’s defects could be attributed squarely to thioesterase chemistry. The results were unambiguous. Both foreign enzymes restored the mutant’s resistance to the detergents sodium dodecyl sulfate and Triton X-100, its tolerance of osmotic and oxidative stress, and its normal low permeability to fluorescent dyes. Adding purified DSF signal to the mutant, by contrast, did nothing for detergent sensitivity, proving that the membrane protection comes from thioesterase activity itself rather than from quorum sensing.

Microscopy drove the point home visually. Under scanning electron microscopy, untreated cells of the mutant looked normal, but exposure to detergent caused the rpfF deletion strain to undergo extensive lysis, spilling its intracellular contents, while wild-type cells and thioesterase-complemented strains remained largely intact. Transmission electron microscopy showed cytoplasmic aggregation and loss of electron density in the mutant under detergent stress, changes that were markedly milder in the complemented strains. Consistent with a repaired membrane, the mutant’s abnormal leakage of extracellular proteins, including cellulase, xylanase, and lipase, fell back toward wild-type levels when any of the three thioesterases was expressed, and an engineered green fluorescent protein that had escaped from the mutant’s cells was once again retained inside.

The lipid analysis explained why. In Gram-negative bacteria, membrane integrity depends on a delicate stoichiometric balance between phospholipids and lipopolysaccharide (LPS), as well as on the precise mix of fatty acyl chains within those lipids. Pulse-labeling experiments with radioactive acetate showed that the rpfF mutant accumulated excess phospholipids, particularly phosphatidylethanolamine and cardiolipin, while its LPS content dropped sharply: the lipid A-to-phospholipid ratio fell from 0.117 in the wild type to 0.037 in the mutant. Gas chromatography-mass spectrometry further revealed a skewed fatty acid profile, with the unsaturated C16:1 species rising and the saturated C16:0 and branched iso-C17:0 species falling. Expression of rpfF, tesA, or fatB1 corrected both the phospholipid-to-LPS ratio and the fatty acid composition.

The deepest defect turned out to be in the rate of fatty acid synthesis itself. Radiolabel incorporation showed that the rpfF mutant manufactured fatty acids at only about one-third of the wild-type rate, and specific synthesis rates measured by scintillation counting confirmed the slowdown. Complementation with any of the thioesterase genes restored synthetic flux to near-wild-type levels. The authors propose that when RpfF is absent, medium-chain acyl-ACPs accumulate inside the cell and feed back to inhibit the FASII machinery, throttling the supply of fatty acids needed to build phospholipids and LPS. By hydrolyzing excess acyl-ACPs, the thioesterase relieves this feedback inhibition and keeps membrane biogenesis running.

Crucially, the thioesterase also rescued virulence. In leaf-clipping assays on susceptible rice plants, the wild-type strain produced lesions averaging 17.8 centimeters at fourteen days after inoculation, while the rpfF mutant produced lesions of only 2.1 centimeters, an 88 percent reduction. Expressing the E. coli or plant thioesterase restored lesion lengths to 12.5 and 10.1 centimeters respectively, approaching the 13.3 centimeters achieved by restoring the native rpfF gene. Bacterial counts within lesions were only marginally lower in the mutant, indicating that the virulence defect reflects failed tissue colonization rather than poor growth inside the plant. Notably, exopolysaccharide production, which collapsed fourfold in the mutant, was restored only by native rpfF and by DSF signaling, not by the foreign thioesterases, showing that EPS is governed by quorum sensing while most other virulence traits ride on membrane health.

The study also showed that the thioesterase function is conserved across the genus and is itself wired into quorum sensing. Deleting rpfF in the related pathogen Xanthomonas campestris pv. campestris likewise caused detergent sensitivity and a skewed phospholipid-to-LPS ratio, and a foreign thioesterase repaired the damage. Moreover, overproducing the sensor kinase RpfC in wild-type Xoo mimicked the mutant’s detergent hypersensitivity, and the isolated receiver domain of RpfC inhibited purified RpfF’s thioesterase activity in vitro, extending to Xoo the known mechanism by which RpfC physically blocks RpfF in X. campestris. The authors propose a dual-function model in which RpfF simultaneously maintains lipid homeostasis through its thioesterase activity and produces DSF signals through its dehydratase activity. As cell density rises and DSF accumulates, RpfC’s grip on RpfF loosens, accelerating fatty acid synthesis and remodeling the membrane to withstand host-imposed stresses. In this way, the pathogen synchronizes virulence factor production with adaptive membrane restructuring, and the divergent phenotypes long observed between rpfF mutants of different Xanthomonas species are reinterpreted as the interplay of a conserved membrane-maintenance function with species-specific quorum-sensing circuitry. The work opens a potential avenue for anti-virulence strategies: disrupting RpfF’s thioesterase activity could strip the pathogen of both its signals and its structural resilience at once.

Subject of Research: The dual dehydratase and thioesterase activities of the RpfF enzyme in Xanthomonas oryzae pv. oryzae and their roles in fatty acid synthesis, membrane integrity, and virulence

Article Title: The RpfF Acyl-ACP thioesterase of major rice pathogen Xanthomonas oryzae pv . oryzae is essential for maintenance of membrane function and virulence

Article References: Luo, L., Hu, Z., Yan, M., Cronan, J. E., & Wang, H. (2026). The RpfF Acyl-ACP thioesterase of major rice pathogen Xanthomonas oryzae pv. oryzae is essential for maintenance of membrane function and virulence. PLOS Pathogens, 22(10), e1014669. https://doi.org/10.1371/journal.ppat.1014669

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014669

Keywords: Xanthomonas oryzae, RpfF, thioesterase, fatty acid synthesis, membrane integrity, quorum sensing, DSF, virulence, rice bacterial leaf blight, phospholipids, lipopolysaccharide, RpfC