In Xanthomonas campestris pv. campestris (
Xcc), the
proteins encoded by the rpf (regulator of
pathogenicity factor) gene cluster produce and sense a
fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an
acyl-CoA ligase. We report that RpfB activates a wide range of
fatty acids to their
CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial
acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the
Xcc genome results in a strain unable to utilize
fatty acids as
carbon sources. An essential RpfB function in the
pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic
enzyme, restored normal growth to this strain. RpfF is a dual function
enzyme that synthesizes DSF by
dehydration of a 3-hydroxyacyl-acyl
carrier protein (ACP)
fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block
membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as
free fatty acids. This lack of acyl chains for
phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the
free fatty acids to give acyl-CoAs that can be utilized to restore
membrane lipid synthesis. Heterologous expression of a different
fatty acid activating
enzyme, the Vibrio harveyi
acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of
free fatty acids.