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Lysobacter species are emerging as novel sources of antibiotics, but the regulation of these antibiotics has not been thoroughly elucidated to date. In this work, we identified a small diffusible signaling factor (DSF) molecule (Lb DSF) that regulates the biosynthesis of a novel Xanthomonas -specific antibiotic compound (XSAC) in Lysobacter brunescens OH23. Lb DSF was isolated from the culture broth of L. brunescens OH23, and the chemical structure of the molecule was determined by NMR and MS. The Lb DSF compound induced GUS expression in a reporter strain of Xanthomonas campestris pv. campestris FE58, which contained the gus gene under the control of a DSF-inducible engXCA promoter. Lb DSF production was found to be linked to the enoyl-CoA hydratase RpfF and dependent on the two-component regulatory system RpfC (hybrid sensor histidine kinase)/RpfG (response regulator), and Lb DSF production was increased 6.72 times in the Δ rpfC compared to wild-type OH23. Lb DSF-regulated XSAC production was dramatically decreased in Δ rpfF , Δ rpfC , and Δ rpfG. Additionally, a significant reduction in surface motility and a number of changes in colony morphology was observed in the Δ rpfF , Δ rpfC , and Δ rpfG compared to the wild-type OH23. The exogenous Lb DSF significantly increased XSAC production in wild-type OH23 and recovered the XSAC biosynthetic ability in Δ rpfF. Taken together, these results showed that Lb DSF is a fatty-acid-derived DSF that positively regulates XSAC biosynthesis, cell morphology, and surface motility. Moreover, the RpfC/RpfG quorum-sensing signal transduction pathway mediates XSAC biosynthesis. These findings may facilitate antibiotic production through genetic engineering in Lysobacter spp. [ABSTRACT FROM AUTHOR]