The ppGpp-HpaR1-gum regulatory pathway modulates exopolysaccharides production in Xanthomonas campestris pv....

Exopolysaccharides (EPS) are critical components of the biofilm matrix, and ppGpp has been demonstrated to positively influence biofilm formation. Here, we elucidate the underlying mechanism by which ppGpp regulates EPS production by facilitating HpaR1 to modulate the expression of the gum cluster in the phytopathogen Xanthomonas campestris pv. campestris (Xcc). ppGpp affected the yield of EPS without influencing its primary or advanced structure, as confirmed by Fourier transform infrared spectroscopy and scanning electron microscopy. Expression of the gum cluster, which governs EPS biosynthesis in Xcc, was down-regulated in the ppGpp-deficient mutant (ΔrelAΔspoT) compared to the wild type (WT). Comparison of EPS production between knock-out mutants of the gum cluster and ppGpp-deficient mutant revealed that the gum cluster was a key determinant of EPS production, with ppGpp acting upstream of the gum cluster. Transcriptomic and qPCR analyses indicated that ppGpp modulated global transcription in Xcc, positively regulating expression of hpaR1, which encodes the transcription factor for the gum cluster. This regulatory role was further substantiated by electrophoretic mobility shift assays, which showed that ppGpp enhanced the DNA-binding activity of HpaR1. Furthermore, genetic complementation with hpaR1 restored EPS production, confirming its functional role in this regulatory pathway. In summary, these findings provide novel insights into the molecular mechanisms linking ppGpp signaling to EPS production in X. campestris pv. campestris.