Genetic loci involved in rubrifacine production in the walnut pathogen Brenneria rubrifaciens

Brenneria rubrifaciens produces a unique red pigment known as rubrifacine that has been hypothesized to play a role in pathogenesis on walnut. Analysis of DNA flanking the Tn5 insertion site in 20 rubrifacine minus (pig(-)) mutants identified three regions required for rubrifacine production. The first region was homologous to nonribosomal peptide synthetases (NRPS), the second was homologous to autoinducer synthase genes (expI homologs), and the third region was homologous to the slyA gene of Candidatus blochmania and Escherichia coli. Pigment production was not necessary for elicitation of the hypersensitive response (HR) in tobacco and had little impact on virulence in tissue-cultured walnut plants. The expI-interrupted mutants exhibited reduced virulence on walnut and were HR negative on tobacco. Pigment production was restored in Br-212 when grown in the presence of wild-type B. rubrifaciens, E. coli carrying the cloned expI-like gene, or introduction of the cloned wild-type copy of the expI-like gene. Two Brenneria spp., B. nigrifluens and B. salicis, also restored pigment production in Br-212. These results demonstrate that rubrifacine production and virulence of B. rubrifaciens on walnut are under the control of a quorum-sensing system and are sensitive to signal molecules from other Brenneria spp.