Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans

Abstract

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.