Molecular genetics of albicidin biosynthesis in Xanthomonas albilineans

Abstract

Albicidins produced by Xanthomonas albilineans are key pathogenesis factors in the systemic development of leaf scald disease in sugarcane (Saccharum spp. hybrids). They are also potent bactericidal antibiotics inhibiting prokaryote DNA replication. Low yields have slowed studies into the chemical structure of albicidins, their application as tools to study DNA replication, and their development as clinical antibiotics. Genetic analysis of albicidin biosynthesis may help to overcome these limitations. Previous work used Tn5 mutagenesis to demonstrate that albicidin biosynthetic genes were in at least two gene clusters spanning more than 60 kb in the genome of X. albilineans. This project investigated the molecular genetic basis for albicidin biosynthesis, initially by cloning and characterizing the Tn5-tagged genes essential for albicidin production in X. albilineans.In order to efficiently clone and sequence DNA flanking transposon Tn5 insertions, a simple inverse PCR method was first developed. The method has advantages over published ones, using four paired inverse PCR reactions to distinguish the target amplicon from non-specific products, and to demonstrate the copy number of Tn5 insertions without Southern blot analysis. This approach proved effective for amplification and cloning of Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in X. albilineans.Three albicidin biosynthetic genes designated xabA, xabB and xabC were cloned and characterized by sequence analysis and functional analysis.nn