At least two separate gene clusters are involved in albicidin production by Xanthomonas albilineans

Abstract

Transposon mutagenesis was used to obtain mutations affecting production of the toxin albicidin in Xanthomonas albilineans, which causes leaf scald disease of sugarcane and is also pathogenic to corn. Transposon Tn5-gusA inserted randomly into genomic DNA of X. albilineans Xa23R1 at a frequency of 10(-4) to 10(-5) per recipient after conjugal transfer from Escherichia coli. Fifty prototrophic mutants defective in albicidin production were isolated from 7,100 Tn5-gusA insertional derivatives tested for toxin production by an antibiosis bioassay. EcoRI fragments containing Tn5 flanking sequences from two mutants (AM15 and AM40) were cloned and used to probe a wild-type Xa23R1 DNA library by colony hybridization. Nine cosmids showed homology to the AM15 probe, and six showed homology to the AM40 probe. Four cosmid clones hybridized to both probes. Forty-five of the 50 defective mutants were restored to albicidin production with two overlapping cosmid clones. Restriction mapping showed that these mutants span a genomic region of about 48 kb. At least one other gene cluster is also involved in albicidin production in Xa23R1. DNA fragments from the 48-kb cluster proved to be very specific to X. albilineans. Some mutants affected in albicidin production retain their ability to colonize sugarcane cultivated in vitro.