Molecular Analysis of Albicidin Resistance in Xanthomonas Albilineans

Abstract

Xanthomonas albilineans is the causal pathogen of leaf scald disease of sugarcane. This devastating disease has been a major problem in most cane growing regions and continues to pose a worldwide economic threat to the sugarcane industry.Chlorosis inducing strains of the pathogen produce several toxic compounds. The major component, albicidin, inhibits DNA replication in sugarcane proplastids, resulting in blocked chloroplast development. Albicidin also inhibits prokaryotic DNA replication and is bactericidal to a range of Gram positive and Gram negative bacteria at concentrations as low as 1 ng ml-1. Albicidin has been partially characterized as a low molecular weight antibiotic that contains 38 carbon atoms with several aromatic rings. Albicidin deficient mutants of X. albilineans fail to cause chlorosis or any other symptom of leaf scald disease in inoculated sugarcane, indicating that albicidin phytotoxins play an import role in disease development. Therefore, resistance to albicidin may provide leaf scald disease resistance in sugarcane. X. albilineans is of interest as a potential source of these resistance genes. This project set out to investigate the genetics and mechanisms of albicidin resistance in X. albilineans.Two strategies were employed to isolate resistance genes from X. albilineans. No albicidin sensitive X. albilineans mutants were generated using transposon insertion mutagenesis; possibly because multiple redundant resistance genes contribute to albicidin resistance in X. albilineans, or because mutations in the resistance gene are lethal, even in Tox- mutants.The second strategy involved the construction of two X. albilineans genomic DNA cosmid libraries, followed by screening for clones conferring albicidin resistance in Escherichia coli. Albicidin resistant clones from the first library in E. coli DH5a proved unstable. One of these clones, E. coli JBDH93, conferred a 350-fold increase in albicidin resistance. However, the resistance gene was not transferrable on the cosmid. The mechanism of resistance did not involve export, binding, enzymatic detoxification or inactivation of the antibiotic, but did show a reduced uptake of the antibiotic. It is known that mutations in the tsx gene can prevent active uptake of the toxin in E. coli. Clone E. coli JBDH93 showed a reduced susceptibility to phage T6, which is characteristic of mutations in the tsx gene. PCR and Southern analysis showed that there had been some rearrangement in the tsx region. However, E. coli JBDH93 showed greater albicidin uptake than previously characterized albicidin resistant tsx mutants, and was resistant to approximately 10-fold higher albicidin concentration than previously characterized tsx mutants. After plating E. coli DH5a cells onto albicidin, no spontaneous mutants were obtained with albicidin resistance comparable to that of E. coli JBDH93. A gene from X. albilineans may be acting in combination with a tsx mutation to increase this clone's resistance to albicidin.The second genomic cosmid library was transformed into PMC 107, a multiple recombination deficient E. coli strain designed to minimise cosmid rearrangements. Several albicidin resistance clones were isolated. One of these clones showed at least a five-fold increased resistance to albicidin. The mechanism of resistance did not involve exclusion, binding, enzymatic detoxification or inactivation of the antibiotic. The 23 kb insert was subcloned and a putative 1487 bp albicidin resistance gene, designated albF was further isolated using PCR methods. In the vector pBlueScript II KS+ with induction of the lac promoter, only clones containing the ORF in the reverse orientation relative to the promoter could be generated, indicating that strong expression of albF is lethal to E. coli cells. Clones with the ORF in the forward orientation could only be maintained with repression of the lac promoter. Under conditions of lac repression, albF increased the minimum inhibitory concentration of albicidin by at least 3000-fold in the E. coli strains PMC107 and DH5a. No resistance to other antibiotics was observed.n n