THE RPFA GENE OF XANTHOMONAS-CAMPESTRIS PATHOVAR CAMPESTRIS, WHICH ISINVOLVED IN THE REGULATION OF PATHOGENICITY FACTOR PRODUCTION, ENCODES AN ACONITASE

Xanthomonas campestris pv campestris (Xcc) is a plant pathogenic bacte rium that controls the production of pathogenicity factors in part by a cluster of genes designated rpf (regulation of pathogenicity factors ). Sequence analysis of one of these genes (rpfA) revealed an open rea ding frame with amino acid sequence similarity to aconitases from othe r bacteria. Aconitase activity was lower in cellular extracts of an rp fA::Tn5 mutant than in those from the wild type. A zymogram of aconita se activity after native gel electrophoresis showed the presence of tw o distinct aconitases in Xcc; the major aconitase was absent in the rp fA::Tn5 mutant. This mutant also had reduced levels of extracellular e nzymes and extracellular polysaccharide (EPS). Supplying rpfA in trans to the rpfA::Tn5 mutant restored both the major aconitase activity an d the synthesis of these pathogenicity factors. The transcription of t he genes for two extracellular enzymes (prtA, encoding a serine protea se, and engXCA, encoding endoglucanase) was reduced in the rpfA mutant background. Because some eukaryotic aconitases are also involved in i ron regulation, we explored a possible connection between rpfA and iro n metabolism. Intracellular iron levels in the mutants were lower than in the wild type as assessed by sensitivity to the iron-activated ant ibiotic, streptonigrin. Wild-type bacteria grown in iron-deficient con ditions had a similar sensitivity to streptonigrin as the aconitase mu tant. Overall, these results suggest that a prokaryotic aconitase can also act as a regulator of gene expression and that the regulation is possibly related to changes in intracellular iron levels.