The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide

Abstract

Xanthomonas campestris pv. citrumelo strain 3048 is the causal agent of citrus bacterial leaf spot disease and has a wide host range that includes rutaceous and leguminous plants. A spontaneous prototrophic mutant of strain 3048 (strain M28) that had lost virulence on citrus but retained virulence on bean plants was recovered. Growth studies in planta showed that M28 cells died rapidly in citrus leaves but grew normally in bean leaves. In addition to the loss of citrus-specific virulence, M28 displayed the following mutant phenotypes in culture: decreased growth rate, reduction of the amount of exopolysaccharide (to ca. 25% of the amount in 3048), loss of capsules, and significant alterations of the two 3048 lipopolysaccharide (LPS) bands visualized by silver stain on polyacrylamide gels, consistent with a defect(s) in LPS assembly. A 38-kb DNA fragment from a 3048 total DNA library that complemented the mutant phenotypes of M28 was identified. The 38-kb fragment did not hybridize to two similarly sized fragments carrying different hrp (hypersensitive response and pathogenicity) genes cloned from 3048. Subcloning, DNA sequence analyses, and gene disruption experiments were used to identify a single gene, opsX (for outer-membrane polysaccharide), responsible for the mutant phenotypes of M28. At least one other gene downstream from opsX also affected the same phenotypes and may be part of a gene cluster. We report here the DNA sequence and transcriptional start site of opsX. A search of protein sequence data bases with the predicted 31.3-kDa OpsX sequence found strong similarity to Lsi-1 of Neisseria gonorrhoeae and RfaQ of Escherichia coli (both are involved in LPS core assembly). The host-specific virulence function of opsX appears to involve biosynthesis of the extracellular polysaccharide and a complete LPS. Both may be needed in normal amounts for protection from citrus, but not bean, defense compounds.