Site-directed mutagenesis of specific genes in a Pierces disease strain of Xylella fastidiosa

• Author(s): Azad, H.; Cooksey, Donald; Dumenyo, C.; Hernandez, Rufina;
• Abstract: Xylella fastidiosa (Xf) is a xylem-limited, fastidious, non-flagellated Gram-negative plant pathogenic bacterium. Strains of X. f. cause many diseases including Pierces disease of grapes and citrus variegated chlorosis (CVC). The exact disease mechanisms are not known but bacterial surface and extracellular factors have been implicated. Functions were assigned to only 47% of the predicted genes in the genome of CVC strain. Among others, some genes were predicted to encode adhesins, hemagglutinins, proteases, endoglucanases, polygalacturonase and the extracellular polysaccharide Gum. Lack of molecular genetic tools such as site-directed mutagenesis has hampered functional genomic analysis through which assigned gene functions can be genetically confirmed and functions can be assignment to the remaining genes. We used the gum operon to develop a method for site-directed mutagenesis of specific genes in X. f. A cosmid library of strain A05 was constructed in pCPP47 and screened with PCR using gumB-specific primers. The ends of the insert DNA in the gum+ cosmid were sequenced and compared with the published genomic sequence to determine the size of the insert and the genomic region cloned. The cosmid was randomly mutagenized in vitro with EZ::TNTM system and the insertion mutants were screened for insertion into specific gum genes. Numerous attempts to transform X. f. with the inactivated cosmids failed. Therefore, the transposon along with the flanking cosmid DNA for each insertion were PCR-cloned into pUC129. One of the resulting plasmids carrying EZ::TNTM insertion into gumH was electroporated into electrocompetent X. f. cells and transformants were selected on PD3 agar medium supplemented with kanamycin at 10 mug/mul. The transformants were confirmed by PCR to have an insertion in gumH sequence and by Southern analyses to contain the EZ::TNTM transposon sequence within gumH sequence in the genome. This simple method should provided the critical molecular tool required for the genetic analysis of the more than 50% of the X. f. genes with unknown functions and the other half with assigned but unconfirmed functions.
• Publication Date: Jan 2003
• Journal: Abstracts Of The General Meeting Of The American Society For Microbiology