Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan r ). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan r gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.Xylella fastidiosa is a fastidious gram-negative, xylem-limited bacterium (26) that causes a range of economically important plant diseases, including citrus variegated chlorosis (CVC) (3, 23); Pierce's disease (PD) of grapevine; alfalfa dwarf; leaf scorch of almond, coffee, elm, sycamore, oak, plum, mulberry, maple, and oleander; and periwinkle wilt (for reviews, see references 19 and 20).CVC is a major problem in Brazil, where over 70 million sweet orange trees (34%) are affected. The disease also occurs in Argentina, under the name "pecosita" (7, 9). CVC affects all commercial sweet orange varieties. Affected fruits are small and hardened and thus of no commercial value. Rapid dissemination of CVC comes from the use of infected nursery trees and transmission of X. fastidiosa by several xylem-feeding sharpshooter insect vectors.The genome sequence of the CVC strain of X. fastidiosa, clone 9a5c, was recently determined, and the nature of genes that were identified by annotation suggests a number of potential pathogenicity mechanisms, such as cell-wall hydrolysis, adhesion, intervessel migration, and toxic...
Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for -galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to -galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.Xylella fastidiosa is a fastidious, gram-negative, xylem-limited bacterium (20) that causes a range of economically important plant diseases, including citrus variegated chlorosis (CVC) (2, 17); Pierce's disease (PD) of grapevine; alfalfa dwarf; leaf scorch of almond, coffee, elm, sycamore, oak, plum, mulberry, maple, and oleander; and periwinkle wilt (15,16). Despite the importance of the X. fastidiosa CVC strain in phytopathology, our understanding of the physiology and genetics of this bacterium is still poor. Genetic tools to study the biology of X. fastidiosa are limited due to the difficulty in culturing and transforming this fastidious organism. Production of mutants is an important and necessary way to identify and study genes and then the mechanisms involved in different processes, such as pathogenicity. Several methods could be used to produce mutants: insertion-duplication mutagenesis (IDM), allelic exchange (AE), and transposon mutagenesis. Recently, random mutagenesis by transposition was used to produce mutants in X. fastidiosa PD strains (7). However, this method could not be used to directly inactivate specific genes, which is achieved by homologous recombination (IDM and AE). IDM has been used to disrupt genes in a variety of other organisms, such as Mycobacterium smegmatis (1), Neisseria gonorrhoeae (8), Streptococcus pneumoniae (12), and Lactobacillus sake (13). This mutagenesis involves circular integration, by a single crossover event, between the targeted chromosomal gene and a truncated copy of this gene cloned in a transient suicide or replicative plasmid, resulting in integration of the entire plasmid and duplication of the target sequence. AE results in the replacement of the endogenous gene by its copy disrupted by a selectable marker. This approach involves homologous recombination with two crossovers. We previously reported the transformation of X. fastidiosa with artificial plasmids carrying the X. fastidiosa chromosomal origin of replication (oriC) and a kanamycin resistance gene under the control of the X. fastidiosa 16S rRNA promoter (14). These X. fastidiosa oriC plasmids were found to be integrated in the chromosome at the rRNA promoter site by homologous recombination involving one crossover (14), suggesting that gene disruption by homologous recombination is possible in X. fastidiosa. Here we report the disruption of genes by homologous recombination involving one crossover (IDM) or two crossovers (AE) as tools to produce mutants in X. fastidiosa by site-directed gene disruption.Construction of plasmi...
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