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Applied and Environmental Microbiology, March 2009, p. 1679-1687, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.00024-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Chromosome-Based Genetic Complementation System for Xylella fastidiosa{triangledown}

Ayumi Matsumoto,1,2 Glenn M. Young,2 and Michele M. Igo1*

Department of Microbiology,1 Department of Food Science and Technology, University of California, Davis, One Shields Avenue, Davis, California 956162

Received 6 January 2009/ Accepted 8 January 2009

Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

* Corresponding author. Mailing address: Department of Microbiology, University of California, Davis, One Shields Avenue, Davis, CA 95616. Phone: (530) 752-8616. Fax: (530) 752-9014. E-mail: mmigo{at}ucdavis.edu

{triangledown} Published ahead of print on 16 January 2009.

Applied and Environmental Microbiology, March 2009, p. 1679-1687, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.00024-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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