SURROGATE GENETICS FOR XYLELLA FASTIDIOSA: REGULATION OF EXOPOLYSACCHARIDE AND TYPE IV PILUS GENE EXPRESSION

• Author(s): Stewart, Valley; Kirkpatrick, Bruce;
• Abstract: Xylella fastidiosa presents a formidable challenge to the molecular geneticist. There are no published methods available for the basic operations of genetic exchange, mutant isolation, and complementation. The slow generation time, poor plating efficiency and requirement for complex culture media are further complications. Surrogate genetics (Maloy & Zahrt, 2000) provides a means to at least partially bypass these challenges. Here, one creates a hybrid organism, transplanting genes of interest from the poorly-studied species (e.g., Xyella fastidiosa) into a well-studied surrogate host (e.g., Escherichia coli). Given sufficiently related hosts, one expects the transplanted genes to function in the surrogate essentially as they do in the original. One may then exploit the advantageous properties of the surrogate to perform a large number of experiments, making and discarding hypotheses to define various aspects of gene function. Once gene function in the surrogate has been thoroughly explored, one can perform a focused set of experiments, informed by the results from the surrogate, to examine function in the native host. The use of E. coli as a surrogate host for studying gene regulation would open a range of experimental approaches that are currently unavailable in X. fastidiosa, and lead to more rapid advances in understanding the control of key pathogenicity determinants. We are analyzing the transcriptional regulation determinants for genes whose products may be involved in pathogenesis (e. g., gum genes, encoding exopolysaccharide; and pil genes, encoding type IV pili) as well as housekeeping genes involved in central metabolism (e.g., amino acid biosynthesis). During infection, X. fastidiosa produces an extracellular matrix that is hypothesized to contribute significantly to disease symptoms (see Bevan, 2000). The X. fastidiosa 9a5c genome encodes a gumBCDEFHJK operon homologous to the corresponding Xanthomonas campestris operon (Simpson et al., 2000). Note however that Xan. campestris pv. campestris gum null mutants exhibit at most a 50% reduction in virulence index when assayed on cabbage (Katzen et al., 1998). Type IV pili are responsible for twitching motility, natural transformation, and adherence in various species. The X. fastidiosa 9a5c genome encodes the various pil genes necessary for synthesis, secretion and assembly of type IV pili (Simpson et al., 2000).
• Publication Date: Dec 2001
• Journal: 2001 Pierce's Disease Research Symposium