CHARACTERIZATION OF REGULATORY PATHWAYS CONTROLLING VIRULENCE IN XYLELLA FASTIDIOSA Project Leader:

We are pursuing a strategy to identify traits important in virulence of Xylella fastidiosa (Xf) through the mutagenesis of “global” regulatory genes, which are known to broadly regulate virulence functions in other microbes. In addition to phenotypic characterization of such mutants, we are using whole-genome microarrays to identify which genes are regulated by these global regulators and examine these genes as putative virulence factors. Here we report a specific example of this approach that has helped to define genes involved in aggregation, biofilm formation, and virulence of Xf. In previous work with X. fastidiosa, we mutated the global regulatory gene rsmA, and found a number of genes that were over-expressed in this mutant when grown in vitro, implying that these genes are normally repressed by the post-transcriptional regulator RsmA in the wild-type. In addition, the rsmA mutant formed much more biofilm than wild type. Among the genes repressed by rsmA was another regulatory gene, algU, which regulates important virulence factors in Pseudomonas. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, biofilm formation, and lower virulence in grapevines. DNA microarray analysis showed that 42 genes had significantly lower expression in algU::nptII than wild type, including several genes which could contribute to cell aggregation and biofilm formation, as well as other physiological processes that could contribute to virulence and survival. Thus, rsmA appears to control biofilm formation and other traits partly through its repression of the positive regulator, algU. INTRODUCTION Many virulence genes in bacterial pathogens are coordinately regulated by “global” regulatory genes. The gene rsmA, for example, is known to regulate pathogenicity and secondary metabolism in a wide group of bacteria (Blumer and Haas, 2000; Mukherjee et al., 1996). Conducting DNA microarray analysis with mutants for such genes, compared with wild-type, can help to refine the list of genes that may contribute to virulence. We have reported on such an analysis with an rsmA mutant of Xylella fastidiosa (Xf), and identified a number of genes that were overexpressed in the mutant (Cooksey, 2004). Among those were pil genes that have been subsequently confirmed to be important in twitching motility and long-distance spread of Xf in grapevines (Hoch and Burr, 2005; Meng et al., 2005), as well as enzymes or other structural proteins. In addition, a few genes controlled by rsmA were “secondary” regulatory genes, such as algU, which controls exopolysaccharide production in certain human and plant pathogens and contributes to virulence (Schnider-Keel, et al., 2001; Yu et al., 1995; Yu et al., 1999). AlgU is a an alternative sigma factor whose role in regulation of biosynthesis of the exopolysaccharide (EPS) alginate has been extensively studied in Pseudomonas aeruginosa and P. syringae. Alginate functions as a virulence factor in P. aeruginosa during infection of cystic fibrosis patients (May and Chakrabarty, 1994), and also contributes to both virulence and epiphytic survival of the plant pathogen P. syringae (Yu et al., 1999). In P. aeruginosa, AlgU activates AlgU-dependent promoters of algD and algR. AlgR regulates algC and algD in cooperation with AlgU (Martin et al., 1994). mucD is a negative regulator of algU activity in P. aeruginosa. Homologs of algU (PD1284), algZ (PD1154), algS (PD0347), algR (PD1153), algC (PD0120), algH (PD1276) and mucD (PD1286) were detected in the Xf genome (Simpson et al., 2000; Van Sluys et al., 2003), but there are no homologs of the alginate biosynthesis genes algA, algD, algG, algF, algI and algJ. The alginate homolog genes in Xf are therefore probably not involved in alginate biosynthesis, but may be involved in synthesis of other EPS or of lipopolysaccharide (LPS), which could play a role in biofilm formation and cell attachment. In P. aeruginosa, the algC gene encodes a bifunctional enzyme that is involved in alginate production (phosphomannomutase activity) and lipopolysaccharide (LPS) production (phosphoglucomutase activity) (Coyne et al., 1994). We have constructed an insertional mutation in algU in X. fastidiosa, which reduced cell-cell aggregation, attachment, biofilm formation, and virulence. DNA microarray analysis of the algU mutant was then conducted to determine which genes it regulates.