Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2013
Publication Date: 5/15/2013
Citation: Lee, M., Tan, C.C., Rogers, E.E., Stenger, D.C. 2013. Functional characterization of two toxin-antitoxin systems of Xylella fastidiosa. American Phytopathological Society Annual Meeting. 103:S2.78.
Technical Abstract: Xylella fastidiosa (Xf) encodes multiple toxin-antitoxin (TA) system homologues, including relE/dinJ and mqsR/ygiT. Phylogenetic analyses indicate these two Xf TA systems have distinct evolutionary histories. Comparisons among Xf subspecies/strains reveal TA systems are often embedded in prophage sequences, suggesting acquisition via horizontal transfer. Tagged proteins of both TA systems were over-expressed, purified, and evaluated for activity. Toxins MqsR and RelE are ribonucleases with distinct cleavage sites (MqsR at G'CU, less often G'CC; RelE lacked specificity in vitro). YgiT and DinJ antitoxins inhibit ribonuclease activity of cognate toxins by direct binding. Single (toxin or antitoxin) and double (toxin and antitoxin) knock-out mutants were constructed in Xf strain Temecula. Both antitoxin mutants displayed reduced planktonic cell density, as measured by optical density, relative to wild type. However, biofilm phenotypes differed: crystal violet staining assays indicated that the dinJ- mutant produced less biofilm, whereas the ygiT- mutant produced more biofilm, relative to wild type. Planktonic cell density of both toxin mutants was unaffected, with biofilm reduced only slightly. Although planktonic cell density and biofilm formation of each double mutant was similar to wild type, significant differences were observed in cell viability. Viable cell counts from biofilm and plankton for the mqsR-/ygiT- double mutant were consistently two orders of magnitude greater than wild type. In contrast, cell viability of the relE-/dinJ- double mutant was variable, possibly due to culture nutritional status. Collectively, the results indicate that Xf mqsR/ygiT and relE/dinJ are functional TA systems affecting growth, biofilm formation and cell viability.