Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/8/2014
Publication Date: 7/17/2014
Citation: Lee, M., Tan, C.C., Rogers, E.E., Stenger, D.C. 2014. Toxin-antitoxin systems mqsR/ygiT and dinJ/RelE of Xylella fastidiosa. Physiological and Molecular Plant Pathology. 87:59-68. Interpretive Summary: Xylella fastidiosa is the casual agent of Pierce’s disease of grapevines. Successful colonization of grapevine vascular tissues and insect vector foreguts requires X. fastidiosa to grow as both planktonic cells and as a biofilm. The role of two toxin-antitoxin systems (MqsR/YgiT and DinJ/RelE) encoded by X. fastidiosa on bacterial cell growth was examined. Sequence comparisons indicate that X. fastidiosa acquired the two TA systems via horizontal transfer from different sources. Genome structure indicated that TA system genes are embedded within phage sequences integrated into the X. fastidiosa chromosome. Biochemical analyses demonstrated that toxins MqsR and RelE are ribonucleases inhibited by direct binding of antitoxins YgiT and DinJ, respectively. Growth characteristics of X. fastidiosa bearing single (toxin or antitoxin) or double (toxin and antitoxin) knockout mutants of each TA system indicated that MqsR/YgiT, but not DinJ/RelE, promotes biofilm formation while at the same time reducing biofilm cell viability. DinJ/RelE likely serves as a regulator of bacterial growth in response to a specific, albeit unidentified, environmental stress. Collectively, the results indicate that both toxin-antitoxin systems are functional regulators of cell growth that may enhance fitness of X. fastidiosa as a plant pathogen.
Technical Abstract: The plant pathogen 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. Genomic comparisons among Xf subspecies/strains reveal TA systems are often embedded in prophage sequences, suggesting acquisition via horizontal transfer. Tagged proteins of each TA system were over-expressed, purified, and evaluated for activity. Toxins MqsR and RelE are ribonucleases with distinct cleavage sites (MqsR at GCU, less often GCC; RelE lacked specificity in vitro). YgiT and DinJ anti-toxins inhibited ribonuclease activity of the cognate toxin by direct binding. Single (toxin or antitoxin) and double (toxin and antitoxin) knockout mutants were constructed in Xf strain Temecula. Both antitoxin mutants displayed reduced planktonic growth relative to wild type. However, biofilm phenotypes differed: the dinJ- mutant produced less biofilm, whereas the ygiT- mutant produced more biofilm, relative to wild type. Planktonic growth of both toxin mutants was unaffected, with biofilm reduced only slightly. Although planktonic growth and biofilm formation of each double mutant did not differ from wild type, significant differences were observed in cell viability. The mqsR-/ygiT- double mutant exhibited increased numbers of viable cells in biofilm as compared to both wild type and the relE/dinJ double mutant. Collectively, the results indicate that Xf mqsR/ygiT and relE/dinJ are functional TA systems. How TA system activity may lead to enhanced fitness and/or affect pathogenicity of Xf remain to be determined.