Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 16, 2011
Publication Date: January 1, 2012
Citation: Lee, M.W., Rogers, E.E., Stenger, D.C. 2012. Xylella fastidiosa plasmid-encoded PemK toxin is an endoribonuclease.. Phytopathology. 102:32-40. Interpretive Summary: Xylella fastidiosa is the causal agent of Pierce’s disease of grape. As with other bacteria, X. fastidiosa harbors plasmids that encode genes not resident on the chromosome and which may be transferred among bacteria. Plasmids often encode genes conferring stable inheritance, thereby ensuring persistence of the plasmid in the bacterium. Recently, a novel plasmid was isolated from mulberry-infecting strains of X. fastidiosa. Stable inheritance of the plasmid is achieved through expression of the PemI/PemK plasmid addiction system. Functional analyses revealed that PemK is a toxin inhibiting bacterial cell growth. PemK toxin inhibits cell growth by degrading RNA, rendering the cell incapable of expressing proteins required for continued growth. The ability of PemK to degrade bacterial RNA is blocked by binding of PemI antitoxin to PemK toxin in a reversible manner. Because PemK toxin is more stable than PemI antitoxin, daughter cells that do not inherit plasmid are killed by residual PemK toxin. This improved understanding of the mechanism of plasmid maintenance will facilitate ongoing efforts to develop stable plasmid shuttle vectors to deliver DNA to X. fastidiosa.
Technical Abstract: Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. PemK toxin and PemI antitoxin were over-expressed in Escherichia coli and activities of each were assessed. Purified PemK toxin specifically degraded single-stranded RNA but not double-stranded RNA, double-stranded DNA, or single-stranded DNA. Addition of PemI antitoxin blocked nuclease activity of PemK toxin. Purified complexes of PemI bound to PemK exhibited minimal nuclease activity; removal of PemI antitoxin from the complex restored nuclease activity of PemK toxin. Sequencing of 5’ RACE products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU. Nine single amino acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, nuclease activity, and PemI binding. Three PemK point substitution mutants (R3A, G16E, and D79V) that lacked nuclease activity also did not inhibit growth. All nine PemK mutants retained the ability to bind PemI antitoxin. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the prototype pemI/pemK plasmid addiction system of E. coli pR100.