Submitted to: American Phytopathology Society
Publication Type: Abstract Only
Publication Acceptance Date: March 15, 2011
Publication Date: June 1, 2011
Citation: Lee, M.W., Rogers, E.E., Stenger, D.C. 2011. PemK toxin encoded by the Xylella fastidiosa IncP-1 plasmid pXF-RIV11 is a ribonuclease. Phytopathology. 101:S99. Technical Abstract: Stable inheritance of the IncP-1 plasmid 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. Here, 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 RNAs digested with PemK revealed a preference for cleavage between U and A residues of the trinucleotide UAC. 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 did not inhibit growth. All nine PemK mutants retained the ability to bind PemI antitoxin. Collectively, the results indicate that mechanism of stable inheritance conferred by the pXF-RIV11 pemI/pemK plasmid addiction system is similar to that of the prototype pemI/pemK addiction system of E. coli plasmid pR100.