Xylella fastidiosa Plasmid-Encoded PemK Toxin Is an Endoribonuclease

Abstract

Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK toxin-antitoxin (TA) system. PemK toxin inhibits bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK by direct binding. PemK and PemI were overexpressed 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 inhibited 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′ rapid amplification of cDNA ends products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU and UACG. Nine single amino-acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, ribonuclease 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. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the R100 pemI/pemK TA system of E. coli.