Xylella fastidiosa plasmid-encoded PemK toxin is an endoribonuclease

Area of Science:

• Bacteriology
• Molecular Biology
• Genetics

Background:

• The pXF-RIV11 plasmid in Xylella fastidiosa ensures stable inheritance through a toxin-antitoxin (TA) system.
• The pemI/pemK TA system consists of the PemK toxin, which inhibits bacterial growth, and the PemI antitoxin, which neutralizes PemK.

Purpose of the Study:

• To investigate the biochemical activities of PemK toxin and PemI antitoxin from Xylella fastidiosa.
• To elucidate the mechanism by which the pemI/pemK TA system confers stable inheritance.
• To compare the X. fastidiosa pemI/pemK system with known TA systems in other bacteria.

Main Methods:

• Overexpression and purification of PemK toxin and PemI antitoxin in Escherichia coli.
• In vitro assays to assess the ribonuclease activity of PemK and the inhibitory effect of PemI.
• Site-directed mutagenesis of PemK to identify key residues for activity and binding.
• RNA sequencing (5' rapid amplification of cDNA ends) to determine PemK cleavage sites.

Main Results:

• Purified PemK toxin demonstrated specific degradation of single-stranded RNA, with a preference for cleavage at U-A sites within UACU and UACG sequences.
• PemI antitoxin effectively inhibited the nuclease activity of PemK.
• PemK mutants lacking nuclease activity failed to inhibit bacterial growth, indicating the essential role of ribonuclease activity in toxicity.
• All tested PemK mutants maintained their ability to bind PemI.

Conclusions:

• The pemI/pemK TA system in X. fastidiosa functions mechanistically similarly to the well-characterized R100 pemI/pemK system in E. coli.
• Stable plasmid inheritance is mediated by the RNA degradation activity of PemK, which is regulated by PemI.
• The study provides insights into the molecular mechanisms underlying plasmid stability in plant pathogenic bacteria.