RESUMEN
Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.
Asunto(s)
Agrobacterium tumefaciens/genética , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Sistemas Toxina-Antitoxina/genética , Agrobacterium tumefaciens/enzimología , ADN/genética , Desoxirribonucleasas/genética , Escherichia coli/genética , ARN/genética , Ribonucleasas/genética , Ribosomas/genéticaRESUMEN
The ribosome-dependent E. coli (Ec) mRNase toxin YoeB has been demonstrated to protect cells during thermal stress. Agrobacterium tumefaciens (At), a plant pathogen, also encodes a YoeB toxin. Initial studies indicated that AtYoeB does not impact the growth of Ec, but its expression is toxic to the native host At. The current work examines this species-specific effect. We establish the highly similar structure and function of Ec and AtYoeB toxins, including the ability of the AtYoeB toxin to inhibit Ec ribosomes in vitro. Comparison of YoeB sequences and structures highlights a four-residue helix between ß-strands 2 and 3 that interacts with mRNA bases within the ribosome. This helix sequence is varied among YoeB toxins, and this variation correlates with bacterial classes of proteobacteria. When the four amino acid sequence of this helix is transplanted from EcYoeB onto AtYoeB, the resulting chimera gains toxicity to Ec cells and lessens toxicity to At cells. The reverse is also true, such that EcYoeB with the AtYoeB helix sequence is less toxic to Ec and gains toxicity to At cultures. We suggest this helix sequence directs mRNA sequence-specific degradation, which varies among proteobacterial classes, and thus controls growth inhibition and YoeB toxicity.
