"A novel family of mutagenic bacterial toxins with unprecedented DNA deaminase activity"

Abstract:

When bacterial cells come into direct contact, antagonism mediated by the delivery of a diverse array of potent toxins frequently ensues. The reported outcomes of such assaults include cell death, growth inhibition, or survival in resistant populations. The potential for interbacterial toxins to have long-term consequences in recipient cells has not been investigated. In this work, we examined the physiological effects of intoxication by DddA, a double-strand DNA-specific cytosine deaminase delivered via the type VI secretion system (T6SS) of Burkholderia cenocepacia. Moreover, we harnessed the biological activity of DddA to generate the first generation of precise genome editing tools for the mitochondrial genome. We find that when expressed in E. coli, DddA leads to cell death by chromosome degradation and arrest of DNA replication. Despite the lethal potential of DddA, several species of bacteria resist killing when confronted by DddA delivered by the T6SS of B. cenocepacia cells. Surprisingly, these targeted cells accumulate mutations characteristic of those induced by the toxin, indicating that even in the absence of killing, interbacterial toxins can have profound consequences on target cell populations. Motivated by the diversity of toxin members in the deaminase superfamily, we investigated whether DNA deaminase activity is a common feature in this group. We discovered that highly divergent deaminases act on DNA, including a novel single-stranded DNA deaminase with a markedly divergent structure. Additionally, in this work, we engineered split-DddA halves bound to programmable DNA-binding proteins, resulting in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyze C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. In total, our work reveals that mutagenic activity is a common feature of bacterial toxins in the deaminase superfamily, and it shows that a surprising consequence of antagonistic interactions in microbial communities can be the generation of genetic diversity. Furthermore, we harnessed the DNA modifying potential of DddA to create the first generation of genome editing tools for mtDNA.