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A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS

Abstract

Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

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Figure 1: GhoT increases persistence.
Figure 2: GhoT is toxic, and GhoS reduces this toxicity.
Figure 3: GhoS adopts a ferredoxin-like fold and Arg28 is important for its cleavage activity.
Figure 4: GhoS cleavage of native and altered ghoT transcripts.

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Acknowledgements

This work was supported by the US National Institutes of Health (R01 GM089999 to T.K.W.) and the US National Science Foundation (CAREER award MCB 0952550 to R.P.). X.W. is partially supported by the 1000-Youth Elite Program from China. We are grateful for the Keio and ASKA strains provided by the Genome Analysis Project in Japan and for the initial growth studies conducted by X. Yan and T. Benefield. We also thank S. Vitha for assistance with microscope imaging and Y. Hu for assistance with western blotting. T.K.W. is the Biotechnology Endowed Professor at the Pennsylvania State University.

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T.K.W., X.W., R.P., W.P. and M.J.B. designed the experiments. X.W., H.-Y.C., D.M.L., S.H.H., D.O.O., V.S.-T. and C.Q. performed the in vivo and in vitro assays for the functional studies of GhoT and GhoS and for the regulation of the ghoST operon. K.Z. and D.M.L. purified GhoS, and D.M.L. and W.P. completed the NMR structure with help from T.H. with the structure calculations and refinement. T.K.W. and X.W. authored the nonstructural parts of the manuscript, and R.P. and W.P. wrote the structural sections. All authors discussed the results and commented on the manuscript.

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Correspondence to Rebecca Page or Thomas K Wood.

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Wang, X., Lord, D., Cheng, HY. et al. A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nat Chem Biol 8, 855–861 (2012). https://doi.org/10.1038/nchembio.1062

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