Tatsuaki Kurata, a , 1 Chayan Kumar Saha, a , b , 1 Jessica A. Buttress, c Toomas Mets, d Tetiana Brodiazhenko, d Kathryn J. Turnbull, e Ololade F. Awoyomi, f Sofia Raquel Alves Oliveira, d Steffi Jimmy, g Karin Ernits, h Maxence Delannoy, i Karina Persson, h Tanel Tenson, d Henrik Strahl, c Vasili Hauryliuk, a , b , d , j , 2 and Gemma C. Atkinson a , b , 2


"aDepartment of Experimental Medicine, University of Lund, 221 84 Lund, Sweden;
bDepartment of Molecular Biology, Umeå University, 901 87 Umeå, Sweden;
cCenter for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne NE2 4AX, United Kingdom;
dUniversity of Tartu, Institute of Technology, 50411 Tartu, Estonia;
eDepartment of Clinical Microbiology, Rigshospitalet, 2200 Copenhagen, Denmark;
fDepartment of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
gCenter for Structural Systems Biology, Deutsches Elektronen-Synchrotron, 22607 Hamburg, Germany;
hDepartment of Chemistry, Umeå University, 901 87 Umeå, Sweden;
iDépartement Génie Biologique, Campus SophiaTech, Université Nice Sophia Antipolis, 06000 Nice, France;
jLaboratory for Molecular Infection Medicine Sweden, Umeå University, 901 87 Umeå, Sweden
2To whom correspondence may be addressed. Email: [email protected] or [email protected]
Edited by Gunnar von Heijne, Department of Biochemistry and Biophysics, Stockholms Universitet, Stockholm, Sweden; received May 7, 2021; accepted December 2, 2021, by Editorial Board Member Robert J. Collier
Author contributions: T.K., C.K.S., T.M., K.P., T.T., H.S., V.H., and G.C.A. designed research; T.K., C.K.S., J.A.B., T.M., T.B., K.J.T., O.F.A., S.R.A.O., S.J., K.E., M.D., and H.S. performed research; C.K.S. contributed new reagents/analytic tools; T.K., C.K.S., T.M., H.S., V.H., and G.C.A. analyzed data; and V.H. and G.C.A. wrote the paper.

1T.K. and C.K.S. contributed equally to this work."


Medical Research, Molecular Biology


"Toxin–antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

DOI: 10.1073/pnas.2102212119


Microscopy was performed using a Nikon Eclipse Ti equipped with a Nikon Plan Apo 100×/1.40 Oil Ph3 objective, CoolLED pE-4000 light source, Photometrics BSI sCMOS camera, and Chroma 49002 (excitation [EX] 470/40, dichroic mirror [DM] 495 lpxr, and emission [EM] 525/50) and Semrock Cy5-4040C (EX 628/40, DM 660 lp, EM 692/40) filter sets.

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