
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.26.609590
preprint
1
Article
AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid
Chen Daphne F. http://orcid.org/0000-0002-5571-3548

Roe Leah T. http://orcid.org/0000-0002-2487-5587

Li Yuping http://orcid.org/0000-0003-2057-3271

Borges Adair L. http://orcid.org/0000-0002-1477-7908

Zhang Jenny Y.
Babbar Palak
Maji Sourobh
Stevens Maisie G.V. http://orcid.org/0009-0004-9732-0349

Correy Galen J. http://orcid.org/0000-0001-5155-7325

Diolaiti Morgan E. http://orcid.org/0000-0001-5900-3060

Smith Dominique H.
Ashworth Alan http://orcid.org/0000-0003-1446-7878

Stroud Robert M. http://orcid.org/0000-0003-2083-5665

Kelly Mark J.S. http://orcid.org/0000-0003-3209-1018

Bondy-Denomy Joseph http://orcid.org/0000-0002-4909-9481

Fraser James S. http://orcid.org/0000-0002-5080-2859

26 8 2024
2024.08.26.609590https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.08.26.609590
nihpp-2024.08.26.609590.pdf
Abstract

Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems to allow phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors, and while enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present an NMR structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa . Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that diverse macrodomains can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11’s enzymatic activity to its potent and specific effects in vivo , clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.
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