A nucleotide-sensing endonuclease from the Gabija bacterial defense system

Abstract

The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.

Recruitment of Mobile Genetic Elements for Diverse Cellular Functions in Prokaryotes

“…Similarly, OLD is the archetypical member of a family of ABC-ATPases fused to a TOPRIM nuclease domain that is found in diverse defense contexts ( Aravind et al, 1998 ; Krishnan et al, 2020 ). In combination with a UvrD-family helicase and RNAseH-family exonuclease, OLD is part of a widespread system that provides cells with innate immunity against several distinct bacteriophages ( Doron et al, 2018 ; Cheng et al, 2021 ). The old and fun superinfection exclusion genes are just two examples of innate immune systems found in prokaryotic genomes associated with mobile genetic elements ( Makarova et al, 2013 ), reflecting a broad evolutionary pattern of continuous back and forth gene shuffling.…”

A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host

“…We propose a model where NixI functions as a nicking endonuclease, introducing site-specific nicks into ICP1's genome. Anti-phage defense systems that encode nicking endonucleases have recently been characterized suggesting nicking is an effective strategy to destroy invading viral DNA (32,53). We show here that NixI is the most inhibitory PLE-encoded product characterized thus far, inhibiting ICP1 progeny production to the same extent as is seen with PLE, and reducing ICP1 replication by an order of magnitude.…”

“…As HNH nucleases require a divalent metal co-factor for activity (typically magnesium, manganese or zinc (28-31)), we investigated NixI's metal requirement with a panel of divalent metals at differing concentrations and determined NixI can utilize Mg 2+ or Mn 2+ for catalysis, but not Zn 2+ or Ca 2+ (Supplementary Figure S1). Although Mn 2+ can stimulate nuclease activity of NixI, work with other HNH nucleases, including a sequence specific nicking endonuclease (32), has shown that Mn 2+ can replace Mg 2+ , leading to non-specific activity (33,34). Therefore, we carried out our nuclease reactions in a buffer supplemented with 10mM Mg 2+ .…”

Comparative Genomics of Xylella fastidiosa Explores Candidate Host-Specificity Determinants and Expands the Known Repertoire of Mobile Genetic Elements and Immunity Systems

“…The Gabija system is composed of the GajA and GajB proteins that contain ATPase, nuclease and helicase domains [ 73 , 94 ]. Gabija gives immunity to various phages when it was cloned in B. subtilis , and to phage T7 when it was cloned in E. coli [ 73 ].…”