To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

Anti-phage defence systems in bacterial genomes have a vast intra- and inter-species diversity. Here we investigated whether combinations of defence systems are selected for or against due to the effects of anti-phage protection. Analysis of defence system co-occurrence in Escherichia coli genomes revealed that positive and negative co-occurrences are common, and surprisingly both can provide synergistic anti-phage activities. Furthermore, we did not find conserved patterns of defence system co-occurrence across bacterial taxa. This suggests that the selection of strains carrying defence system combinations is more likely a consequence of environmental conditions than mechanistic incompatibilities between defence systems. Additionally, we showed that Gabija and Zorya type II can sense the anti-restriction protein Ocr to activate defence, and that phages also make use of these shared sensing mechanisms to evade multiple defence systems with a single escape strategy. Together, our results highlight the complex co-evolutionary dynamics of phage defence and anti-defence.

Bacteria have evolved numerous defence systems to combat phage infection, but our understanding of the underlying mechanisms is limited beyond their antiviral function. Here, we provide mechanistic insights into the Kiwa defence system, which is composed of two proteins, the transmembrane protein KwaA and the DUF4868 domain-containing protein KwaB. Using Kiwa from Escherichia coli ECOR8 and ECOR12, we found that KwaA detects phage infection by sensing inhibition of the host RNA polymerase. Consequently, the inhibitory effect of KwaA on the effector protein KwaB is relieved, which results in decreased phage DNA replication efficiency through a RecBCD-dependent pathway. Notably, Kiwa restores RecBCD activity even against phages that encode anti-RecBCD proteins. Our findings also reveal that KwaB has antiphage activity in the absence of both KwaA and phage trigger, but the conserved two-protein Kiwa operon implies that strict regulation of KwaB activity by KwaA is necessary to prevent potential auto-immunity.