ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/046/ERR11749546/ERR11749546.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/035/ERR11749535/ERR11749535.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/043/ERR11749543/ERR11749543.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/041/ERR11749541/ERR11749541.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/037/ERR11749537/ERR11749537.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/038/ERR11749538/ERR11749538.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/044/ERR11749544/ERR11749544.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/042/ERR11749542/ERR11749542.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/036/ERR11749536/ERR11749536.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/039/ERR11749539/ERR11749539.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/040/ERR11749540/ERR11749540.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/047/ERR11749547/ERR11749547.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/045/ERR11749545/ERR11749545.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR117/034/ERR11749534/ERR11749534.fastq.gzprimaryOK200GenomicsUniversity of LiverpoolLIVhttps://www.ebi.ac.uk/ena/browser/view/PRJEB61545The constant arms race between bacteria and their phages has resulted in a large diversity of bacterial defence systems1,2, with many bacteria carrying several systems3,4. In response, phages often carry counter-defence genes5–9. If and how bacterial defence mechanisms interact to protect against phages with counter-defence genes remains unclear. Here, we report the existence of a novel defence system, coined MADS (Methylation Associated Defence System), which is located in a strongly conserved genomic defence hotspot in Pseudomonas aeruginosa and distributed across Gram-positive and Gram-negative bacteria. We find that the natural co-existence of MADS and a Type IE CRISPR-Cas adaptive immune system in the genome of P. aeruginosa SMC4386 provides synergistic levels of protection against phage DMS3, which carries an anti-CRISPR (acr) gene. Previous work has demonstrated that Acr-phages need to cooperate to overcome CRISPR immunity, with a first sacrificial phage causing host immunosuppression to enable successful secondary phage infections10,11. Modelling and experiments show that the co-existence of MADS and CRISPR-Cas provides strong and durable protection against Acr-phages by disrupting their cooperation and limiting the spread of mutants that overcome MADS. These data reveal that combining bacterial defences can robustly neutralise phage with counter-defence genes, even if each defence on its own can be readily by-passed, which is key to understanding how selection acts on defence combinations and their coevolutionary consequences. doi:10.1101/2023.03.30.534895ENAfalseMethylation Associated Defence System in Pseudomonas aeruginosa SMC4386 strain using PacBio dataMethylation Associated Defence System2023-04-282023-04-28PRJEB61545