Supported by: Paper citation: Payne, L.J., Todeschini, T.C., Wu, Y., Perry, B.J., Ronson, C.W., Fineran, P.C., Nobrega, F.L., Jackson, S.A. (2021) Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Research, 49, 10868-10878. • We have developed PADLOC, a tool to identify many widespread defence systems, including recently discovered systems. • Using PADLOC, we have discovered additional proteins frequently associated with several known defence systems and classified these into new system subtypes. • We have identified several phages that are inhibited by these new defence systems. • PADLOC is available as a standalone package (https://github.com/padlocbio/ padloc) and as a webserver (https://padloc.otago.ac.nz), which allow analysis of user-supplied genomes. Summary • When expressed heterologously in E. coli, the new systems limit phage infection. • Several phages exhibited reduced ability to form plaques on bacterial lawns (efficiency of plaquing; EOP) and collapse of liquid culture was prevented at a multiplicity of infection (MOI) below 0.1. New systems provide defence against phage infection Clone defence system Transform into E. coli Infect with phage Measure infection efficency Control Zorya III Hachiman II Lamassu II EOP relative to control (%) Control Zorya III Hachiman II Lamassu II Phage T1 T3 T4 T7 LambdaVir PVP-SE1 0 20 40 60 80 100 120 *** * *** ** ** * ** * ** ********* 0 300 600 900 0.0 0.5 1.0 1.5 2.0 2.5 MOI = 0 OD600 0 300 600 900 MOI = 0.01 Time (min) 0 300 600 900 MOI = 0.1 strong weak Identify loci with at least two genes of the same system Cluster genes in the neighbourhood of defence loci Assess the frequency of association Genomes 1 2 3 n Select novel variants with frequently associated genes 36,395 loci 41,470 genomes 73,063 clusters 225,898 genes 6 variants + 1 new system 730,430 associations ThsA ThsB HamA HamB PtuA PtuB ZorA1 ZorB1 LmuA LmuB ThsC HamC LmuC DruE2 DruF2 PtuC ZorG ZorF DruL Canonical Associated Association frequency 0.5 0.75 1.0 • By analysing these associations, we discovered several new system subtypes and a novel system comprising a Helicase, Methylase and ATPase. • To demonstrate how PADLOC can be used to address biological questions, we systematically identified proteins frequently associated with known systems. Identification and classification of new defence systems • Shown is a subset of all defence systems identified with PADLOC in ~200,000 RefSeq genomes. • More than 100 types of defence systems can currently be identified using PADLOC, including CRISPR-Cas, Abi, and many recently discovered systems. 0 2.5 5 7.5 >10 Average percentage of genomes in each species encoding a system Bacteria Archaea 10 1 1 1 1 6 4 4 6 1 2 1 2 2 3 2 1 6 1 15 5 1 12 3 8 2 16 5 3 14 5 9 3 5 5 3 3 5 11 2 2 3 1 2 12 3 1 4 1 1 7 7 1 1 12 14 1 1 4 21 17 9 17 34 13 3 3 9 1 2 1 21 8 2 4 2 1 22 7 4 5 1 2 5 5 16 5 2 7 5 5 20 8 1 2 4 6 4 1 1 13 2 1 1 1 2 15 3 2 2 8 5 24 1 6 3 15 6 10 3 1 2 2 3 3 1 8 1 31 1 8 1 1 13 13 13 3 8 3 19 2 3 9 7 1 2 1 2 17 7 6 4 1 1 13 3 3 11 7 2 7 4 3 2 4 5 3 36 5 14 14 8 8 3 1 2 1 1 1 1 16 4 1 4 2 5 1 3 4 3 8 10 1 8 4 3 1 1 16 7 1 5 3 10 6 7 12 9 13 1 1 13 1 2 2 2 16 11 11 5 2 6 9 4 36 20 15 2 9 3 2 1 16 6 3 3 3 12 1 5 6 9 3 6 3 22 22 25 1 1 9 5 5 10 1 5 37 5 3 5 1 6 12 4 5 4 32 4 11 1 4 7 15 29 38 17 4 4 9 10 12 4 6 59 19 2 1 13 6 4 17 12 1 12 12 6 6 12 12 12 11 5 3 2 3 10 12 12 6 3 15 9 21 11 7 3 4 1 1 1 3 1 20 7 1 3 4 3 1 16 1 6 3 1 8 1 1 5 3 11 4 2 2 6 9 2 3 1 4 4 3 11 5 3 5 12 10 3 6 2 13 18 1 3 6 18 14 8 2 2 2 8 2 2 12 4 4 Thermoproteota Thermoplasmatota Methanobacteriota Halobacteriota Verrucomicrobiota_A Verrucomicrobiota Thermotogota Synergistota Spirochaetota Proteobacteria Planctomycetota Patescibacteria Nitrospirota Myxococcota Marinisomatota Fusobacteriota Firmicutes_F Firmicutes_E Firmicutes_C Firmicutes_B Firmicutes_A Firmicutes Fibrobacterota Desulfobacterota Deinococcota Deferr