{"paper_id":"050dad19-7dab-4917-b673-dc729bb591dc","body_text":"1 \nPglZ from Type I BREX phage defence systems is a metal -dependent nuclease 1 \nthat forms a sub-complex with BrxB 2 \nJennifer J. Readshawa,*, Lindsey A. Doyleb,*, Maria Puiua, Abigail Kellya, Andrew Nelsonc, Alex J. Kaiserb, 3 \nSydney McGuired, Julieta Peralta-Acostad, Darren L. Smith c, Barry L. Stoddardb, Brett K. Kaiser d,†, Tim 4 \nR. Blowera,e,†  5 \n 6 \naDepartment of Biosciences, Durham University, Stockton Road, Durham, DH1 3LE, UK. 7 \nbDivision of Basic Sciences, Fred Hutchinson Cancer Center, 1100 Fairview Ave. N. Seattle WA 98019, 8 \nUSA. 9 \ncDepartment of Applied Sciences, University of Northumbria, Newcastle Upon Tyne NE1 8ST, UK. 10 \ndDepartment of Biology, Seattle University, 901 12th Ave. Seattle WA 98122, USA. 11 \neNew England Biolabs, 240 County Road, Ipswich, MA 01938, USA. 12 \n*These authors contributed equally. 13 \n†To whom correspondence may be addressed. Email: kaiserb@seattleu.edu, 14 \ntimothy.blower@durham.ac.uk, tblower@neb.com. 15 \n 16 \nKeywords: Phage defence, Bacteriophage exclusion, nuclease, metal-dependent, BREX 17 \n 18 \n 19 \n 20 \n 21 \n 22 \n 23 \n 24 \n 25 \n 26 \n 27 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n2 \nABSTRACT  28 \nBREX (Bacteriophage Exclusion) systems, identified through shared identity with  Pgl (Phage Growth 29 \nLimitation) systems, are a widespread, highly diverse group of phage defen ce systems found 30 \nthroughout bacteria and archaea. The varied BREX Types harbour multiple protein subunits (between 31 \nfour and eight) and all encode a conserved putative phosphatase (PglZ aka BrxZ) and an equally 32 \nconserved, putative ATPase (BrxC).  Almost all BREX systems also contain a site -specific 33 \nmethyltransferase (PglX aka BrxX). Despite having determined the structure and fundamental 34 \nbiophysical and biochemical behavio urs for the PglX methyltransferase, the BrxL effector, the BrxA 35 \nDNA-binding protein and the BrxR transcriptional regulator, the mechanism by which BREX impedes 36 \nphage replication remains largely undetermined. In this study, we identify a stable BREX sub-complex 37 \nof PglZ:BrxB, validate the structure and dynamic behavio ur of that sub-complex, and assess the 38 \nbiochemical activity of PglZ, revealing it to be a metal -dependent nuclease. PglZ can cleave cyclic 39 \noligonucleotides, linear oligonucleotides, plasmid DNA and both non -modified and modified linear 40 \nphage genomes. PglZ nuclease activity has no obvious role in BREX-dependent methylation, but does 41 \ncontribute to BREX phage defence. BrxB binding does not impact PglZ nuclease activity. These data 42 \ncontribute to our growing understanding of the BREX phage defence mechanism. 43 \n  44 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n3 \nINTRODUCTION  45 \nUp to 10% of bacterial and archaeal genes are dedicated to phage defen ce (1). The mechanisms 46 \nemployed to defend against phage infection are diverse and include prevention of viral entry, 47 \ninduction of cell dormancy or death upon infection, and mechanisms that degrade viral genomes or 48 \nblock viral DNA replication  (2, 3). Phages combat these systems through the evolution of elaborate 49 \ncountermeasures that block their action, leading to viral resistance and a continuous arms race 50 \nbetween phage and bacterial population s (4). Recent analyses have demonstrated that bacteria 51 \nencode far more phage defence systems than just the most well-studied ‘first responder’ systems such 52 \nas restriction endonucleases and CRISPR  (5–10). Furthermore, many of these newly discovered 53 \nbacterial systems display obvious similarities to human innate viral defense systems, implying 54 \ncommon evolutionary origins and related mechanisms of action (11). 55 \n 56 \nOriginally discovered in the early 1980s  (12), Phage Growth Limitation (Pgl) (13, 14)  and 57 \nrelated Bacteriophage Exclusion (BREX) systems are widespread in bacterial and archaeal species (15). 58 \nBREX systems are encoded by single operons, often within genetic defence islands, and are currently 59 \ncategorised into at least six types based on the number of genes in each system (typically four to eight) 60 \nand on the sequence -based functional annotation of those individual genes and putative translated 61 \nprotein subunits (15); Type I systems, the most common subtype, comprise six conserved genes and 62 \ncan readily be assayed for the two phenotypes of phage defence and BREX -dependent methylation 63 \nthough the mechanisms are unknown. 64 \n 65 \nPgl and BREX systems have two genes in common. The first is named pglZ (aka brxZ) and the 66 \nsecond is brxC (15). The PglZ domain of a two-component signalling system response regulator, PorX, 67 \nhas recently been shown to degrade cyclic nucleotides (16), but any equivalent activity within BREX 68 \nhas not yet been explored. Beyond PglZ and BrxC, most BREX systems include a gene encoding a site-69 \nspecific methyltransferase, termed PglX (aka BrxX). The identity and order of the remaining genes in 70 \neach BREX type vary significantly: various BREX systems encode protein subunits with domains that 71 \ndisplay recognisable homology to kinases, phosphatases, DNA and/or nucleotide binding domains, 72 \nDNA modification enzymes, chambered AAA+ ATPases, and/or DNA helicases. Several BREX subunits 73 \nare quite large, with significant regions of unknown structure -function properties and behavio urs 74 \nflanking domains with well -annotated putative functions. Type I BREX systems, like their related 75 \ncounterparts, do not contain any readily identifiable DNA nuclease domains or subunits and appear 76 \nto likely restrict phage by inhibiting phage DNA replication within the infected bacterial cell. 77 \n 78 \nDespite having previously determined the high resolution structures and biochemical 79 \nactivities of the PglX methyltransferase (17, 18),  BrxR (a WYL -domain helix-turn-helix DNA binding 80 \ntranscriptional regulator) (19, 20), BrxA (a small DNA binding protein)  (21), and BrxL (a chambered 81 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n4 \nAAA+ ATPase and dsDNA binding protein) (22), the mechanism by which BREX systems function to 82 \nrestrict phage replication and to protect the host genome from the system’s activit y is still largely 83 \nunknown. We have performed analyses to further characterise several Type I BREX systems we have 84 \npreviously investigated: those from Salmonella Typhimurium (17, 23) and Escherichia fergusonii (24) 85 \n(Fig. 1A), and from Acinetobacter (20, 22). Using these systems, in vivo pull-down and co-expression 86 \nanalysis identified larger BREX compl exes and a stable sub -complex formed by PglZ and BrxB. 87 \nComputational models of the PglZ:BrxB interactions and their likely conformation and dynamic 88 \nbehaviour when bound to one another was validated through single -particle cryoEM analy ses. 89 \nSubsequent biochemical analysis has identified that PglZ recapitulates PorX activity and is a metal -90 \ndependent nuclease that can cleave not only a broad range of cyclic and linear oligonucleotides, but 91 \nalso plasmid and linear dsDNA. The BrxB interaction does not impact PglZ nuclease activity, nor is a 92 \nnuclease required for BREX -dependent methylation. Nuclease activity does, however, contribute to 93 \nBREX phage defence. These data contribute to our growing understanding of the elusive BREX 94 \nmechanism.     95 \n 96 \n 97 \n 98 \n 99 \n 100 \n 101 \n 102 \n 103 \n 104 \n 105 \n 106 \n 107 \n 108 \n 109 \n 110 \n 111 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n5 \n 112 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n6 \nMATERIALS AND METHODS   113 \nBacterial strains and culture conditions 114 \nE. coli strains DH5α (Invitrogen), ER2796 (New England Biolabs) (25), ER2566 (New England Biolabs), 115 \nRosetta 2 (DE3) pLysS (Novagen) , BL21 (DE3) pRARE (Novagen) , BL21 (DE3) RIL (Novagen)  and T7 116 \nExpress (New England Biolabs) were routinely grown at 37 C, either on agar plates or shaking at 150 117 \nrpm for liquid cultures. 2x Yeast Extract Tryptone (YT) was used as the standard growth media for 118 \nliquid cultures, and Luria Broth (LB) was supplemented with 0.35% (w/v) or 1.5% (w/v) agar for semi-119 \nsolid and solid agar plates, respectively. When necessary, growth media was supplemented with 120 \nampicillin (Ap, 100 μg/ml), chloramphenicol (Cm, 25 μg/ml), kanamycin ( Km,  100 μg/mL, 121 \nspectinomycin ( Sp, 100 μg/mL), isopropyl--D-thiogalactopyranoside (IPTG, 1 mM). Growth was 122 \nmonitored using a spectrophotometer (WPA Biowave C08000) measuring optical density at 600 nm 123 \n(OD600).  124 \n 125 \nDNA isolation and manipulation 126 \nPlasmid DNA was purified from transformed DH5 cells using an NEB Monarch® Plasmid MiniPrep kit 127 \nfollowing the manufacturer’s instructions. Larger amounts of negatively supercoiled plasmid pSG483 128 \n(26) DNA for assays was purified from transformed DH5  cells using a Machery -Nagel NucleoBond 129 \nXtra Midi Plus EF kit following the manufacturer’s instructions. Plasmid  DNA was eluted in MiliQ and 130 \nstored at -20 C. Plasmids are described in Supplementary Table S1.  131 \n 132 \nPhage genomic DNA was purified by incubating 450 μl phage lysate with 4.5 μl DNase I (1 133 \nmg/ml; Sigma-Aldrich) and 12.5 μl RNase A (10 mg/ml; ThermoFisher) for 30 min at 37 C. The lysate 134 \nwas further incubated with 2.25 μl proteinase K (20 mg/ml; Sigma-Aldrich) and 23 μl of 10% (w/v) SDS 135 \nfor 30 min at 37 C. The sample was mixed with 500 μl UltraPure phenol:chloroform:isoamyl alcohol 136 \n(25:24:1; v/v/v) (ThermoFisher) and centrifuged at 16,000 x g for 5 min at 4 C. The aqueous layer was 137 \nremoved carried forward, and the previous step was repeated. The resulting aqueous layer was mixed 138 \nwith 500 μl chloroform:isoamyl alcohol (24:1; v/v) and centrifuged at 16,000 x g for 5 min at 4 C. The 139 \naqueous layer was carried forward and incubated with 45 μl 3 M sodium acetate pH 5.2 and 500 μl 140 \nisopropanol for 15 min at room temperature, before being centrifuged at 16,000 x g for 20 min and 4 141 \nC. The supernatant was removed, and the pellet was washed with 70% ethanol by gentle aspiration 142 \nbefore being dried at room temperature. The dry pellet was soaked in 50 μl of MiliQ and incubated 143 \novernight at 4 C. The gDNA was analysed on a 0.75% 1x TAE agarose gel by agarose gel 144 \nelectrophoresis, and stored at -20 C.  145 \n 146 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n7 \nPreparation of nicked, linear, and relaxed form pSG483 147 \nLinear pSG483 was obtained through incubation of 10 μg pSG483 with 10 units of BamHI -HF® (New 148 \nEngland Biolabs) in 1x CutSmart buffer (New England Biolabs) for 1 h at 37 C. The enzyme was 149 \ndeactivated by incubation at 65 C for 10 min. Nicked pSG483 was obtained by incubating 10 -50 μg 150 \npSG483 with 10 units Nb.Bpu10I (ThermoFisher) in 1x Buffer R ( ThermoFisher) for 4 h at 37 C. The 151 \nreaction was terminated by incubation at 80 C for 20 min.  152 \n 153 \nFor production of relaxed pSG483, 50 μg nicked pSG483 was further incubated with 1 mM ATP 154 \nand 10 units T4 ligase ( New England Biolabs) for 16 h at room temperature. After ligation, an equal 155 \nvolume of UltraPure phenol:chloroform:isoamyl alcohol (25:24:1; v/v/v) (ThermoFisher) was added 156 \nto the reaction mixture, vortexed briefly, and centrifuged at 16 ,000 x g for 2 min. The resulting 157 \naqueous layer was removed and carried forward. An equal volume of chloroform was added to the 158 \naqueous layer before centrifugation at 16,000 x g for 2 min. The resulting aqueous layer was carried 159 \nforward and 1/10 volume of 3 M sodium acetate pH 5.2 was added, followed by 2 volumes of 100% 160 \nethanol. The sample was mixed by pipetting and stored at -80 C for 30 min. The sample was 161 \ncentrifuged at 16,000 x g for 20 min at 4 C. The ethanol was removed, and the DNA pellet dried at 162 \nroom temperature. The DNA pellet was resuspended to 300 ng/μl with MiliQ. All DNA products were 163 \nanalysed by agarose gel electrophoresis prior to storage at -20 C. 164 \n  165 \nBacterial growth assays 166 \nT7 Express E. coli cells were transformed with: empty vectors pETDuet, pCDFDuet and pCOLADuet (i), 167 \npTRB710 (His-SUMO-BrxB and PglZ (BZ)) (ii), pTRB759 (BrxC and PglX (CX)) (iii), pTRB758 (BrxA and 168 \nBrxL (AL)) (iv) and combinations of BZ/CX (v), BZ/AL (vi), CX/AL (vii) and BZ/CX/AL (viii). Colonies were 169 \ninoculated and grown overnight in 5 ml 2x YT with respective antibiotics at 37 C shaking at 180 rpm. 170 \nCultures were re-seeded 1:100 (v/v) in 100 ml 2x YT with the relevant antibiotics and grown at 37 C 171 \nuntil OD600 reached ~0.4. ODs of all cultures were then normalised to OD600 of ~1.0. Cultures were then 172 \nserially diluted 10-1 to 10-7 and spotted on LB agar plates containing the relevant antibiotics +/ - IPTG 173 \nfor induction of each complex combination. Plates were then incubated overnight at 37 C and imaged 174 \nfor colony counting and CFU/ml determination. 175 \n 176 \nProtein expression and purification 177 \nFor large-scale expression of Salmonella PglZ or co-expression of Salmonella His-SUMO-BrxB and PglZ, 178 \nE. coli ER2566 was transformed with pSALMZ and pTRB710, respectively. For large-scale expression of 179 \nE. fergusonii  proteins for biochemistry, E. coli ER2566 was transformed with pTRB449 (PglZ) and 180 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n8 \npTRB444 (BrxB). E. coli Rosetta (DE3) pLysS was also transformed with pTRB444. E. fergusonii mutant 181 \nderivatives were expressed by transforming E. coli ER2566 with plasmids pTRB729 ( PglZ T538A), 182 \npTRB730 (PglZ H741A), pTRB763 ( PglZ T538A/H741A), pTRB727 ( BrxB W135A), and pTRB726 ( BrxB 183 \nR46A), and transforming E. coli Rosetta (DE3) pLysS with pTRB724 (BrxB E47A), pTRB725 (BrxB S133A), 184 \npTRB726 (BrxB R46A), pTRB727 (BrxB W135A), and pTRB728 (BrxB E89A).  185 \n 186 \nThe same procedures were used for both Salmonella and E. fergusonii proteins. Single colonies 187 \nwere used to inoculate 70 ml 2x YT for overnight growth at 37 C shaking at 180 rpm. Starter cultures 188 \nwere re-seeded 1:100 (v/v) into 1 L 2x YT containing the relevant antibiotic(s) in 2 L baffled flasks and 189 \nincubated at 37 C until the OD600 reached ~0.4. At this point, the incubation temperature was reduced 190 \nto 18  C for overnight incubation and expression was induced with IPTG. Cells were harvested by 191 \ncentrifugation at 4,200 x g for 20 min at 4 C. Cell pellets were resuspended on ice in ice -cold A500 192 \n(20 mM Tris HCl pH 7.9, 500 mM NaCl, 10% (v/v) glycerol, 10 mM imidazole). Resuspended cells were 193 \ndisrupted by sonication (45% amplitude, 10 s on 20 s off pulse intervals, 2 min) and clarified by 194 \ncentrifugation at 45,000 x g for 45 min at 4 C. Clarified cell lysate was loaded onto a 5 ml HisTrap HP 195 \ncolumn (Cytiva) pre-equilibrated in A100 (20 mM Tris HCl pH 7.9, 100 mM NaCl, 10% (v/v) glycerol, 10 196 \nmM imidazole). The HisTrap column was then washed with 50 ml A100, and bound proteins were 197 \neluted directly onto a pre-equilibrated 5 ml HiTrap Q HP column using B100 (20 mM Tris HCl pH 7.9, 198 \n100 mM NaCl, 10% (v/v) glycerol, 250 mM imidazole). The Q HP column was washed with 50 ml A100 199 \nand transferred to an  Åkta Pure (Cytiva), and the target protein was eluted by anion exchange 200 \nchromatography (AEC) using a salt gradient from 100% A100 to 60% C1000 (20 mM Tris HCl pH 7.9, 1 201 \nM NaCl, 10% (v/v) glycerol). Chromatographic peak fractions were collected, pooled, and incubated 202 \novernight in the presence of human sentrin/SUMO -specific protease 2 (hSENP2) to facilitate the 203 \ncleavage of the His-SUMO tag at 4 C. The following day, the SENP -treated sample was applied to a 204 \nsecond His-Trap HP column pre -equilibrated in A100. The flow -through containing untagged target 205 \nprotein was collected and concentrated by centrifugation using the appropriate MWCO Vivaspin 206 \nconcentrator (Sartorius). Concentrated protein samples were applied to a HiPrep 16/60 Sephacryl® 207 \nS-200 HR column (S -200; Cytiva) pre -equilibrated with 1.2 column volumes (CV) of sizing buffer (50 208 \nmM Tris HCl pH 7.9, 500 mM KCl, 10% (v/v) glycerol) for further purification by size exclusion 209 \nchromatography (SEC). SEC peak fractions were pooled and analysed by SDS-PAGE, then concentrated 210 \nas described previously and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher). 211 \nFinal purified samples for biochemical analysis were resuspended in a 1:2 mixture of protein 212 \nsample:storage buffer (50 mM Tris HCl pH 7.9, 500 mM KCl, 70% (v/v) glycerol) and flash frozen in 213 \nliquid nitrogen for storage at -80 C.  214 \n 215 \nAcinetobacter proteins were expressed as follows: plasmids encoding PglZAci with a C-terminal, 216 \nthrombin-cleavable Twin-Strep Tag (pET15b.PglZ-TST) and untagged BrxBAci (pET24d.BrxB) were used 217 \nto co-transform E. coli BL21 (DE3) RIL. Overnight cultures (25 ml, LB) were diluted 100-fold into 1 L of 218 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n9 \nLB and grown to an OD 600 of 0.6, at which time IPTG was added to 200 µM. Cultures were incubated 219 \nat 16 °C for 18 h, pelleted by centrifugation at 4,200 x g for 45 min, and the pellets were stored at -20 220 \n°C. Pellets were lysed in Buffer W (100 mM Tris -HCl pH 8.0, 150 mM NaCl , 1 mM EDTA), centrifuged 221 \nfor 25 min at 18,000 x g in an SS34 rotor at 4  °C, and the supernatant was filtered through a 5 µm 222 \nsyringe filter. The soluble lysate was bound to streptactin resin, washed with 10 CV of Buffer W and 223 \neluted in Buffer E (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin). Samples 224 \nwere concentrated in a 30 kDa MWCO Amicon filter (EMD Millipore) and purified by SEC on a SEC650 225 \ncolumn (BioRad) equilibrated in 25 mM Tris-HCl pH 7.5, 200 mM NaCl.  226 \n 227 \nSDS-PAGE electrophoresis 228 \nProtein samples were analysed by SDS -PAGE. For protein purity analysis, 4 μg of PglZ and derivative 229 \nmutants, and 4 μg of BrxB and derivative mutants were made up to 10 μl with A100 and mixed with 5 230 \nμl 3x sample buffer (187.5 mM Tris HCl pH 6.8, 6% (w/v) SDS, 30% (v/v) glycerol, 0.03% (w/v) 231 \nbromophenol blue, 150 mM DTT) and denatured for 10 min at 95 C. Protein samples were loaded 232 \nonto and resolved in 15% (v/v) and 12% (v/v) poly -acrylamide gels, respectively, in 1x Tris-glycine 233 \nrunning buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS) at 180 V for 1h 15 min. For PglZ and BrxB 234 \ninteraction analysis post analytical SEC, 30 μl of fractions for analysis were mixed with 6 μl of 6x sample 235 \nbuffer (375 mM Tris HCl pH 6.8, 12% (w/v) SDS, 60% (v/v) glycerol, 0.06% (w/v) bromophenol blue, 236 \n300 mM DTT) and resolved in 15% (v/v) poly-acrylamide gels as described previously. Gels were 237 \nstained with Quick Coomassie (Protein Ark) and destained with MiliQ. Gel images were obtained on a 238 \nChemiDoc Imaging System on the Coomassie brilliant blue setting (BioRad). 239 \n 240 \nProtein pull-down assays 241 \nHis-strep tagged BrxB (expressed from 2HR-T, addgene #29718) was used as bait for pull-down assays 242 \nof BREX components expressed from pCOLA DUET1 in E. coli BL21 (DE3) pRARE. Overnight cultures 243 \nwere used to inoculate 25 ml of 2xYT with the relevant antibiotics to OD 0.1 before growth at 37 oC 244 \n180 rpm to OD ~0.8. Cultures were induced with 1 mM IPTG and incubated at 18 oC with shaking 245 \novernight. Cells were harvested at 4,200 x g for 15 min before freezing at -70 oC. Pellets were defrosted 246 \nand resuspended in 10 ml 100 mM Tris pH 7.9 150 mM NaCl before sonication (5 min of 10 s pulses at 247 \n30% power). Lysates were clarified by centrifugation at 45,000 x g for 10 min at 4 C. Clarified lysates 248 \nwere incubated with 200 µl pre-equilibrated Strep-Tactin Sepharose High Performance resin (Cytiva) 249 \nat 4 oC for 90 min with rolling before application to a Proteus Mini Spin column (ProteinArk). The resin 250 \nwas washed three times with 100 mM Tris pH 7.9 150 mM NaCl, before incubation of the resin in the 251 \ncolumn with 50 µl 100 mM Tris pH 7.9 150 mM NaCl, 2.5 mM desthiobiotin. The protein was eluted 252 \nfrom the column by centrifugation at 12,000 x g for 1 min . The 50 µl eluate was re -applied and re -253 \nincubated with the resin before a second centrifugal elution step. Pull-down products were separated 254 \nand visualised on a 4- 15% SDS-PAGE gel. 255 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n10 \n 256 \nHis-SUMO (27) tagged BrxB was used as bait for pull -down assays following induced 257 \nexpression of all BREX components. Plasmids were co-expressed in T7 E. coli cells for protein complex 258 \nformation in the following way: empty vector pETDuet1 as control for His-SUMO-BZ (i), empty vectors 259 \npETDuet1 and pCDFDuet1 as control for BZ/CX (ii), empty vectors pETDuet1, pCDFDuet1 and 260 \npCOLADuet1 as control for BZ/CX/AL (iii) BZ on its own (iv), BZ along with CX (v) and BZ with CX and 261 \nwith AL (vi). Single colonies were inoculated in 20 m l 2x YT for overnight growth at 37 C shaking at 262 \n180 rpm. Started cultures were then re-seeded into 1 L 2x YT containing the relevant antibiotic(s) in 2 263 \nL baffled flasks and incubated at 37 C until the OD600 reached ~0.4. Cultures were then incubated at 264 \n18 C overnight and expression was induced with IPTG. Cells were harvested by centrifugation at 4,200 265 \nx g for 20 min at 4 C. Cell pellets were resuspended on ice in ice-cold A500. Resuspended cells were 266 \ndisrupted by sonication (45% amplitude, 5 s on 10 s off pulse intervals, 5 min) and centrifuged at 267 \n45,000 x g for 30 min at 4 C. Clarified cell lysate was loaded onto a 5 m l HisTrap HP column (Cytiva) 268 \npre-equilibrated in A100. The HisTrap column was then washed with 50 m l A100, and transferred to 269 \nan Åkta Pure (Cytiva) for complex elution using an imidazole gradient from 10 mM imidazole to 250 270 \nmM using B100. Peak fractions were collected accordingly and concentrated by centrifugation using 271 \nthe MWCO Vivaspin concentrator (Sartorius) of appropriate size. Concentrated complexes were then 272 \nloaded on a HiPrep 16/60 Sephacryl® S-200 HR column (S-200; Cytiva) pre-equilibrated with 1.2 CV 273 \nof sizing buffer for size exclusion chromatography (SEC) purification. SEC peak fractions were pooled 274 \nand analysed by SDS -PAGE, then concentrated and quantified using a NanoDrop 2000 275 \nSpectrophotometer (Thermo Fisher). 276 \n 277 \nBis(4-nitrophenyl) phosphate phosphodiesterase activity assay 278 \nEDTA treated PglZ (PglZ EDTA) was prepared by incubating PglZ with 1 mM EDTA for 15 min at room 279 \ntemperature. The EDTA was removed by centrifuging in a 30 kDa MWCO Vivaspin ultrafiltration spin 280 \ncolumn (Cytiva) at 12,000 x g at 4 C, until the volume < 100 μl. The sample was resuspended in ~400 281 \nμl A100 and centrifuged again. This was repeated twice. PglZ and PglZ EDTA (2.2 μM) were incubated 282 \nwith 1x PglZ buffer (“ZB”: 50 mM Tris HCl pH 8.0, 150 mM NaCl) with (PglZ EDTA) or without 0.5 mM 283 \nMgCl2, MnCl 2, or CaCl 2, for 30 mins at room temperature. The phosphodiesterase reaction was 284 \ninitiated by adding 10 μl 25 mM bis(4-nitrophenyl)phosphate (bis-pNPP, Merck) to 90 μl PglZ reaction 285 \nmix, and monitoring the release of reaction product, p-nitrophenol, for 2 hours at 37 C by measuring 286 \nthe absorbance at 405 nm on a SPECTROstar® Nano microplate reader (BMG Labtech). PglZ  derived 287 \nmutants were also assayed for activity as described. Triplicate reactions were performed per assay, 288 \nand the assay was completed in triplicate. Control reactions comprised 1x ZB with and without 0.5 289 \nmM MgCl2, MnCl2, or CaCl2 in the presence of bis-pNPP.  290 \n 291 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n11 \nNucleotide cleavage assay 292 \nPglZ and derivative mutants (2 μM) were incubated with 10 μM ZnCl2 or MnCl2 in 1x ZB with 10 μM of 293 \nthe following nucleotides: cyclic hexa -adenosine monophosphate (cA6); cyclic tetra -adenosine 294 \nmonophosphate (cA4); cyclic tri-adenosine monophosphate (cA3); 3 ,5-cyclic di-adenylate (cA2); 5-295 \nphosphoadenylyl-(3-5)-adenosine (pApA); 5 -phosphoadenylyl-(3-5)-guanosine (pApG); 5 -296 \nphosphoguanylyl-(3-5)-guanosine (pGpG); 3 ,5-cyclic di -guanylate (cG2); 3 ,5-cyclic adenosine 297 \nmonophosphate (cAMP); 3 ,5-cyclic uridine monophosphate (cUMP); 3 ,5-cyclic thymidine 298 \nmonophosphate (cTMP); 3 ,5-cyclic cytidine monophosphate (cCMP); 3 ,5-cyclic guanosine 299 \nmonophosphate (cGMP); 2 3-cyclic uridine monophosphate (2 3 cUMP); 2 3-cyclic adenosine 300 \nmonophosphate (23 cAMP); 23-cyclic guanosine monophosphate (23 cGMP); cyclic adenosine-(3-301 \n5)-monophosphate adenosine -(3-5)-monophosphate guanosine -(3-5)-monophosphate 302 \n(c[A(35)pA(35)pG(35)p]); cyclic adenosine -(2-5)-monophosphate guanosine -(3-5)-303 \nmonophosphate (c[A(2 5)pG(35)p]); cyclic adenosine -(3-5)-monophosphate guanosine -(3-5)- 304 \nmonophosphate (c-ApGp); P1-(5-adenosyl) P4-(5-adenosyl) tetraphosphate (Ap4A); P 1-(5-adenosyl) 305 \nP4-(5adenosyl) triphosphate (Ap3A); or P 1-(5-adenosyl) P 4-(5- guanosyl) tetraphosphate (Ap4G). 306 \nReactions were carried overnight at 37 C in a total volume of 50 μl. PglZ (2 μM) was also incubated 307 \nunder the same conditions in the presence of BrxB (10 μM) and BrxB R46A (10 μM). Reactions were 308 \ncentrifuged at 12,000 x g for 10 min at 4 C to remove precipitants and 2 μl was loaded onto an Aeris 309 \n5 m PEPTIDE XB-C18 (150 x 4.6 mm) reversed phase high-performance liquid chromatography (HPLC) 310 \ncolumn (Phenomenex) at a flow rate of 1.5 ml/min and a linear gradient of 0-30% buffer 2 in 12 column 311 \nvolumes (CV), using buffer 1 (10 mM triethylammonium acetate pH 8.0) and buffer 2 (80% (v/v) 312 \nacetonitrile, 10 mM triethylammonium acetate pH 8.0) in 12 CV. P rotein sample in the absence of 313 \nnucleotide, and nucleotide in the absence of protein sample were used as controls. Standard mixes 314 \ncontained 10 μM of nucleotide(s) made up to 50 μl in 1x ZB and stored at 4 C.  315 \n 316 \nInductively coupled plasma mass spectrometry (ICP-MS) 317 \nTotal metal contents of protein samples were determined via ICP-MS (Thermo Scientific iCAP RQ ICP-318 \nMS) under KED mode (Kinetic Energy Discrimination) utilised with helium. Protein samples were 319 \ndiluted into 2.5% nitric acid containing 10 μg/l berylium, indium and silver as internal standards. 320 \nConcentrations determined via comparison to matrix-matched elemental standard solutions. 321 \n 322 \nAnalytical size exclusion chromatography  323 \nAnalytical size exclusion chromatography (SEC) was performed on an Åkta Pure FPLC system 324 \n(Cytiva). Protein samples were made up to 10 μM in a 100 μl final volume with analytical SEC buffer 325 \n(20 mM Tris HCl pH 7.9, 150 mM NaCl). BrxB was loaded onto a Superdex 75 increase 10/300 GL SEC 326 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n12 \ncolumn (S -75i; Cytiva). PglZ and derivative mutants were loaded onto a Superdex  200 increase 327 \n10/300 GL SEC column (S200i; Cytiva). PglZ and mutant derivatives pre -incubated with BrxB and 328 \nmutant derivatives at equimolar concentrations for 15 min at room temperature were also loaded 329 \nonto an S200i. All columns were pre -equilibrated with 1.2 CV analytical SEC buffer. Samples were 330 \nloaded onto a 100 μl capillary loop using a 100 μl Hamilton syringe. The loop was washed with 500 μl 331 \nnuclease-free water followed by 500 μl analytical SEC buffer before and between each run using a 500 332 \nμl Hamilton syringe. Samples were loaded onto the column by running 500 μl of analytical SEC buffer 333 \nthrough the capillary loop at a flow rate of 0.5 ml/min, and samples were resolved on the column 334 \nusing 1.2 CV analytical SEC buffer. In cases where the content of chromatogram peaks required 335 \nverification by SDS -PAGE or mass spectrometry, 0.5 ml fractionation was performed, and fractions 336 \nwere collected in 96-well deep-plate blocks.  337 \n 338 \nCalibration curves were generated by plotting the elution volumes ( Ve) of controls from 339 \ncalibration kits (GE healthcare) against their respective known molecular weights ( Mr). Calibration 340 \nsamples were prepared in 2 individual mixtures, Mix A (3 mg /ml RNase A, Ferritin, Conalbumin, 341 \nCarbonic Anhydrase) and Mix B (3 mg/ml RNase A, Aldolase, Aprotinin, 4 mg/ml Ovalbumin) and made 342 \nup to a final volume approximately equal to 0.5% geometric column volume. For determination of 343 \ncolumn void volume ( Vo), 1 mg /ml Blue Dextran was applied to the column as above, with elution 344 \nvolume directly proportional to Vo. Elution volumes (Ve) were calculated using the Peaks function in 345 \nUnicorn 7 (Cytiva) and converted to partitioning coefficients (Kav) using the following equation: 346 \n𝐾𝑎𝑣 =  𝑉𝑒 − 𝑉𝑜\n𝑉𝑐 − 𝑉𝑜\n 347 \nMolecular weight and Stokes radius calibration curves were subsequently plotted using Prism 348 \n(GraphPad) as Kav vs Log 10(Mr,kDa) and Log 10(Rst,Å) vs Kav, respectively. Observed Rst values were 349 \ngenerated by performing linear regression on respective plots using the following equations: 350 \n𝑀𝑟 = 10∧  (𝐾𝑎𝑣 − 𝑐\n𝑚 ) 351 \n 352 \n𝑅𝑠𝑡 = 10∧ ((𝑚(𝐾𝑎𝑣) + 𝐶) 353 \nObserved values were compared against calculated hydrodynamic radii. Radius calculations 354 \nof inputted AlphaFold predictive models were performed using the HullRad tool (Fluidic Analytics). 355 \n 356 \nMass photometry 357 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n13 \nMass Photometry experiments were undertaken on the TwoMP ( Refeyn)  instrument, using the 358 \nAcquire 2024R1.1 and Discover 2024R1.0 software for data acquisition and analysis , respectively. 359 \nDiscoverMP v2024 R2.1 was used to make figures. The autofocus function was used to find the focus 360 \nplane using 19 µl of PBS on uncoated glass slides (Refeyn). Thyroglobulin monomer and dimer peaks, 361 \nconalbumin and aldolase were used as standards. Stocks of each protein or complex to be tested were 362 \nprepared at 100  nM immediately before dilution 1:19 into PBS and collection of a 1 min video. 363 \nGaussian fits were used for most measurements, with PgZ:BrxB and PglZ:BrxBE74A measurements also 364 \nmaking use of the interval function. 365 \n 366 \nMass spectrometry 367 \nCollected BrxB peaks were buffer exchanged into 10 mM ammonium bicarbonate using a 10 kDa 368 \nMWCO spin concentrator and submitted for positive ion electrospray time -of-flight mass 369 \nspectrometry (ES+-ToF MS) at a final concentration of 0.5 mg /ml. Analysis was performed at our in-370 \nhouse Durham University Chemistry Department facility by Mr Peter Stokes using a Xevo QToF 371 \n(Waters, UK) mass spectrometer. 372 \n 373 \nRelevant protein bands were excised from SDS -PAGE gels for their identity to be confirmed 374 \nvia trypsin digest and mass spectrometry by Dr. Adrian Brown at the Department of Biosciences, 375 \nDurham University. 376 \n 377 \nPacific biosciences sequencing 378 \nLibraries for methylation sequencing were prepared using the SMRTbell HiFi 96 Prep kit  (Pacific 379 \nBiosciences). Bacterial gDNA was sheared using Qiagen Tissue Lyser II at 30 Hz for 240 s to produce 380 \nDNA fragments with a mean size of 8 –10 kb. The DNA was damage and end repaired. SMRT -bell 381 \nadapters were then ligated. Exonuclease treatment removed non-incorporated SMRT -bell DNA. 382 \nSequencing was performed on a PacBio Revio (Pacific Biosciences). Data were analysed using PacBio 383 \nSMRTAnalysis on SMRTLink_25.1 software Base Modification Analysis for Sequel data, to identify DNA 384 \nmodifications and their corresponding target motifs. 385 \n 386 \nThermal shift assays (TSAs) 387 \nThermal shift assays (TSAs )were performed to determine the ability of proteins to bind divalent 388 \nmetal cations. Samples of PglZ, PglZ incubated with 1 mM EDTA (PglZ + EDTA), and PglZ treated with 389 \nEDTA (removed, as described previously; PglZ - EDTA) were incubated with 4 x 10-3 μl SPYRO 390 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n14 \nOrange protein dye (ThermoFisher) per 1 μl protein for 1 h at 4 C. Reactions containing 5 μM 391 \nprotein in 1x ZB were incubated with (PglZ - EDTA) or without 0.5 mM MgCl2, MnCl2, CaCl2, CuCl2, 392 \nNiSO4, and ZnCl2 for 15 mins at RT, made up to 20 μl with nuclease-free water in a sealed 96-well 393 \nsemi-skirted PCR plate (Starlab). Samples were centrifuged and inserted into a CFX connect real-time 394 \nqPCR machine for thermal shift analysis. The fluorescence was measured in 0.5 C increments from 395 \n25 to 95 C. Deconvolution of thermal shift isotherms was performed using NAMI python tool (28), 396 \nand thermal shift graphs were generated using Prism (GraphPad).  397 \n 398 \nNuclease assays 399 \nThe ability of PglZ to degrade DNA and RNA was analysed using plasmid DNA, phage gDNA, and phage 400 \nRNA. Prior to any assays, PglZ was treated with EDTA, as described previously, to ensure consistency 401 \nin the metal binding between samples. For titration experiments, 0, 12, 24, 48, 96, 192, 384, 768, and 402 \n1536 nM of purified PglZ were incubated with 6 nM pSG483 supercoiled plasmid DNA, 6 nM 403 \npSG483BREX KO supercoiled plasmid DNA (E. fergusonii BREX site mutated), 200 ng pBrxXL WT plasmid 404 \nDNA, and 200 ng pBrxXL -pglX plasmid DNA. Purified PglZ at 0, 48, 96, 192, 384, 768, and 1536 nM 405 \nwas also incubated with 200 ng T4 gDNA, 200 ng Pau gDNA, 6 nM M18mp13 ssDNA, and 6 nM 406 \nMS2 RNA. Reactions were incubated for 1 h at 37 C with 1x ZB and 0.5 mM MnCl2. Control reactions 407 \neither eliminated the metal or included 1 mM ATP in the reaction mix. The activity of PglZ derived 408 \nmutants against supercoiled pSG483 were tested at 384, 768, and 1536 nM in the presence of 1x ZB 409 \nand 0.5 mM MnCl2 at 37 C for 1 h.  410 \n 411 \nTo test the activity of PglZ in the presence of various metals, PglZ (768 nM) was incubated with 412 \nsupercoiled pSG483 (6 nM) in 1x ZB at 37 oC for 1 h in the presence of 0.5 mM MgCl 2, MnCl2, CaCl2, 413 \nZnCl2, CuCl2, and NiSO4. Control reactions contained no divalent cations. To test the inhibition of PglZ 414 \nby various nucleotides, PglZ (768 nM) was incubated supercoiled pSG483 (6 nM) in 1x ZB and 0.5 mM 415 \nMnCl2 with and without 1 mM ATP, GTP, CTP, UTP, dATP, dGTP, dTTP, dCTP, ADP, AMP, or AMP -PNP 416 \nfor 1 h at 37 C. Control reactions contained no nucleotide or no PglZ.  417 \n 418 \nTo test the activity of PglZ in the presence of BrxB, PglZ (768 nM) was incubated with pSG483 419 \n(6 nM) with BrxB at 0.35, 0.7, 1.5, 3.0, and 6.1 μM for 1 h at 37 oC. Reactions comprised of 1x ZB and 420 \n0.5 mM MnCl2 and were completed in the presence and absence of 1 mM ATP. PglZ was also incubated 421 \nwith BrxB mutants W135A and R46A. Control reactions contained no protein, PglZ only (768 nM), and 422 \nBrxB or derivative mutants only (6.1 μM). 423 \n 424 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n15 \nAll reactions were made up to 20 μl. Reactions were stopped by the addition of 2 μl stopping 425 \nbuffer (5% SDS (v/v), 125 mM EDTA) followed by 4 μl TriTrack loading dye (ThermoFisher). Samples 426 \nwere analysed by agarose gel electrophoresis in 1.4% (w/v) gels for pSG483 analysis, or 0.8% (w/v) 427 \ngels for pBrxXL, phage gDNA, and RNA analysis using 1x TAE buffer and running at 45 V for ~16 h. 428 \nAgarose gels were post stained in 1x TAE containing 0.5 μg/ml ethidium bromide and destained in 1x 429 \nTAE. Gel images were obtained on a ChemiDoc  Imaging System on the ethidium bromide setting 430 \n(BioRad). Gel images were analysed using Fiji (ImageJ; v 2.1.0) with background subtracted. For 431 \npSG483 assays, the supercoiled, nicked, and linear DNA band intensity was measured per lane and 432 \ncalculated as a percentage of the total DNA in the respective lane. For pBrxXL assays, the DNA band 433 \nintensity of all bands per lane were measured independently and compared as a percentage against 434 \nthe corresponding DNA band in the ‘0’ PglZ control lane. For phage gDNA/RNA assays, intact phage 435 \ngDNA/RNA band intensity was measured in each lane and compared as a percentage against the ‘0’ 436 \nPglZ control. Mean values and standard deviation were calculated from triplicate data. Data were 437 \nplotted in Prism (GraphPad). 438 \n 439 \nEfficiency of plating (EOP) 440 \nE. coli  bacteriophages were isolated from freshwater sources in Durham city centre and the 441 \nsurrounding areas, as described previously  (23). E. coli DH5 were transformed with pTRB563 442 \n(pBrxXL), pTRB564 (pBrxXL -pglX), pTRB744 (pBrxXL -brxB W135A), pTRB745  (pBrxXL-pglZ H741A), 443 \npTRB746 (pBrxXL-brxB E47A), pTRB747 (pBrxXL-brxB E89A), pTRB748 (pBrxXL-brxB S133A), pTRB749 444 \n(pBrxXL-brxB R46A), pTRB750 ( pBrxXL-pglZ T538A), or  pTRB766 (pBrxXL -pglZ T538A/H741A) and 445 \ngrown overnight. Serial dilutions of phage Pau were produced in phage buffer (10 mM Tris HCl pH 7.4, 446 \n10 mM MgSO4, 0.01% (v/v) gelatin). 200 μl of overnight culture and 10 μl of phage dilution were added 447 \nto a sterile 8 ml plastic bijoux with 3 ml of 0.35% (w/v) LB-agar and poured onto LB plates. Plates were 448 \nincubated overnight at 37 C before plaque forming units (pfu) were counted on each plate. EOP 449 \nvalues were calculated by determining the phage titre on a test strain divided by the titre on a control 450 \nstrain. EOP data were collected in triplicate and the mean value was plotted in GraphPad Prism.  451 \n 452 \nInitial single particle screening of Salmonella  PglZ:BrxB   453 \nNegative stain grids were prepared by applying 4 μl of size exclusion chromatography (SEC) purified 454 \nPglZ:BrxB sample at a concentration of approximately 0.04 mg /ml to a glow -discharged 455 \nFormvar/Carbon 400 mesh Copper grid (Ted Pella). The sample was allowed to absorb for 30 s 456 \nfollowed by wicking excess solution with filter paper. The grid was quickly washed two times in 30 μl 457 \ndrops of water and once in a 30 μl drop of 2% uranyl formate (UF) followed by a final staining for 30 s 458 \nwith another 30 μl drop of 2% UF. The grids were air dried for at least 1 hr. Grids were screened on an 459 \nin-house Talos L120C transmission electron microscope (Thermo Fisher), operating at 120 kV and 460 \nequipped with a 4k x 4k Ceta  CMOS high -resolution 16M camera (Thermo Fisher). The sample 461 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n16 \ndistributed homogeneously and at random orientations over the surface of the prepared negative 462 \nstained grids.  463 \n 464 \nCryoEM hybrid model determination for  Salmonella  PglZ:BrxB   465 \nFlow charts and summary of data collection of the methods described below are shown in 466 \nSupplementary Figure S2. Grids were prepared for cryoEM by applying 3 μl of SEC purified sample at 467 \na concentration of 0.25 or 0.5 mg/ml (diluted in 20 mM Tris pH 8.0, 300 mM KCl) to a glow-discharged 468 \nC-Flat 1.2/1.3 holey carbon film coated copper grid (Electron Microscopy Sciences). The grids were 469 \nblotted for 5 s at a tension of 0, and plunge-frozen into liquid ethane using a Mark IV Vitrobot (Thermo 470 \nFisher). Two datasets of 4686 (dataset 1) and 4731 (dataset 2) movies were collected at a super 471 \nresolution pixel size of 0.56 Å using a Glacios 200 kV electron microscope (Thermo Fisher) equipped 472 \nwith a Gatan K3 direct electron detector. Preprocessing of datasets was performed in WARP (29) 473 \nwhere pixels were binned to 1.122 Å. Datasets were imported into CryoSPARC (30) and particles in 474 \ndataset 1 were picked by automated searching for Gaussian signals, extracted and Fourier cropped to 475 \na box size of 300 and 100 pixels, respectively, and filtered with multiple rounds of 2D classification and 476 \nselection. Final particles from dataset 1 were lowpass filtered to 20 Å and used as a template for 477 \nparticle picking in dataset 2. Picked particles from dataset 2 were then extracted and filtered as in 478 \ndataset 1. Final particles from both datasets were combined into a single Ab-initio 3D reconstruction 479 \njob with 4 classes, resulting in a single class with full particles (124,721) and the remaining classes with 480 \nfragments or junk particles. The particles contained in the single class were reextracted without 481 \nFourier cropping to a box size of 300 pixels followed by homogeneous and non -uniform refinement 482 \n(31) resulting in a map with GSFSC resolution of 4.45 Å.  The resulting volumes were evaluated in 483 \nChimeraX (32). 484 \n 485 \nModel fitting  486 \nPredicted models of the Salmonella PglZ:BrxB sub-complex was generated by AlphaFold (33) resulting 487 \nin high per-atom confidences. Initial placement of models was accomplished in ChimeraX (32) using 488 \nthe Fit in Map tool. Domains were then further fit into the volume individually. The predicted PglZ:BrxB 489 \ninterface was preserved by treating PglZ residues 1 -98 as part of the BrxB domain. The models were 490 \nthen further refined in the Phenix suite (34) using Cryo_fit (35) and Real Space Refine (36). No 491 \nrebuilding was performed due to lack of detail in the volumes. 492 \n 493 \n 494 \n 495 \n 496 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n17 \nRESULTS 497 \nSalmonella  BREX components form larger complexes in vivo   498 \nHaving previously performed characterisation of independent core Type I BREX components BrxA, 499 \nPglX and BrxL (17, 18, 21, 22), we turned to examining interactions between BREX proteins. The BREX 500 \nlocus from Salmonella Typhimurium strain D23580 (Fig. 1A) had already been sub-cloned and shown 501 \nto be active in phage defence (17, 23). Salmonella genes brxA, brxB, brxC and pglX were cloned into 502 \none multiple cloning site of pCOLA DUET1, and genes pglZ and brxL were cloned into the second 503 \nmultiple cloning site. A compatible vector  based on 2HR -T (addgene #29718)  was generated that 504 \nexpressed His -strep-BrxB. Combining these two vectors, and appropriate vector -only controls, we 505 \nobserved robust expression of the His-strep-BrxB fusion in the absence of the full BREX locus (Fig. 1B). 506 \nWhen then expressed in cells also expressing the full Salmonella BREX locus we observed co -507 \npurification of BREX proteins BrxC, PglX, PglZ and BrxL with His -Strep-BrxB, indicating formation of 508 \nhigher order complexes (Fig. 1B ). The indicated bands were confirmed for identity by mass 509 \nspectrometry (Fig. 1B). The most abundant protein after His-Strep-BrxB was PglZ. In order to produce 510 \nlarger quantities of the BREX complex(es) the six BREX genes were cloned as three pairs into 511 \ncompatible DUET vectors and co -purification was performed on strains containing increasing 512 \ncombinations of expression vectors ( Fig. 1C). For these experiments, the His -strep tag was replaced 513 \nwith a His-SUMO tag to aid later purification. We saw robust His-SUMO-BrxB co-purification with PglZ, 514 \nand then with PglZ, BrxC and PglX, and finally again PglZ, BrxC, PglX and BrxL. No BrxA was co-purified 515 \n(Fig. 1C). We noted that certain combinations of vector caused poor growth of cells and so performed 516 \nviable counts (Fig. 1D and Supplementary Fig. S1A). Expression of BrxC and PglX was toxic in E. coli, 517 \nbut this was in part negated by co-expression of His-SUMO-BrxB and PglZ, or BrxA and BrxL, or all six 518 \nproteins. Due to the robust expression and co -purification of His -SUMO-BrxB and PglZ we chose to 519 \npursue this sub-complex for further study. Large scale co-expression and co-purificaiton of His-SUMO-520 \nBrxB and PglZ yielded a clean sample of native PglZ:BrxB sub-complexes (Supplementary Figs. S1B-C). 521 \n 522 \nHaving identified PglZ:BrxB as a strong pairwise protein-protein interaction in Salmonella, we 523 \nfurther tested this observation using a previously characteri sed Type I BREX system found in 524 \nAcinetobacter (20). Using that system, it was also found that PglZ and BrxB interact strongly and co -525 \neluted from affinity -based and size exclusion columns in a 1:1 ratio ( Supplementary Fig. S1 ). 526 \nInterestingly, and unlike its behavio ur in Salmonella, BrxB from Acinetobacter was found to require 527 \nthe co-expression and corresponding presence of bound PglZ in order to remain soluble in vitro. These 528 \ndata indicate that the PglZ:BrxB interaction is generalisable and reproducible. 529 \n 530 \nSalmonella  PglZ:BrxB complexes show dynamic movement  531 \nSize exclusion chromatography (SEC) of native PglZ, BrxB and co-expressed and co-purified PglZ:BrxB 532 \nexpected sub-complexes demonstrated an altered elution profile for PglZ:BrxB, indicating formation 533 \nof a larger sub-complex (Fig. 2A). The Salmonella PglZ:BrxB sub-complexes were used to perform  534 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n18 \n 535 \n 536 \n 537 \n 538 \n 539 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n19 \nstructural studies through negative stain transmission electron microscopy, followed by cryo-electron 540 \nmicroscopy (cryoEM) (Fig. 2B and Supplementary Fig. S2). The final model had a Gold Standard Fourier 541 \nShell Correlation ( GSFSC) resolution of 4.45 Å ( Supplementary Fig. S2 ). At this resolution it was 542 \npossible to make use of AlphaFold outputs for PglZ:BrxB to generate a final hybrid model of the 543 \nPglZ:BrxB sub-complex. BrxB is itself a globular protein and was shown to be bound to the N-terminal 544 \ndomain (residues 1-96) of PglZ (Fig. 2B). EMBL PISA (37) identified BrxB residues R49, N135 and W137, 545 \nand PglZ residues K58, E62 and D88, as important for binding (Fig. 2B, inset). PglZ forms an “S” shape, 546 \nwith a central domain (residues 98-292) and a large C -terminal PglZ domain (residues 30 4-748) that 547 \ncontains the metal -binding site identified within PorX (16), and a final extension including a seven 548 \nsheet β-barrel (residues 749-867) (Fig. 2B). Comparison of the Salmonella PglZ:BrxB AlphaFold model 549 \nalone against our cryoEM hybrid model shows two distinct points of movement (Supplementary Fig. 550 \nS3). The PglZ N -terminal domain and BrxB, and the C -terminal β-barrel extension have both made 551 \nlarge movements between the two models, whereas the central and PglZ domains remain fixed 552 \n(Supplementary Fig. S3). This dynamic flexibility is likely the cause of our data being limited to lower 553 \nresolution. Nevertheless, this model confirms the presence of a flexible but stable PglZ:BrxB complex.  554 \n 555 \nPglZ can cleave cyclic and linear oligonucleotides in a metal -dependent 556 \nmanner 557 \nNext, we performed biochemical characterisation of PglZ in isolation, in preparation for later 558 \ninvestigation of the PglZ:BrxB sub-complex. As experimentation began we noted that the Salmonella 559 \nPglZ homologue had a tendency to precipitate during tests. As such, we chose to use PglZ from E. 560 \nfergusonii, a system we had previously characterised (24), as a substitute biochemical model.  561 \n 562 \nA superposition of the AlphaFold output for the PglZ domain from E. fergusonii PglZ (residues 563 \nV474-L759) with the PglZ domain from PorX (PDB: 7PVK, residues 213 -518) produced an RMSD of 564 \n2.822 (over 1096 atoms) ( Figs. 2B and 3A). Residues identified as important for PorX metal binding 565 \nand oligonucleotide cleavage activity, T272 (mutated to T272A in PDB 7PVK) and H500 (16) correspond 566 \nto E. fergusonii PglZ residues T538 and H741, respectively (Fig. 3A). Mutant proteins E. fergusonii PglZ 567 \nT538A, H741A and a double mutant T538A/H741A were expressed and purified, and shown to have 568 \nsimilar mass photometric and SEC profiles as E. fergusonii PglZ WT (Supplementary Fig. S4).  569 \n 570 \nPglZ wild type (WT) and mutants were tested for metal content following purification, using 571 \ninductively coupled plasma mass spectrometry (ICP-MS). There was a clear abundance of zinc in PglZ 572 \nWT samples, and levels were greatly lowered in the mutant samples ( Fig. 3B ). Following EDTA 573 \ntreatment to remove metals and subsequent purification to remove EDTA, E. fergusonii PglZ WT and 574 \nmutants were tested for stability in the presence of a range of metals using thermal shift assays (TSAs) 575 \n(Fig. 3C and Supplementary Fig. S5). PglZ proteins were destabilised by copper and, surprisingly, zinc,  576 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n20  577 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n21 \nbut were stabilised by magnesium, manganese and nickel  (Fig. 3C  and Supplementary Fig . S5). 578 \nCalcium had no effect, likely because it could not bind ( Fig. 3C). Mutants PglZ T538A and PglZ H741A 579 \nwere stabilised by manganese, indicating some metal binding could occur ( Supplementary Fig. S5). 580 \nDouble mutant PglZ T538A/H741A was not stabilised by any metal indicating that metal binding in the 581 \nactive site was no longer possible ( Supplementary Fig. S5 ). The double mutant was, however, still 582 \ndestabilised by copper and zinc, suggesting effects for copper and zinc seen with both this mutant and 583 \nalso PglZ WT are due to non-specific binding (Supplementary Fig. S5).  The melting temperatures for 584 \nPglZ WT and mutants indicated that T538A reduces overall stability, but H741A has less of an impact 585 \n(Supplementary Fig. S5E).  586 \n 587 \nInitial tests for  potential phosphodiesterase activity using bis-pNPP as a substrate with PglZ 588 \nWT and additional zinc resulted in precipitation, and so magnesium, manganese, and calcium were all 589 \ntested as alternates, with manganese showing the greatest levels of activity (Supplementary Fig. S6). 590 \nManganese was  therefore selected as an alternate metal  in bis-pNPP phosphodiesterase activity 591 \nassays. Having stripped metals from the samples and restored manganese, E. fergusonii  PglZ WT 592 \nshowed robust production of p-nitrophenol from bis-pNPP, at levels greater than for untreated PglZ 593 \nWT that contained the zinc remaining after purification (Fig. 3D). In contrast, when all mutants were 594 \nEDTA treated, re-purified, and then provided manganese, PglZ H741A had reduced activity, and both 595 \nPglZ T538A and the double mutant T538A/H741A lacked activity, demonstrating levels similar to those 596 \nobserved for the PglZ WT sample that was without  metal following EDTA treatment ( Fig. 3D). This 597 \nresult indicated that PglZ, like PorX, can cleave bis-pNPP in a metal -dependent manner, and that 598 \nmutations interfering with the likely metal binding site caused reduced activity. 599 \n 600 \n Cleavage of cyclic oligonucleotides was then tested by incubating E. fergusonii PglZ WT with 601 \ncA6 and analysing the resulting products by high performance liquid chromatography (HPLC). Having 602 \ntested a range of metals it was noted that zinc was the preferred metal in these assays, and was used 603 \nat a reduced concentration to prevent protein destabilisation and precipitation. E. fergusonii PglZ WT 604 \nrobustly linearised cA6 and sequentially cleaved nucleo tide products, indicated by a trace for each 605 \nlinear species down to AMP ( Fig. 3E ). PglZ cleavage  activity was ablated by EDTA treatment, and 606 \nmutant PglZ T538A showed no appreciable activity ( Fig. 3F). Mutant H741A appeared able to cleave 607 \ncA6 but did not produce further cleavage products ( Fig. 3F). PglZ was then tested against a broader 608 \nrange of nucleotides ( Supplementary Figs . S7 and S8 ). PglZ was observed to cleave  cyclic 609 \noligonucleotides (cA4, cA3, cA2, and cG2) and linear oligonucleotides (pApA, pApG, pGpG, c(ApGp), 610 \nand c[A(35)pA(35)pG(35)]) containing both adenosine and guanosine, including an oligonucleotide 611 \nwith 2-5 rather than 3 -5 phosphodiester linkages (c[G(25)pA(35)p]). PglZ was unable to cleave 612 \ncyclic mononucleotides (cAMP, cGMP, cTMP, cUMP, cCMP, 23 cAMP, 23 cGMP, and 23 cUMP) or 613 \ndinucleotide polyphosphates (Ap3A, Ap4A, and Ap4G) in the presence of Zn. Following this analysis 614 \nwe returned to testing metal usage and noted that at low manganese concentrations we were also 615 \nable to observe PglZ -dependent cleavage of both cA6 and pApA ( Supplementary Fig . S7C). 616 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n22 \nCollectively, these data show robust metal -dependent cyclic and linear oligonucleotide cleavage by 617 \nPglZ from a BREX system.   618 \n 619 \nPglZ is an endonuclease that can cleave dsDNA  620 \nHaving established that PglZ has nuclease activity against oligonucleotides we were curious as to 621 \nwhether PglZ could cleave dsDNA. Plasmid pSG483, a pUC19 derivative that can be easily prepared as 622 \nsupercoiled (S), nicked (N), relaxed (R) or linear (L) dsDNA, was selected as a suitable substrate to be 623 \ntested against E. fergusonii PglZ. Initial assays indicated that manganese would be the preferred metal 624 \nin this context, but supplementing with zinc did allow some PglZ nuclease activity (Supplementary Fig. 625 \nS9A). Incubation of pSG483 with a titration of E. fergusonii  PglZ WT revealed both nicking and 626 \nlinearisation activities, which were metal-dependent and could be inhibited by ATP ( Fig. 4A). This 627 \nconfirmed that PglZ can nick and cut dsDNA, and is an endonuclease. Due to the observed inhibition 628 \nby ATP, a range of mononucleotides were then tested. All NTPs, dNTPs and AMP-PNP inhibited PglZ 629 \nnuclease activity, but AMP did not (Supplementary Fig. S9B). The PglZ mutants were then tested for 630 \nnuclease activity (Fig. 4B). E. fergusonii PglZ H741A had increased nicking but decreased linearisation 631 \nactivity, whereas PglZ T538A and the double mutant PglZ T538A/H741A were both ablated for activity 632 \n(Fig. 4B). This follows the previous observed trend for activity ( Fig. 3F) and indicates T538A prevents 633 \nmetal binding and therefore activity, whilst H741A reduces metal binding and activity. 634 \n 635 \n The BREX methyltransferase, PglX, determines sequence recognition for host methylation and 636 \nphage defence (17). E. fergusonii PglX recognises the sequence GCTAAT, and there is 1 copy of this 637 \nmotif in pSG483. A mutant pSG483 was generated (pSG483BREX KO) with the GCTAAT motif mutated to 638 \nGCTATT to allow testing of whether PglZ cleavage is dependent on BREX motifs. When a titration of E. 639 \nfergusonii PglZ WT was titrated against pSG483 BREX KO there was no observed difference to the result 640 \nwith pSG483 ( Figs. 4A and 4C ). We then considered whether BREX methylation might impact PglZ 641 \nnuclease activity. We prepared pBREXxl WT, a plasmid encoding the full E. fergusonii locus and the 642 \nmutant pBREXxl-ΔpglX. Each plasmid has previously been shown to be BREX methylated and lacking 643 \nmethylation, respectively (24). E. fergusonii  PglZ WT caused equal degradation of both plasmids, 644 \nindicating BREX methylation does not impact PglZ activity under these isolated conditions 645 \n(Supplementary Figs. S9C-D).   646 \n 647 \n Phage Pau was shown to be susceptible to E. fergusonii BREX in an earlier study (24). Phage 648 \nPau does not have modified DNA (23), unlike phage T4, which has modified cytosines and so is 649 \ninherently resistant to BREX. When tested, E. fergusonii PglZ was able to cleave genomic DNAs from 650 \nboth these phages (Fig. 4D). The cleavage did not produce a distinct pattern, rather a faint smear of 651 \nproducts, demonstrating that PglZ is likely a sequence-independent endonuclease (Fig. 4D). It was also 652 \ninteresting that T4 cytosine modifications did not impact PglZ cleavage when in isolation, though T4  653 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n23 \n 654 \n 655 \n 656 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n24 \n(and other modified phages) are resistant to BREX phage defence. E. fergusonii PglZ WT could also 657 \ncause sequence -independent cleavage of ssDNA, using M18mp13 genomic DNA as substrate 658 \n(Supplementary Fig. S10A ). There was no activity, however, on MS2 phage genomic RNA 659 \n(Supplementary Fig. S10B).  660 \n 661 \nFinally, to ensure our biochemical data and structural study are aligned, we confirmed that 662 \nSalmonella PglZ WT also demonstrated metal -dependent nicking and linearisation of pSG483  663 \n(Supplementary Fig. S10C ). Salmonella PglZ showed  a preference for zinc or magnesium  and in 664 \ncontrast to E. fergusonii PglZ, Salmonella PglZ could also use calcium and copper , and could not use 665 \nmanganese (Supplementary Fig. S10C). 666 \n 667 \nPglZ:BrxB interactions can be ablated by interface mutations  668 \nAs both E. fergusonii and Salmonella PglZ were shown to be nucleases, we also wanted to demonstrate 669 \nthat E. fergusonii  PglZ and BrxB also form sub -complexes as observed for the Salmonella and 670 \nAcinetobacter homologues (Figs. 1 and 2, Supplementary Fig. S1). Our hybrid model identified several 671 \nSalmonella BrxB residues important for the PglZ:BrxB interaction (Fig. 2B). A suite of E. fergusonii BrxB 672 \nWT and mutant proteins were expressed and purified ( Supplementary Fig. S11A ). None of the 673 \nproteins contained any metals after purification, as analysed by ICP -MS (Supplementary Fig. S11B). 674 \nSEC analysis of E. fergusonii  BrxB WT showed that it formed both monomer and dimer peaks 675 \n(Supplementary Figs. S11C and D ), as confirmed by native mass spectrometric analysis 676 \n(Supplementary Fig. S11E). Analytical SEC was performed using E. fergusonii BrxB WT, PglZ and BrxB 677 \nWT with PglZ (Fig. 5A). Incubating BrxB WT with PglZ caused higher order complexes to form, as shown 678 \nby elution profiles and corresponding SDS-PAGE analysis of the peaks (Fig. 5A). This indicated that E. 679 \nfergusonii PglZ:BrxB sub-complexes were also forming, though perhaps with higher order forms being 680 \nproduced beyond those observed for Salmonella homologues (Fig. 2A).  681 \n 682 \nIn contrast, co-incubation of E. fergusonii PglZ WT with BrxB R46A and BrxB W135A failed to produce 683 \nPglZ:BrxB complexes (Figs. 5B and C ). BrxB mutants E47A, E89A and S133A generated intermediate 684 \nelution profiles, indicating some complexes were forming, but to lesser extent than with BrxB WT 685 \n(Supplementary Fig. S12). Mass photometric analysis of E. fergusonii PglZ incubated with BrxB WT and 686 \nmutants showed similar trends, in that complexes formed with BrxB WT, none formed with BrxB R46A 687 \nor BrxB W135A, or BrxB S133A in these conditions, and complexes formed but less robustly with BrxB 688 \nE89A and BrxB E47A (Fig. 5D).  689 \n 690 \nFinally, we also examined by analytical SEC whether any of the E. fergusonii PglZ mutants 691 \nT538A, H741A or T538A/H741A would impact BrxB WT binding and formation of higher order  692 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n25 \n 693 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n26 \ncomplexes. As expected, due to these mutations being distant from the BrxB binding site (Figs. 2B and 694 \n3A), no impact on complex formation was observed (Supplementary Figure S13). Together, these data 695 \nsupport comparisons between our two model homologues, as both were shown to have nuclease 696 \nactivity, and also now both have been shown to form sub-complexes.  697 \n 698 \nInteraction with BrxB impacts neither PglZ  nuclease activity nor inhibition of 699 \nnuclease activity by ATP  700 \nGel-based nuclease activities were then used to investigate the impact of BrxB interactions on PglZ 701 \nactivity. In these assays, E. fergusonii BrxB had no identifiable nicking or linearisation activity (Fig. 6A). 702 \nTitration of E. fergusonii BrxB against E. fergusonii PglZ had no appreciable impact on PglZ nicking and 703 \nlinearisation activities until the highest BrxB concentration ( Fig. 6A). Comparisons of the AlphaFold 704 \nmodel for the BrxB structure using DALI (38) indicated some similarity to nucleotide binding regions 705 \nof AAA+ proteins, but BrxB appears to be lacking key Walker motif residues. As BrxB had the potential 706 \nfor binding ATP, we investigated whether BrxB might impact the observed inhibition of PglZ activity 707 \nby ATP (Fig. 4A). When the same PglZ to BrxB titration was performed in the presence of ATP there 708 \nwas no indication that BrxB could overcome inhibition of PglZ activity by ATP  (Fig. 6A ).  For 709 \ncompleteness, we also tested whether non -interacting E. fergusonii  BrxB mutants impacted PglZ 710 \nactivity, but no effect was observed (Fig. 6B).  Next, we used HPLC analysis of oligonucleotide cleavage 711 \nas another measure of BrxB impact. Neither BrxB WT nor BrxB R46A altered the ability of E. fergusonii 712 \nPglZ to cleave cA6 or pApA ( Fig. 6C). This indicates that the role of BrxB, at least in these isolated 713 \nconditions, is independent of PglZ  nuclease activity, and likely has more relevance in the context of 714 \nlarger BREX complexes.  715 \n 716 \n 717 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n27 \n 718 \n 719 \n 720 \n 721 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n28 \nPglZ nuclease activity contributes to BREX phage defence but not BREX -722 \ndependent methylation  723 \nWith the E. fergusonii PglZ and BrxB mutations now characterised biochemically we examined their 724 \nimpact on the two measurable BREX phenotypes, phage defence and BREX -dependent methylation. 725 \nMutations were constructed in the context of pBrxXL, which encodes the full E. fergusonii BREX locus 726 \nunder native promoters (24). The suite of mutants were tested for defence against phage Pau from 727 \nthe Durham collection (23), measured by Efficiency of Plating (EOP), using an appropriate vector 728 \ncontrol. The positive and negative controls pBrxXL and pBrxXL -ΔpglX provided strong and no phage 729 \ndefence, respectively ( Fig. 7A). BrxB mutant constructs S133A and W135A, and the PglZ H741A 730 \nconstruct all showed a small reduction in phage defence activity, around 10-fold (Fig. 7A). PglZ T538A 731 \nand double mutant T538A/H741A constructs showed a large reduction in defence of around 3 logs, 732 \nbut remained impressively active ( Fig. 7A). These data indicate that mutations preventing PglZ:BrxB 733 \ninteractions or ablating PglZ nuclease activity have an impact but can be compensated for in vivo.  734 \n 735 \n Genomic DNA was extracted from each strain and PacBio sequencing analysis was performed 736 \nto investigate BREX-dependent methylation, examining N6mA methylation on the fifth adenine of the 737 \nGCTAAT motif ( Fig. 7B). Positive control  pBrxXL WT had 99.4 % methylation of BREX motifs, and 738 \nnegative control pBrxXL-ΔpglX had no detectable methylation, as observed previously (24). In all cases, 739 \nthe current mutations in pBrxXL had no impact on BREX-dependent methylation (Fig. 7B), indicating 740 \nPglZ nuclease activity works independently from BREX methylation.  741 \n 742 \n 743 \n 744 \n 745 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n29 \n 746 \n 747 \n 748 \n 749 \n 750 \n 751 \n 752 \n 753 \n 754 \n 755 \n 756 \n 757 \n 758 \n 759 \n 760 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n30 \nDISCUSSION  761 \nType I BREX systems perform phage defence and BREX-dependent methylation, but the mechanisms 762 \nfor each activity have proven difficult to uncover. Having previously performed individual studies of 763 \nBREX components and regulators (17–22), in this study we examined what higher order BREX 764 \ncomplexes form in cells and focussed on characterisation of PglZ, BrxB and the resulting stable 765 \nPglZ:BrxB sub-complex.  766 \n 767 \n Using Salmonella BrxB as bait we observed pull -down of BrxB with BrxC, PglX, PglZ and also 768 \nBrxL ( Fig. 1 ). Although BrxL is the effector protein needed for phage defence with the E. coli  and 769 \nAcinetobacter BREX systems (20, 39), we previously noted that Salmonella BREX can provide phage 770 \ndefence without BrxL (23), and there is potential for BrxL having a regulatory role in this system (40). 771 \nIt was therefore curious that BrxL readily associates with other Salmonella BREX components . 772 \nHowever, low levels of BrxL can also be seen in similar pull-down experiments performed using  E. coli 773 \nBREX (18). Using BrxL as bait does also pull -down other BREX components, though the data do show 774 \nthat the more robust complex appears to be formed of BrxB, BrxC, PglX and PglZ (18). We noted strong 775 \nassociation between PglZ and BrxB, also seen with E. coli BREX (18), and so chose to examine this sub-776 \ncomplex as a step towards understanding the structure and function of higher order BREX complexes. 777 \n 778 \n Our resulting hybrid model of Salmonella PglZ:BrxB (Fig. 2) uses 4.45 Å resolution density 779 \nobtained by cryoEM to fit AlphaFold (33) predicted domains of each protein. Notably, our model 780 \ndemonstrates points of structural flexibility that allow movement of the PglZ:BrxB interaction domain 781 \nand the PglZ C-terminal β-barrel domain (Supplementary Fig. S3). This flexibility is the likely cause of 782 \nthe limited resolution observed for the corresponding cryoEM analysis. 783 \n 784 \n The first evidence of biochemical activity for PglZ domains, originally considered an alkaline 785 \nphosphatase (15), came from demonstration that the PglZ domain of PorX , a two -component 786 \nsignalling system response regulator, could act as a phosphodiesterase and linearise cyclic nucleotides 787 \n(16). PorX activity i s zinc -dependent. Having examined the structure of Salmonella PglZ:BrxB, we 788 \nswitched to E. fergusonii PglZ and BrxB for biochemical characterisation as the proteins behaved more 789 \nreproducibly under assay conditions. After substantial efforts to identify a preferred metal and 790 \nconcentration, it could be demonstrated that E. fergusonii PglZ has similar activity to PorX, cleaving a 791 \nwide range of cyclic nucleotides, but not cyclic mononucleotides or dinucleotide polyphosphates (Fig. 792 \n3 and Supplementary Fig. S4-8).  793 \n 794 \n We hypothesised that our observed E. fergusonii PglZ activity could impact dsDNA. When 795 \ntested, E. fergusonii PglZ nicked and linearised dsDNA ( Fig. 4). This activity was applicable to ssDNA, 796 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n31 \nbut not dsRNA, and was independent of BREX motifs, appeared sequence -independent, was not 797 \nimpacted by BREX methylation and was not impacted by larger DNA modifications such as glucose 798 \nmodifications to hydroxymethylated cytosines in phage T4 genomic DNA (Fig. 4 and Supplementary 799 \nFigs. S9 and S10). Initial investigations of BREX activity indicated little digestion of invading phage DNA, 800 \nmerely an inhibition of phage genome replication, making BREX a classic “restriction” system (15). 801 \nNicking is a hallmark of multiple other phage defence systems, including Shedu, Lamassu, Dnd and 802 \nGabija (41–45). In the latter case, Gabija activity is also regulated by the detection and degration of 803 \nnucleotides (45). As we observed inhibition of PglZ nicking activity in the presence of ATP (Fig. 4), we 804 \ncannot rule out analogous regulatory activity by PglZ in the context of a full BREX mechanism . It is 805 \nunclear whether ATP might be competing for the PglZ domain catalytic site, or have another inhibitory 806 \nbinding site that alters activity. We also cannot dismiss a potential role for PglZ-dependent nicking in 807 \nprotecting from invading DNA, perhaps as a precursor licensing step to allow  further inhibition of 808 \nreplication. 809 \n 810 \n The role of BrxB has thus far remained hypothetical, yet the predicted fold mimics AAA+ 811 \nnucleotide binding domains. We were able to demonstrate binding of E. fergusonii BrxB to PglZ, and 812 \npinpoint residues required for stable complex formation (Fig. 5 and Supplementary Fig. S12 and S13). 813 \nWhen we then tested whether BrxB might bind and therefore alter the observed inhibition of PglZ by 814 \nATP, nothing changed (Fig. 6). Alternatively, and due to the observed data in Salmonella (Fig. 1) and 815 \nE. coli (18), we postulate that the role of BrxB might be as a scaffold protein, participating within and 816 \nallowing connections between multiple BREX components within higher order complexes.   817 \n 818 \n When assaying the two phenotypes of phage defence and methylation we saw that ablation 819 \nof PglZ:BrxB interactions made only a small impact on phage defence (Fig. 7A), perhaps because within 820 \na higher order complex other interactions occur to support function.  In contrast, removal of PglZ 821 \nnuclease activity by mutation had a stronger impact though still did not remove all phage defence 822 \nactivity, indicating that the overall BREX mechanism can compensate for loss or reduction in PglZ 823 \nfunction (Fig. 7A). Whilst deletion of pglZ prevents BREX phage defence and methylation (17, 20, 39),  824 \nour mutations of either PglZ nuclease activity or BrxB binding had no impact on methylation (Fig. 7B), 825 \nindicating PglZ likely plays a role in formation of the BREX methylation complex, but not in that specific 826 \nactivity. 827 \n 828 \n A working model for BREX activity was recently posited, wherein a BREX-BCXZ complex would 829 \nform and move along DNA to allow methylation (18). Our data support formation of this complex (Fig. 830 \n1), and contrary to data indicating PglX alone is sufficient for methylation (46), we could not see 831 \nmethylation using Salmonella or the same E. coli homologue (17, 18)and expression of PglX alone in 832 \ncells also does not result in methylation (39). A BREX -BCXZ complex would have to be able to 833 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n32 \ndistinguish between DNA templates containing BREX methylation on one strand following host 834 \ngenome replication, and target invading DNA containing no BREX methylation. When invading DNA is 835 \nrecognised, we envision a role for the PglZ nuclease in which nicking of target DNA licenses the BREX-836 \nBCXZ complex to switch from methylation surveillance to restriction. This could include recruitment 837 \nof BrxL and, for instance, movement of the BREX complex to stall replication forks. Nevertheless, there 838 \nremains many questions as to the specifics of BREX activity and our data indicate clear next steps in  839 \nthe characterisation of higher order BREX complexes.  840 \n 841 \n 842 \n 843 \n 844 \n 845 \n 846 \n 847 \n 848 \n 849 \n 850 \n 851 \n 852 \n 853 \n 854 \n 855 \n 856 \n 857 \n 858 \n 859 \n 860 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n33 \nACKNOWLEDGEMENTS  861 \nThis research was supported by the Electron Microscopy Shared Resource,  RRID:SCR_022611, of the 862 \nFred Hutch/University of Washington/Seattle Children’s Cancer Consortium (P30 CA015704). CryoEM 863 \nmolecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for 864 \nBiocomputing, Visualization, and Informatics at the University of California, San Francisco, with 865 \nsupport from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and 866 \nComputational Biology, National Institute of Allergy and Infectious Diseases. 867 \n 868 \n Author contributions: J.J.R. and L.A.D. contributed equally. J.J.R. expressed all E. fergusonii 869 \nproteins and performed biochemical analyses. L.A.D. performed cryoEM practical aspects, data 870 \ncollection and data processing. M.P. produced Salmonella proteins for biochemistry and performed 871 \nco-expression analyses and phage assays. A.K. performed in vivo  pull-down analyses  and mass 872 \nphotometry. A.N. performed PacBio sequencing and analysis. A.J.K., S.M. and J.P-A. produced and 873 \nanalysed Acinetobacter proteins. T.R.B. produced Salmonella protein for cryoEM. D.L.S., B.L.S., B.K.K., 874 \nand T.R.B. supervised the project and obtained funding. All authors contributed to data analysis and 875 \nwriting the manuscript. 876 \n 877 \nSUPPLEMENTARY DATA  878 \nSupplementary data have been provided and comprise 13 Figures and one Table. 879 \n 880 \nCONFLICT OF INTEREST  881 \nT.R.B. is an employee of, and B.L.S. is a paid consultant for, New England Biolabs, which provided 882 \nfunding support for this study and which develops a wide variety of phage restriction systems for 883 \ncommercial sale. 884 \n 885 \nFUNDING 886 \nThis work was supported  by a Biotechnology and Biological Sciences Research Council Newcastle -887 \nLiverpool-Durham Doctoral Training Partnership studentship [grant number BB/T008695/1] to J.J.R., 888 \na Biotechnology and Biological Sciences Research Council  responsive mode grant [grant number 889 \nBB/Y003659/1] to M.P., a Lister Institute Prize Fellowship to A.K.  and T.R.B., New England Biolabs 890 \n(NEB), the Fred Hutchinson Cancer Center (FHCC) and the NIH for both BLS (R01 GM105691) and BKK 891 \n(R15 GM140375). 892 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n34 \n 893 \n For the purpose of open access, the authors have applied a CC BY public copyright licence to 894 \nany Author Accepted Manuscript version arising from this submission. 895 \n 896 \nDATA AVAILABILITY  897 \nThe cryoEM model and corresponding maps for PglZ complexed with BrxB have been deposited in the 898 \nRCSB PDB database (ID code 9NV3) and in the EMDB (ID code EMD -49827). All other data needed to 899 \nevaluate the conclusions in the paper are present in the paper and/or Supplementary Data. 900 \n 901 \n 902 \n 903 \n 904 \n 905 \n 906 \n 907 \n 908 \n 909 \n 910 \n 911 \n 912 \n 913 \n 914 \n 915 \n 916 \n 917 \n 918 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n35 \nREFERENCES  919 \n1. Tesson,F., Hervé,A., Mordret,E., Touchon,M., d’Humières,C., Cury,J. and Bernheim,A. (2022) 920 \nSystematic and quantitative view of the antiviral arsenal of prokaryotes. Nat Commun, 13, 921 \n2561. 922 \n2. Labrie,S.J., Samson,J.E. and Moineau,S. (2010) Bacteriophage resistance mechanisms. Nat Rev 923 \nMicrobiol, 8, 317–327. 924 \n3. 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Nucleic Acids Res, 52, 8580–1046 \n8594. 1047 \n  1048 \n 1049 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n39 \nFIGURE LEGENDS   1050 \nFigure 1. BREX proteins form higher order complexes. ( A) Schematic of the BREX phage defence 1051 \nislands from Salmonella and Escherichia fergusonii. The Salmonella island encodes BREX and PARIS 1052 \n(ariA, ariB) defence systems. The E. fergusonii island encodes BREX and a Type IV restriction enzyme 1053 \nof the GmrSD family, BrxU. (B) Pull-down of Salmonella BREX complexes. E. coli BL21 (DE3) pRARE 1054 \nwas transformed with an inducible plasmid expressing His-strep tagged BrxB (or vector control), and a 1055 \nsecond plasmid expressing the six BREX genes brxA, brxB, brxC, pglX, pglZ and brxL (or vector 1056 \ncontrol). Pull-down samples were analysed by SDS-PAGE and indicated bands were identified by mass 1057 \nspectrometry. (C) Expression of Salmonella BREX proteins in pairs from pET DUET -based vectors, 1058 \npulled down with His -SUMO-BrxB. Pull-down samples were analysed on SDS -PAGE and indicated 1059 \nbands were identified by mass spectrometry. ( D) Toxicity during expression of Salmonella BREX 1060 \nproteins, measured as viable counts. Error bars represent the standard deviation of the mean from 1061 \ntriplicate data. 1062 \n 1063 \nFigure 2. Structure of the Salmonella PglZ:BrxB stable sub -complex. ( A) Size exclusion 1064 \nchromatography traces of independent Salmonella PglZ and BrxB purifications, and the PglZ:BrxB co -1065 \nexpression sample used for cryoEM, show that PglZ and BrxB form a stable complex. ( B) A cryoEM 1066 \nmap generated from single particles of the Salmonella PglZ:BrxB complex validates the interaction 1067 \nbetween both proteins, as well as the location of the PglZ:BrxB interaction surfaces and identity of 1068 \nresidues in the protein -protein interface (inset box). The resulting model is closely related to the 1069 \ncorresponding AlphaFold prediction for the Salmonella PglZ:BrxB complex, albeit with slight 1070 \nrearrangements corresponding to a small rotation of the N-terminal domain of PglZ and associated BrxB 1071 \nrelative to the larger core of PglZ (Supplementary Figure S3). 1072 \n 1073 \nFigure 3. PglZ can cleave cyclic nucleotides in a metal-dependent manner. (A) Overlay of AlphaFold2 1074 \nE. fergusonii PglZ predicted structure with PorX from Porphyromonas gingivalis (PDB: 7PVK). (B) ICP-1075 \nMS of PglZ WT and mutants showing divalent cations bound following purification. Plotted data 1076 \nrepresent mean values ± SD. Metal content is plotted as a percentage of the total protein in the sample. 1077 \n(C) Thermal shift assays performed upon PglZ WT (5 μM) following incubation with EDTA or metals 1078 \n(0.5 mM). Mean changes in melting temperature ( ΔTm) are plotted by comparison to PglZ WT in the 1079 \nabsence of EDTA or metal, which is set as ‘0’. Error bars represent standard deviation (6 replicates) 1080 \n(D) Bis(4-nitrophenyl) phosphate (2.5 mM) phosphodiesterase assays using PglZ WT (2 μM) in the 1081 \npresence and absence of EDTA and Mn, and mutants in the presence of Mn. The Mn is supplied by 0.5 1082 \nmM MnCl2. Plotted data represent mean values ± SD (9 replicates). Absorbance (A405nm) represents the 1083 \namount of reaction product p-nitrophenyl phosphate. ( E) HPLC analysis of cyclic hexa -adenosine 1084 \nmonophosphate (cA6) cleavage by PglZ WT (2 μM) in the presence of Zn (10 μM). (F) HPLC analysis 1085 \nof cyclic hexa-adenosine monophosphate (cA6)  cleavage by PglZ WT (2 μM) treated with EDTA, and 1086 \nmutants T538A and H741A in the presence of Zn (10 μM). Control reactions are represented by cA6 1087 \nalone, and standard mixes are comprised of pApA and AMP. All nucleotides in the reaction mixes are 1088 \nat 10 μM. Presented traces are representative of triplicate data.  1089 \n 1090 \nFigure 4. PglZ is a metal-dependent nuclease that does not recognise BREX sites. (A) PglZ nicks and 1091 \nlinearises supercoiled plasmid pSG483 in a metal -dependent manner that is inhibited by ATP. ( B) 1092 \nMutation of metal binding site alters nuclease activity, either by shifting activity away from linearisation 1093 \nand towards nicking (H741A) or eliminating activity (T538A and T538A/H741A). ( C) PglZ can linearise 1094 \nplasmid pSG483 DNA with a mutated BREX site. (D) PglZ does not appear to have site-specific nicking 1095 \nactivity on linearised phage DNA, nor be impacted by DNA modifications such as those found in T4. 1096 \nPglZ was titrated against constant supercoiled pSG483 plasmid DNA (6 nM) or phage gDNA (200 ng) 1097 \nin the presence and absence of MnCl2 (0.5 mM) and ATP (1 mM). Control lanes represent supercoiled 1098 \n(S), nicked (N), linear (L), and relaxed (multiple topoisomers; R) plasmid DNA, or contain the 1099 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint \n\n \n \n \n \n40 \nappropriate DNA in the absence of protein. Assays are presented on 1.4% (w/v) or 0.8% (w/v) agarose 1100 \n1x TAE gels post -stained with ethidium bromide. Assays shown are representative of triplicate 1101 \nexperiments. Data points and error bars represent the mean ± SD of triplicate data. 1102 \n 1103 \nFigure 5. E. fergusonii BrxB mutants fail to form complexes with PglZ. Analytical SEC (S200i) and SDS-1104 \nPAGE analysis of PglZ:BrxB complexes formed with BrxB WT ( A), BrxB\nR46A\n (B), and BrxB\nW135A\n (C). 1105 \nSamples of PglZ (10 μM) with equimolar BrxB WT and mutants were made up to 100 μl and pre-1106 \nincubated for 15 min prior to loading on the S200i. The expected elution volumes (Ve) of various complex 1107 \nconformations are highlighted by black or red dotted lines, and the elution profile of PglZ incubated with 1108 \nBrxB is shown as a dark green solid line. Control elution profiles of PglZ alone (light green dashed line) 1109 \nand BrxB alone (dark blue dashed line) are also shown. Fractionated peak samples were resolved on 1110 \n15% (v/v) polyacrylamide gels for 1 h 15 min in tris -glycine running buffer and stained with Quick 1111 \nCoomassie. Protein identities are highlighted with black arrows. (D) Mass photometry of PglZ with BrxB 1112 \nWT and mutants. Counts were acquired for 60 s with BrxB (5 nM) or samples of PglZ (5 nM) pre -1113 \nincubated with equimolar BrxB WT and mutants in phosphate buffered saline (PBS).  1114 \n 1115 \nFigure 6. BrxB interacting with PglZ does not appreciably alter PglZ nuclease activity. ( A) Incubation 1116 \nof PglZ (768 nM) with a titration of BrxB WT in the absence or presence of MnCl2 (0.5 mM) and ATP (1 1117 \nmM). (B) Incubation of PglZ WT (768 nM) with a titration of BrxB mutants R46A and W135A in the 1118 \nabsence or presence of MnCl2 (0.5 mM). Control lanes represent supercoiled (S), nicked (N), linear (L), 1119 \nand relaxed (multiple topoisomers; R) plasmid DNA, or contain DNA in the absence of protein. Assays 1120 \nare presented on 1.4% (w/v) agarose 1x TAE gels post -stained with ethidium bromide. Assays shown 1121 \nare representative of triplicate experiments. (C) PglZ (2 μM) incubated in the presence of BrxB (10 μM) 1122 \ndoes not prevent cleavage of cA6 or pApA (10 μM). Control reactions are comprised of the nucleotide 1123 \nin the absence of protein. Standard mixes are comprised of pApA and AMP. Presented traces are 1124 \nrepresentative of triplicate data.  1125 \n 1126 \nFigure 7. PglZ:BrxB mutants have small impact on BREX phage defence and no impact on BREX 1127 \nmethylation. (A) EOP results of phage Pau tested against E. fergusonii BREX constructs. Error bars 1128 \nrepresent standard deviation from the mean of triplicate data. ( B) PacBio sequencing results showing 1129 \nthe percentage of BREX motif methylation in each strain. 1130 \n 1131 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.26.645558doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}