{"paper_id":"a8a557e2-5259-4a1a-b6c8-07fb7655db4a","body_text":"1 \nStructure and rational engineering of the PglX methyltransferase and 1 \nspecificity factor for BREX phage defence 2 \nSam C. Wenta, David M. Pictona, Richard D. Morganb, Andrew Nelsonc, David T. F. Drydena, Darren L. 3 \nSmithc, Nicolas Wennerd, Jay C. D. Hintond, Tim R. Blowera,* 4 \n 5 \naDepartment of Biosciences, Durham University, South Road, Durham, DH1 3LE, UK. 6 \nbNew England Biolabs, 240 County Road, Ipswich, MA 01938, USA. 7 \ncFaculty of Health and Life Sciences, Northumbria University, Newcastle Upon Tyne, NE1 8ST, UK. 8 \ndInstitute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, L69 7ZB, 9 \nUK. 10 \n*To whom correspondence may be addressed . E mail: timothy.blower@durham.ac.uk, tel: 11 \n+44(0)1913343923. 12 \n 13 \nKeywords: BREX, phage defence, PglX, methyltransferase, Ocr 14 \n 15 \n 16 \n 17 \n 18 \n 19 \n 20 \n 21 \n 22 \n 23 \n 24 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n2 \nABSTRACT  25 \nBacteria have evolved a broad range of systems that provide defence  against their viral predators, 26 \nbacteriophages. Bacteriophage Exclusion (BREX) systems recognize and methylate 6 bp non -27 \npalindromic motifs within the host genome , and prevent replication of non-methylated phage DNA 28 \nthat encodes these same motifs. How BREX recognizes cognate motifs has not been fully understood. 29 \nWe have characterised BREX from pathogenic Salmonella and generated the first X -ray 30 \ncrystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes 31 \nthe methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the 32 \nstructure of PglX bound to the phage -derived DNA mimic, Ocr , an inhibitor of BREX activity.  Our 33 \nanalyses propose modes for DNA -binding by PglX and indicate that larger BREX complexes are 34 \nrequired for methyltransferase activity and defence.  Through rational engineering  of PglX , we  35 \nbroadened both the range of phages targeted, and the host motif sequences that are methylated by 36 \nBREX. Our data demonstrate that PglX is the sole specificity factor for BREX activity, provid ing motif 37 \nrecognition for both phage defence and host methylation. 38 \n 39 \n 40 \n 41 \n 42 \n 43 \n 44 \n 45 \n 46 \n 47 \n 48 \n 49 \n 50 \n 51 \n 52 \n 53 \n 54 \n 55 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n3 \nINTRODUCTION  56 \nBacteria have evolved a diverse range of defences to protect from bacteriophages (phages) and mobile 57 \ngenetic elements 1,2. Classic examples of host defence mechanisms include restriction -modification 58 \n(RM) 3, abortive infection 4,5 and CRISPR-cas 6. Genes encoding these systems tend to co-localise into 59 \n“defence islands” 7. Analysis of defence islands using a “guilt-by-association” approach have resulted 60 \nin significant expansion of predicted and validated defence systems 8,9, including Bacteriophage 61 \nExclusion (BREX) 10, CBASS 11, BstA 12, retrons 13, viperins 14 , pycsar 15 and PARIS 16. Whilst the 62 \ncombinations of phage defence systems encoded in any island can differ, there is ev idence that 63 \nconserved regulatory systems, such as the BrxR family, control defence expression perhaps mediating 64 \nrobust defence against a broad spectrum of invaders 17–19.  65 \n 66 \nBREX genes are found in 10% of bacterial and archaeal genomes 10. BREX is related to Phage Growth 67 \nLimitation (Pgl) (22) and was first identified through analysis of genes neighbouring pglZ, performed 68 \nto locate likely defence genes 10. Together with gmrS/gmrD, which encode a Type IV restriction 69 \nenzyme, BREX genes form one of the most common defence island pairings 7,21. We have recently 70 \ndemonstrated that a defence island encoded on a multidrug -resistant plasmid of Escherichia 71 \nfergusonii provides complementary phage defence using  BREX and a GmrSD homologue, BrxU 22. 72 \nThere are six BREX sub-types, and type I BREX contains six genes; brxA, brxB, brxC, pglX, pglZ and brxL 73 \n10. BrxA is a DNA-binding protein 23, and BrxL is a DNA-stimulated AAA+ ATPase 24. PglX has sequence 74 \nand structural homology to methyltransferases and is hypothesised to methylate non-palindromic 6 75 \nbp sequences (BREX motifs) on the N6 adenine at the fifth position of the motif  10,22,25, allowing 76 \ndiscrimination between self and non-self DNA. Interestingly, it has been shown that Ocr from phage 77 \nT7, a protein that mimics dsDNA 26, can inhibit BREX activity through binding to PglX 27. Whilst 78 \nreminiscent of RM systems, the mechanism of BREX activity remains unclear. 79 \n 80 \nThe stySA locus from Salmonella enterica serovar Typhimurium 28, (also known as SenLT2III), was 81 \nrecently re-constructed in an attenuated lab strain of S. Typhimurium (LT2) and shown to have BREX 82 \nactivity 29. In 2017, invasive non-typhoidal Salmonella (iNTS) disease was responsible for 77,500 deaths 83 \nglobally, of which 66,500 deaths occurred in sub -Saharan Africa 30. A high proportion of African iNTS 84 \ncases are caused by S. Typhimurium ST313 31,32. Representative ST313 strain D23580 31 encodes a BREX 85 \nlocus that is closely-related to the LT2 BREX locus (Fig. 1a), comprising a defence island formed from 86 \nan amalgamation of the type I BREX system and PARIS 16. The D23580 BREX defence island lacks the 87 \nadditional upstream and regulatory genes observed in the E. fergusonii type I BREX defence island 22. 88 \n 89 \nThe relative simplicity of the Salmonella BREX system and the clinical relevance of the host strain 90 \nprompted us to test the effects of the D23580 BREX defence island against environmental Salmonella 91 \nphages. The D23580 BREX phage defence island was then characterised through systematic gene 92 \ndeletions in an E. coli background, to allow use of the Durham phage collection 33 in identifying the 93 \ndeterminants of phage defence and PglX -dependent host methylation. We present the first X-ray 94 \ncrystallographic structural characterisation of PglX . We also present the first X -ray crystallographic 95 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n4 \nstructural characterisation of PglX bound by the DNA mimic Ocr. Through rational engineering of PglX 96 \nit was possible to alter the BREX motif recognised for methylation and phage defence. Our structural 97 \nand biochemical analyses support PglX being the BREX methyltransferase and suggest modes of DNA-98 \nbinding. Our data also definitively show PglX is the sole specificity factor in BREX phage defence, 99 \nproviding motif recognition for both phage targeting and host methylation.    100 \n 101 \n 102 \n 103 \n 104 \n 105 \n 106 \n 107 \n  108 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n5 \nRESULTS 109 \nThe Salmonella  D23580 BREX phage defence island provides protection 110 \nagainst environmental Salmonella  phages 111 \nThe BREX phage defence island from Salmonella enterica serovar Typhimurium ST313 strain D23580 112 \n(referred to as D23580 from now on) encodes two phage defence systems, type I BREX 10, and PARIS 113 \n16, collectively “BREX Sty” (Fig. 1a). The SalComD23580 RNA -seq-based gene expression compendium  114 \n(http:/bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl?_HL) shows that the defence island is expr essed 115 \nconstitutively at the transcriptional level during exponential growth in LB and minimal media , and 116 \nwithin murine macrophages 34. Differential RNA -seq (dRNA -seq) was used to identify a promoter 117 \nupstream of brxA (STMMW_44431) at location 4773879 on the D23580 chromosome, which drives 118 \ntranscription of the BREX-PARIS island 34 (Fig. 1a). 119 \n 120 \nAlso known as StySA 28, the ~15.7 kb D23580 BREXSty phage defence island has two synonymous point 121 \nmutations in pglX compared to the model S. Typhimurium ST19 strain LT2. The BREX island has 122 \nrecently been studied in the S. Typhimurium-derived strain ER3625. Phage transduction was used to 123 \nconstruct ER3625 as a genetic hybrid between S. Abony 803 strain and S. Typhimurium in the 1960’s, 124 \nand the strain has recently been sequenced 35. In comparison to D23580, the defective BREX phage 125 \ndefence island of S. Typhimurium strain ER3625 ha d a further 12 point mutations, of which 7 were 126 \ndistributed throughout pglZ, and 5 in the 3′-terminal section of brxC 29.  127 \n 128 \nThe contiguous PARIS defence systems mediate an abortive infection response in the presence of the 129 \nanti-BREX and anti -restriction protein Ocr  16. The co -localisation of the PARIS gene s ariAB within 130 \nBREXSty raises the possibility that the BREX and PARIS defences work together in S. Typhimurium. Our 131 \nfirst aim was to confirm BREXSty activity in D23580. 132 \n 133 \nTo assess phage defence in D23580 we needed to isolate Salmonella phages. As phages isolated on 134 \nD23580 wild type (WT) would be inherently resistant to BREXSty, we first used a genetic approach to 135 \ngenerate a strain of D23580 that lacked BREXSty. The ST313 strain D23580 encodes 5 prophages that 136 \nencode their own antiphage systems, including the prophage BTP1 -encoded BstA  12.  To reduce 137 \ninterference from other antiphage systems, we began with the D23580Δφ mutant strain that lacks the 138 \nfive major prophages. The entire BREXSty defence island, including PARIS, was then removed from 139 \nD23580Δφ using scar-less λ red recombination (Fig. S1) 36, resulting in strain D23850ΔφΔBREX 37. 140 \n 141 \nSewage effluent was obtained direct from source with the assistance of Northumbrian Water, and was 142 \nused for phage enrichment on D23850ΔφΔBREX. A range of plaques were obtained after these 143 \nenrichments, and 8 phage lysates were prepared following rounds of purification from visually distinct 144 \nplaques. Activity of the D2 3580 BREX defence island was confirmed using EOP assays with the 8 145 \nSalmonella phage isolates, testing the ability of the phages to plaque on D23580Δφ, with 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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n6 \nD23850ΔφΔBREX as the control ( Fig. 1b). An EOP value of less than 1 indicates that a phage is less 147 \nefficient at forming plaques on the test strain compared to the control.  Phages KMP, SB58 and SL2K 148 \nhad an EOP of <1, with a reducetion in plaquing of ~ 100-fold compared to controls, indicating 149 \nsensitivity to BREXSty (Fig. 1b). Phage DB1 was more weakly affected, with an EOP of 0.13 (Fig. 1b). The 150 \nremaining four phages appeared unaffected by activity of BREXSty, with EOPS ~1 (Fig. 1b). These data 151 \nconfirm that the BREX Sty defence island of D23580Δφ can provide active anti-phage activity  in 152 \nSalmonella. 153 \n 154 \nImpact of  Salmonella  D23580 BREX phage  defence island gene deletions on 155 \nphage defence and methylation  156 \nHaving investigated the impact of the D23580 BREX phage defence island, BREXSty, in the original 157 \nSalmonella host, we investigated BREX Sty in an E. coli  background. The motivation for using this 158 \nheterologous host was to allow direct comparison with the previously characterised BREX phage 159 \ndefence island from E. fergusonii 22, and use of our Durham collection of phages 33. E. coli is also a 160 \nmore tractable experimental model for future experiments within this study. BREXSty was sub-cloned 161 \nin sections and then combined into plasmid pGGA by Golden Gate Assembly (GGA) 38, yielding plasmid 162 \npBrxXLSty that contained the entire BREX and PARIS defence island, namely the eight genes from brxA 163 \nto brxL as depicted (Fig. 1a), under the control of the native promoters ( Fig. S2). Plasmid pTRB507 is 164 \nan equivalent empty vector control. Liquid cultures of E. coli DH5α WT, or cultures transformed with 165 \neither pBrxXLSty or pTRB507, were infected with Durham phage TB34  33, or lab phage T7 (ATCC BAA -166 \n1025-B2) (Figs. 2a-c). Infected control cultures were lysed by both phages; the T7 -infected cultures 167 \ndid not recover, whereas the TB34-infected cultures began to grow again at 10-12 hrs post-infection, 168 \npresumably due to the selection of spontaneous TB34 -resistant mutants  (Figs. 2a and b). In the 169 \npresence of pBrxXLSty, however, cultures infected with TB34 grew similarly to uninfected controls, 170 \nwhilst cultures infected with T7 were lysed (Fig. 2c). These findings show that BREXSty is active in an E. 171 \ncoli background, and demonstrates that pBrxXLSty provides defence against TB34, but not against T7. 172 \n 173 \nTo investigate the role of each phage defence gene in protection against TB34 infection, we generated 174 \nindividual deletions of each D23580 BREX /PARIS gene in pBrxXL Sty, and a double mutant that lacked 175 \nboth the ariA and ariB genes of the PARIS system ( Fig. S2). E. coli DH5α cells were transformed with 176 \nthe mutant plasmids and liquid cultures of resulting strains were subsequently infected with TB34 and 177 \nT7 (Figs. 2d-l). Deletion of brxA, brxB, brxC, pglX and pglZ abolished defence against TB34 (Figs. 2d-h). 178 \nOur finding that deletion of  brxL did not impact protection against TB34  revealed that BrxL is not 179 \nrequired for the phage defence activity of BREXSty against TB34 ( Fig. 2i). Deletion of aria and ariB, 180 \neither singly or together, also did not alter defence against TB34 (Figs. 2j-l). 181 \n 182 \n 183 \n 184 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n7 \n 185 \n 186 \n 187 \n 188 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n8 \nProtection from infection by TB34 and T7 was then monitored using the quantitative EOP assay ( Fig. 189 \n3a). BREXSty encoded on pBrxXLSty provided a moderate 100-fold reduction in TB34 plating efficiency 190 \nand had no appreciable impact on T7 ( Fig. 3a). The 100-fold reduction matches the scale of phage 191 \ndefence observed in Salmonella D23580Δφ against Salmonella phages (Fig. 1b). Therefore, plasmid 192 \npBrxXLSty and BREXSty in the natural host chromosome provide a similar level of defence. Consistent 193 \nwith results obtained with liquid cultures, deletion of brxA, brxB, brxC, pglX and pglZ ablated phage 194 \ndefence in the EOP assay (Fig. 2; Fig. 3a). However, whereas deletion of brxL did not appear to impact 195 \nprotection in liquid cultures (Fig. 2i), the EOP measurements revealed 10,000 -fold enhancement of 196 \ndefence against TB34 in the absence of brxL compared to cells carrying pBrxXLSty WT (Fig. 3a). 197 \nIndividual deletion of PARIS genes ariA and ariB caused a 10-fold increase in phage defence, while the 198 \ndouble ariA, ariB deletion had no additional impact (Fig. 3a). Collectively, these data demonstrate that 199 \nTB34 is targeted by type I BREX in the BREXSty D23580 BREX defence island, and that unlike the E. coli 200 \nand Acinetobacter BREX systems 17,25, BrxL is not necessarily a requirement for phage defence. 201 \n 202 \nThe EOP results of TB34 when tested against the brxL deletion and ariA, ariB double deletion strains 203 \nprompted us to test a wider range of phages. Using the Durham collection of 12 coliphages 33, we re-204 \ntested all phages against pBrxXL Sty, pBrxXL Sty-ΔbrxL and pBrxXL Sty-ΔariAΔariB (Fig. S3). Phages TB34, 205 \nAlma, BB1, CS16, Mav and Sipho had 10 - to 100-fold reduced EOPs on pBrxXLSty, compared to empty 206 \nvector controls ( Fig. S3). The brxL deletion caused a range of impacts . In some cases we observed 207 \nenhanced defence  (TB34, Alma, Sipho), but in other case s there was no difference to an already 208 \nsusceptible phage (BB1, CS16, Mav) ( Fig. S3). With phage Pau, against which BREX Sty WT had little 209 \neffect, the brxL deletion enhanced defence (Fig. S3). Other phages unaffected by the WT pBrxXL Sty 210 \nplasmid were also no t impacted by pBrxXLSty-ΔbrxL (Fig. S3 ). In contrast, the pBrxXLSty-ΔariAΔariB 211 \nconstruct generally produced similar EOP values  compared to pBrxXLSty WT, though there was an 212 \napproximate ten-fold further reduction in EOP for phages Alma and Sip ( Fig. S3), and there was one 213 \nmajor difference where the ariA, ariB double deletion massively reduced the EOP of BB1 compared to 214 \npBrxXLSty WT (Fig. S3). These data show that the PARIS system was itself not active against any tested 215 \nphage, and that deletion of brxL has phage-dependent impacts on defence (Fig. S3). 216 \n 217 \nHaving performed systematic analysis of gene deletions on phage defence, we then investigated a 218 \nsecond BREX phenotype; DNA methylation. PglX methyltransferases from type I BREX loci generate 219 \nN6-methylated adenines (N6mA) at the fifth position within 6-bp non-palindromic motif sequences of 220 \nhost DNA 10,22,25. Restoring active function of the Salmonella LT2 StySA BREX system identified GATCAG 221 \nas the target motif sequence 29. W e explored the use of the MinION next -generation sequencing 222 \nsystem to detect N6mA methylation patterns. Previously, we performed this type of analysis using 223 \nmethylation-deficient E. coli ER2796 39 in order to reduce background methylation. However, we were 224 \nunable to transform strain E. coli ER2796 with our pBrxXLSty constructs, perhaps because the defence 225 \nisland impacted upon bacterial fitness in the absence of methylation. We therefore used E. coli DH5α 226 \nstrains, noting that the background GATC methylation might interfere with detection of the proposed 227 \nGATCAG BREX methylation motif. Total genomic DNA was extracted from each strain and sequenced 228 \nby MinION. E. coli DH5α pBrxXLEferg, encoding the BREX phage defence island from E. fergusonii, was  229 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n9 \n 230 \n 231 \n 232 \n 233 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n10 \nused as an initial positive control to ensure the methylation detection procedure was working. We 234 \nsuccessfully identified the GCTAAT methylation motif (Fig. S4a), as previously reported 22. To confirm 235 \nthe Salmonella BREX motif we used a baseline control, wherein the pBrxXLSty WT sample was subjected 236 \nto whole genome amplification (WGA), which should remove DNA modifications. The WGA sample 237 \ncontained the lowest detectable level of methylated GATCAG sequences, 12.87%, whilst pBrxXLSty WT 238 \nshowed GATCAG methylation at 78.78% of sites, confirming that D23580 BREX produces N6mA at 239 \nGATCAG sequences (Fig. 3b; Fig. S4b). The brxA, brxB, brxC, pglX and pglZ mutants showed reduced 240 \nnumbers of GATCAG methylation sites (Fig. 3b), indicating that all five gene products are required for 241 \nmethylation. This finding is consistent with results involving the Acinetobacter BREX 17, but differs from 242 \nthose obtained with E. coli BREX; the E. coli brxA was not required for methylation in conditions of 243 \narabinose-induced BREX expression 25. In S. Typhimurium BREX, deletion of brxL did not reduce 244 \nmethylation ( Fig. 3b) and the ariA, ariB and double mutants showed approximately WT levels of 245 \nmethylation (Fig. 3b). 246 \n 247 \nThe observed changes in methylation levels identified the genetic requirements for BREX -mediated 248 \nmethylation. However, the data did not agree with quantitative data  on BREX methylation obtained 249 \npreviously from Pacific Biosciences (PacBio) sequencing 22. To perform a direct comparison, we used 250 \nthe same 12 strains to generate samples for PacBio sequencing (Fig. 3b). The PacBio results were more 251 \nrobust than those from MinION, with 0% of motifs modified in the WGA sample and 100% of motifs 252 \nmodified with pBrxXLSty WT. The BREX mutants also showed either no, or near-saturated, methylation 253 \n(Fig. 3b). The PARIS deletions resulted in  close to WT levels of methylation by PacBio ( Fig. 3b), 254 \nindicating that PARIS is not involved in the observed methylation. These data show the genetic 255 \nrequirements for D23580 BREX -dependent host methylation and demonstrate the utility of two 256 \nsequencing platforms when examining N6mA modifications. 257 \n 258 \nStructure of PglX shows SAM binding for methyltransferase activity  259 \nIt has not been understood how BREX systems recognize their cognate motifs. The likely candidate 260 \nprotein, shown to be essential for methylation and defence, was the conserved PglX putative 261 \nmethyltransferase. The closest structural homologue to the Alphafold predicted stru cture of PglX in 262 \nthe PDB database is the Type IIL RM enzyme, MmeI 40, though domains are missing . As a result, in 263 \norder to  learn more about BREX motif recognition , the structure of Salmonella PglX was sought 264 \nthrough X-ray crystallography. Following crystallization and data collection, an Alphafold model of PglX 265 \nwas used as a search model for molecular repl acement, assisting the solution and refinement of the 266 \ncrystallographic structure of Salmonella PglX bound to S-adenosyl-L-methionine (SAM), a co-factor for 267 \nmethylation, to 3.4 Å (Fig. 4; Table 1). 268 \n 269 \nThe crystal structure contains two copies of PglX in the asymmetric unit , the smallest repeating unit 270 \nof the crystal . However, the arrangement of the two copies allows only weak interactions that are 271 \nlikely formed due to interactions within the crystal rather than being biologically significant. The  272 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n11 \n 273 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n12 \narchitecture of PglX presents two distinct domains, N -terminal and C -terminal, linked by a central 274 \nshort hinge region (residues 659 – 654) (Figs. 4a and b). Due to absence of available density, two short 275 \nloop regions were unable to be modelled (residues 53 – 56 and 418 – 420), but otherwise the full PglX 276 \nprotein was resolved. SAM was also resolved bound within PglX (Fig. 4c).  277 \n 278 \nThe closest structural homologue for the solved PglX structure , as designated by the DALI server  41, 279 \nremains the Type IIL restriction-modification system, MmeI ( PDB 5HR4; Z-score 20.3). MmeI 280 \ndemonstrates both N6mA DNA methyltransferase and DNA restriction activity 40 but the MmeI 281 \nstructure only has 60.8% sequence coverage against PglX, (1225 residues and 745 residues for PglX 282 \nand MmeI, respectively) , and aligns to PglX with an RMSD of 7.13 Å  (Fig. S5a). The majority of this 283 \nalignment falls within the N-terminal domain of PglX and bridges the hinge region, extending into the 284 \nC-terminal domain. The MmeI structure shows a methyltransferase domain bound to the SAM analog 285 \nsinefungin 40, and in our PglX structure SAM binds within the same pocket  (Fig. 4 ). Within this 286 \nhomologous domain of PglX (residues 227 – 661) sit the amino-methyltransferase motif I GxG residues 287 \nimplicated in SAM binding (residues 315  – 317), and adenine specific motif IV responsible for 288 \ninteracting with a flipped-out adenine base from the target DNA (NPPY; residues 509-512) (Fig. 4; Fig. 289 \nS5b). The presence and organisation of these motifs around the SAM molecule  (Fig. 4c) is indicative 290 \nof a γ-class amino-methyltransferase 42, consistent with its homology to MmeI 40. Though MmeI has 291 \nboth methyltransferase and restriction activities the MmeI nuclease domain (residues 1-155) was not 292 \nresolved in the MmeI structure 40. The nuclease domain of MmeI is separated by a helical linker. The 293 \nN-terminal domain of PglX contains a similar linker and an N-terminal helical bundle (residues 1 – 227), 294 \nbut no nuclease domain (Figs. 4a and b). Assessing conservation between homologs in the UniRef 295 \ndatabase using ConSurf  43, the MmeI -like DNA methyltransferase region of PglX appears highly 296 \nconserved compared to the N-terminal helical bundle domain  (Fig. S 5c). Using DALI to search for 297 \nstructural homologues of the C -terminal domain alone (residues 672 – 1221) returns Type I RM 298 \nspecificity subunits. The immediate section of the C-terminal domain of PglX aligns with target 299 \nrecognition domains (TRD) required for motif binding (residues 662 – 849). This is followed by two 300 \nlong spacer helices (residues 850 – 960) that mimic dimeri zed spacers found in specificity factors of 301 \nType I DNA methyltransferases such as EcoKI 44 (Fig. 4a and b).  The spacers lead to a final C-terminal 302 \nregion of unknown function (residues 961 – 1225). Interestingly, the spacer and C-terminal regions 303 \nextend 320 residues beyond the end of the alignment with MmeI and show a high degree of 304 \nconservation (Fig. 4a and b; Fig. S5a and c). This might suggest a specialised function conserved to 305 \nallow BREX activity, perhaps as a binding surface for other BREX components. As a result, t he PglX 306 \nstructure, and lack of nuclease motifs and potential aligned catalytic residues, supports PglX acting as 307 \na methyltransferase only, and not acting as a restriction enzyme. 308 \n 309 \nWith expression and purification methods established, and the structure supporting PglX as the BREX 310 \nmethyltransferase (Fig. 4), a SAM-dependent methyltransferase assay was performed to assess the 311 \nability of purified PglX to methylate DNA in vitro. Using E. coli DH5α genomic DNA known to contain 312 \nthe target BREXSty motif as a substrate, PglX was added and incubated for 30 min at room temperature 313 \nin a buffer containing SAM. M ethyltransferase acti vity was measured indirectly via the reaction 314 \nproduct, S -adenosyl-L-homocysteine (SAH). No methylation was apparent from PglX under these 315 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n13 \nconditions (Fig. S6). We hypothesize that PglX methyltransferase activity likely requires the presence 316 \nof other BREX components. 317 \n 318 \nSalmonella  BREX can be inhibited by Ocr homologues through binding PglX  319 \nOcr is the T7-encoded restriction system inhibitor that blocks phage defence activity of the E. coli BREX 320 \nsystem 27. Additionally, Ocr triggers Abi by the type II PARIS phage defence system  16. BREXSty also 321 \nencodes a homolog of PARIS (Fig. 1a). Though notably, no activity was observed for BREXSty against 322 \nphage T7 (Fig. 2 and Fig. 3a). Following the production of individual gene knockouts, it was possible 323 \nto individually assay inhibition of BREX and activation of PARIS by Ocr. To determine whether Ocr 324 \ninhibited BREX, vector pBAD30-ocr was generated. EOP assays were then carried out with E. coli DH5α 325 \npBrxXLSty-ΔariAΔariB pBAD30-ocr and showed that expression of Ocr fully inhibited BREX defence (Fig. 326 \n5a). As Ocr is a product of T7, a coliphage, this experiment was also repeated using an Ocr homologue, 327 \nGp5, encoded by Salmonella phage Sp6 45. Homology was inferred by protein sequence searches using 328 \nBLAST (NP_853565.1: 78.6% sequence similarity, 88% coverage) followed by predictive modelling 329 \nfrom protein sequence using AlphaFold  46. The structures of Ocr and Gp5 aligned with an RMSD of 330 \n0.91 Å. We again selected TB34 as a model phage  and tested Gp5 activity. Results showed that Gp5 331 \nalso fully inhibited the phage defence mediated by pBrxXLSty (Fig. 5a). 332 \n 333 \nAs we had demonstrated inhibition of BREX by overexpression of the inhibitors Ocr and Gp5, it was 334 \npostulated that the same experimental system might elicit phage defence mediated by  the PARIS 335 \nsystem. This time, the pBrxXLSty-ΔpglX strain was used for co-expression of Ocr or Gp5, as this strain is 336 \ndeficient for BREX phage defence but retains the PARIS system. The resulting EOP assays did not show 337 \nPARIS-dependent defence activity against TB34 (Fig. S7). We are therefore yet to find conditions that 338 \nstimulate activity of the Salmonella PARIS system.  339 \n 340 \nWe then aimed to recreate a PglX :Ocr complex 27, using our purified Salmonella PglX. The solution 341 \nstate of native PglX was determined using analytical SEC. PglX eluted from the SEC column at 15.55 ml 342 \n(Fig. S8a), which indicated a size of ~150 kDa, matching the 143 kDa calculated weight of PglX. These 343 \ndata indicate that PglX exists as a monomer in solution, supporting our conclusions from the PglX-SAM 344 \nstructure (Fig. 4). Analytical SEC was then performed to determine whether Ocr directly interacts with 345 \nthe Salmonella PglX. The Ocr sample was first examined by analytical SEC in isolation (Fig. S8a). Whilst 346 \nthe Ocr SEC profile appeared to have multiple species, there was a dominant peak at 15.9 ml and a 347 \nshoulder at 18 ml. Ocr is known to be a dimer in solution 26,47, which would be 27.6 kDa and correspond 348 \nto the 18 ml peak, leaving the 15.9 ml peak unidentified. Purity of the Ocr sample had previously been 349 \nconfirmed by mass spectrometry and SDS-PAGE (Fig. S8b and c). PglX and Ocr were then combined at 350 \na 1:2 molar ratio prior to SEC (Fig. S8a). The combined sample produced additional peaks beyond 351 \nthose from the individual PglX and Ocr samples (Fig. S8a). Of particular interest was the peak at an 352 \nelution volume of 14.2 ml  that indicated a large complex of approximately ~379 kDa , potentially 353 \ncomprised of at least two copies of PglX, and Ocr dimers (Fig. S8a). Elution volume is dependent on  354 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n14 \n 355 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n15 \nprotein molecular weight, and can also reflect the shape and size of the protein molecule itself. The 356 \nhydrodynamic radius of the PglX -Ocr complex seen by analytical  SEC can be calculated from the 357 \nobserved K av value 48, allowing comparison to the calculated hydrodynamic radius of predicted 358 \nPglX:Ocr complex models produced by AlphaFold 49. A model of two monomers of PglX and one Ocr 359 \ndimer produced by AlphaFold produced a predicted hydrodynamic radius of 58.3 Å , compared to a 360 \ncalculated hydrodynamic radius of 63.9 Å for the observed A -SEC peak . This suggested that the 361 \nadditional peak eluting at 14.2 ml represented a PglX-Ocr heterotetramer in solution. 362 \n 363 \nPglX forms a heterotetrameric complex with inhibitor Ocr  364 \nTo investigate the mechanism of BREX inhibition by Ocr, efforts were made to produce a structural 365 \nmodel via X-ray crystallography. PglX-SAM and Ocr were mixed at a 1:2 molar ratio and incubated 366 \nprior to setting crystallisation trials. After data collection and merging, and using our previously 367 \nderived PglX-SAM structure (Fig. 4) and the PDB structure of  Ocr (1S7Z) as search models, the PglX -368 \nSAM:Ocr structure was solved to 3.5 Å (Figs. 5b and c; Table 1).  369 \n 370 \nWithin the asymmetric unit, PglX -SAM binds to a protomer of Ocr as a 1:1 complex, with the single 371 \nprotomer of Ocr binding along the negatively charged region of the C -terminal domain of PglX. Data 372 \non the solution state of Ocr (a dimer), coupled with our predictions of complex size by analytical SEC, 373 \nindicated that PglX:Ocr should form a larger complex. Indeed, when we searched for crystallographic 374 \nsymmetry mates that showed packing of PglX -SAM:Ocr, the predicted complex was visible ( Figs. 5b 375 \nand c). In this complex, the Ocr protomers perfectly align and abut each other, forming the equivalent 376 \nof a solution state dimer , and the size matches our analytical SEC . We therefore concluded that this 377 \nheterotetrameric form represented the solution state of the PglX-SAM:Ocr complex (Figs. 5b and c).  378 \n 379 \nWithin PglX, there were again two regions of the sequence which could not be mo delled due to 380 \ninsufficient density (residues 54 – 55 and 413 – 420). The latter is an extended gap in the same region 381 \nas a smaller gap in the PglX -SAM structure (D418  – F420), suggesting flexibility in this region. Also 382 \nvisible in the PglX-SAM:Ocr structure is a bound SAM molecule, in the same ligand binding position as 383 \nseen in the PglX -SAM structure ( Figs. 4 and 5). The exact orientation of ribose and methionine 384 \ncomponents of the molecule varied slightly, though this is likely due to variation in manual positioning 385 \nof the molecule during refinement, as well as the resolution. The PglX molecules from the PglX -SAM 386 \nand PglX-SAM:Ocr structures align closely with an RMSD of 1.34 Å, suggesting that binding of Ocr does 387 \nnot elicit any substantive domain movement (Fig. S9). Important residue interactions for Ocr binding 388 \nwere inferred using EMBL PISA 50. The complex is stabilised by a number of hydrogen bonds between 389 \nOcr and the C-terminal domain of PglX (Fig. 5d). Six salt bridges are produced between R79, N35, N42, 390 \nN62, N76 and Q109 of Ocr and N1213, K1201, K1097, K1070, K1110, and K516 of PglX, respectively 391 \n(Fig. 5d). Though no movement is observed in PglX, the binding of Ocr to Type I RM complexes elicits 392 \ndomain movement similar to DNA binding, suggesting either that PglX domain movement is reliant on 393 \ninteractions with other BREX components, or that DNA binding occurs along the C -terminal domain 394 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n16 \nprior to movement towards the methyltransferase N-terminal domain. If other BREX components are 395 \nrequired for such movement, th e finding would be consistent with the lack of methyltransferase 396 \nactivity in vitro in the absence of other BREX components ( Fig. S6) or the lack of methyltransferase 397 \nactivity from PglX alone in vivo 25. Collectively, these data suggest that Ocr acts as a DNA mimic, 398 \ncapable of sequestering PglX and therefore blocking BREX activity by preventing recognition of target 399 \nDNA. 400 \n 401 \nStructural comparisons show multiple potential modes of DNA binding by 402 \nPglX 403 \nOcr mimics the structure of 20-24 bp of bent B-form DNA 26, as shown by the binding of both molecules 404 \nto the EcoKI methyltransferase complex  44. Using the DNA -bound (PDB 2Y7H) and Ocr -bound (PDB 405 \n2Y7C) complexes of EcoKI, the Ocr and DNA molecules were superimposed onto each other . As a 406 \nresult, the Ocr molecule in the PglX -SAM:Ocr structure was aligned with the Ocr molecule in 2Y7C, 407 \neffectively aligning the B -form DNA from 2Y7H to the Ocr molecule in PglX -SAM:Ocr structure ( Fig. 408 \nS10a). There does appear to be enough space for an extended DNA molecule to pass through the 409 \ngroove in the hinge region in this orientation, but Ocr is not long enough to extend through this region 410 \n(Figs. 6a and b; Fig. S10b). This implicates the C-terminal domain in DNA binding, though raises the 411 \npossibility of an alternative DNA binding orientation. 412 \n 413 \nThe surface charge of PglX was calculated using APBS software plugin 51 and modelled in PyMOL 52 to 414 \nattempt to predict a lternate DNA binding position s (Fig. S11a). Notably, PglX displayed a large 415 \npositively charged surface area in the hinge region between the N-and C-terminal domains, extending 416 \nfurther along the inside of the C -terminal domain. As MmeI was solved in a DNA -bound state (PDB 417 \n5HR4), we could s uperpose these two structures and remov e MmeI, leaving the DNA molecule s at 418 \nwithin th e positively charged hinge region  of PglX  (Fig. 6b; Fig. S 11b). Notably, the angle of the 419 \nsuperimposed DNA molecule from the MmeI structure (PDB 5HR4) differs from the previously 420 \nidentified angle of the 2Y7C DNA molecule (Fig. 6b). Further to this, the DNA molecule from the MmeI 421 \nstructure contained an adenine base which had been flipped out of the DNA molecule, in preparation 422 \nfor methyl transfer. Looking at the position of the superimposed MmeI DNA molecule, this adenine 423 \nbase is positioned close to the SAM m olecule in PglX ( Fig. S11b). Together, these data suggest that 424 \nPglX might bind DNA within the hinge region in a similar conformation to that seen with MmeI, though 425 \nthe exact orientation of the DNA molecule may shift around the position of the adenine base.  In 426 \nsupport of this prediction, the donated methyl group of the SAM is not quite positioned correctly for 427 \ntransfer to the flipped adenine (Fig. S11b). In this model, unlike for Ocr mimicking DNA, the distal C-428 \nterminal region of PglX remains largely removed from the DNA molecule, though binding of DNA may 429 \nrequire, or produce, a conformational change in PglX that brings this domain closer to the DNA. 430 \n 431 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n17 \n 432 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n18 \nPglX can be rationally engineered to alter phage target and methylation 433 \nmotif 434 \nRational engin eering of PglX could potentially allow for a BREX system to be targeted against a 435 \ndifferent set of phages, and for the generation of specific methylation patterns. To this end, p rotein 436 \nsequences from BREX -related methyltransferases with assigned DNA recogn ition motifs were 437 \ncollected and added to the sequences of BREX methyltransferases identified in the REBASE RM 438 \ndatabase 53. BLASTp was then used to find 32 distin ct sequences that displayed high sequence 439 \nsimilarity scores to PglX (<E100)  (Fig. S12). Most of the predicted motifs from REBASE were inferred 440 \nby matching the BREX methyltransferase to an N6mA modification  observed in genomic sequencing 441 \ndata. MmeI is the closest structural homologue of PglX and the residues essential for motif recognition 442 \nhave been identified from structural data 40. As with PglX, MmeI recognises a 6 bp motif (TCCRAC) and 443 \nproduces N6mA modifications at the 5th adenine base. Structural alignments of MmeI and PglX 444 \nallowed identification of the residue s of PglX that aligned with the residues involved in MmeI motif 445 \nrecognition and suggested regions in which to focus the search for covariation in BREX 446 \nmethyltransferase sequence alignments. Candidate residues and alterations were then chosen based 447 \non these alignments. For example, for motif position -1 (relative to the modified adenine base); lysine 448 \nwas conserved at residue 802 for enzymes recognising cytosine at this position, or histidine was 449 \nconserved at residue 838 for enzymes recognising guanosine at t his position, or asparagine was 450 \nconserved at residue 838 for enzymes recognising adenine at this position (Fig. S12). We designed 23 451 \nmutants that altered all five of the non-modified base positions in the PglX recognition motif 452 \n(Supplementary Table S 1). The regions targeted for mutation were overlaid on our structures and 453 \nshown to gather mainly within the TRD of PglX (between residues 684 – 838), with one additional loop 454 \n(residues 591 – 600) within the methyltransferase domain (Fig. 6c). 455 \n 456 \nFollowing the design of the PglX mutants, an assay system was required to test function. Generating 457 \neach of the mutants individually in the 17.9 kb pBrxXL Sty plasmid would have been costly and time 458 \nconsuming. Instead, a complementation system was designed that utili zed the pBrxXL Sty-ΔpglX 459 \nconstruct. The BREX Sty pglX gene was cloned into pBAD30. Complement ation of the pBrxXLSty-ΔpglX 460 \nconstruct with the pBAD30 -pglX plasmid in EOP assays provided phage defence against TB34, albeit 461 \nslightly lower than that seen  from the E. coli DH5α pBrxXLSty construct (Fig. 7a). Next, a marker was 462 \nrequired to indicate whether the recognition motif had been modified. Again, it was preferable to 463 \ninitially test this through functional EOP assays as sequencing for methylation chang es caused by all 464 \n23 mutants would be laborious and expensive. Fortunately, the activity of pBrxXLSty had already been 465 \ncharacterised against the Durham Phage Collection and phages in this collection had been sequenced 466 \nto allow enumeration of BREX recognition motifs 33. This allowed the identification of one phage, Trib, 467 \nwhich was susceptible to  E. coli and E. fergusonii BREX systems but contained no native Salmonella 468 \nD23580 BREX recognition motifs and therefore was not impacted by BREX Sty (Fig. 7a) 33. Trib did, 469 \nhowever, encode all of the predicted re -engineered motifs ( Supplementary Table S 1). This finding 470 \nallowed us to first screen all mutants for phage defence activity against phage Trib  before 471 \ndetermination of the recognition motif of any active mutants by sequencing. 472 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n19 \n 473 \n 474 \n 475 \n 476 \n 477 \n 478 \n 479 \n 480 \n 481 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n20 \nEOP assays were carried out in triplicate for all 23 pBAD30-pglX mutants co -expressed with the 482 \npBrxXLSty-ΔpglX construct in E. coli DH5α (data not shown). Mutant 3 appeared to provide around 10-483 \nfold protection against Trib (Fig. 7a), similar to phage defence levels provided by BREXEferg against this 484 \nphage 33. Mutants 8, 10, 15 and 22 showed sporadic reductions in EOP, usually around two -fold. 485 \nMutant 4 consistently produced poor overnight growth and failed to provide sufficient bacterial lawns 486 \nfor plaque enumeration, even after increasing the inoculum volume. Remaining mutants 487 \ndemonstrated no noticeable reduction in plaquing efficiency. To co nfirm the BREX system remained 488 \nfunctional against other targets, mutants 3, 8, 10, 15 and 22 were also assayed against phage TB34. 489 \nMutant 3 caused a reduction in EOP for TB34 similar to that shown against Trib, though around two-490 \nfold higher than produced by the E. coli DH5α pBrxXLSty strain (Fig. 7a). The remaining 18 mutants did 491 \nnot show any reduction in EOP against TB34 , despite TB34 encoding the expected re -engineered 492 \nmotifs, and were deemed to be inactive. The re was also a small reduction in BREX activity in the 493 \ncomplemented system (Fig. 7a). Accordingly, the T802A and S838N mutations of mutant 3 were also 494 \ngenerated directly within the pglX gene of pBrxXLSty, resulting in pBrxXL Sty(pglX mut.3) that did not 495 \nrequire complementation. This new construct was assayed against both TB34 and Trib. Now in context 496 \nwithin the BREX locus, EOP values were reduced further for both TB34 and Trib against E. coli DH5α 497 \npBrxXLSty(pglX mut.3), though still not quite as low as shown by the activity of the WT BREX system 498 \nagainst TB34 (Fig. 7a). 499 \n 500 \nNext, the host genomes of E. coli  DH5α pBrxXL Sty(pglX mut.3) and E. coli  DH5α pBrxXL Sty-ΔpglX + 501 \npBAD30-pglX(mut.3) strains were sequenced and genomic methylation levels were assessed by PacBio 502 \nsequencing, alongside the WT strains (Fig. 7b). The E. coli DH5α pBrxXLSty-ΔpglX + pBAD30-pglX control 503 \nhad almost 100% methylation at GATC AG sites, demonstrating that the  complementation system 504 \nmediated efficient methylation in comparison to pBrxXLSty (Fig. 7b). Analysis of the mutant 3 strains 505 \nrevealed methylation at almost 100% of GATMAG motifs (Fig. 7b). This indicated that the mutations 506 \nof mutant 3, T802A and S838N, had not altered the recognised motif to GATAAG as predicted, but had 507 \nbroadened recognition to include both  the original GATC AG motif and also GATA AG. These data 508 \ncollectively demonstrate the successful re-engineering of PglX to target BREX against new phages, and 509 \nto methylate altered DNA sequence motifs. The experiments also demonstrated that PglX is the sole 510 \nspecificity factor in the BREX phage defence  system, providing motif recognition for b oth phage 511 \ntargeting and host methylation. 512 \n 513 \n 514 \n 515 \n 516 \n 517 \n 518 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n21 \nDISCUSSION  519 \nThis study provides microbiological, genetic and epigenomic characterisation of the BREX phage 520 \ndefence island within Salmonella D23580. We present the first structures of the putative PglX  521 \nmethyltransferase, bound to SAM and in complex with the phage -derived inhibitor Ocr. Finally, we 522 \ndemonstrate successful rational engineering of BREX, opening up the potential for tailored phage 523 \ntargeting and generation of specific N6mA motifs. This work identifies PglX as the sole specificity factor 524 \nfor methylation and phage defence within BREX. 525 \n 526 \nClustered phage d efence systems can provide additive 22 or even synergistic  54 protection. The 527 \nSalmonella D23580 BREX phage defence island has an embedded PARIS system (Fig. 1a), suggesting a 528 \ncomplementary relationship; PARIS has been shown to cause abortive infection upon encountering 529 \nthe phage encoded anti -restriction protein, Ocr, which in turn inhibit s BREX defence in E. coli 16,27. 530 \nUsing an E. coli model, we saw no activity from the Salmonella BREX phage defence island against Ocr-531 \nencoding phage T7 (Fig. 2). The reason that BREXSty had no impact was because T7 does not encode 532 \nany GATC AG motifs . PARIS also did not respond to Ocr ( Fig. 2 ). Using an Ocr homolog from a 533 \nSalmonella phage also did not activate PARIS ( Fig. S7), and so we can only conclude that the PARIS 534 \nsystem may provide protection, but that a susceptible phage has not yet been tested. 535 \n 536 \nAs with previous studies,  Salmonella brxB, brxC, pglX and pglZ proved essential for both restriction 537 \nand methylation  (Fig. 3 ) 17,25. However, brxA was required  for phage defence  and methylation in 538 \nSalmonella BREX ( Fig. 3 ) and Acinetobacter BREX 17, but was shown to be dispensable for both 539 \nactivities in E. coli BREX 25. BrxA is a DNA-binding protein 23 with an unknown role in BREX activity, so 540 \nwe are yet to understand the variable requirement for brxA. Salmonella brxL was demonstrated to be 541 \ndispensable for host methylation (Fig. 3b) and this matches the observed phenotype in Acinetobacter 542 \nand E. coli 17,25. Curiously, whilst brxL was essential for phage defence in both E. coli and Acinetobacter 543 \nBREX systems 17,25, it was not required for Salmonella BREX (Fig. 3a). BrxL was recently shown to form 544 \na dimer of hexameric rings, forming a barrel-like structure that binds and translocates along DNA 24. 545 \nThus, BrxL had been considered to have an essential role as the “effector” for BREX phage defence. 546 \nClearly this is not the case in the Salmonella BREX system, which is made more apparent by EOP results 547 \nfor E. coli DH5α pBrxXLSty-ΔbrxL tested against the Durham phage collection (Fig. S3) 33. Deletion of 548 \nbrxL enhanced protection by several orders of magnitude  for certain phages  (Fig. S3). It is possible 549 \nthat Salmonella BrxL modulates or regulates BREX activity in some way . RM systems are often 550 \nassociated with restriction alleviation proteins that activate in times of stress, reducing restriction 551 \nactivity and increasing methylation activity; a phenotype characteristic of Type I RM systems 55–57. It is 552 \npossible that BrxL plays an analogous role to restriction alleviation proteins within  BREX and that 553 \ndefence activity increases in the absence of BrxL. However, if that were the case, why is this phenotype 554 \nnot observed for brxL deletions in E. coli or Acinetobacter BREX systems? Overexpression of a C -555 \nterminal fragment of BrxL has been shown to upregulate several genes elsewhere in the Salmonella 556 \ngenome, including certain prophage genes 29. It was postulated that because the corresponding Lon-557 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n22 \nlike domain in th e C-terminal BrxL fragment has similarity to the Lon -related C-terminal domain of 558 \nRadA that is required for DNA branch migration in homologous recombination 58, BrxL may inhibit 559 \nphage DNA replication at DNA forks. This would be somewhat in keeping with the  model of BrxL 560 \ncomplexes translocating along DNA. The brxL deletion data provide additional insight to this model as 561 \nthey suggest that whilst BrxL -dependent BREX defence may interrupt replication forks, other BREX 562 \ncomponents have another activity sufficient to prevent phage DNA replication. 563 \n 564 \nTo better understand the activity of other BREX components we produced the first structure of PglX, 565 \ndemonstrating that the N -terminal domain has a methyltransferase fold, and binds SAM ( Fig. 4). In 566 \ncontrast, fold, conserved residues, and surface properties of the C-terminal domain suggest a role in 567 \nDNA recognition and binding. Despite repeated efforts w e could not crystallize PglX with DNA. We 568 \nhypothesised that Ocr binding might provide insight into DNA binding by PglX . We showed that Ocr 569 \nand Salmonella homolog Gp5 both impacted BREX phage defence (Fig. 5a), and produced stable 570 \ncomplexes of PglX:Ocr (Fig. S8a). The resulting structure involved the interaction of an Ocr dimer with 571 \ntwo PglX monomers ( Figs. 5b and c). The structure of PglX in th e Ocr-bound complex varied little in 572 \ncomparison to the PglX -SAM structure, and there  was no movement of domains upon Ocr binding. 573 \nUsing these two structures, we developed two models for DNA binding by PglX, via (i) alignment with 574 \na 20 bp DNA molecule represented by Ocr and (ii) alignment via DNA bound to MmeI (Figs. 6a and b; 575 \nFig. S 10). As t he Ocr -bound structure only allows placement of a short, 20 bp , DNA molecule, it 576 \ninteracts with the C-terminal domain but does not enter the hinge region between N-terminal and C-577 \nterminal domains. The MmeI-bound DNA is positioned to interact with the hinge and TRD. Our data 578 \nshould aid the design of oligos for future structural studies of  PglX bound to DNA , and supported 579 \nefforts to engineer BREX activity (Fig. 6c). 580 \n 581 \nRational engineering of PglX broadened motif recognition, allowing the Salmonella BREX to target new 582 \nphages and methylate new BREX motifs ( Fig. 7).  We were able to switch recognition for position -1 583 \n(relative to the point of methylation). MmeI recognises guanine at this position using R810 to form a 584 \nhydrogen bond with guanine in the major groove, and an A774L mutant was shown to prevent binding 585 \nof an A-T base pairing at position -1 through steric interference, switching specificity from R:Y to G:C 586 \n40,59. The T802A and S838N mutations in PglX mutant 3 correspond to the positions of the A774 and 587 \nR810 residues in MmeI, respectively, and are within the TRD. As rapid adaptability and evolution are 588 \nvital factors in the phage -bacteria arms race that increase survivability of the local population  60, it 589 \nfollows that PglX would be the target of variability as a means to alter BREX defence specificity. Indeed, 590 \nphase variation is common in pglX genes, but not other BREX components 10,61. 591 \n 592 \nThe inability of PglX to perf orm methylation during our in vitro reaction, nor when recombinantly 593 \nexpressed in the absence of other BREX genes  in vivo 25, implies higher order BREX complexes might 594 \nbe required. Such complexes could induce domain movements that would provide agreement with 595 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n23 \nboth proposed models of DNA binding. The arrangement of PglX monomers in the Ocr -bound 596 \nstructure is also potentially interesting, as a larger BREX complex might scan both sides of a dsDNA for 597 \nthe non-palindromic BREX motif by employing two PglX monomers, akin to the use by Type III and 598 \nsome dimeric Type II RM systems. Clearly, further work is needed on BREX components and complexes 599 \nto uncover mechanistic details. The current study demonstrates that PglX is the sole BREX specificity 600 \nfactor, responsible for both the recognition and targeting of individual BREX motifs for host 601 \nmethylation and the resulting prevention of phage replication.  602 \n 603 \n 604 \n 605 \n 606 \n 607 \n 608 \n 609 \n 610 \n 611 \n 612 \n 613 \n 614 \n 615 \n 616 \n 617 \n 618 \n 619 \n 620 \n 621 \n 622 \n 623 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n24 \nMATERIALS AND METHODS 624 \nBacterial strains  625 \nStrains used in this study are shown in Supplementary Table 2 . We have described the  Salmonella 626 \nD23850Δφ strain previously  62. The Salmonella D23850ΔφΔBREX strain was generated as described 627 \npreviously 37, using scarless lambda red recombination (Fig. S1). Unless stated otherwise, E. coli strains 628 \nDH5α (Invitrogen), BL21 ( λDE3, Invitrogen) and ER2796 (NEB) were grown at 37 °C, either on agar 629 \nplates or shaking at 220 rpm for liquid cultures. Luria broth (LB) was used as the standard growth 630 \nmedia for liquid cultures, and was supplemented with 0.35% w/v or 1.5% w/v agar for semi-solid and 631 \nsolid agar plates, respectively. Growth was monitored using a spectrophotometer (WPA Biowave 632 \nC08000) measuring optical density at 600 nm (OD 600). When necessary, growth media wa s 633 \nsupplemented with ampicillin (Ap, 100 µg/ml) or chloramphenicol (Cm, 25 µg/ml). Protein was 634 \nexpressed from pSAT1 or pBAD30 plasmid backbones by addition of 0.5 mM isopropyl-β-D-635 \nthiogalactopyranoside (IPTG) or 0.1% L-arabinose, respectively. 636 \n 637 \n 638 \nUse of envi ronmental phages   639 \nPhages used in this study are shown in Supplementary Table 2 . Coliphages in the Durham phage 640 \ncollection have been d escribed previously 33. For Salmonella phages, sewage effluent was collected 641 \nfrom a sampling site in Durham, courtesy of Northumbrian Water Ltd. Filtrates were supplemented 642 \nwith 10 ml of LB, and inoculated with 10 ml of D23580ΔφΔBREX. Cultures were grown for 3 days before 643 \na 1 ml aliquot s were transferred to sterile microcentrifuge tubes and centrifuged at 12000 x g for 5 644 \nmin at 4 °C. The supernatants were transferred to new microcentrifuge tubes and 100 μl of chloroform 645 \nwas added to kill any remaining bacteria. Phage isolation was then carried out as previously described 646 \n33. 647 \n 648 \nPlasmid constructs and cloning  649 \nPrimers used in this study are shown in Supplementary Table 3, and plasmids used in this study are 650 \nshown in Supplementary Table 4 . Ligation independent cloning (LIC) was utili zed to create protein 651 \noverexpression plasmids from pSAT1-LIC and pBAD30-LIC, as described previously 63. This allowed the 652 \nexpression of fusion proteins with cleavable tags for efficient purification of recombinant proteins.  653 \nThe pBrxXL Sty plasmid was created previously 33 and contains the entire Salmonella D23580 BREX 654 \ncoding region, including the regio n 508 bp directly upstream of the brxA start codon to ensure that 655 \nany promoters and transcriptional regulatory sites required for BREX expression and function were 656 \nincluded. The creation of individual gene knockouts utili zed Gibson Assembly (Gibson Assembl y) 64. 657 \nIndividual gene knockouts were designed within the context of the pBrxXL Sty vector to allow direct 658 \ncomparison on the same plasmid backbone. PCR primers were designed to amplify the pBrxXL Sty 659 \nplasmid sequence either side of the gene to be removed ( Supplementary Table 3). Primers were 660 \ndesigned with overlapping regions to allow ligation of the amplicons via GA. GA designs consisted of 661 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n25 \n2-3 fragments of pBrxXLSty produced by PCR with primers containing 20 bp homologous overlaps from 662 \nupstream and downstream of the gene to be removed. Knockouts were designed for each of the six 663 \nBREX gene s, each of the two PARIS system genes, ariA and ariB, alongside an additional double 664 \nknockout of both PARIS system genes. PCR -amplified and gel -purified fragments were pooled in an 665 \nequimolar ratio to a final volume of 5 μl and added to 15 μl of assembly master mix. Reaction mixtures 666 \nwere incubated at 50 °C for 1 hr, then visualized on and gel purified from agarose gels. Resulting 667 \nproducts which displayed the correct size were used to transform E. coli DH5α and cells were plated 668 \non Cm agar plates and incubated at 37 °C overnight. Plasmids from resulting colonies were extracted 669 \nand sequenced (DBS Genomics) to confirm correct assembly. Gene knockouts for which GA was not 670 \nsuccessful were instead synthesised by Genscript.  Primers for GA protocols were synthesised by IDT 671 \nand were designed using the Benchling cloning design software, available online (benchling.com).  672 \n 673 \nDNA sequencing 674 \nAll genomic DNA extraction steps in this study were carried out using either a Zymo Miniprep Plus kit 675 \n(Cambridge Biosciences) or a Monarch gDNA extraction kit (NEB). Bacterial genomic sequencing was 676 \nperformed by either MinION Mk1C nanopore sequencing or PacBio sequencing.  677 \n 678 \nFor MinION sequencing, DNA repair and end prep, barcode ligation and adapter ligation steps were 679 \ncarried out accord ing to Oxford Nanopore protocols (available at: community.nanopore.com) using 680 \nthe NEBNext Companion Module (New England Biolabs), Native Barcoding Expansions (EXP -NBD104 681 \nand EXP-NBD114) and ligation sequencing kit (SQK-LSK109), respectively. Sequencing was carried out 682 \nusing a MinION Flow cell (R9.4.1) on a MinION Mk1C. Following generation of raw sequencing data, 683 \nbasecalling was performed by the Guppy basecalling package 684 \n(github.com/nanoporetech/pyguppyclient) either during sequencing or post sequencing and data was 685 \ndeconvoluted using the ont_fast5_api package (github.com/nanoporetech/ont_fast5_api). 686 \nMegalodon was used for the detection of modified bases and the estimation of genomic methylation 687 \nlevels, with a 0.75 probability threshold for both modified and canonical bases for read selection and 688 \naverage percentage methylation calculations. 689 \n 690 \nLibraries for sequencing were prepared using the SMRTbell Template Prep kit 3.0 (Pacific Biosciences). 691 \nBacterial gDNA was sheared using gTubes (Covaris) to produce DNA fragments with a mean size of 5–692 \n10 kb. The DNA was damage repaired and end repaired. SMRT -bell adapters were then ligated. 693 \nExonuclease treatment removed Non SMRT-bell DNA. Sequencing was performed on a PacBio Sequel 694 \nIIe (Pacific Biosciences). Data were analysed using PacBio SMRTAnalysis on SMRTLink_9.0 software 695 \nBase Modification Analysis for Sequel data, to identify DNA modifications and their corr esponding 696 \ntarget motifs. 697 \n 698 \nGrowth and infection curves 699 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n26 \nPhage growth and infection curves were carried out to monitor phage resistance conferred by 700 \npBrxXLSty WT and pBrxXLSty mutants in liquid culture. Growth was carried out in 200 μl culture volumes 701 \nat 37 °C with shaking in a 96 -well plate format, with OD 600 measurements taken every 5 min. Initial 702 \nscreening of inoculation and infection conditions produced optimal results with  initial inoculation 703 \nfrom overnight culture to OD 600 0.1 and phage multiplicity of infection (MOI) of 10 -6. As well as 704 \ninfection with phage TB34, a negative control – phage T7 – and a positive control (uninfected culture) 705 \nwere also run for each strain. All strains other than E. coli DH5α WT were grown with 25 μg/ml Cm. 706 \n 707 \nEfficiency Of Plating assays. 708 \nEfficiency of plating (EOP) assays were carried out to assess the plaquing ability of phages in the 709 \nDurham Phage Collection against E. coli DH5α pBrxXLSty and BREX knockout strains relative to control 710 \nstrains. We used serial dilutions of high titre lysates in phage buffer and dilutions were mixed with 711 \novernight culture and molten 0.3% w/v agar, poured onto a 1% agar plate, dried and incubated 712 \novernight at 37 °C. For strains containing pBAD30 vectors, overnight cultures were induced with 0.2% 713 \nw/v L-arabinose and incubated at 37 °C for 30 min prior to plating and both top and bottom agar layers 714 \nincluded 0.2% w/v L-arabinose to induce continuous expression over the course of lawn growth. The 715 \nEOP was calculated by dividing the pfu (plaque forming units) of the test strain by the pfu of the control 716 \nstrain. Data shown are the mean and the standard deviation of at least 3 biological and technical 717 \nreplicates.    718 \n 719 \nProtein expression and purification  720 \nAll large-scale protein expression was performed in 1 L volumes of 2x YT broth in 2 L flasks with shaking 721 \nat 180 rpm. In all cases, colonies from fresh transformation plates were used to inoculate 5 ml of 2x 722 \nYT broth and grown overnight at 37 °C. This culture was then used to seed a 65 ml volume of 2x YT 723 \nbroth at 1 : 100 v/v and grown overnight at 37 °C to produce a second overnight culture. This culture 724 \nwas then used to seed 1 L of 2x TY at a 1 : 200 ratio, cultures were grown at 37 °C until exponential 725 \ngrowth phase (OD600 0.3 – 0.7), induced, and protein was expressed at 18 °C overnight. 726 \n 727 \nAll purification st eps were performed either on ice or at 4 °C. Fast protein liquid chromatography 728 \n(FPLC) steps were carried out at 4 °C using an Akta Pure protein chromatography system (Cytiva). 729 \nProtein purity was assessed using SDS -PAGE. Cells were harvested by centrifugation at 4000 rpm for 730 \n15 min at 4 ⁰C  and then resuspended in ice-cold A500 buffer (20 mM Tris HCl pH 7.9, 500 mM NaCl, 731 \n30 mM imidazole, 10% glycerol) . Cells were lysed by sonication using a Vibracell VCX500 732 \nultrasonicator, the soluble fraction was separated from insoluble cell material by centrifugation at 733 \n20000 x g for 45 minutes at 4 °C and the supernatant was removed to a fresh, chilled tube for 734 \npurification. Soluble cell lysate was applied to a 5 ml pre-packed Ni-NTA His-Trap HP column (Cytiva) 735 \nusing a benchtop peristaltic pump at around 1.5 ml/min to allow binding of the 6xHis tag to the nickel 736 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n27 \nresin. Columns were then washed with between 5 – 10 column volumes (CVs) of A500 to remove 737 \nresidual unbound protein and isocratic elution steps were performed using A500 buffer with imidazole 738 \nconcentrations adjusted to 30 mM, 50 mM, 90 mM, 150 mM and 250 mM. Clean samples were pooled, 739 \ndialysed into low salt A100 buffer ( 20 mM Tris HCl pH 7.9, 100 mM NaCl, 10 mM imidazole, 10% 740 \nglycerol) and applied to a 5 ml HiTrap Heparin HP column (Cytiva), allowing separation of proteins with 741 \naffinity for DNA. Bound protein was then washed with 5 – 10 CV of A100 and eluted using a salt 742 \ngradient with C1000 buffer (20 mM Tris HCl pH 7.9, 1 M NaCl, 10% glycerol). Clean fractions were then 743 \npooled and digested with of human sentrin/SUMO-specific protease 2 (hSENP2) overnight at 4 °C to 744 \nremove purification tags. Samples were then applied to a second Ni-NTA His-Trap HP column, this time 745 \nallowing the now untagged protein of interest to flow through and removing remaining nickel binding 746 \ncontaminants. Successful tag cleavage and subsequent protein purity was assessed by SDS-PAGE, with 747 \ntag cleavage visible as a noticeable reduction in protein molecular weight relative to tagged protein.  748 \nFinally, size exclusion chromatography (SEC) was used to separate proteins by size, using a HiPrep 749 \n16/60 Sephacryl S -200 SEC column (Cytiva) connected to the FPLC system. Protein samples were 750 \ndialysed overnight at 4 °C into S500 buffer  (50 mM Tris HCl pH 7.9, 500  mM KCl, 10% glycerol ) and 751 \nconcentrated to a 500 μl volume. The column was pre-equilibrated in S500, and the sample was loaded 752 \nthrough a 500 μl volume capillary loop at 0.5 ml/min. Sample was eluted over 1.2 CVs at 0.5 ml/min 753 \nand fractionated into 2 ml vol umes for analysis by SDS -PAGE. Purified protein from SEC was 754 \nconcentrated to around 6 mg/ml and diluted in storage buffer  (50 mM Tris HCl pH 7.9, 500 mM KCl, 755 \n70% glycerol) at a 1 : 2 ratio of protein to buffer, respectively, giving a final concentration of around 2 756 \nmg/ml. Samples were split into appropriately sized aliquots, snap frozen in liquid nitrogen and stored 757 \nat -80 °C for future use. 758 \n 759 \nProtein crystallization and structure determination  760 \nHighly pure protein samples were used for crystallisation screen ing. Samples were either used 761 \nimmediately following purification or thawed on ice from -80 °C storage. Samples were dialysed into 762 \ncrystal buffer ( 20 mM Tris HCl pH 7.9, 150 mM NaCl, 2 .5 mM DTT ) and concentrated to 12 mg/ml. 763 \nProtein concentration determinat ion was performed using Nanodrop One (Thermofisher). Crystal 764 \nscreens were set using the sitting drop vapour diffusion method either by hand or using a Mosquito 765 \nXtal3 liquid handling robot (SPT Labtech). Crystal screens were incubated at 18 °C. All commerci ally 766 \navailable crystal screens were produced by Molecular Dimensions. For PglX and SAM samples, PglX 767 \nwas incubated with 1 mM SAM (Sigma) for 30 minutes on ice prior to addition to screens. For PglX -768 \nSAM:Ocr samples, PglX underwent the SAM incubation as above plus an additional 30 minute 769 \nincubation on ice with 2.74 mg/ml of Ocr. Ocr was recombinantly expressed and purified as previously 770 \ndescribed 26,47. PglX -SAM crystallized in 0.2 M potassium bromide, 0.1 M Tris pH 7.5, 8% w/v PEG 771 \n20000, 5% w/v PEG 500. PglX-SAM:Ocr crystallized in 0.1 M sodium/potassium phosphate pH 6.2, 14% 772 \nw/v PEG 4000, 6% MPD. Crystallization was confirmed by microscopy, with larger crystals extracted 773 \nfor X-ray diffraction. To harvest, 20 μl of screen condition was mixed with 20 μl of cryo buffer (25 mM 774 \nTris HCl pH 7.9, 187.5 mM NaCl, 3.125 mM DTT, 80% glycerol) and the solution was mixed thoroughly 775 \nby vortexing. This solution was then added directly to the crystal drop at a 1 : 1 ratio.  Crystals were 776 \nextracted using nylon cryo loops and stored in liq uid nitrogen until shipment.  Data collection was 777 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n28 \ncarried out remotely at Diamond Light Source, Oxford, UK on beamlines I04 and I24, using their 778 \n“Generic Data Acquisition” software (opengda.org). 779 \n 780 \nInitial data processing was performed by automated processes on iSpyB (Diamond Light Source) using 781 \nthe Xia2-DIALS X-ray data processing and integration tool 65.  The same program was used to merge 782 \nmultiple datasets and provide initial data on the space groups and unit cell sizes. Further data 783 \nreduction and production of dataset statistics was carried out using AIMLESS within CCP4i2 66. Merged 784 \ndatasets were first processed in CCP4i2 using BUCCANEER and REFMAC 66, and then iteratively built 785 \nand refined in Coot 67 and Phenix 68, respectively. Quality of the final model was assessed using a 786 \ncombination of CCP4i2, Phenix, Coot and the wwPDB validation server. Visualisation and structural 787 \nfigure generation was performed in PyMol 52. For PglX, the crystal structure was solved by molecular 788 \nreplacement in Phaser 69 using the PglX predicted model produced by AlphaFold 46. The SAM molecule 789 \nwas downloaded from the PDB ligand repository and placed manually in Coot and similarly iteratively 790 \nbuilt and refined. The structure of the PglX -SAM:Ocr heterodimer complex was solved by molecular 791 \nreplacement in Phaser  69 using the PglX structure solved previously and the structure of Ocr ( PDB 792 \n1S7Z). 793 \n 794 \nAnalytical Size Exclusion Chromatography  795 \nAnalytical SEC was performed on a Superose 6 10/300 GL SEC column (Cytiva, discontinued) connected 796 \nto an Akta Pure protein chromatography system (Cytiva). The column, system and loading loop were 797 \nwashed between each run and equilibrated with 1.2 CVs of A-SEC buffer (20 mM Tris-HCl pH 7.9, 150 798 \nmM NaCl ). Protein samples were buffer exchanged into A -SEC buffer and concentrated. Final 799 \nconcentration ranged between 1 μM and 5 μM, as required to give a distinct measurable elution peak. 800 \nProtein was loaded onto the system via a 100 μl capillary loop loaded using a 100 μl Hamilton syringe. 801 \nFor PglX-SAM:Ocr samples, PglX was incubated with each on ice in the same process as that used for 802 \ncrystallisation screening. Protein in capillary loops was injected onto the column with 1.5 ml of A-SEC 803 \nbuffer and eluted over 1.2 CVs with A -SEC buffer at 0.5 ml/min. For estimation of protein molecular 804 \nweight, relative to elution volume (Ve), a calibration curve was produced from commercially available 805 \nhigh and low molecular weight pr otein calibration kits (Cytiva). Peaks were identified using the 806 \nUnicorn 7 software package (Cytiva).  807 \n 808 \nVe values were converted into the partitioning coefficient (Kav) for each sample using the equation: 809 \n 810 \nKav = Ve- Vo\nVc- Vo\n 811 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n29 \n 812 \nThe molecular weight calibration curve is then plotted as Kav against Log10(Mr, kDa). The Stokes radius 813 \ncalibration curve plotted as Log 10(Rst, Å) against Kav, allowing calculation of sample Stokes radius 814 \nmeasurements. Estimated stokes radius calculations were carried out using the HullRad Stokes radius 815 \nestimation server 49. 816 \n 817 \nMethyltransferase assay  818 \nSAM-dependant N6mA DNA methylation activi ty of PglX was probed in vitro  using an MTase -Glo 819 \nMethytransferase Assay kit (Promega). The kit allows indirect measurement of SAM -dependent 820 \nmethyltransferase activity via production of the SAH reaction product. Through a proprietary two step 821 \nreaction, SAH is used to produce ADP then ATP, which in turn is used by a luciferase reporter enzyme 822 \nto generate a measurable luminescence signal. Signal can then be correlated to that produced by a 823 \nSAH standard curve. The methyltransferase assay was carried out as per manufacturer’s instructions 824 \nin a 96-well plate format. PglX was buffer exchanged into the methyltransferase assay reaction buffer 825 \n(80 mM Tris pH 8.8, 200 mM NaCl, 4 mM EDTA, 12 mM MgCl2, 4 mM dithiothreitol (DTT) and 826 \nconcentrated to 1 μM. As a substrate, 100 ng of E. coli DH5α genomic DNA was used per reaction as 827 \nthis should provide ample Salmonella BREX recognition motifs for methylation. The reaction mix was 828 \nthen combined with the protein samples at a 1 : 1 ratio with 10 μM of SAM and the reaction was 829 \nincubated at room temperature for 30 minutes. The SAH standard curve  was prepared by two -fold 830 \nserial dilutions of a 1 μM SAH stock in methyltransferase reaction buffer. Luminescence was measured 831 \non a Biotek Synergy 2 plate reader.   832 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n30 \nDATA AVAILABILITY  833 \nThe crystal structures of PglX-SAM and PglX-SAM:Ocr have been deposited in the Protein Data Bank 834 \nunder accession number s 8C45 and 8Q56, respectively . All other data needed to evaluate the 835 \nconclusions in the paper are present in the paper and/or Supplementary Data. MinION and PacBio 836 \ndata that support the findings of this study have been deposited in the European Nucleotide Archive 837 \n(ENA) at EMBL-EBI under accession number PRJEB71369. 838 \n 839 \nFUNDING 840 \nThis work was supported by an Engineering and Physical Sciences Research Council Molecular Sciences 841 \nfor Medicine Centre for Doctoral Training studentship [grant number EP/S022791/1] to S.C.W., a 842 \nBiotechnology and Biological Sciences Research Council Newcastle -Liverpool-Durham Doctor al 843 \nTraining Partnership studentship [grant number BB/M011186/1] to D.M.P., and a Lister Institute Prize 844 \nFellowship to T.R.B. This work was supported in part by a Wellcome Trust Senior Investigator award 845 \n[grant number 106914/Z/15/Z] to J.C.D.H. For the purpose of open access, the authors have applied a 846 \nCC BY public copyright licence to any Author Accepted Manuscript version arising from this 847 \nsubmission. 848 \n 849 \nACKNOWLEDGEMENTS  850 \nWe gratefully acknowledge Diamond Light Source for time on beamlines I04 and I24 under proposal 851 \nMX24948. 852 \n 853 \nCOMPETING INTERESTS  854 \nThe authors declare no competing interests. 855 \n 856 \nCONTRIBUTIONS  857 \nAnalysed data: S.C.W., D.M.P., R.D.M., A.N., N.W. and T.R.B. Designed research: S.C.W., D.M.P., 858 \nR.D.M., A.N., D.T.F.D., D.L.S., N.W., J.C.D.H. and T.R.B. Performed research: S.C.W., D.M.P., R.D.M., 859 \nA.N., N.W. AND T.R.B. Wrote the paper: S.C.W., D.M.P., A.N., D.T.F.D, J.C.D.H and T.R.B . Funding 860 \nacquisition: J.C.D.H. and T.R.B. Supervised the study: D.L.S., J.C.D.H. and T.R.B. 861 \n 862 \n 863 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n31 \nREFERENCES  864 \n1. Hampton, H. G., Watson, B. N. J. & Fineran, P. C. The arms race between bacteria and their 865 \nphage foes. Nature 577, 327–336 (2020). 866 \n2. Stern, A. & Sorek, R. The phage-host arms race: shaping the evolution of microbes. Bioessays 867 \n33, 43–51 (2011). 868 \n3. Tock, M. R. & Dryden, D. T. F. The biology of restriction and anti-restriction. Current Opinion 869 \nin Microbiology vol. 8 466–472 (2005). 870 \n4. Blower, T. R. et al. 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Genetics of the phage growth limitation (Pgl) system of 999 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n38 \nStreptomyces coelicolor A3(2). Mol. Microbiol. 44, 489–500 (2002). 1000 \n62. Owen, S. V. et al. Characterization of the Prophage Repertoire of African Salmonella 1001 \nTyphimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1. 1002 \nFront. Microbiol. 8, 235 (2017). 1003 \n63. Cai, Y. et al. A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis 1004 \nthrough inactivation of tRNA acceptor stems. Sci. Adv. 6, eabb6651 (2020). 1005 \n64. Gibson, D. G. et al. 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Crystallogr. 40, 658–674 (2007). 1016 \n 1017 \n 1018 \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint \n\n \n \n \n \n39 \nTABLE 1019 \n 1020 \n 1021 \n 1022 \n 1023 \n 1024 \n 1025 \n 1026 \n 1027 \n 1028 \n 1029 \n 1030 \n 1031 \n 1032 \n 1033 \n 1034 \n 1035 \n 1036 \n 1037 \n 1038 \n 1039 \n 1040 \n 1041 \n 1042 \nTable 1. X-Ray data collection and refinement statistics  \nStructure PglX-SAM PglX-SAM:Ocr \nPDB Code 8C45 8Q56 \nWavelength 0.9795 0.9795  \nResolution range 48.98 - 3.402 (3.523 - \n3.402) \n59.61 - 3.5 (3.625 - 3.5)  \nSpace group P 41 21 2 C 1 2 1  \nUnit cell, a b c (Å), \nα β γ (°)  \n138.539 138.539 \n407.956 90 90 90 \n238.458 60.786 146.637 \n90 114.889 90  \nTotal reflections 104405 47094 (8532)  \nUnique reflections 55611 (5460) 24556 (2426)  \nMultiplicity 1.9 1.9  \nCompleteness (%) 87.15 (15.55) 97.84 (80.53)  \nMean I/sigma(I) 8 (0.1) 3.8 (0.3)  \nRmerge 0.047 0.028  \nRmeas 0.067 (2.142) 0.092 (0.756)  \nCC1/2 0.999 (0.214) 0.995 (0.378)  \nReflections used in refinement 48492 (849) 24038 (1957)  \nReflections used for Rfree 2444 (43) 1922 (144)  \nRwork 0.2745 (0.4253) 0.2462 (0.4074)  \nRfree 0.2992 (0.4026) 0.2917 (0.4202)  \nNumber of non-hydrogen atoms 19848 10776  \nmacromolecules 19848 10747  \nligands 98 49  \nsolvents 0 2  \nProtein residues 2432 1318  \nRMS (bonds, Å) 0.005 0.004  \nRMS (angles, °) 0.91 0.78  \nRamachandran favored (%) 90.36 91.6  \nRamachandran allowed (%) 9.64  8.4 \nRamachandran outliers (%) 0 0 \nAverage B-factor 169.33 138.5  \nmacromolecules 169.33 138.54  \nligands  104 139  \nsolvent  N/A 113.43  \nValues in parenthesis are for the highest resolution shell  \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 April 13, 2024. ; https://doi.org/10.1101/2024.04.12.589231doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}