1 Supporting information 2 Materials and methods 3 4 Preparation of anti-MrgA IgY 5 The mrgA gene was amplified by using primers SA1941-f(Eco)Exp and SA1941-r(Sal), and cloned 6 into EcoRI – SalI site of pGEX5X-1(amersham pharmacia biotech). MrgA protein was expressed as 7 a fusion protein with GST, and purified according to the manufacture’s instruction. GST was cleaved 8 off by Factor Xa treatment. The digested proteins were fractionated by SDS-PAGE and visualized 9 by CBB. The gel slices containing about 1.5 mg MrgA was used to immunize a chicken as 10 previously described (Morikawa et al., 2003). 11 12 Western blot analysis 13 Whole cell extract was prepared using lysostaphin (1µg in 80 µL PBS pH 7.4) as described 14 previously (Morikawa et al., 2003). 15 The whole cell extract (equivalent to 250 µL culture of OD600 0.7) or the proteins from 16 chromatography were submitted to SDS polyacrylamide gels electrophoresis (15% gel) and 17 transferred to immune-Blot PVDF Membrane (BIORAD, 0.2 µm). MrgA detection was carried out 18 as previously described, using 1000-fold diluted anti-MrgA IgY (Morikawa et al., 2003). 19 20 Purification of dodecamer MrgA and MrgA* 21 The mrgA gene was amplified by primers Dps-S and Dps-AS2 using N315 genomic DNA as a 22 template. The mrgA* was amplified by mrgA*-y10-F and mrgA*-y11-R from N315mrgA* genomic 23 DNA (Table S1). These fragments were cloned into the NcoI and XhoI sites of pET-28b to generate 24 pET-28b-mrgA and pET-28b-mrgA*, respectively. These vectors express MrgA or MrgA* without 25 histidine tag. 26 MrgA and MrgA* were expressed in E. coli as described above. E. coli BL21 (DE3) harboring 27 each plasmid was grown at 37 °C in 200 mL of LB medium supplemented with 25 μg mL-1 28 kanamycin with shaking at 150 r.p.m for 24h. 50 mL of the saturated culture was inoculated into 1 L 29 of fresh LB medium supplemented with 25 μg mL-1 kanamycin and cultured for 2 h followed by 30 keeping at 4 °C for 2 h. IPTG was added to a final concentration of 0.25 mM, and further cultured 31 for overnight at 25 °C. 32 Cells were harvested by centrifugation at 4500×g for 10 min at 4 °C, and disrupted using a 33 sonicator (Branson, Danbury, CT) in 50 mM sodium phosphate (pH 8.0), 50 mM NaCl. The cell 34 debris was removed by centrifugation at 40000×g for 1 h at 4 °C, and the supernatant was loaded 35 onto a HiTrap Q HP column (GE Healthcare Biosciences AB, Sweden) pre-equilibrated with 20 mM 36 Tris-HCl (pH 8.0) for MrgA and 20 mM Tris-HCl (pH 9.0) for MrgA*. High pH value was 37 preferable for efficient recovery of MrgA*. Following the washing with the same buffer, adsorbed 38 protein was eluted with 125 mL of 10–500 mM gradient of NaCl in 20 mM Tris-HCl (pH 8.0 or 9.0). 39 Each protein fractions (about 120 mL) were filtered through 0.22 m membrane, then further 40 purified by HiLoad 26/60 Superdex 200-pg with 2 mL injection or HiPrep 16/60 Sephacryl S-200 41 column with 200 L injection, with 20 mM Tris-HCl (pH 8.0), 200 mM NaCl. Dodecamer fractions 42 were collected (Fig. S3). About 1mg of MrgA or MrgA* was prepared with 6-8 times or 25-30 times 43 injection, respectively. Proteins were concentrated by Amicon 50K. If necessary, gel exclusion 44 chromatography was repeated to obtain pure dodecamer preparation. The purity was more than 95% 45 in SDS-PAGE stained with Coomassie brilliant blue (Fig. 3m). 46 47 Observation of DNA-MrgA or MrgA* complexes 48 Supercoiled plasmid (4.5 ng pMK3, 7.2 kbp) was incubated with or without MrgA or MrgA* (75 ng 49 dodecamer) at 37 °C for 10 min, in 15 µl of 10 mM HEPES (pH 7.5) buffer containing 50 mM NaCl 50 and 2 mM MgCl2. The DNA/ MrgA or MrgA*-dodecamer molar ratio was 1:395. The samples were 51 deposited onto mica coated with 1 mM spermidine. After 5 min, the surface was washed by 500 L 52 of MilliQ, dried with nitrogen, and submitted to AFM analyses. 53 54 In vitro DNA protection assay 55 Plasmid DNA (300 ng pMK3) was pre-incubated with or without MrgA, or MrgA*-dodecamer (10 56 and 20 g ) in 30 l of 20 mM Tris-HCl (pH8.0) buffer containing 200 mM NaCl at 37 °C for 1 h. 8 57 l of 1.5 mM fresh ferrous ammonium sulfate solution was added and incubated for 5 min at 25 °C, 58 followed by the addition of 2 µl of 200 mM H2O2 (final conc. 10mM). After 5 min at 25 °C, the 59 reaction was mixed with 10 µl of 10% SDS and 50 l of CIA (chloroform isoamyl alcohol=24:1). 60 Supernatant was recovered after centrifugation at 15000 r.p.m for 5 min at 4 °C. 20 l of supernatant 61 was separated on 1% agarose gel in TAE buffer and DNA was visualized by ethidium bromide 62 staining. BSA and lysozyme were used as negative controls. 63 64 Supplemental figures captions 65 66 Fig. S1 67 Multiple sequence alignment of Dps family proteins. Residues identical to S. aureus MrgA are 68 shaded in yellow. Accession number in GenBank or Protein Deta Bank from top to bottom: S. aureus 69 MrgA (NP375246), B. brevis (1N1QD), B. anthracis Dps1 and Dps2 (AAM18635 AAM18636), B. 70 subtilis Dps and MrgA (NP390943 NP391178), L. innocua Dps (NP470279), L. monocytogenes Dps 71 (2IY4L), L. lactis DpsA (NP268182), S. suis Dpr (YP001199055), S. mutans Dpr (NP720977), S. 72 pyogenes Dpr (NP269604), T. elongates DpsB (NP683259), D. radiodurans Dps (NP295984), M. 73 arborescens Dps (AAZ06850), M. smegmatis Dps (YP890680), B. melitensis Dps (NP540897), A. 74 tumefaciens Dps (NP355427), E. coli Dps (CAA49169), S. enterica subsp. enterica serovar 75 Enteritidis Dps (YP002242918), S. enterica serovar Thyphimurium Dps (NP459808), T. elongates 76 DpsA (NP681404), H. salinarum Dps (YP001690176), H. pylori NAP (BAK64090), C. jejuni Dps 77 (YP002344906), B. burgdorferi NapA (NP212824), P. gingivalis Dps (YP001930152), V. cholerae 78 Dps (NP229797), L. lactis DpsB (YP001174737). His29, His41, Glu45, Glu60 and Asp56 residues 79 in S. aurus MrgA are represented by red stars. 80 not have conventional ferroxidase centers (Ingmer, 2010). The exceptions are LlDpsA and LlDpsB, which do 81 82 Fig. S2 83 Growth curves of S. aureus strains N315, N315∆mrgA, and N315mrgA*. Cells were grown in BHI 84 medium at 37˚C with shaking at 180 r.p.m. There were no difference in growth rate and yield among 85 tested strains. 86 87 Fig. S3 88 Elution profile of MrgA dodecamer (a) or MrgA* dodecamer (b) through HiLoad 26/60 Superdex 89 200-pg gel chromatography. The positions of size markers were indicated by arrows. The calculated 90 molecular weight of MrgA dodecaer is 200.4 kDa, and the major peak was judged as dodecamer 91 form. When minor peaks (as seen in MrgA*) were detected, they were eliminated by repeating 92 gel-exclusion chromatography for further analysis. Dodecamers can be stored stably at 4 ˚C at least 93 for 3 months. 94 95 Fig. S4 96 In vitro DNA protection assay. (a) Representative gel image of DNA protection assay. Lanes 1 and 9: 97 plasmid DNA without oxidative stress (Fe2+/H2O2). Lanes 2 and 10: plasmid DNA treated with 98 oxidative stress. Lanes 3~8 and 11~12: Plasmid DNA was pre-incubated with 10 µg (lanes 3, 5, 7, 99 11) or 20µg (lanes 4, 6, 8, 12) of MrgA, MrgA*, BSA, or lysozyme prior to the Fe2+ addition. SC: 100 supercoil, N: nicked. (b) Relative amount of supercoiled plasmid DNA. Agarose gel image was 101 scanned by conventional scanner, and the signal intensities of supercoil DNA was measured by using 102 NIH imageJ software. Mean and SD values of no protein (n=9), MrgA (n=4), MrgA* (n=3), BSA 103 (n=6), and lysozyme (n=4) are shown. All proteins are 10 g. *: p < 0.05. NS: p >0.25. 104 105 References 106 Ingmer H (2010) Dps and Bacterial Chromatin. Bacterial Chromatin,(Dame R.T. DCJ, ed.) pp. 175– 107 201. Springer Science+Business Media B.V., Netherlands. 108 Morikawa K, Inose Y, Okamura H, Maruyama A, Hayashi H, Takeyasu K & Ohta T (2003) A new 109 staphylococcal sigma factor in the conserved gene cassette: functional significance and 110 implication for the evolutionary processes. Genes Cells 8: 699-712. 111