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