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Supplementary Materials and Methods
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Protein separation and identification SM fraction
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Cell, SM and RM proteins were dissolved in a solubilizing buffer (40 mM Tris
4
base, 7 M urea, 2 M thiourea, and 4% CHAPS). Protein concentrations were
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measured by Bradford method using bovine serum albumin as standard. 15%
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acrylamide resolving gel was used to separate the proteins (Laemmli, 1970) (Fig. 2-
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B). After staining the gel with Coomassie brilliant blue (Bio-Rad laboratories),
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characteristic bands were excised. Gel slices were reduced and alkylated using DTT
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and iodoacetamide, respectively, then digested with trypsin based on the protocol of
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Shevchenko et al (Shevchenko et al., 1996). Extracted peptides were stored at -20ºC
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until use.
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LC-MS/MS analysis was performed using the method and criteria for protein
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identification described previously and based on the whole genome sequence of M.
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magneticum strain AMB-1 (Matsunaga et al., 2005, Tanaka et al., 2006). The
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annotation of proteins was conducted by BLAST searches against the DNA Data
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Bank of Japan (DDBJ) database; domain prediction was performed by the SMART
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(Schultz et al., 1998, Letunic et al., 2006). When the whole sequence of the MamY
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protein (aa1-aa389) was uploaded onto the SMART program, we received failed
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results. However, when a partial sequence of the protein (aa15-aa228) was
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uploaded, domain predictions were attained. Centaurinß2, which has a highly
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conserved region within other BAR proteins (aa1-aa222) (Peter et al., 2004) and
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MamY, were analyzed using a multiple alignment program, ClustalW. Identical,
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highly similar and similar amino acids are indicated as (*), (:) and (.), respectively.
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The secondary structure of the protein was predicted using the PSIPRED program
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(McGuffin et al., 2000).
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1
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Protein expression and purification of CentaurinBAR and MamY
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centaurinBAR was amplified by PCR with BamHI fused primer sets
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(centaurinBAR_F_BamHI, centaurinBAR_R_BamHI) from a human cDNA library
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(KIAA0041) that was a gift from the Kazusa DNA Research Institute, Chiba, Japan,
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while the mamY was amplified with BamHI fused primer sets (mamY_F_BamHI,
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mamY_R_BamHI) from M. magneticum AMB-1 whole genome. Recombinant
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proteins were expressed in E. coli as GST-fusion proteins using the pGEX6P-1 vector
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according to the manufacturer’s protocol (GE healthcare). Proteins were purified
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with glutathione-Sepharose, cleaved from the GST-tag using Prescission protease
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(GE healthcare), and purified by size-exclusion and ion-exchange chromatography.
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Purity of the proteins was verified by SDS-PAGE.
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Nonpolar deletion of mamY
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The mamY sequence was obtained from NCBI (YP_420381.1). The
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counterselectable suicide vector for in-frame mamY gene deletion was constructed
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with following methods. Five hundred bases upstream and downstream of mamY
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were
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MYdw_F_XbaI and MYdw_R_HindIII (Table S1). The PCR products were contained
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the first six and the last six codons of mamY for in-frame deletion. These amplified
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fragments were digested and ligated in pK19mobsacB (Schafer et al., 1994),
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generating the pK19mobsacBMYupMYdw. Thus the gentamicin resistance gene
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(pBBR1MCS5) (Kovach et al., 1995) is also amplified, digested and ligated to the
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XbaI
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pK19mobsacBMYupGmrMYdw (pK19MYGmr).
amplified
site
by
in
using
these
primers,
MYup_F_EcoRI,
pK19mobsacBMYupMYdw,
2
MYup_R_XbaI,
generating
the
1
Before the trial for the mamY gene deletion, whether the gentamicin
2
resistance gene (Gmr) expression using mamY promoter (PmamY) is enough for
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positive selection of transformation was verified. MYupGmrMYdw gene fragment
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containing PmamY and Gmr was amplified with PmamY_mamY_F_SpeI and
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gentamicin_R_XbaI primers and ligated into SspI site of pUMG (6.4 kbp, Apr) which
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is shuttle vector of E. coli and M. magneticum AMB-1 (Okamura et al., 2003). The
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vector pUMGMYupGmrMYdw was transformed and grown in the presence of
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Gentamicin (2.5 g/ml) and wild type was used as negative control.
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The pK19MYGmr vector was conjugated into AMB-1 by using S17-1 (Simon,
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1983) as the donor strain, and colonies were grown in the presence of Gentamicin
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(2.5 g/ml) and checked for the deletion construct. To select for recombinants that
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lost the integrated plasmid, cells were grown for three massages in 25 ml MSGM
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without Gentamicin and the plated on MSGM containing the filtered 1% sucrose.
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The obtained colonies were checked for the presence of the deletion marker and the
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absence of mamY by using PCR and sequencing to find the ΔmamY strain.
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Expression of MamY-GFP and MamY-6H fusion proteins in magnetotactic bacteria
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For the construction of the MamY protein expression vector, a double strand
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DNA fragment (SpeI_NsiI_NheI_6H_Stop) was first inserted within the SspI site of
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the pUMG (6.4 kbp, Apr) plasmid. The constructed plasmid was designated as
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pUMG_6H. The DNA fragment comprising of the PmamY and the downstream mamY
22
gene
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(PmamY_mamY_F_SpeI) and a NheI fused reverse primer (mamY_R_NheI) and was
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cloned into pUMG_6H to construct the pUMPmamY_mamY_6H plasmid. For the
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expression of MamY-GFP fusion protein, gfp gene (pAcGFP1, clontech) was
was
amplified
by
PCR
with
3
a
SpeI
fused
forward
primer
1
amplified with a NheI fused forward primer (gfp_F_NheI) and a NheI fused reverse
2
primer (gfp_R_NheI). The constructed plasmids, pUMPmamY_mamY_6H and
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pUMPmamY_mamY_gfp, were transformed into the ΔmamY strain by electroporation.
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Supplementary references
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Peterson, (1995) Four new derivatives of the broad-host-range cloning vector
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Laemmli, U. K., (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680-685.
Letunic, I., R. R. Copley, B. Pils, S. Pinkert, J. Schultz & P. Bork, (2006) SMART 5: domains in
the context of genomes and networks. Nucleic Acids Res 34: D257-260.
Matsunaga, T., Y. Okamura, Y. Fukuda, A. T. Wahyudi, Y. Murase & H. Takeyama, (2005)
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Matsunaga, (2003) Design and application of a new cryptic-plasmid-based shuttle vector
for Magnetospirillum magneticum. Appl Environ Microbiol 69: 4274-4277.
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