Yamashita et al.

Supplementary Material
Single molecule imaging on living bacterial cell surface
by high-speed AFM
Hayato Yamashita1, 5, Azuma Taoka2, 3, 5, Takayuki Uchihashi1, 3, Tomoya Asano4, Toshio
Ando1, 3, 6, Yoshihiro Fukumori2, 3, 6, *1

School of Mathematics and Physics, College of Science and Engineering, Kanazawa

University, Kakuma-machi, Kanazawa 920-1192, Japan. 2School of Natural System, Collegeof Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan. 3Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa
University, Kakuma-machi, Kanazawa 920-1192, Japan. 4 Division of Functional Genomics,
Advanced Science Research Center, Kanazawa University

Corresponding author:*Yoshihiro Fukumori, Department of Life Science, Graduate School of Natural Science andTechnology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. tel.: +81 76
264 6231; fax: +81 76 264 6230; e-mail: fukumor@staff.kanazawa-u.ac.jp

Footnotes: 5 H.Y. and A.T. contributed equally to this work. 6 T.A. and Y.F. contributed
equally to this work.

Yamashita et al.

Supplementary figures

Supplementary figure 1

Supplementary figure 1: Phase contrast microscopic image of M. magneticum
AMB-1 cells attached on mica treated with poly-L-lysine and glutaraldehyde.

Yamashita et al.

Supplementary figure 2BA

20 nm

20 nm

Supplementary figure 2: High magnification AFM images of cells surface of M. magneticum
cultiveted in high (21.8 mM) (A) and low (1.8 mM) (B) iron concentration media. AFM images wererecorded at imaging rates of 2.0 s/frame (A) and 0.5 s/frame (B) with (A and B) 200 × 200 pixels.

Yamashita et al.
band no.

gene ID1amb2490

Outer membrane protein/protective antigen OMA87

1

amb2690

nitrate reductase

2amb3086

nitrous oxide reductase

3amb0684

flagellin-like protein

3amb3772

hemolysin activation/secretion protein

4amb4436

TPR repeat-containing protein

124amb1395

nitrite reductase

4amb4141

FOF1-type ATP synthase, alpha subunit

34
5
6
7894

amb3086

nitrous oxide reductase

5amb3421

hypothetical protein

6

amb2245

TolC like outer membrane protein

6amb0303

periplasmic protease

6amb3421

hypothetical protein

7amb3211translocation protein TolB8amb1448

hypothetical protein

8

amb0941

carbohydrate-selective porin

9amb0025

Porin 4113

amb1085

outer membrane protein domain-containing protein

13

amb3210outer membrane protein and related peptidoglycan-associated lipo protein

13

amb0602

outer membrane protein and related peptidoglycan-associated protein

13

amb0511

parvulin-like peptidyl-prolyl isomerase

13

amb3083

cytochrome c553

14

amb1085

outer membrane protein domain-containing protein

250 kDa
150 kDa
100 kDa
75 kDa50 kDa37 kDa131425 kDa
20 kDa15 kDa14
14

amb0602amb3083

annotation

outer membrane protein and related peptidoglycan-associated protein
cytochrome c553

Supplementary figure 3: List of proteins identified in the outer membrane fraction of M. magneticum AMB-1 byusing MS/MS analysis.

Yamashita et al.

Supplementary figure 4

Simulation

Observed image
HS-AFM image

3nm

Pseudomonas aeruginosa OprP
extracellular
periplasmic

E. coliextracellular
periplasmic

3nm

3nm

3nm

Delftia acidovorans Omp32
extracellular
periplasmic

Rhodopseudomonas blasticus
extracellular
periplasmic

3nm

Salmonella typhi OmpF

3nm

3nm3nmextracellular

3nm

3nm

Salmonella enterica Sucrose-specific porin

periplasmic

3nm

extracellular

3nm

periplasmic

3nm

Neisseria meningitidis PorB
extracellular
periplasmic

3nm

3nm

Supplementary figure 4: Comparison of observed triangular outer membrane protein by AFM(left) with simulated images (midle and right) constructed from crystal structural data of theknown porins: Escherichia coli OmpF (PDB: 2OMF), Rhodopseudomonas blasticus porin (PDB:2POR), Salmonella typhi OmpF (PDB: 3NSG), Neisseria meningitides PorB (PDB: 3A2R),
Pseudomonas aeruginosa OprP (PDB: 2O4V), Delftia acidovorans Omp32 (PDB: 2FGQ) andSalmonella enterica Sucrose-specific porin (PDB: 1OH2).

SUPPLEMENTARY MOVIE

Movie S1
Cell surface imaging by HS-AFM. Images were obtained at 1.0 s/frame with number ofpixels of 200 200.

Movie S2
HS-AFM observation of molecular dynamics on living cell surface. Images wereobtained at 0.5 s/frame with number of pixels of 200 × 200.

Movie S3
HS-AFM observation of cell surface immobilized with glutaraldehyde.
Images were obtained at 0.5 s/frame with number of pixels of 150 × 150.

Movie S4
Nano-dissection procedure of honeycomb-like structure in isolated outermembrane patch by a HS-AFM scanning. After dissection honeycomb-likestructure was dissociated to triagular molecules. Images were obtained at 1.0
s/frame with number of pixels of 100 × 100.