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Spatially Resolved Chemical Analysis of G. sulfurreducens Cell Surface Nikolai Lebedev, Rhonda M. Stroud, Matthew D. Yates, and Leonard Martin Tender ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b02032 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Spatially Resolved Chemical Analysis of G. sulfurreducens Cell Surface Nikolai Lebedev,*† Rhonda M. Stroud,‡ Matthew D. Yates,† Leonard Martin Tender*†

† Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC 20375, USA ‡ Materials Science and Technology Division, US Naval Research Laboratory, Washington, DC 20375, USA

Abstract: G. sulfurreducens is of interest for the highest efficiency of power generation and

extremely long extracellular electron transfer (EET) between the bacterium and electrodes. Despite more than 15 years of intensive molecular biological research there is still no clear answer which molecules are responsible for these processes. In the present work we look at the problem from another (atomic) perspective; we identify location and shape of the compounds that are known to be conductive, particularly Fe atoms. By using highly sophisticated energy dispersive X-ray spectroscopy (EDXS) combined with high angle annular dark field (HAADF) transmission electron microscopy enabling detection, identification, and localization of chemical compounds

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on the surface at nearly atomic spatial resolution, we analyze Fe spatial distribution within Geobacter sulfurreducens community. We discover the presence of small Fe-containing particles on the surface of the bacterium cells. The size of the particles (diameter 5.6 nm) is highly reproducible and comparable with the size of a single protein. The particles cover about 2% of cell surface that is similar to expected for molecular conductors responsible for electron transfer through the bacterium cell wall. We find that G. sulfurreducens filaments (“bacterial molecular wires”) also contain Fe atoms in their bundles. We observe that the bacterium enable changing the distance between the Fe-containing bundles in the filaments from separated to attached (the latter is needed for the efficient electron transfer between the Fe containing particles) depending on the bacterium metabolic activity and attachment to extracellular substrates. These results show what type of Fe containing particles are involved in bacterial extracellular communication. They can be used for the design and construction of artificial bio-molecular wires and bio-inorganic interfaces.

Keywords: Geobacter sulfurreducens, extracellular electron transfer, energy dispersive X-ray spectroscopy, atomic force microscopy, chemical topography, bacterial cell surface

Understanding mechanisms, components and factors controlling energy and information transfer at bio-inorganic interface is critical for the construction of many advanced materials and devices including sensors for environmental monitoring, energy harvesting materials, or brain-machine interfaces. Despite extreme importance, no clear identification of the molecules responsible for efficient long range electron transfer in bacterial communities has been achieved so far. In the present work we look at the problem from another (atomic) perspective, we identify the location and structure of components that are known to be conductive and can be involved in electron


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transfer, particularly Fe. Fe atoms are crucial for efficient electron coupling at bio-inorganic interface and are the main components of all known biological systems. 1-3 Fe-containing proteins are involved in energy harvesting, conversion, and transfer processes, in regulation of cell metabolic activity and gene expression.

4, 5

These proteins usually have one or few Fe atoms in

their catalytic centers, and the chain of these atoms is either incorporated in a single multi-Fe protein or as an associate of single Fe-containing proteins forms so-called “conductive channels” in multiprotein complexes.

2, 6, 7

Electronic energy levels in these proteins are discrete and the

entire molecules are generally electrically neutral. On the other hand, metallic electrodes have a continuum of energy levels and require applying high bias voltage (electrostatic potential) for inducing electron transfer (ET). Recently, we have shown that the high efficiency of ET through Fe-containing proteins is due to a) the matching of the atomic energy levels of Fe atoms and the electrode Fermi level, b) the efficient coupling between this level and the electrode, and c) the appropriate protein molecular orbital delocalization reducing the barrier of ET through space. 8, 9 To achieve this high efficiency ET, Fe must be properly localized in space at atomic spatial resolution. 10, 11 Among biological systems, Geobacter sulfurreducens has the highest efficiency of long range extracellular ET (EET) and the highest ability for sustainable energy production.

12, 13

By using

electrochemical techniques it was shown that the current generated by G. sulfurreducens biofilm can reach 300 µA/cm2; and be transferred over a distance of 100 µm.

14-16

With molecular

biological techniques, about a hundred proteins that might be involved in ET process have been identified and locations of some of them in the cell were predicted. 17, 18 By using an immunogold labeling transmission electron microscopy (TEM) it was shown that of a hundred of cytochromes identified in G. sulfurreducens genome,

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only some are surface exposed and thus might


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participate in EET.

19-22

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Scanning tunneling microscopy (STM) and conductive atomic force

microscopy (AFM) indicate that several bacterial cytochromes and bacterial filaments (pili) are electrically conductive ex situ and thus might be potential candidates for a “molecular wire” involved in the long-range EET.

12, 23-27

However, their identity and participation in the

extracellular bacterial conductivity still needs to be estimated. 24, 28 There are only two methods permitting direct in situ detection and chemical identification of atoms at very high spatial resolution, Electron Energy Loss Spectroscopy (EELS) and Energy Dispersive X-ray (EDX) Spectroscopy (EDXS) combined with High Angle Annular Dark Field (HAADF) Transmission Electron Microscopy (TEM). 29 However, EELS works well only for thin samples (10-100 nm) that makes impossible it utilization for studying whole bacterial cells having size more than 0.5 µm. On the other hand, EDXS is easier for quantifying the relative elemental abundances in multi-element samples, and it works well on thick and thin samples. In the present work, for identification and localization of Fe atoms in G. sulfurreducens cells we use the most sensitive EDX spectroscopic and imaging techniques. To confirm the intactness of our EDX samples we compare their structures to that of similar samples assayed by AFM, a technique allowing for structural analysis of living bacterial cells in native environment (though without chemical identification) at the same high spatial resolution. 30 With the EDX technique we discover on the surface of G. sulfurreducens cell very small Fecontaining particles having size about 5.6 nm that is comparable to the size of single proteins. The size of these particles is highly reproducible and they cover about 2% of the cell surface. In addition, at suboptimal cultivation temperature (250C) we detect on the bacterium cell surfaces bigger Fe-containing proteins (FeCPs) of irregular size and shape similar to observed before. 31-33 Location, Fe content, and the density of the discovered small particles are consistent with the


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expected for G. sulfurreducens outer membrane (OM) conductors. We also detected Fe in the bundles of G. sulfurreducens filaments which some authors assume to be electrically conductive. 34

We observe that G. sulfurreducens can vary the distance between the Fe-containing bundles and

the bundles can be either separated, or in direct contact needed for the efficient ET between them. Our results are the direct experimental demonstration of the presence and localization of small FeCPs on the surface of bacterial cells and in bacterial filaments. They show what type of FeCPs are involved in ET through the bacterium cell wall; that conductivity of G. sulfurreducens filaments might be related to the presence of Fe in their bundles; and that the bacterium can regulate ET through the filaments by filament contraction. RESULTS For proving that the sample preparation for TEM imaging does not damage the cell structures we compare them to the untreated cells from the initial culture assayed with AFM (Fig S3 A, B). We saw no difference in G. sulfurreducens cell size, shape, contact areas, intercellular space, and even the attached EPS. In both specimens the cell size is about 1.5 x 0.6 µm with relatively smooth surface composed of highly dense-packed particles. EPS was attached to the isolated washed cells as a rather narrow relatively transparent layer (Fig S3 A, B) in which sometimes we observe filaments of about 9 nm diameter (see section Fe in G. sulfurreducens Filaments). The only difference between STEM and AFM topo images was the presence of round e-dense bodies in HAADF (Fig. S3 A). The absence of similar structures in AFM topo images indicates that they are more likely located inside the cells. Detection and Identification of Nucleoids To identify the origin of these bodies we measure EDX atomic maps of the cells. We observe that the bodies are enriched in O, P, Ca, Mg, Na, and K, but relatively deficient in C and N (Fig.


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S3 small panels). Fe, S, and Cl are more or less equally distributed in the bodies and through the rest of the cell. Comparison of the bodies atomic composition to expected for the main classes of biomolecules

35

(Fig. S3) indicates that they more likely are nucleoids.

36, 37

Indeed, they are

enriched in O and P that is specific for nucleic acids and molecules like ATP, and deficient in C and N that is specific for proteins. They are relatively high in Ca and Mg which is specific for catalysts,

35

but deficient in C indicating that they are not enriched in polysaccharides that is

specific for energy storage inclusion bodies and EPS. 38 The presence of high amounts of Na and K might be an indicator that the bodies are involved in active membrane transport (like in buoyancy vacuoles), but Cl (counter ion for Na and K) is not present either in the bodies or the area surrounding them. Approximately the same amounts of Fe and S in the bodies and in the surrounding material indicate that the bodies are not specifically active in energy conversion and electron transfer. Detection of FeCPs on G. sulfurreducens cell surface Measurement of EDX spectra at various parts of an individual G. sulfurreducens cell confirms the identification based on EDX mapping (Fig. 1 A, B). It shows that C:N:O ratio (see Fig. S1) of the cell surface is about 74:14:11, comparable to expected ratios for proteins based their sequences (Table S3). In addition, the cell surface has some specific reproducible amounts of Cu, Na, Mg, P, S, K, but is deficient in Ca (Fig. 1 B). Interestingly, that all specimens of bacterial surface show very low/no amounts of Cl that might be expected since G. sulfurreducens is a Gram-negative bacterium. Contrary to the cell surface, nucleoids are substantially enriched in Mg, P, Ca, while the amount of all these elements in the part of EPS bound to the cell is relatively low (Fig. 1 B). In addition to these elements, EDX spectra also reveal some amounts of Fe on the cell surface. To test if this Fe is equally distributed over the cell surface or if it is clustered to specific areas, we


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A

B Cell surface EPS

Nucleoid

500 nm

C

D Fe-containing particles Cell surface

5 nm

Figure 1. (A) EDX image of an individual G. sulfurreducens cell and (B) EDX spectra at various parts of the cell. The areas where the spectra are collected are indicated by green and yellow squares on panel A. (C) EDX image of a fragment of the G. sulfurreducens cell (outlined by dark blue square in panel A) with small e-dense particles (pointed by arrows). (D) EDX spectra of the particles shown in panel C (red) and the empty cell surface (grey). The presence of three Fe bands in the EDX spectra confirms high fidelity of Fe identification. measure HAADF image and EDX spectra of various parts of the cell surface at higher spatial resolution (~50 000x). Under this magnification we observe that the surface of the cells has small e-dense particles that are enriched in Fe (Fig. 1 C, D). EDX spectra show that the C:N:O ratio in


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these particles are about the same as expected for protein. The size of the FeCPs is highly reproducible (average diameter 5.6 nm; Fig. 2 A, C; Fig. S4), similar to cytochromes, FeS proteins, or Fe-transporters (Table S3). 1, 39 Compared to the cell surface, FeCP are enriched in Cu (Fig. 1D) that is specific for Fe oxidases. 40 The density of these particles on the bacterial surface is relatively high (they cover about 2% of cell surface); and they are more or less equally distributed over the cell (Fig. 2 A). Similar density on G. sulfurreducens cell surface was observed by immunolabelling for some ET proteins like OmcS and OmcZ cytochromes. 19, 41 In addition to the small FeCPs described above, HAADF images also show that planktonic G. sulfurreducens cells cultivated at +300C have large e-dense particles which are also enriched in Fe (Fig. 2 A, Object 231), though we rarely observe these large particles on the surface of the cells cultivated at this temperature. The large particles have irregular shape and size and their origin is not clear. Based on their shapes, they might be either associates of the small FeCP or the initial

+300C Object 234 Object 231

Object 232

Object 233

A

+250C Object 250 Object 252

B

C

Object 254

Object 249 Object 253

Figure 2. (A and B) EDX images of the surface of a G. sulfurreducens cell cultivated at +300C and at +250C, respectively. (C) Size of individual Fe-containing particles on the surface of the cells cultivated at +250C (blue bars) and +300C (red bars).


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steps of the formation of big Fe aggregates detected before on G. sulfurreducens cell surface. 31, 33 It was demonstrated that planktonic G. sulfurreducens cells are able to acquire Fe from the surrounding medium and use it for extracellular respiration.

39, 40

This effect is temperature-

dependent. 24 To test the role of temperature in the formation of FeCPs we performed HAADF and EDXS analyses of the cells cultivated at 250C. We observe that after cultivation of planktonic G. sulfurreducens cells for 3-5 days at 250C the average size of FeCPs observed on the cell surface indeed increases to 14 nm without substantial changes in the cell surface coverage (Fig. 2 B, C; Fig. S4). Fe in G. sulfurreducens filaments Bacterial pili (type-IV filaments) are very interesting structures that are responsible for cell attachment to inorganic surface,

42, 43

to each other, and other organisms.

44, 45

Planktonic G.

sulfurreducens clusters might use pili for keeping the cells together and also for acquiring soluble the surrounding areas (grey lines).The areas used for EDX spectra collection are shown in panel C by blue and gray rectangles, respectively. (D) EDX Fe map of the G. sulfurreducens filament. The light blue arrows in panels C and E point to the bundles on G. sulfurreducens filaments. The light blue circles in panel D surround the areas of high Fe signal along the filament. (E) AFM image of G. sulfurreducens filament on mica. Fe and transferring it to the cell surface. 40, 46 Some data suggest that pili of G. sulfurreducens are conductive and responsible for ET from the cells to the electrode (extracellular respiration). 12 The presence of Fe in pili might substantially improve their conductivity. 8, 9 To test the possibility of Fe attachment to G. sulfurreducens pili we analyzed Fe content in the filaments by EDXS. G. sulfurreducens cells have filaments with a diameter of about 8-10 nm. 34, 47-49 These filaments can be observed by AFM around G. sulfurreducens cells deposited on mica (Fig. 3 E). We observe


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EPS

Cell

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A

B

Filament

C

E

20 nm

D 100 nm

20 nm

Figure 3. (A) HAADF and (C) EDX images and (B) EDX spectra of the filament (blue lines) and the surrounding areas (grey lines).The areas used for EDX spectra collection are shown in panel C by blue and gray rectangles, respectively. (D) EDX Fe map of the G. sulfurreducens filament. The light blue arrows in panels C and E point to the bundles on G. sulfurreducens filaments. The light blue circles in panel D surround the areas of high Fe signal along the filament. (E) AFM image of G. sulfurreducens filament on mica. similar filaments at G. sulfurreducens cells by HAADF (Fig. 3 A). Though the size of the filament is specific for pili (Fig. 4 E,G,H)

34, 47-49

, to exclude the possibility of DNA contamination we

compare their atomic composition (C:N:O ratio) with expected for DNA and proteins and compare them to the data collected for the nucleoids (Table. 1, Figure S1). EDXS analysis clearly shows


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similarity of N:O ratios between expected for PilA and observed for filaments on one hand and expected for DNA and observed for nucleoids on the other allowing us clear distinguish these two molecules. Compared to N and O, the amount of C in our experiments is overestimated. This might be due to the presence of lacey carbon in the TEM grids. Indeed, the enhancement of C amounts in all our experiments is about the same. Particularly, for filaments and nucleoids it is 1.54 compared to expected for pure molecules (Table 1). Appropriate correction (x 0.65) gives very consistent results for all our experiments.

Table 1 Expected and experimentally observed C:N:O atomic composition of DNA, nucleoid, PilA, and the G. sulfurreducens filament. C

N

O

DNA (expected) 38

12

26

Nucleoid

60.4

11.5 25.9

PilA (expected)

49.6

12

Filament

73.4

12.4 12.7

14.7

The EDX spectrum of the filaments detected in HAADF also shows the presence of other atoms (Na, Mg, P, S, K) typical for proteins (Fig. 3 B). In addition compared to the surrounding cell surface, EDX spectra of the filament show the increased amounts of Ca and Mg (these atoms present in contracting proteins) and some amounts of Fe (visible both at 0.8 and 6.3 keV; Fig. 3 B). At first glance, the presence of Fe is rather surprising since pilA sequence indicates presence


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neither His, nor Cys (the amino acids usually responsible for Fe-chelating by proteins 1). To approach this paradox we analyze Fe distribution along the filaments. Detailed analysis of filament structure by AFM shows that G. sulfurreducens filaments have thick areas (bundles) randomly distributed along the filaments (Fig. 3 E). Similar bundles we detected in HAADF images (Fig. 3 C). The origin of these bundles is not clear, but they are proposed to be either attached cytochromes or other proteins. 47, 50 When we measure Fe distribution along the filament by EDX mapping, we observe that Fe is also not equally distributed along the filament, but form a pattern with maxima correlated with the position of the bundles (Fig. 3 D). This leads us to the assumption that another non-PilA protein might be attached to PilA that is responsible for Fe binding to the entire pili. Temperature-induced alteration of the shape of G. sulfurreducens filaments Now the question is: What is the role of Fe in G. sulfurreducens filaments? The distance between some bundles in the observed sample is rather long for direct e-hopping between them. 7, 25 Since the G. sulfurreducens pili contraction has not been directly measured yet, we assume that the presence of bundles on G. sulfurreducens pili 34 will allow us to use them as markers for pili length. To do this we measured the structure of the filaments deposited on mica by topo AFM. When we examine G. sulfurreducens planktonic cells grown at 250C we found that their filaments have bundle periodicity (top to top distance) of about 18-25 nm and thus the bundles that are about 10 nm in diameter are separated from each other (Fig. 4 A,C,F, Fig. S5). Consistent with previous results, 28 we also observe that some of these filaments have large particles attached to their ends (Fig. 4 A). The size and shape of these particles are similar to the large FeCPs that we observe in HAADF images on G. sulfurreducens cell surface (Fig. 1 D). When we perform similar AFM examination for the filaments of G. sulfurreducens planktonic cells grown at 300C we observe that


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+250C

A

+300C

B

100 nm

100 nm

C

D

20 nm

20 nm

E

G

F

Filament at 250C: h = 3.5-4 nm; w = 14 nm; bundle periodicity = 18-25 nm (with small in-between step ~ 7 nm) Filament at 300C: h = 2-3 nm; w = 10 nm; bundle periodicity = 9-14 nm (in contact to each other)

H

6.0 3.6 1.2 nm

9.0

18.0

27.0

36.0

45.0

54.0

63.0

72.0

81.0

90.0 nm

Figure 4. AFM images of G. sulfurreducens filaments on mica at low (A, B) and high (C, D) spatial resolution. The areas shown at high spatial resolution are depicted by rectangles in panels A and B. The bacteria were cultivated at 250C (A, C) and 300C (B, D). Bars in panels A, B = 100


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nm; in panels C, D = 20 nm. (E, G, H) Cross sectional profile and metrics for the filament of the bacteria cultivated at 250C along the red line depicted in panel A. (F) Parameters of filaments for bacteria cultivated at 250C and 300C along the gray lines shown in panels C and D (see Fig. S5 for profiles). their bundles are in contact with each other (their top to top distance is about 9-14 nm; Fig. 4 B,D,F, Fig. S5) allowing for direct ET between them. Isolated G. sulfurreducens filaments with similar structure were observed before and was shown to be electrically conductive.

34

Interestingly, we did not observe particles at the ends of the filaments of the cells grown at 300C (Fig. 4 B). What is specific for EPS? EPS is a viscous substance surrounding bacterial cells and allowing planktonic cells to keep in close proximity to each other. 38 This substance consists mainly of polysaccharides and lipids, but can also include proteins, DNA, and ions. 38, 51 From an ET perspective viscosity of this substance should eliminate/prevent simple diffusion of ET mediators, but allows for sequential ET hoping through immobilized components if they are present. 52 For analyzing EPS atomic composition we isolate it from supernatant obtained after precipitation of G. sulfurreducens cell. For the collection of this (loosely bound to the cells fraction of EPS) we use Amicon concentrator (95% collection of compounds with Mw >12 kDa). We found that the obtained material is enriched in C and O indicating that the main components of this material are polysaccharides and lipids (Table 2). Rough estimation of polysaccharide/lipid ratio based on the experimentally observed ratio of C to O gives 94% to 6%. Though the exact amount of C in this ratio might be overestimated due to the scattering from lacey carbon, even taking into account the correction factor x0.65 (see Table 1 and discussion in section Fe in G. sulfurreducens filaments) the corrected result (91% to 9%) indicates


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that isolated EPS fraction consists mainly of polysaccharides. In this isolated EPS fraction we observe high concentration of ions (Na, K) and especially Cl (Fig. 5), which we did not detect on the surface of washed G. sulfurreducens cells. The amount of N in isolated EPS is extremely low, indicating its depletion of proteins (Table. 2). Isolated EPS also did not show the presence of Fe (Fig. 5). Compared to the results described above, a relatively narrow EPS layer attached to G. sulfurreducens and isolated together with the cells (Figs. 2B and 4B) has a) a substantial reduction in the amounts of ions, b) enrichment in N that is specific for proteins (Table 2), and c) enrichment in Fe (Fig. 5) that is consistent with the presence of ET components. 52

Figure 5. EDX spectra of EPS layer tightly bound to G. sulfurreducens cells (EPS-A, blue line) and loosely bound to the cells (isolated from the supernatant after cell precipitation, EPS-I, green line). Table 2 Expected C:N:O atomic composition of pure polysaccharides and lipids, and experimentally observed for isolated and tightly bound to Geobacter cells EPS.

Lipids (expected)

C

N

O

55

-

6


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Polysaccharides (expected)

6

EPS-I (isolated)

51.6 1.4

34.8

EPS-A (attached)

78.1 9.9

11.9

-

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CONCLUSIONS By applying experimental techniques for chemical identification with extremely high, close to single atomic level spatial resolution (EDX spectroscopy and mapping) we detect small (average diameter 5.6 nm) Fe-containing particles on the surface of individual G. sulfurreducens planktonic cells. The diameter of the particles is highly reproducible and comparable to the diameter of bacterial ET cytochromes (OmcS and OmcZ) and iron transporters (HasA). These particles are randomly distributed over the bacterium cell surface and cover about 2% of it. Proteinaceous filaments (so called “bacterial molecular wires”) surrounding G. sulfurreducens cells also contain Fe in their bundles. The distance between the Fe-containing bundles in Geobacter filaments can vary depending on the bacterium metabolic activity (cultivation conditions) and interaction with inorganic substrates. The obtained results are the direct experimental demonstration of the presence and localization of small FeCPs on the surface of bacterial cells and in bacterial filaments. They show what type of FeCPs are involved in ET through the bacterium cell wall; that conductivity of G. sulfurreducens filaments might be related to the presence of Fe in their bundles; and that the bacterium can regulate ET through the filaments by filament contraction.

METHODS Material Experiments were performed with planktonic culture of G. sulfurreducens strain DL1 (ATCC#51573). The cells were cultivated anaerobically (N2:CO2, 80:20%) with ultrapure (18


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GW) water-based growth medium containing fumarate (40 mm) and acetate (20 mm) using previously described methods.

26, 53, 54

The NB medium content per liter was 2.0 g sodium

bicarbonate (NaHCO3), 0.1 g potassium chloride (KCl), 0.25 g ammonium chloride (NH4Cl), 0.6 g sodium phosphate dibasic (NaH2PO4), and 10 mL DL mineral mixture. 53 Na-acetate (20 mM) is added as a reductant and 40 mM Na-fumarate as electron acceptor. We use a soluble electron acceptor to avoid any possible artificial contamination of the cell surface with insoluble ironcontaining particles. After cultivation at 300C for 8-12 days (+ 3-5 days at 250C when indicated as 250C culture) the cells were precipitated at 3 000 g x3 min, diluted with ultrapure H2O, drop-casted on TEM Au-grid coated with lacey carbon (SPI Cat #3820G-MB), and immediately dried in N2 flow. 55-57 After curing in vacuum (10-6 Torr) at 1400C for 8 hrs these samples were analyzed by HAADF microscopy, and used for EDX spectroscopy and mapping. For AFM experiments the same washed cells were deposited on freshly cleaved mica (Electron Microscopy Science, Cat. # 71855-10), dried in N2 flow, and immediately tested. The fraction enriched in EPS (loosely bound EPS, EPS-I) was obtained from the supernatant left after cell precipitation by concentration with Amicon Microcon-30 at 14 000 g x 10 min (allowing for 90% collection of compounds with Mw >12 kDa). This fraction was prepared for TEM and AFM experiments the same way as the cell fraction. HAADF imaging and EDX spectroscopy We use lacey carbon coated 200 mesh gold grid (SPI supplies, West Chester, PA, USA; Fig. S1 B) to hold the biological specimens in our work. This grid keeps the cells suspended in the holes between carbon (Fig S2 A, B) that eliminates any (except some carbon) atomic contamination. A Nion UltraSTEM200-X scattering transmission electron microscope (STEM) equipped with a Bruker XFlash 6j100 UHV compatible, windowless silicon drift detector was used in this study.


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The geometric solid angle of the detector is calculated to be 0.7 sr. The microscope was operated at 60 kV, with a nominal probe size of 0.15 nm and probe current of 50–120 pA. Spectra were collected by using the Espirit 1.93 software and exported as EMSA files for analysis. High angle annular dark field (HAADF) images were collected by using Gatan Digital Micrograph 2.3 software. The spectra and images processing (averaging, smoothing, peak identification) were performed with Gatan and Bruker software. Calculation of C:O:N atomic composition of various objects is based on K-series peaks collected for 3-5 identical objects in each sample preparation (Fig. S1 A). Since spatial resolution of EDX images (which require separate sets of different pixels for identification of each chemical element) is much lower than HAADF (Fig. S2 A,B) we collected both types of images and use the results gained with EDX images for chemical identification and mapping and HAADF images for particle and filament structural analysis (Fig. S3). Consistent with this, the images presented in the paper are in low resolution when used for discussion of elemental analysis and in high resolution HAADF mode when used for structural analysis. Atomic Force Microscopy The AFM examination of the structure of individual Geobacter cells deposited on mica was performed with JSPM-5200 microscope (JEOL-USA, Peabody, MA, USA) in tapping amplitudemodulation mode.

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The experiments were performed in air at 22 °C immediately after the cell

drying in N2 on freshly cleaved mica. A Multi75G Si AFM probe (Budget Sensors, Sofia, Bulgaria; force constant 3 N/m) was used at an oscillation frequency of 78 kHz at scan rate 3 mm/s using a 1.20–1.60 Hz feedback filter. Image processing (line averaging and background subtraction) was done with JEOLSPM software. ASSOCIATED CONTENT


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Supporting Information. Full scale EDX spectra of G.sulfurreducens filaments and nucleoids; TEM image of a blank lacey carbon in Au grid; comparison of EDX and HAADF images of the same specimen; EDX image and atomic map of a G. sulfurreducens planktonic cell; comparison of EDX image to AFM image of the cells from the same culture before EDX sample preparation; numerical data for particle size distribution on the surface of G. sulfurreducens cells; detailed statistical analysis of height profiles of elongated and contracted G.sulfurreducens filaments; comparative data for C:N:O compositions and sizes of some known surface exposed bacterial proteins are available free of charge on the ACS Publications website. Financial Interests The authors declare no conflict of financial interests. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] ORCID Nikolai Lebedev: 0000-0003-1280-2839 Rhonda M. Stroud: 0000-0001-5242-8015 Matthew D. Yates: 0000-0003-4373-3864 ACKNOWLEDGMENT


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We acknowledge Dr. Linda Chrisey (the Office of Naval Research) for financial support (Awards N0001415WX01038 and N0001415WX00195) and Mrs. Galina Spivak (Capital One) for her help with the Cover Art work. REFERENCES 1.

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55. Jamroskovic, J.; Shao, P. P.; Suvorova, E.; Barak, I.; Bernier-Latmani, R., Combined Scanning Transmission X-Ray and Electron Microscopy for the Characterization of Bacterial Endospores. FEMS Microbiol. Lett. 2014, 358, 188-193. 56. Tanzil, A. H.; Sultana, S. T.; Saunders, S. R.; Dohnalkova, A. C.; Shi, L.; Davenport, E.; Ha, P.; Beyenal, H., Production of Gold Nanoparticles by Electrode-Respiring Geobacter sulfurreducens Biofilms. Enzyme Microb. Technol. 2016, 95, 69-75. 57. Tugarova, A. V.; Vetchinkina, E. P.; Loshchinina, E. A.; Burov, A. M.; Nikitina, V. E.; Kamnev, A. A., Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles. Microb. Ecol. 2014, 68, 495-503. 58. Lebedev, N.; Strycharz-Glaven, S. M.; Tender, L. M., High Resolution Afm and SingleCell Resonance Raman Spectroscopy of Geobacter sulfurreducens Biofilms Early in Growth. Frontiers Energy Res. 2014, 2, 1-8.

TOC picture (For Table of Content Only): Fe-containing particles

5 nm Cell surface


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