Download Shewanella oneidensis MR-1 chemotaxis proteins and electron

Electron Transfer at the Microbe–Mineral Interface
Shewanella oneidensis MR-1 chemotaxis proteins
and electron-transport chain components
essential for congregation near insoluble electron
acceptors
H. Wayne Harris*, Mohamed Y. El-Naggar† and Kenneth H. Nealson‡1
*Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, U.S.A., †Department of Physics and Astronomy, University of
Southern California, Los Angeles, CA 90089, U.S.A., and ‡Department of Earth Sciences, University of Southern California, Los Angeles, CA 90089, U.S.A.
Abstract
Shewanella oneidensis MR-1 cells utilize a behaviour response called electrokinesis to increase their speed
in the vicinity of IEAs (insoluble electron acceptors), including manganese oxides, iron oxides and poised
electrodes [Harris, El-Naggar, Bretschger, Ward, Romine, Obraztsova and Nealson (2010) Proc. Natl. Acad.
Sci. U.S.A. 107, 326–331]. However, it is not currently understood how bacteria remain in the vicinity of the
IEA and accumulate both on the surface and in the surrounding medium. In the present paper, we provide
results indicating that cells that have contacted the IEAs swim faster than those that have not recently
made contact. In addition, fast-swimming cells exhibit an enhancement of swimming reversals leading
to rapid non-random accumulation of cells on, and adjacent to, mineral particles. We call the observed
accumulation near IEAs ‘congregation’. Congregation is eliminated by the loss of a critical gene involved
with EET (extracellular electron transport) (cymA, SO_4591) and is altered or eliminated in several deletion
mutants of homologues of genes that are involved with chemotaxis or energy taxis in Escherichia coli. These
genes include chemotactic signal transduction protein (cheA-3, SO_3207), methyl-accepting chemotaxis
proteins with the Cache domain (mcp_cache, SO_2240) or the PAS (Per/Arnt/Sim) domain (mcp_pas,
SO_1385). In the present paper, we report studies of S. oneidensis MR-1 that lend some insight into how
microbes in this group can ‘sense’ the presence of a solid substrate such as a mineral surface, and maintain
themselves in the vicinity of the mineral (i.e. via congregation), which may ultimately lead to attachment
and biofilm formation.
EET (extracellular electron transport)
Before the discovery of EET [1,2], electron transport was
considered to be an intracellular phenomenon, occurring
in the cytoplasm or on the cytoplasmic (or photosynthetic) membranes of mitochondria, bacteria and archaea.
EET was first discovered in bacteria, referred to as
DMRB (dissimilatory metal-reducing bacteria), a discovery
that required a change in thinking, with the realization that
bacteria (and archaea) are capable of electron transfer to
solid substrates such as manganese and/or iron oxides and
oxyhydroxides [1,2]. The mineral–microbe interface thus
became the focus of intense efforts with regard to unravelling
the mechanism(s) whereby microbes transport electrons
across the outer membrane to IEAs (insoluble electron
acceptors) that cannot be transported into the cell. In the
subsequent years, it has been hypothesized that a number
of electron-transfer mechanisms are used by cells, including:
Key words: chemotaxis, electrokinesis, energy taxis, microbe–mineral interaction, microbial fuel
cell (MFC), Shewanella oneidensis MR-1.
Abbreviations used: EET, extracellular electron transport; GFP, green fluorescent protein; IEA,
insoluble electron acceptor; MCP, methyl-accepting chemotaxis protein; PAS, Per/Arnt/Sim; pmf,
protonmotive force; TMAO, trimethylamine N-oxide.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2012) 40, 1167–1177; doi:10.1042/BST20120232
(i) direct reduction of minerals via extracellular multihaem
cytochromes [3–7]; (ii) indirect reduction of minerals via
soluble redox molecules (i.e. electron shuttles) [8–10]; (iii)
electron transfer along extracellular appendages known as
microbial nanowires [11–13]; and (iv) extracellular matrices
containing conductive or semi-conductive minerals [14].
Given that survival of the cells may well be dependent upon
EET, it would be surprising if a number of solutions to
this problem had not evolved; one might expect more to be
discovered. This seems especially true when one considers
that almost everything that is known about EET comes from
studies of only two model systems, Shewanella [15,16] and
Geobacter [17].
One area that has attracted only minimal attention is how
microbes can locate IEAs. The observation that microbes
utilize motility to accumulate on and in the vicinity of metal
oxide particles [18–20] implies that there are mechanisms
for sensing and taxis. But, to date, there are no detailed
explanations of how an organism ‘recognizes’ an IEA, and
whereas the redox potential of a surface may be ideal in terms
of electron flow, how does a microbe know that an IEA is
present? It is this question that we focus on in the present
paper.
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Chemotaxis and energy taxis
In chemotaxis, cells swim up gradients of attractants
using MCPs (methyl-accepting chemotaxis proteins) as
receptors. These receptors bind the attractants directly
at periplasmic ligand-binding domains or indirectly, using
periplasmic binding proteins. Sensory information is routed
through a two-component signal transduction system that
includes a histidine protein kinase, CheA, and a response
regulator, CheY, to the flagellar motor [21]. Responses require
neither transport nor metabolism of the chemoattractant [21–
23]. Taxis to oxygen and soluble anaerobic electron acceptors,
often referred to as energy taxis, involves a variation
of bacterial chemotaxis, and has been observed in several other
bacteria species [22,24–27].
Energy taxis in Shewanella is not yet fully explained,
and studies have shown that the so-called ‘chemical-inplug’ assay can be unreliable for determining that the
cells directly sense the electron acceptors using receptors
[20,22,28,29]. Instead of using receptors that bind the
chemoattractants as ligands, the cells may respond to a
change in some energetic parameter, e.g. the redox state of
an electron-transport protein or a change in the electrochemical gradient associated with the pmf (protonmotive
force). Shewanella oneidensis MR-1 is rich in chemotaxisrelated genes, suggesting that this bacterium is capable
of a wide range of behavioural responses, and, indeed,
S. oneidensis MR-1 has been shown to respond to a variety
of different electron acceptors [19,30]. So far, only one of
three putative CheA proteins, CheA-3, has been shown to
be necessary for behavioural responses to anaerobic electron
acceptors, including nitrate, nitrite, fumarate, DMSO,
TMAO (trimethylamine N-oxide) and Fe(III) citrate [28,30].
A mutant lacking cheA-3 has been shown to be smoothswimming, i.e. unable to change the direction of rotation of its
flagellum [30]. An MCP with a Cache domain (SO_2240) has
also been shown to be necessary for behavioural responses
to a number of electron acceptors (TMAO, DMSO, nitrite,
nitrate and fumarate), although deletion of this gene did
not completely abolish the tactic responses [28]. Because
deletion of the SO_2240 mcp resulted in loss of responses
to a range of anaerobic electron acceptors, it was suggested
that this MCP is an energy taxis receptor. Mutants lacking
any one of the four mcp genes that encode MCPs with
PAS (Per/Arnt/Sim) domains (SO_0584, SO_1385, SO_2123
and SO_3404) showed near-wild-type tactic responses to
soluble electron acceptors in chemical-plug-in-pond assays
[28]. However, double mutants lacking both SO_2240 and
any one of the four MCPs that have PAS domains had
slightly stronger phenotypes, perhaps indicating that the cells
monitor more than one energetic parameter [28].
Thus it appears that S. oneidensis MR-1 cells have the
necessary equipment for sensing and responding to soluble
electron acceptors, but there is as yet no explanation for how
they can sense and respond to insoluble metal oxides and
electrodes.
Other reports suggest that riboflavin excreted by Shewanella cells is an attractant that mediates energy taxis [9]. In
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electron acceptor-limited cells to create a chemical gradient
for the taxis [10]. In general, unless the cells themselves
create the gradient, chemical gradients do not agree with our
observations of a rapid behavioural response around poised
electrodes, since the latter do not release diffusing chemicals
[18]. As noted above, there have been severe criticisms of
the chemical-plug-in-pond and swim plate techniques used
in previous studies [29]: criticisms that have motivated us to
use video microscopy and cell tracking methods to study this
process.
In a previous study, we used video microscopy to show
that S. oneidensis MR-1 cells swim faster in the vicinity
of IEAs (both metal oxides and charged electrodes) [18], a
response perhaps indicative of a direct connection between
electron acceptor reduction and pmf generation. In the
present paper, we report, in addition to the observed increase
in speed, an increase in flagellar reversal frequency, which
leads to an accumulation of cells around metal oxide particles
and electrodes, a response we refer to as ‘congregation’. A
hypothetical model is presented to explain the phenomenon:
a model that involves swimming speed enhancement upon
contact with the IEA, and flagellar reversal at high swimming
speeds, resulting in accumulation of cells in the vicinity of the
electron acceptor, or congregation. We also report genes that,
when deleted, results in phenotypes with defective abilities
to congregate.
Methods
Cultivation and strains
S. oneidensis MR-1 and several deletion mutants originating from S. oneidensis MR-1 were examined in our
study (Table 1, and Supplementary Figures S1 and S2
at http://www.biochemsoctrans.org/bst/040/bst0401167add.
htm). All experiments were carried out using a previously
described defined minimal medium, containing 18 mM
sodium lactate as an energy source [31] (see Supplementary Table S1 at http://www.biochemsoctrans.org/bst/040/
bst0401167add.htm). Strains were inoculated from freezer
stocks on LB (Luria–Bertani) plates and then grown
overnight at 30 ◦ C. Individual colonies were then selected
and inoculated into defined minimal medium (M1). All
strains and mutants were inoculated into 5 ml of M1
medium inside airtight 15 ml tubes (VWR International
LLC) and incubated horizontally in a shaker at 180 rev./min
for 48 h at 30 ◦ C (Amerex Instruments). Attenuance
was measured using a spectrophotometer (Unico 1100RS
spectrophotometer). Cells were sampled at a D600 of 0.48–
0.52 (after ∼48 h). For the metal oxide particle experiments,
5 μl of suspended mineral particles [300 mg/ml MnO2
or Fe(OH)3 ] was then mixed with the cell culture.
Aerobic cells and minerals were mixed by inversion three
times, and were then loaded by capillary action into
rectangular capillary tubes (0.02 mm×0.20 mm) (Vitrocom).
These tubes were then sealed (zero time) with silicone vacuum
Electron Transfer at the Microbe–Mineral Interface
Table 1 Relevant genes used in our study
Name (gene name)
Locus number
Physical description
Role
Reference(s)
S. oneidensis MR-1
Wild-type strain
fccA
SO_0970
Tetrahaem flavocytochrome,
fumarate reductase
Fumarate reduction
[1,39]
[40]
cymA
SO_4591
Tetrahaem cytochrome c
Necessary for reduction of several
anaerobic electron acceptors,
including metal oxides
[6,41]
mtrA
cheA-3
cheA-1
SO_1777
SO_3207
SO_2121
Decahaem cytochrome c
Histidine protein kinase
Histidine protein kinase
Extracellular metal oxide respiration
Chemotactic signal transduction
Unknown
[5,42]
[30]
[30,43]
mcp_pas
mcp_pas4x
SO_1385
SO_1385, SO_0584,
SO_2123, SO_3404
MCP with PAS domains
MCPs with PAS domains
Unknown
Unknown
[28,30,43]
[28,30,43]
mcp_cache
SO_2240
MCP with a Cache domain
‘Energy taxis’ in response to soluble
electron acceptors
[28]
grease (Dow Corning) and then observed by microscopy.
Metal oxides were synthesized as described in the Supplementary Online Data (at http://www.biochemsoctrans.
org/bst/040/bst0401167add.htm).
Miniature electrochemical cell and coated
electrode
For electrode experiments, cells were loaded into a miniature
electrochemical observation device and sealed with silicone
vacuum grease at zero time (see Supplementary Figure S3
at http://www.biochemsoctrans.org/bst/040/bst0401167add.
htm) [18]. The device was similar to a system described
previously [18], but with several additional features (see
Supplementary Figure S3). First, the graphite fibre electrode
was coated with Teflon and then cut to expose a defined area
of conductive graphite. For details regarding coated graphite
electrodes, mineral synthesis, soluble electron acceptor
chemicals and GFP (green fluorescent protein) time-lapse
photography, see the Supplementary Online Data. Cells were
placed in an electrochemical cell and the electrode first poised
at + 700 mV after 15 min or, alternatively, the potential was
stepped up in 50 mV increments from 0 mV to + 700 mV at
3 min intervals (i.e. 0, 50, 100, 150, etc. mV).
Hand-tracking analysis of cell movements
Bacterial swimming tracks (both computer and manual
tracks) were calibrated using a microscope scale ruler
(100 μm). From each experiment, the overall swimming activity within the video frame, equivalent to a 107 μm×193 μm
field of view, was recorded and the video was time-normalized
to give swimming speeds in μm/s. Several measurements were
made for each bacterial swimming track: the total distance
moved, the time of track (between when the bacteria first
appear and disappear), the number of reversals, the distance
between each reversal and the IEA, and the distance between
the IEA and the start of bacteria track (see the Supplementary
Online Data).
Analysis
The starting position of the bacteria with respect to the
nearest IEA surface was logged, and each bacterial reversal
event was identified and logged with regard to the distance
from the nearest IEA surface (Figures 1A and 1B). For a
known time of swimming activity, the swimming cells were
divided into two groups for analysis: cells that swam within
2 μm of a particle were considered ‘contacting’ and those
that did not swim within 2 μm from the particle surface were
considered ‘non-contacting’. In addition to the hand-tracking
methods described above, some experiments (such as those
in Figure 3) utilized a computer-tracking algorithm [32] (see
the Supplementary Online Data for details).
S. oneidensis MR-1 cells swim faster in
proximity to metal oxide particles and
poised electrodes
Cells that contacted IEAs (either metal oxides or charged
electrodes) swam at significantly higher speeds than cells
that did not contact these surfaces (Figures 1A and 1B and
Supplementary Movie S1 at http://www.biochemsoctrans.
org/bst/040/bst0401167add.htm). Figure 2(A) shows that cell
swimming speeds near the exposed tip of a Teflon-coated
electrode poised at + 700 mV compared with the Ag/AgCl
electrode are similar to the swimming speed in response
to a Fe(OH)3 or MnO2 mineral (Table 2 and Supplementary Movie S2 at http://www.biochemsoctrans.org/bst/040/
bst0401167add.htm). Cells lack a swimming response during
0–500 mV applied potential (Supplementary Figure S2 and
Supplementary Movie S3 at http://www.biochemsoctrans.
org/bst/040/bst0401167add.htm). The coated electrode, with
a defined area (∼700 μm2 ) of conductive graphite at the tip
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Figure 1 Swimming S. oneidensis MR-1 congregate around IEAs because of reversals in swimming direction
Swimming tracks of S. oneidensis MR-1 around a stationary particle of manganese oxide (A) or an electrode poised at
+ 700 mV compared with Ag/AgCl (B) were analysed. Particles of MnO2 were mixed with S. oneidensis MR-1 cells and then
sealed in a capillary tube with lactate as the carbon source (A). Video data were recorded after the cells consumed all of
the dissolved O2 . The grey lines indicate the path of each individual bacterium that swam in 10 s. The red star signifies the
location where a cell reversed direction. The distance from the start of the bacteria track and from any reversal event to
the mineral was recorded (dash-dotted line and dotted line respectively). (C) Time-lapse fluorescent microscopy showing
motile S. oneidensis MR-1 cells labelled with GFP around MnO2 (outlined with red dotted oval in leftmost frame). Each
frame was taken 1 h apart (0–4 h, from left to right) and was photographed immediately after irreversibly photobleaching
the protein at zero time. Scale bar, 100 μm. There appears to be accumulation of motile bright cells (from outside of the
frame) near the mineral and attached to the surface.
(Figure 1B and Supplementary Figure S3B), allowed us to
approximate the particle size and redox potential of small
metal oxide particles found in sediment. Unlike the previous
study that recorded only swimming speeds [18], our analysis
(Figures 1A and 1B) includes the starting position, and the
position of all reversal events (red star) of each individual cell
(dot-dashed line and dotted line respectively).
Swimming S. oneidensis MR-1 cells
congregate and then attach to the MnO2
surface
Using strains labelled with GFP, it was demonstrated that
many of these swimming cells eventually (in 0.5–4 h) become
attached to mineral surface and electrode (Figure 1C and
Supplementary Figure S4 at http://www.biochemsoctrans.
org/bst/040/bst0401167add.htm). The time course in Figure 1(C) starts when the GFP-labelled cells in the tube are
photobleached at zero time (leftmost frame). Then new motile
cells move from outside of the bleached area into the darkened
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red dotted oval in leftmost frame).
In the proximity of metal oxides and
charged electrodes, S. oneidensis MR-1
cells exhibits more swimming reversals
Changes in swimming speeds alone do not account for
the congregation response. Video microscopy and manual
tracking of cell motility around mineral surfaces and charged
electrodes revealed that direction of swimming was also
altered. Because S. oneidensis MR-1 has a single polar
flagellum, reversal of swimming direction is accomplished
simply by reversal of flagellar rotation. At 30 min after the
aerobic cells were added to the capillary containing an IEA,
most cells cease swimming, whereas a small percentage,
located near the minerals continue swimming (Figures 3A
and 3B, and Supplementary Movies S1, S4 and S5 at http://
www. biochemsoctrans . org/ bst / 040 / bst0401167add . htm).
Video microscopy and manual tracking of cell motility
around manganese oxide particles (at a 30 min time
Electron Transfer at the Microbe–Mineral Interface
Figure 2 Congregation behaviour of swimming S. oneidensis was characterized by increased swimming speed and increased reversal
frequency of cells near IEAs
(A) Elevated swimming speeds of bacteria occur within 60 μm of the IEA surface [, MnO2 ; 䊊, Fe(OH)3 ; ×, electrode poised
at 700 mV compared with Ag/AgCl]. (B) The reversal timing allowed bacteria to congregate around the acceptor surface
rather than swimming away. (C) A plot of average reversal frequency against binned speed of swimming S. oneidensis
MR-1 indicates that stimulated cells (25–45 μm/s) reverse direction more often than less-stimulated cells (5–25 μm/s).
(D) Total number of data points collected for each type of experiment, which were then used to generate the graphs
(A–C). Overlapping S.D. values in (A–C) indicate a similar reversal and speed response in the vicinity of MnO2 , Fe(OH)3
minerals or electrode poised at + 600 mV compared with Ag/AgCl. This specialized reversal timing allowed the bacteria
to congregate and attach to electron-accepting surfaces. The contacting S. oneidensis cells can transfer electrons directly to
IEAs via specialized outer membrane cytochromes.
point), showed that cells, which contacted the MnO2
particle, had a significantly higher reversal frequency than
cells swimming further away from the particle. Figure 2
shows hand-tracked data for S. oneidensis MR-1 cells
swimming around a large MnO2 particle. The contacting
cells and non-contacting cells for each experiment can
be found in Supplementary Table S2 at http://www.
biochemsoctrans.org/bst/040/bst0401167add.htm. The reversal frequency of the contacting cells was 0.944 ± 0.53
reversals/s, whereas the reversal frequency of the noncontacting cells was 0.627 ± 0.73 reversals/s. The increased
reversal frequency of cells surrounding the IEA then
provides a mechanism for the congregation.
Electron-accepting electrodes also induce
changes in the behaviour of S. oneidensis
MR-1 cell swimming
A previous study showed that + 700 mV compared with
the Ag/AgCl electrode, a potential that mimics the redox
potential of the Mn(IV)/Mn(II) couple, induced a rapid
swimming response in S. oneidensis MR-1 cells [18].
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Figure 3 Number of swimmers declines to near zero except for wild-type strains in the presence of IEAs
Mean (± 4 S.D.) number of swimming bacteria (per s) after being sealed in a capillary (zero time) in the vicinity of insoluble
MnO2 (A) or Fe(OH)3 (B) mineral particle or with no mineral added (C). The strains are labelled S. oneidensis MR-1 (䊉),
SO_2240 (䊐), SO_1385 (), SO_3207 (×) and cymA (䊊). The mcp_cache (SO_2240), cheA-3 (SO_3207)
and mcp_pas (SO_1385) mutants formed less effective congregations compared with wild-type around Fe(OH)3 . This
was apparent when comparing cell swimming 30 min after being sealed with the mineral. The cheA-3 and mcp_cache
strains were entirely unable to congregate around the mineral.
Table 2 Comparison of reversal frequency and speed of swimming S. oneidensis within a population: those that contact insoluble
electron acceptor surface compared with those which have not contacted
The data were collected after bacteria and mineral were sealed in the capillary for 15 min. The swimming tracks within the same experiment are
sorted into two separate groups based on swimming path: those that contact insoluble electron acceptor surface (swam within 2 μm) compared
with those which have not contacted (2 μm). Results are means ± 2 S.D. The pairs of letters highlight measurements that are significantly different
(P < 0.05) from each other; those without are not statistically different. These paired measurements also correspond to the observation that strains
successfully congregated around mineral after 30 min.
Reversal frequency (reversals/s)
Speed (μm/s)
Conditions
2 μm
2 μm
2 μm
2 μm
MnO2
0.972 ± 0.58a
0.328 ± 0.48a
24.37 ± 6b
19.26 ± 11.2b
Fe(OH)3
+ 700 mV (15 min)
+ 700 mV (1 h)
0.745 ± 0.5c
0.216 ± 0.39c
18.12 ± 5.4d
0.518 ± 0.5e
0.042 ± 0.08e
19.54 ± 11.8f
0.551 ± 0.51g
0.301 ± 0.33g
16.56 ± 6.7
12.6 ± 5.4d
8.28 ± 3.27f
16.23 ± 16.9
+ 700 mV (uncoated)
0.481 ± 0.62
0.392 ± 0.61
38.31 ± 12.3
This redox potential was therefore selected for additional
cell tracking to determine changes in reversal frequency.
Figure 1(B) shows the congregation response of S. oneidensis
MR-1 cells swimming around an electrode poised at
+ 700 mV after 1 h. However, if + 700 mV is applied only
15 min after the cells are sealed in an anaerobic capillary, the
congregation response is intensified (Table 2). Analysis of
the tracks, after 15 min, indicated that swimming cells in the
contacting group swam at a speed of 19.54 ± 11.8 μm/s with
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a reversal frequency of 0.551 ± 0.51 reversals/s, whereas noncontacting cells swam more slowly (8.28 ± 3.27 μm/s) with
far fewer reversals (0.042 ± 0.08 reversals/s) (Table 2). Applied
voltages of 550–800 mV elicited the maximum swimming
response and reversal timing conducive to congregation in
wild-type, whereas 0–500 mV applied resulted in fewer
swimmers (Supplementary Figure S2). As can be seen, when
the distance from the electrode increases, the swimming speed
and the reversal frequency decrease (Figure 2).
Electron Transfer at the Microbe–Mineral Interface
Changes in reversal frequency correlate
with changes in swimming speed in
S. oneidensis MR-1 cells around IEAs
We plotted the average reversal frequencies against swimming
speeds, grouped in 5 μm/s increments. This plot revealed
a correlation between the frequency of reversals and the
swimming speed: the faster the cells swam, the more often
those cells reversed (Figure 2C). This relationship was seen
for cells around the electrode at + 700 mV, and around MnO2
and Fe(OH)3 mineral particles (Figure 2 and Table 2).
Cell response varies with time
Figures 3(A) and 3(B) depict swimming and motility after
being sealed inside an anaerobic capillary tube where oxygen
was consumed and cell motility persisted only around the
minerals. As mentioned above, if cells were exposed
to the charged electrode at 15 min after the capillary was
sealed, the response was far greater than if the cells were
allowed to sit anaerobically for 1 h before charging the
electrode (Table 2, rows 3 and 4). This can also be seen directly
in the Supplementary Movies, where in one experiment, the
+ 700 mV compared with Ag/AgCl potential was applied
15 min after bacteria were sealed inside the capillary tube
(Supplementary Movie S2), and in another the charge was
applied 1 h after bacteria were sealed inside a capillary tube
(Supplementary Movie S3). These data are summarized in
Table 2.
This difference in timing is critical when mutant studies
are being carried out, as many of these strains survive poorly
in the absence of an electron acceptor, and if they cannot
congregate or they cannot respire via EET, they will die
rapidly. After 15 min, the cells had consumed the oxygen
(as determined using an electrode), and most strains showed
some motility around the IEAs; however, by 30 min, several
strains, including cymA (SO_4591) and mcp_cache
(SO_2240), were completely non-motile around MnO2 ,
Fe(OH)3 and poised electrodes (Figure 3). Interestingly, the
mcp_pas mutant (SO_1385) found to be present in many
Shewanella strains, exhibited wild-type levels of motility and
reversals with regard to MnO2 , but irregular or no response
to Fe(OH)3 particles or poised potentials (Supplementary
Movie S5).
Figure 4 Deletion mutants lacking MCPs (SO_1385 or
SO_2240), chemotaxis transduction protein (SO_3207) or EET
cytochromes (SO_4591) are unable to congregate around MnO2
or Fe(OH)3
Reversal frequencies of swimming bacteria within a population: those
that contacted insoluble mineral surface compared to those that did
not. Experiment data, from added MnO2 (A) or Fe(OH)3 (B) to bacteria
cultures of S. oneidensis MR-1, mcp_cache (SO_2240), mcp_pas
(SO_1385), cymA (SO_4591) and cheA-3 SO_3207 were
divided into two subpopulations for analysis. This allowed for a comparison of reversal frequency and speed of swimming bacteria within
a population. A division was made between bacteria that contacted
(light grey) insoluble acceptor surface (i.e. swam within 2 μm)
compared with those that were swimming, but had not contacted the
surface (dark grey). The contacting group was significantly faster (Supplementary Table S2 at http://www.biochemsoctrans.org/bst/040/
bst0401167add.htm) and reversed direction more often than the
non-contacting group, in the following experiments: S. oneidensis
MR-1 with MnO2 (A), mcp_pas (SO_1385) with MnO2 (A) and
S. oneidensis MR-1 with Fe(OH)3 (B). SO_3207 exhibited a
smooth-swimming phenotype with no reversals, and is therefore not
shown. Results are means ± 4 S.D.
Increased reversal frequency after
contacting IEAs is essential for
congregation
The receptor protein and histidine protein kinase required
for energy taxis in S. oneidensis MR-1 have been identified
[28]. Mutants lacking the chemotaxis proteins, i.e. mcp_cache
(SO_2240), mcp_pas (SO_1385) and cheA-3 (SO_3207),
or the EET cytochromes, i.e. cymA (SO_4591), were
screened for their response to MnO2 , Fe(OH)3 and the
poised electrode. In response to Fe(OH)3 , these mutants
showed a significant (P<0.05; Student’s t test) decrease in
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Figure 5 Proposed congregation model
Aerobic cells swim stochastically in all directions, reversing 0.5 times per s (A). Within 10 min of being sealed in the capillary
with metal oxide, motile cells consume all available dissolved O2 and begin to randomly contact mineral, passing electrons
via the specially adapted EET chain and build a pmf. (B) Now anaerobic, the cells making contact congregate by sensing
intercellular pmf (mediated by MCPs SO_2240 and SO_1385). Using appropriately timed reversals, via the chemotaxis
pathway, stimulated cells often return to mineral resulting in congregation (C) and eventual attachment. A model of the
process is shown on the right.
reversal frequency compared with wild-type S. oneidensis
MR-1 (Figure 4). In the case of the cheA-3 mutant, this
behaviour was expected because the mutant is unable to
reverse its direction of motion. In contrast, the SO_2240
cells, although capable of reversal, showed no significant
difference in reversal frequency or swimming speed
compared with those in the non-contacting group (Figure 4
and Supplementary Table S2).
Figure 4 displays reversal frequencies of contacting and
non-contacting cells in response to MnO2 or Fe(OH)3 , which
highlight significant reversal frequency timing irregularities
compared with wild-type. The SO_2240 mutant exhibited
irregular reversal timing around both MnO2 and Fe(OH)3 ,
whereas the SO_1385 reversal phenotype is similar to wildtype around MnO2 , but irregular around Fe(OH)3 (Figure 4).
Neither the SO_2240 nor the SO_1385 mutant responded
with swimming to any voltage, during the applied potential
iterations, compared with the wild-type swimming response
(Supplementary Figure S2). Additional deletion mutants of
genes that contain the PAS domain, SO_0584, SO_2123
and SO_3404 showed no difference in phenotype from
wild-type in response to insoluble acceptors (results not
shown).
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Authors Journal compilation EET is required for congregation in
S. oneidensis MR-1
Mutants unable to perform EET are unable to congregate.
For example, cymA failed to congregate around minerals
and poised electrodes. After 15 min, swimming cymA cells
contacting the MnO2 or Fe(OH)3 did not reverse significantly
more than non-contacting cells (Figure 4). By 30 min,
cymA cells were completely non-motile around MnO2 ,
Fe(OH)3 and poised electrodes. However, fccA mutant
strain (SO_0970) lacking cytochromes which are nonessential for EET did not respond significantly differently
from wild-type.
Model of congregation
The results of our study have led us to propose a
simple hypothetical model that provides an explanation for
how these microbes congregate near to IEAs (Figure 5).
Congregation could be of substantial value in environments where rapid redox cycling occurs, particularly in
sediments, where dissolved oxygen can change dramatically
and quickly [33], not unlike the situation at the beginning
Electron Transfer at the Microbe–Mineral Interface
of our experiment when the capillary was sealed with added
MnO2 .
The simple model, shown in Figure 5, consists of the
following steps. (i) Initially, the cells are highly motile,
utilizing dissolved oxygen as the electron acceptor and seldom
reversing direction (Figure 5A). As oxygen is depleted,
swimming speed decreases, and after 15–30 min, all cells
are non-motile except for those that have incidentally
encountered an electron acceptor (metal oxide or poised
electrode). This is a stochastic process that continues
throughout the experiment. Of the total cells in the capillaries,
only 1–3 % are motile. (ii) These contacting cells interact
with the particle via a series of electron carriers to the outer
membrane (Mtr) protein complexes. The contacting cells
are energized to swim, resulting in the previously described
electrokinesis response [18]. (iii) These fast-swimming cells
undergo rapid flagellar reversal and directional reversal,
characteristic of a monotrichous cell [34], and, by this
response, are maintained in the vicinity of the solid-state
electron acceptor. This is a directed response that occurs
in addition to the stochastic recruitment of new cells. (iv)
Swimming cells continuously attach to the electron acceptor,
eventually forming a biofilm.
Significance of PAS domain MCP (SO_1385)
Several studies have hypothesized that four MCPs with PAS
domains are in some way responsible for energy taxis or
response to IEAs [28–30]. However, up to this point, no
study has found SO_1385 genes to be essential for response
to electron acceptors in Shewanella [28]. Our results, using
analysis of cell swimming tracks, suggest that SO_1385
may have a role in congregation around IEAs, where the
mechanism is fundamentally different from that needed to
locate a soluble electron acceptor. We found that one of
the four putative PAS domain-containing signal transducers
(SO_1385) appears to play a significant role in congregation
around IEAs with low redox potential such as Fe(OH)3 ,
whereas the other three signal transducers (SO_0584,
SO_2123 and SO_3404) appear to play no essential role in
congregation. Genetic analysis shows that the SO_1385 gene
is the most abundant in the 17 Shewanella species (present
in 12 of the 17 Shewanella species analysed) and encodes
a PAS domain-containing receptor [35]. These findings are
noteworthy because transcriptomic analysis of wild-type
S. oneidensis MR-1 revealed specific up-regulation of this
SO_1385 gene under Fe(III)- or Mn(IV)-reducing conditions.
Furthermore, the gene (SO_1385) has been shown to share
58 % homology with Escherichia coli aerotaxis transducer
[35] and is therefore a strong candidate as a flavin-containing
redox/energy taxis transducer in S. oneidensis MR-1.
Previously, this PAS sensory protein was thought to play only
a minor or insignificant role in S. oneidensis MR-1 energy taxis
in response to electron acceptors [28].
The need for EET
One prediction of this model is that it should require EET
in order to activate the cells. Not surprisingly, mutation of
any of the genes involved with EET abolished congregation,
as they had been reported to abolish electrokinesis [18]. In
addition, increasing the IEA surface area, for example by
increasing the conductive surface area of an electrode
by using less insulation coating, but applying the same surface
charge also increased the speed of swimming (Table 2 and
Supplementary Figure S5 at http://www.biochemsoctrans.
org/bst/040/bst0401167add.htm). At first glance, it is tempting to define this congregation behaviour as a variation of
energy taxis, as it is likely to be a metabolism-dependent
response. But, given the possible involvement of the EET
chain and multiple MCP interactions (receptors with both
PAS domains and Cache domains), this behaviour seems to
be distinct from all previously defined behaviours. Therefore
we refrain from making this distinction until further research
can be performed.
The need for a sensing mechanism
Another prediction of the congregation model is that there
must be some sensing mechanism involved that can lead to
control of flagellar reversal. The fact that reversal frequency
is increased in bacteria with higher speed (and thus closer to
IEA) provides a way for the bacteria to congregate around the
IEAs, but what is the sensing mechanism? Experiments with
several mutants known to be involved with chemotaxis in
S. oneidensis [20,28–30] suggest some answers. For example,
in a mutant lacking a functional chemotaxis protein CheA3 (SO_3207), congregation is totally eliminated. A cheA3
mutant in E. coli leads to a phenotype in which flagellar
reversal is inhibited [30]. Assuming this mutant is unable
to reverse flagellar rotation, one would predict that cells
would be stimulated to become motile by random contact
with the electron acceptor, but would almost never return
to the insoluble particle (Figure 4 and Supplementary Figure
S1).
Similarly, a mutant lacking a functional MCP coded for
by the gene SO_2240 was incapable of congregation. This
MCP is a Cache domain-containing protein that is thought
to act by sensing the transmembrane potential in E. coli [28].
It is easy to see how such ability could be coupled to the
congregation response. One possibility is that a rapid increase
in pmf that might occur upon contact with the IEA would
stimulate flagellar reversal: another might be that the pmf is
constantly monitored and, as it increases, the probability of
flagellar reversal also increases. This possibility is now under
investigation (Figure 4 and Supplementary Figure S1).
Mutation of genes coding for PAS domain proteins led
to an interesting incongruity that was seen with regard to
congregation around hydrous ferric oxide, in that one mutant
was observed that blocked the congregation around iron,
but showed no effect on the congregation around manganese
particles. This was the MCP PAS domain-containing protein
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Biochemical Society Transactions (2012) Volume 40, part 6
coded for by SO_1385. Given the low potential of iron oxide
in comparison with MnO2 , this might not be particularly
surprising, but it may also provide some clues about the
interaction of the PAS domain-containing MCPs, which are
now under more detailed study (Figure 4).
Nature, they will almost certainly become rapidly electrondonor-limited. Congregation provides a way to avoid both
electron donor and IEA limitation. In fact, when metabolism
of IEA particles is observed, they are often completely
degraded with minimal cell attachment despite extensive
congregation activity (Supplementary Movie S6 at http://
www.biochemsoctrans.org/bst/040/bst0401167add.htm).
Will this work in E. coli?
Engineered E. coli (E. coli mtrCAB) has been shown to
be capable of EET [36]. Our results show that wild-type
E. coli cannot congregate around IEAs. This raises the
question of whether or not the engineered E. coli strain,
with transplanted mtrCAB genes from Shewanella, is capable
of this behaviour. Our hypothesis is that it would not be
capable of congregation. Despite having mcp_pas, mcp_cache
and mtrCAB (EET cytochromes) genes, it cannot congregate
because the strain lacks a single polar flagellum. On the basis
of our tracking data (Figures 1A and 1B), we believe that this
response must be limited to monotrichous bacteria that will
be capable of returning to the surface of IEAs by a series of
runs and reversals [34]. According to our model, even with
the functioning Shewanella mtrA–mtrC genes expressed in
E. coli, allowing this organism to reduce solid metal oxides
[36], congregation behaviour should not occur. Although an
E. coli with added Shewanella MCPs [30] cannot perform
true congregation around IEAs not only because it lacks the
mtrA–mtrC genes, but also because its response to flagellar
reversal will be to tumble rather than to reverse, and the
probability of returning to the IEA surface will be vanishingly
small.
Why congregation?
The term congregation describes the observed motility driven
accumulation on the surface and in the vicinity of IEAs:
a distinctive type of behaviour that cannot be put into
any of the presently known bacterial response definitions.
Our results, which characterized the response around IEAs,
do not support the idea of chemotaxis towards a small
amount of soluble electron acceptor [37]. Neither do our
data fit the previously defined energy taxis paradigm [27].
As the molecular mechanism(s) involved becomes clear, the
relationship between congregation and other more wellknown tactic responses should become clear.
The biological rationale for this behaviour is also not
yet clear, but it should be considered in the context
of the kind of environment that these microbes encounter, where rapid limitation of either electron donors
and/or electron acceptors can occur. Thus commitment to
either an electron donor or an electron acceptor may
constitute an important regulatory ‘decision’ retaining
the capacity to move from the zone of electron donor
excess to the zone of electron acceptor excess may be
a very positive adaptive trait [27,38]. For example, in
our experiments, we employed a high level of electron
donor (18 mM lactate), which is almost certainly seldom
encountered in Nature. If cells settle on the IEA surface in
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Authors Journal compilation Post-congregation activities
Initial studies with different strains of Shewanella indicate
that congregation is an important first step in the attachment
and biofilm formation by several different microbes and that
these processes are closely linked (H.W. Harris, J.S. McLean,
M.Y. El-Naggar, E.C. Salas and K.H. Nealson, unpublished
work); i.e. a strong congregation response under a given
condition leads to attachment and biofilm formation. If so,
then this mechanism is potentially of great importance with
regard to natural environments where redox chemistry and
electron exchange occur.
Acknowledgements
Special thanks to Mandy J. Ward for advice on research and Jeff
McLean for experiment design. We thank Meaghan Sullivan and
William Tran for their manual tracking analyses. We thank Cécile
Jourlin-Castelli, Samantha Reed, Jun Li and David Culley for supplying
the mcp_cache, mtrB, mtrA, mcp_pas, cheA3 and cymA
mutants.
Funding
This work is supported by an Air Force Office of Scientific Research
Award [grant number FA9550-06-1-0292].
References
1 Myers, C.R. and Nealson, K.H. (1988) Microbial reduction of manganese
oxides: Interactions with iron and sulfur. Geochim. Cosmochim. Acta 52,
2727–2732
2 Lovley, D.R. and Phillips, E.J.P. (1988) Novel mode of microbial energy
metabolism: organic carbon oxidation coupled to dissimilatory reduction
of iron or manganese. Appl. Environ. Microbiol. 54, 1472–1480
3 Meyer, T.E., Tsapin, A.I., Vandenberghe, I., de Smet, L., Frishman, D.,
Nealson, K.H., Cusanovich, M.A. and van Beeumen, J.J. (2004)
Identification of 42 possible cytochrome C genes in the Shewanella
oneidensis genome and characterization of six soluble cytochromes.
OMICS 8, 57–77
4 Mitchell, A.C., Peterson, L., Reardon, C.L., Reed, S.B., Culley, D.E., Romine,
M.R. and Geesey, G.G. (2012) Role of outer membrane c-type
cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell
production, accumulation, and detachment during respiration on
hematite. Geobiology 10, 355–370
5 Myers, C.R. and Myers, J.M. (2002) MtrB is required for proper
incorporation of the cytochromes OmcA and OmcB into the outer
membrane of Shewanella putrefaciens MR-1. Appl. Environ. Microbiol.
68, 5585–5594
6 Myers, J.M. and Myers, C.R. (2001) Role for outer membrane
cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in
reduction of manganese dioxide. Appl. Environ. Microbiol. 67, 260–269
Electron Transfer at the Microbe–Mineral Interface
7 Beliaev, A.S. and Saffarini, D.A. (1998) Shewanella putrefaciens mtrB
encodes an outer membrane protein required for Fe(III) and Mn(IV)
reduction. J. Bacteriol. 180, 6292–6297
8 Lovley, D.R., Coates, J.D., Blunt-Harris, E.L., Phillips, E.J. P. and Woodward,
J.C. (1996) Humic substances as electron acceptors for microbial
respiration. Nature 382, 445–448
9 Li, R., Tiedje, J.M., Chiu, C. and Worden, R.M. (2012) Soluble electron
shuttles can mediate energy taxis toward insoluble electron acceptors.
Environ. Sci. Technol. 46, 2813–2820
10 Marsili, E., Baron, D.B., Shikhare, I.D., Coursolle, D., Gralnick, J.A. and
Bond, D.R. (2008) Shewanella secretes flavins that mediate extracellular
electron transfer. Proc. Natl. Acad. Sci. U.S.A. 105, 3968–3973
11 El-Naggar, M.Y., Gorby, Y.A., Xia, W. and Nealson, K.H. (2008) The
molecular density of states in bacterial nanowires. Biophys. J. 95,
L10–L12
12 Gorby, Y.A., Beveridge, T.J. and Wiley, W.R. (2005), Composition,
Reactivity, and Regulation of Extracellular Metal-Reducing Structures
(Nanowires) Produced by Dissimilatory Metal-Reducing Bacteria, Annual
NABIR PI Meeting, 18–20 April 2005, Warrenton, VA, U.S.A.
13 El-Naggar, M.Y., Wanger, G., Leung, K.M., Yuzvinsky, T.D., Southam, G.,
Yang, J., Lau, W.M., Nealson, K.H. and Gorby, Y.A. (2010) Electrical
transport along bacterial nanowires from Shewanella oneidensis MR-1.
Proc. Natl. Acad. Sci. U.S.A. 107, 18127–18131
14 Okamoto, A., Hashimoto, K. and Nakamura, R. (2012) Long-range
electron conduction of Shewanella biofilms mediated by outer
membrane C-type cytochromes. Bioelectrochemistry 85, 61–65
15 Shi, L., Richardson, D.J., Wang, Z., Kerisit, S.N., Rosso, K.M., Zachara, J.M.
and Fredrickson, J.K. (2009) The roles of outer membrane cytochromes
of Shewanella and Geobacter in extracellular electron transfer. Environ.
Microbiol. Rep. 1, 220–227
16 Fredrickson, J.K., Romine, M.F., Beliaev, A.S., Auchtung, J.M., Driscoll,
M.E., Gardner, T.S., Nealson, K.H., Osterman, A.L., Pinchuk, G., Reed, J.L.
et al. (2008) Towards environmental systems biology of Shewanella.
Nat. Rev. Microbiol. 6, 592–603
17 Lovley, D.R., Holmes, D.E. and Nevin, K.P. (2004) Dissimilatory Fe(III) and
Mn(IV) reduction. Adv. Microb. Physiol. 49, 219–286
18 Harris, H.W., El-Naggar, M.Y., Bretschger, O., Ward, M.J., Romine, M.F.,
Obraztsova, A.Y. and Nealson, K.H. (2010) Electrokinesis is a microbial
behavior that requires extracellular electron transport. Proc. Natl. Acad.
Sci. U.S.A. 107, 326–331
19 Nealson, K.H., Moser, D.P. and Saffarini, D.A. (1995) Anaerobic electron
acceptor chemotaxis in Shewanella putrefaciens. Appl. Environ.
Microbiol. 61, 1551–1554
20 Bencharit, S. and Ward, M.J. (2005) Chemotactic responses to metals and
anaerobic electron acceptors in Shewanella oneidensis MR-1. J.
Bacteriol. 187, 5049–5053
21 Porter, S.L., Wadhams, G.H. and Armitage, J.P. (2011) Signal processing
in complex chemotaxis pathways. Nat. Rev. Microbiol. 9, 153–165
22 Taylor, B.L., Zhulin, I.B. and Johnson, M.S. (1999) Aerotaxis and
other energy-sensing behaviors in bacteria. Annu. Rev. Microbiol. 53,
103–128
23 Rebbapragada, A., Johnson, M.S., Harding, G.P., Zuccarelli, A.J., Fletcher,
H.M., Zhulin, I.B. and Taylor, B.L. (1997) The Aer protein and the serine
chemoreceptor Tsr independently sense intracellular energy levels and
transduce oxygen, redox, and energy signals for Escherichia coli
behavior. Proc. Natl. Acad. Sci. U.S.A. 94, 10541–10546
24 Bibikov, S.I., Barnes, L.A., Gitin, Y. and Parkinson, J.S. (2000) Domain
organization and flavin adenine dinucleotide-binding determinants in the
aerotaxis signal transducer Aer of Escherichia coli. Proc. Natl. Acad. Sci.
U.S.A. 97, 5830–5835
25 Bibikov, S.I., Biran, R., Rudd, K.E. and Parkinson, J.S. (1997) A signal
transducer for aerotaxis in Escherichia coli. J. Bacteriol. 179, 4075–4079
26 Alexandre, G., Greer, S.E. and Zhulin, I.B. (2000) Energy taxis is the
dominant behavior in Azospirillum brasilense. J. Bacteriol. 182,
6042–6048
27 Alexandre, G., Greer-Phillips, S. and Zhulin, I.B. (2004) Ecological role of
energy taxis in microorganisms. FEMS Microbiol. Rev. 28, 113–126
28 Baraquet, C., Théraulaz, L., Iobbi-Nivol, C., Méjean, V. and Jourlin-Castelli,
C. (2009) Unexpected chemoreceptors mediate energy taxis towards
electron acceptors in Shewanella oneidensis. Mol. Microbiol. 73,
278–290
29 Li, J., Go, A., Ward, M. and Ottemann, K. (2010) The chemical-in-plug
bacterial chemotaxis assay is prone to false positive responses. BMC Res.
Notes 3, 1–5
30 Li, J., Romine, M.F. and Ward, M.J. (2007) Identification and analysis of a
highly conserved chemotaxis gene cluster in Shewanella species. FEMS
Microbiol. Lett. 273, 180–186
31 Bretschger, O., Obraztsova, A., Stumm, C.A., Chang, I.S., Gorby, Y.A.,
Reed, S.B., Culley, D.E., Reardon, C.L., Barua, S., Romine, M.F. et al.
(2007) Current production and metal oxide reduction by Shewanella
oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 73,
7003–7012
32 Crocker, J.C. and Grier, D.G. (1996) Methods of digital video microscopy
for colloidal studies. J. Colloid Interface Sci. 179, 298–310
33 Nealson, K.H. (1997) Sediment bacteria: who’s there, what are they
doing, and what’s new? Annu. Rev. Earth Planet. Sci. 25, 403–434
34 Mitchell, J.G. (2002) The energetics and scaling of search strategies in
bacteria. Am. Nat. 160, 727–740
35 Beliaev, A.S., Klingeman, D.M., Klappenbach, J.A., Wu, L., Romine, M.F.,
Tiedje, J.M., Nealson, K.H., Fredrickson, J.K. and Zhou, J. (2005) Global
transcriptome analysis of Shewanella oneidensis MR-1 exposed to
different terminal electron acceptors. J. Bacteriol. 187, 7138–7145
36 Jensen, H.M., Albers, A.E., Malley, K.R., Londer, Y.Y., Cohen, B.E., Helms,
B.A., Weigle, P., Groves, J.T. and Ajo-Franklin, C.M. (2010) Engineering of
a synthetic electron conduit in living cells. Proc. Natl. Acad. Sci. U.S.A.
107, 19213–19218
37 Childers, S.E., Ciufo, S. and Lovley, D.R. (2002) Geobacter metallireducens
accesses insoluble Fe(III) oxide by chemotaxis. Nature 416, 767–769
38 Mitchell, J.G. and Kogure, K. (2006) Bacterial motility: links to the
environment and a driving force for microbial physics. FEMS Microbiol.
Ecol. 55, 3–16
39 Neuman, K.C., Chadd, E.H., Liou, G.F., Bergman, K. and Block, S.M. (1999)
Characterization of photodamage to Escherichia coli in optical traps.
Biophys. J. 77, 2856–2863
40 McLean, J.S., Wanger, G., Gorby, Y.A., Wainstein, M., McQuaid, J., Ishii,
S.I., Bretschger, O., Beyenal, H. and Nealson, K.H. (2010) Quantification
of electron transfer rates to a solid phase electron acceptor through the
stages of biofilm formation from single cells to multicellular
communities. Environ. Sci. Technol. 44, 2721–2727
41 Myers, C.R. and Myers, J.M. (1997) Cloning and sequence of cymA, a
gene encoding a tetraheme cytochrome c required for reduction of
iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1. J.
Bacteriol. 179, 1143–1152
42 Reguera, G., Nevin, K.P., Nicoll, J.S., Covalla, S.F., Woodard, T.L. and
Lovley, D.R. (2006) Biofilm and nanowire production leads to increased
current in Geobacter sulfurreducens fuel cells. Appl. Environ. Microbiol.
72, 7345–7348
43 Min, T.L., Mears, P.J., Chubiz, L.M., Golding, I., Chemla, Y.R. and Rao, C.V.
(2009) High-resolution, long-term characterization of bacterial motility
using optical tweezers. Nat. Methods. 6, 831–835
Received 11 September 2012
doi:10.1042/BST20120232
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Electron Transfer at the Microbe–Mineral Interface
SUPPLEMENTARY ONLINE DATA
Shewanella oneidensis MR-1 chemotaxis proteins
and electron-transport chain components
essential for congregation near insoluble electron
acceptors
H. Wayne Harris*, Mohamed Y. El-Naggar† and Kenneth H. Nealson‡1
*Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, U.S.A., †Department of Physics and Astronomy, University of
Southern California, Los Angeles, CA 90089, U.S.A., and ‡Department of Earth Sciences, University of Southern California, Los Angeles, CA 90089, U.S.A.
Methods
Growth medium
The defined minimal growth medium described in Table S1
was used for the aerobic growth of all strains screened in these
experiments [1]. Initially, NaOH was added to Nanopure
water (Thermo Scientific) to enhance solubility of Pipes
buffer. A working solution was then autoclaved at 121◦ C
for 15 min (Amsco Scientific). All minerals and amino acids
were filter-sterilized with a 0.2 μm PES (polyether sulfone)
vacuum filtration system (Thermo Scientific) and then added
to the working solution in a sterile hood (Labconco). Finally,
the pH was adjusted to 7.15 ± 0.05 by adding sterile NaOH
or HCl as necessary.
Hand-tracking analysis of cell movements
First, the computer and manual tracks were calibrated with a
microscope scale ruler (100 μm). From each experiment, the
overall swimming activity within the video frame, equivalent
to a 107 μm×193 μm field of view, was recorded and
normalized to time (seconds). The first measurements for
each bacterial swimming track were the total distance moved
and the time over which this movement occurred. Next,
the starting position of the bacteria with respect to the
nearest IEA surface was logged. Finally, the location of each
bacterial reversal event was identified, and the distance from
the nearest acceptor surface was recorded (Figure 1A and
1B of the main text). Note that Figures 1(A), 1(B), 2(A)–
2(C), 4 and 5 and Table 2 of the main text, and Figure S1
and Table S2 were all generated from hand-tracking data,
whereas Figure 3 of the main text and Figure S2 were
generated using the tracking algorithm. A sample of tracking
algorithm outputs have been verified, by comparison with
hand tracking, to give the same number of swimming bacteria
(computer reversal frequency, position and speed data were
not used in our study).
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2012) 40, 1167–1177; doi:10.1042/BST20120232
Tracking algorithm
Cells were monitored near metal oxides and working
electrodes in electrochemical cells, using ×100 (optical) light
microscopy [2]. The locations of individual cells and the
subsequent linking of these locations to form trajectories
were based on the particle tracking algorithms of Crocker and
Grier [1]. The tracking algorithm was utilized similarly to the
methods described previously [3] except that an additional
filter was applied to eliminate stationary bacteria from being
counted. The original tracking algorithm (http://physics.
georgetown.edu/matlab/) has been modified and is available
for use and review. The algorithm parameters (expected cell
size, minimum spot intensity and maximum distance travelled
between frames) were adjusted to obtain tracking with an
acceptable level of accuracy. For our study, the acceptable
level was achieved by the program only if it could successfully
detect and assign trajectories to all well-photographed
bacteria, which were in focus and had sharp contrast with the
background, as well as most out-of-focus bacteria (>80%)
in all video samples collected. All computed trajectories were
then checked manually by visual inspection and compared
with video tracked by hand, frame by frame [2]. From our
analysis of bacteria motility, it is worth noting that many wildtype cells remained stationary in the presence of insoluble
electron acceptors (>300 stationary bacteria/20 mm2 ), so they
were excluded from cell tracking measurements.
GFP time-lapse experiments
All strains and mutants were grown aerobically on defined
minimal medium with 50 μg/ml kanamycin (and 18 mM
lactate for 48 h at 30◦ C). Samples of 5 ml from cultures
were taken when the cells reached a D600 of 0.4, mixed
with manganese or iron oxides and introduced to a small
glass capillary that was then sealed using vacuum grease (as
described above) [2]. Using an inverted Nikon Eclipse TI
microscope with a ×40 lens, the GFP-labelled cells were
bleached using maximum light intensity settings, over a
45 min period. To ensure that bleaching occurred,
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Authors Journal compilation Biochemical Society Transactions (2012) Volume 40, part 6
time-lapse screen area (a 107 μm×193 μm field of view)
was captured every 5 min until original cells appeared dark,
whereas surrounding cells remained brightly fluorescent.
Then a time-lapse video of the entire section of tube, with
×20 lens, was captured using Nikon NIS Elements software
and the ‘perfect focus’ feature for the next 4 h. A separate
negative control, with mcp_cache (SO_2240) and GFP,
was also acquired for 4 h. No cells were seen accumulating
into the dark zone in this negative control and neither did
cells recover GFP fluorescence.
Miniature electrochemical cell and coated
electrode
The device was assembled using protocol described previously [3]; however, several additional features were added
to the device (Figure S3). A printed counterelectrode
(5 mm×4 mm) and reference electrode (Ag/AgCl) was
attached to the cathode compartment, supplied by Pine
Research.
Graphite electrode coating
Graphite fibres were cut to 25 mm lengths and sandwiched
between two glass microscope slides (50 mm × 20 mm).
The electrodes were then coated with non-conductive
FluoroPlate® (Crest Coating), to a thickness of ∼3 μm.
The electrodes were inspected under scanning electron
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Authors Journal compilation microscopy for inconsistencies and established to be nonconductive by suspending the electrodes in an electrochemical
cell. The ends of the electrode were then cleaved with a
razor blade to expose graphite at the very tip of the electrode
(Figure S3). For the applied potential experiments, an updated
electrochemical observation cell (Figure S3A) was fabricated
similar to that of Harris et al. [3].
Mineral synthesis
The Fe(OH)3 stock solution was prepared using the method
of Cornell and Schwertmann [4] and then verified by
X-ray diffraction [5]. The preparation of colloidal MnO2
began with 8 g of KMnO4 dissolved in 200 ml of distilled
water. The solution was mixed continuously using a magnetic
stir bar on high speed and heated (until just below boiling
temperature). Then, 5 ml of 10 M NaOH was added to
neutralize the acid produced by the reaction. In a separate
flask, 15 g of MnCl2 was dissolved in 75 ml of distilled water.
Finally, the solution was mixed slowly with the permanganate
solution (in a chemical fume hood) for 75 min. After cooling
the solution, the precipitate was washed by centrifugation
and rinsed at least five times with nanopure water. The final
precipitate was allowed to dry by vacuum filter on a clean
bench and desiccated for 36 h. The resulting minerals were
analysed via X-ray diffraction to confirm the production of
MnO2 [5–7].
Electron Transfer at the Microbe–Mineral Interface
Figure S1 The congregation of S. oneidensis around a particle of MnO2 requires chemotaxis protein CheA-3 (SO_3207)
The elevated reversal frequency of wild-type S. oneidensis MR-1 was associated with high-velocity swimmers
(>25–40 μm/s) compared with the low reversal frequency of the slower non-stimulated cells (swimming at 5–25 μm/s),
which allowed the bacteria to congregate, like a swarm of bees, around a particle of MnO2 . Strain cheA-3 (SO_3207)
cannot reverse direction and therefore cannot congregate around MnO2 . Other genes coding for MCPs (SO_1385) and
chemotaxis protein CheA-1 (SO_2121) are not essential for congregation around MnO2 .
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Authors Journal compilation Biochemical Society Transactions (2012) Volume 40, part 6
Figure S2 The number of swimming cells near electrode during each applied potential (for 3 min each)
The wild-type S. oneidensis MR-1 responded with swimming to 550–800 mV applied potentials, whereas deletion mutant
strains mcp_cache (SO_2240) and mcp_pas (SO_1385) did not respond with swimming. The swimming of mcp_pas
(SO_1385) is shown in red and the swimming of wild-type in blue. The lower of the values is shown in front of the other
for each potential. The data are from two experiments.
C The
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Authors Journal compilation Electron Transfer at the Microbe–Mineral Interface
Figure S3 Miniature electrochemical cell and insulator-coated electrode
(A) Miniature electrochemical cell. (B) Teflon-coated working electrode made of graphite fibre. Insulation of Teflon coating
made only the tip of electrode (*) conductive (700 μm2 ) inside the electrochemical cell.
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Authors Journal compilation Biochemical Society Transactions (2012) Volume 40, part 6
Figure S4 Attached bacteria respired at the poised electrode
Attached bacteria on uncoated graphite poised at + 700 mV compared
with Ag/AgCl were captured at × 20 light microscopy image (A)
followed immediately by a × 20 FITC image of the same location (B). By
adding Redox Sensor Green DyeTM , it was shown that the S. oneidensis
MR-1 cells, attached to the electrode surface, respired at an elevated
respiration rate relative to the surrounding cells (1.5 × 108 cells/ml).
C The
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Authors Journal compilation Electron Transfer at the Microbe–Mineral Interface
Table S1 Composition of media
(a) MR-1 minimal medium
Chemical description
Supplier and catalogue number
Final concentration
in medium (mM)
Pipes buffer
Sigma P-1851
50
Sodium hydroxide
Ammonium chloride
Potassium chloride
Sigma S-5881
Sigma A-5666
Sigma P-4504
7.5
28.04
1.34
Sodium phosphate monobasic, monohydrate
Vitamin solution, 100× stock
Amino acid solution, 100× stock
Sigma S-9638
See below
See below
4.35
Mineral solution, 100× stock
Sodium lactate, 60% (w/w) syrup
See below
Sigma L-1375
18
(b) Vitamin solution
Chemical description
Supplier and catalogue number
Final concentration
in medium (nM)
Biotin (d-biotin)
Folic acid
Sigma B-4639
Sigma F-7876
81.87
45.34
Pyridoxine HCl
Riboflavin
Thiamine HCl
Sigma P-9755
Sigma R-4500
Sigma T-4625
486.38
132.84
140.73
Nicotinic acid
d-Pantothenic acid, hemicalcium salt
Vitamin B12
Sigma N-4126
Sigma P-2250
Sigma V-2876
406.17
209.82
0.74
p-Aminobenzoic acid
Thioctic acid (α-lipoic acid)
Sigma A-9878
Sigma T-5625
364.62
242.37
Chemical description
Concentration
of 100× stock (g/l)
Supplier and
catalogue number
Final concentration
in medium (mg/l)
l-Glutamic acid
l-Arginine
2
2
Sigma G-1251
Sigma A-3909
2
2
dl-Serine
2
Sigma S-4375
2
(c) Amino acid solution
(d) Mineral solution
Chemical description
Supplier and catalogue number
Final concentration
in medium (μM)
Nitrilotriacetic acid (dissolve with NaOH to pH 8)
Sigma N-9877
Magnesium sulfate heptahydrate
Manganese sulfate monohydrate
Sodium chloride
Aldrich 23,039-1
Aldrich 22,128-7
Sigma S-3014
78.49
Ferrous sulfate heptahydrate
Calcium chloride dihydrate
Sigma F-8633
Sigma C-3881
3.60
6.80
Cobalt chloride hexahydrate
Zinc chloride
Cupric sulfate pentahydrate
Sigma C-3169
Sigma Z-3500
Sigma C-6283
4.20
9.54
0.40
Aluminium potassium disulfate dodecahydrate
Boric acid
Sodium molybdate dihydrate
Sigma A-7167
Sigma B-6768
Aldrich 22,184-8
0.21
1.62
1.03
Nickel chloride hexahydrate
Sodium tungstate
Sigma N-6136
Sigma S-0765
1.01
0.76
121.71
29.58
171.12
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Authors Journal compilation Biochemical Society Transactions (2012) Volume 40, part 6
Table S2 S. oneidensis MR-1 mutations involved with congregation near IEAs
Data were collected after bacteria and mineral were sealed in the capillary for 15 min. The swimming tracks within the same experiment were
sorted into two separate groups based on swimming path: those that contacted insoluble metal oxide surface (swam within 2 μm) compared with
those that did not contact. The pairs of letters highlight measurements that are significantly different (P < 0.05) from each other. These groups are
from the strains that are observed to successfully congregate around mineral after 30 min. Deletion mutants mcp_cache (SO_2240), mcp_pas
(SO_1385), cymA (SO_4591) and cheA-3 (SO_3207) were unable to congregate around Fe(OH)3 particles. Whereas mcp_pas (SO_1385)
congregated like wild-type around MnO2 , the strain was unable to congregate around Fe(OH)3 particles. This deficiency is reflected in the comparison
between contacting and non-contacting cells around Fe(OH)3 particles. Results are means ± 4 S.D.
(a) Fe(OH)3
Reversal frequency (reversals/s)
Speed (μm/s)
Strain
2 μm
2 μm
2 μm
2 μm
MR-1
Δmcp_cache
0.745 ± 0.50a
0.016 ± 0.05
0.216 ± 0.39a
0.207 ± 0.50
18.1 ± 5.4b
16.5 ± 5.4
12.6 ± 5.4b
15.0 ± 6.9
Δmcp_pas
ΔcymA
ΔcheA-3
0.043 ± 0.12
0.057 ± 0.15
0±0
0.258 ± 0.69
0.111 ± 0.23
0±0
46.2 ± 24.0
14.6 ± 7.0
0±0
53.0 ± 37.0
13.0 ± 4.9
12.7 ± 4.3
(b) MnO2
Reversal frequency (reversals/s)
Speed (μm/s)
Strain
2 μm
2 μm
2 μm
2 μm
MR-1
Δmcp_cache
Δmcp_pas
0.982 ± 0.60a
0.188 ± 0.20
0.918 ± 0.44c
0.318 ± 0.50a
0.208 ± 0.40
0.204 ± 0.33c
23.45 ± 7.0b
12.6 ± 9.0
21.33 ± 7.4d
19.4 ± 11.2b
14.1 ± 6.4
17.2 ± 5.6d
ΔcymA
ΔcheA-3
0.113 ± 0.21
0±0
0.109 ± 0.24
0±0
18.97 ± 7.3
29.13 ± 31.7
14.5 ± 3.9
30.6 ± 22.8
References
1 Crocker, J.C. and Grier, D.G. (1996) Methods of digital video microscopy for
colloidal studies. J. Colloid Interface Sci. 179, 298–310
2 Vaituzis, Z. and Doetsch, R.N. (1969) Motility tracks: technique for
quantitative study of bacterial movement. Appl. Microbiol. 17, 584–588
3 Harris, H.W., El-Naggar, M.Y., Bretschger, O., Ward, M.J., Romine, M.F.,
Obraztsova, A.Y. and Nealson, K.H. (2010) Electrokinesis is a microbial
behavior that requires extracellular electron transport. Proc. Natl. Acad.
Sci. U.S.A. 107, 326–331
4 Cornell, R.M. and Schwertmann, U. (1996), The Iron Oxides: Structures,
Properties, Reactions, Occurrence and Uses, Wiley VCH, Weinheim
5 Salas, E.C., Berelson, W.M., Hammond, D.E., Kampf, A.R. and Nealson, K.H.
(2010) The impact of bacterial strain on the products of dissimilatory iron
reduction. Geochim. Cosmochim. Acta 74, 574–583
6 Morgan, J.J. and Stumm, W. (1964) Colloid-chemical properties of
manganese dioxide. J. Colloid Sci. 19, 347–359
7 Bretschger, O., Obraztsova, A., Stumm, C.A., Chang, I.S., Gorby, Y.A., Reed,
S.B., Culley, D.E., Reardon, C.L., Barua, S., Romine, M.F. et al. (2007)
Current production and metal oxide reduction by Shewanella oneidensis
MR-1 wild type and mutants. Appl. Environ. Microbiol. 73, 7003–7012
Received 11 September 2012
doi:10.1042/BST20120232
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Authors Journal compilation