Possible Nonconductive Role of Geobacter sulfurreducens Pilus Nanowires in Biofilm Formation
Geobacter sulfurreducens required expression of electrically conductive pili to form biofilms on Fe(III) oxide surfaces, but pili were also essential for biofilm development on plain glass when fumarate was the sole electron acceptor. Furthermore, pili were needed for cell aggregation in agglutination studies. These results suggest that the pili of G. sulfurreducens also have a structural role in biofilm formation.
JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 2125–2127 Vol. 189, No. 5
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Possible Nonconductive Role of Geobacter sulfurreducens Pilus
Nanowires in Bioﬁlm Formation
Gemma Reguera,* Rachael B. Pollina,† Julie S. Nicoll,‡ and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
Received 14 August 2006/Accepted 29 November 2006
Geobacter sulfurreducens required expression of electrically conductive pili to form bioﬁlms on Fe(III) oxide
surfaces, but pili were also essential for bioﬁlm development on plain glass when fumarate was the sole electron
acceptor. Furthermore, pili were needed for cell aggregation in agglutination studies. These results suggest that
the pili of G. sulfurreducens also have a structural role in bioﬁlm formation.
One of the hallmarks of Geobacter species is their ability to
conserve energy from the transfer of electrons to a variety of
extracellular electron acceptors, such as metals [Fe(III),
Mn(IV), and U(VI)], humic acids, and electrodes (5, 6). Es-
tablishing an electrical connection with an extracellular elec-
tron acceptor poses challenges not faced by microorganisms
that reduce soluble electron acceptors within the cell. In con-
trast to other Fe(III) oxide-reducing bacteria (4, 9–11), such as
Shewanella and Geothrix species, Geobacter species do not ex-
crete electron shuttles (8) and require direct contact with the
electron-accepting surface (1, 10). Previous studies (14) have
demonstrated that the pili of Geobacter sulfurreducens are con-
ductive and that expression of pili is required for growth on
Fe(III) oxides. These “microbial nanowires” are not required
for attachment to the insoluble electron acceptor; rather, they
function as electronic conduits to transfer electrons to the
Fe(III) oxides, extending the electron transfer capabilities of
the cell well beyond the outer surface (14). Pilus “nanowires”
also serve as electric conduits to mediate long-range electron
transfer across multilayer bioﬁlms formed on anode elec-
trodes, which is required to maximize current production per
unit of anode surface area (15).
Bioﬁlms on Fe(III) oxide. When G. sulfurreducens (2) was
grown under strictly anaerobic conditions at 30°C in freshwater
medium (7) with acetate (15 mM) as an electron donor and
with Fe(III) oxide coatings [prepared on borosilicate coverslips
(17) and providing 4.3 ⫾ 0.7 mol of Fe(III) per coverslip
(mean ⫾ standard deviation; n ⫽ 3)] as the sole electron
acceptor, a bioﬁlm grew on the Fe(III) coating (measured with
a crystal violet assay ), but planktonic growth was not
supported (Fig. 1A). Viability staining with a BacLight viability
kit (Molecular Probes) and confocal scanning laser microscopy
(CSLM) analyses (14) of 48-h bioﬁlms revealed a structured
bioﬁlm composed of cell clusters approximately 18 ⫾ 1 m
high (Fig. 1B). Control coverslips without the Fe(III) oxide
coatings did not support bioﬁlm growth (Fig. 1C), suggesting
that bioﬁlm growth was not supported by any nutrient carried
over in the inoculum. Viability staining suggested that even
cells at a substantial distance from the Fe(III) oxide surface
remained metabolically active (Fig. 1B). This may be attrib-
uted to long-range electron transfer via the electrically con-
ductive pili, as previously proposed for long-range transfer to
the anode surface of microbial fuel cells (15). In contrast, a
previously described (14) mutant in which the gene coding for
PilA, the pilin structural subunit, was deleted grew poorly on
the Fe(III) oxide coatings (Fig. 1D) and produced 10-fold less
biomass than the wild type produced after 72 h (Fig. 2), and
complementation of the mutation in trans (14) restored the
bioﬁlm phenotype (data not shown). These ﬁndings are con-
sistent with the previous ﬁnding that pili are required for
growth on Fe(III) oxide (14).
Bioﬁlm formation when electron transfer to the Fe(III) ox-
ide surface is not required. Even though pili are not required
for growth with fumarate as an electron acceptor (14), addition
of fumarate (40 mM) to cultures with Fe(III) oxide-coated
coverslips, while having little impact on the bioﬁlm biomass of
the wild type, increased the mutant bioﬁlm biomass to approx-
imately one-half of the wild-type biomass (Fig. 2). More wild-
type biomass accumulated on glass coverslips when fumarate
was provided as the electron acceptor, but the biomass of the
pilin-deﬁcient mutant bioﬁlms remained approximately one-
half that of the wild-type bioﬁlms (Fig. 2). These results dem-
onstrated that pili are required for optimal bioﬁlm develop-
ment even when the surface is not the electron acceptor.
This conclusion was consistent with CLSM images of the
fumarate-grown bioﬁlms (Fig. 3). In the presence of fumarate,
wild-type cells formed pillars that were 19 ⫾ 1.5 and 22 ⫾ 0.5
m high on Fe(III) oxide-coated surfaces and glass surfaces,
respectively. In contrast, the maximum bioﬁlm heights for the
pilin-deﬁcient mutant were 7.6 ⫾ 1.5 and 8.5 ⫾ 1.4 monthe
same surfaces. This difference was not apparent in the ﬁrst
24 h, when both the mutant and wild-type bioﬁlms formed
short microcolonies. However, the wild-type microcolonies
continued to grow, and both the height and width of the col-
onies increased to form mature bioﬁlms (Fig. 3). Viability
staining indicated that cells in all the layers of the wild-type
and mutant bioﬁlms were alive, suggesting that fumarate dif-
* Corresponding author. Present address: Department of Microbi-
ology and Molecular Genetics, 2215 Biomedical Physical Sciences,
Michigan State University, East Lansing, MI 48824-4320. Phone: (517)
355-6463. Fax: (517) 353-8957. E-mail: firstname.lastname@example.org.
† Present address: Infectious Diseases Department, The Mount
Sinai School of Medicine, New York, NY 10029.
‡ Present address: Center for Adaptation Genetics and Drug Resis-
tance, Department of Molecular Biology and Microbiology, Tufts Uni-
versity School of Medicine, Boston, MA 02111.
Published ahead of print on 8 December 2006.
fusion across the bioﬁlms was not a limiting factor, as previ-
ously reported for other bacterial bioﬁlms (16). These results
indicate that the pili of G. sulfurreducens play a role in the
development of the highly structured bioﬁlms of G. sulfurre-
ducens that is unlikely to be related to the electrical conduc-
tivity of the pili.
Geobacter pili promote autoagglutination. Pili in various
bacteria mediate twitching motility during bioﬁlm formation
(12). Other pili, such as the toxin-coregulated pili (TCP) of
Vibrio cholerae (13), and also the pili of G. sulfurreducens (14)
do not appear to be involved in motility. Rather, TCP are a
structural bioﬁlm component that mediate cell interactions
leading to microcolony development during colonization of the
human intestine (3) or during bioﬁlm formation on chitin sur-
faces (13). The ability of TCP to promote bacterial interactions
also enables TCP-expressing cells to autoagglutinate in vitro
(3). Similar agglutination studies were carried out with G.
sulfurreducens by growing cells with fumarate as the electron
acceptor at 25°C to induce pilus formation (14). The degree of
agglutination was assayed by measuring the optical density at
600 nm of the cells that remained in suspension and subtract-
ing the value obtained from the optical density of the culture
after disruption of the aggregates with agitation. After 72 h of
growth the wild-type strain formed large aggregates that set-
tled at the bottom of the culture vessel (Fig. 4). There was no
autoagglutination at 30°C, a temperature at which planktonic
cells do not express pili (14). The mutant in which pilA was
deleted did not agglutinate at 25°C (Fig. 4). Complementation
of the mutation with a wild-type copy of the pilA gene ex-
pressed in trans produced a strain that agglutinated at levels
FIG. 1. Bioﬁlm formation on Fe(III) oxide coatings by G. sulfurreducens. (A) When provided as a sole electron acceptor for growth, the Fe(III)
oxide coating supported the growth of a bioﬁlm, whose biomass increased steadily during the ﬁrst 72 h, but did not support planktonic growth.
, optical density at 600 nm. (B to D) Top view (at a 15
angle) (top panels) and side view (bottom panels) projections generated by CSLM
of 48-h wild-type (B) or pilin-deﬁcient mutant (D) bioﬁlms formed on an Fe(III) oxide-coated surface or of a wild-type bioﬁlm on a control
coverslip without the Fe(III) oxide coating (C). Green indicates live cells, and red indicates dead cells. Yellow regions are areas where the two dyes
overlap. The substratum (coverslip) is at the bottom. Bars, 20 m.
FIG. 2. Average biomasses of mature wild-type (WT) and pilin-
deﬁcient mutant (PilA⫺) bioﬁlms formed on Fe(III) oxide coatings
[Fe(III)] or glass surfaces. Where indicated, the soluble electron ac-
ceptor fumarate also was present in the growth medium. The results
are averages for triplicate samples from two independent experiments.
, optical density at 600 nm.
2126 NOTES J. B
that were much higher than the levels observed for the wild-
type strain (Fig. 4), consistent with the fact that genetic
complementation leads to overproduction of pili (14). These
results suggest that the pili of G. sulfurreducens participate in
cell-cell aggregation necessary for the development of micro-
colonies during bioﬁlm differentiation.
Implications. The results presented here demonstrate that
in addition to serving as electric conduits for electron transfer
to Fe(III) oxides (14) and long-range electron transfer across
anode bioﬁlms in G. sulfurreducens fuel cells (15), the G. sul-
furreducens pili also are required for maximum bioﬁlm growth
even when electron transfer to an electron-accepting surface is
not required. This is an important consideration because the
overall rate of electron transfer to an electron-accepting sur-
face is dependent upon the number of metabolically active
cells that can stack on the surface. Thus, high rates of electron
transfer to an electron-accepting surface require not only the
electronic capabilities of the pili but also their structural at-
tributes that permit cells to stack at high densities on a given
surface. These considerations make it clear that further eval-
uation of the contributions of pili and other outer cell compo-
nents to the bioﬁlm structure is essential in order to better
understand, and perhaps optimize, electron transfer to elec-
This research was supported by grants DE-FG02-02ER63423 and
DE-FC02-02ER63446 from the Ofﬁce of Science (BER), U.S. Depart-
ment of Energy, and by award N00014-03-1-0405 from the Ofﬁce of
Naval Research. G.R. acknowledges support provided by a postdoc-
toral fellowship from the Ministerio de Educacio´n y Ciencia of Spain
and by the European Social Fund.
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FIG. 4. Autoagglutination phenotypes of a wild-type strain (WT), a
pilus-deﬁcient mutant (PilA
), and a genetically complemented mu
tant (pRG5-pilA) grown under pilus-inducing conditions (25°C). The
results are the averages for triplicate samples from two independent
experiments. A600, absorbance at 600 nm.
FIG. 3. CSLM analyses of wild-type (A and C) and PilA
D) bioﬁlms formed on Fe(III) oxide coatings (A and B) or glass
coverslips (C and D) in medium with fumarate. Green indicates live
cells, and red indicates dead cells. Yellow indicates dye overlap. The
images are three-dimensional top views (top panels) and side views
(bottom panels) reconstructed from the ﬂuorescence patterns of the
series of two-dimensional optical sections collected by CSLM. Bars,
VOL. 189, 2007 NOTES 2127