The E. coli CsgB nucleator of curli assembles to β-sheet
oligomers that alter the CsgA fibrillization mechanism
Qin Shua, Scott L. Cricka, Jerome S. Pinknerb, Bradley Fordb, Scott J. Hultgrenb, and Carl Friedena,1
aDepartment of Biochemistry and Molecular Biophysics, andbDepartment of Molecular Microbiology and Center for Women’s Infectious Disease Research,
Washington University School of Medicine, St. Louis, MO 63110
Contributed by Carl Frieden, March 16, 2012 (sent for review January 30, 2012)
Curli are extracellular proteinaceous functional amyloid aggre-
gates produced by Escherichia coli, Salmonella spp., and other en-
teric bacteria. Curli mediate host cell adhesion and invasion and
play a critical role in biofilm formation. Curli filaments consist of
CsgA, the major subunit, and CsgB, the minor subunit. In vitro,
purified CsgA and CsgB exhibit intrinsically disordered properties,
and both are capable of forming amyloid fibers similar in morphol-
ogy to those formed in vivo. However, in vivo, CsgA alone cannot
form curli fibers, and CsgB is required for filament growth. Thus,
we studied the aggregation of CsgA and CsgB both alone and
together in vitro to investigate the different roles of CsgA and
CsgB in curli formation. We found that though CsgA and CsgB
individually are able to self-associate to form aggregates/fibrils,
they do so using different mechanisms and with different kinetic
behavior. CsgB rapidly forms structured oligomers, whereas CsgA
aggregation is slower and appears to proceed through large amor-
phous aggregates before forming filaments. Substoichiometric
concentrations of CsgB induce a change in the mechanism of CsgA
aggregation from that of forming amorphous aggregates to that
of structured intermediates similar to those of CsgB alone. Oligo-
meric CsgB accelerated the aggregation of CsgA, in contrast to
monomeric CsgB, which had no effect. The structured β-strand
oligomers formed by CsgB serve as nucleators for CsgA aggrega-
tion. These results provide insights into the formation of curli
in vivo, especially the nucleator function of CsgB.
biofilm formation|circular dichroism|thioflavin T|
atomic force microscopy
nella, and many other Enterobacteriaceae (1–6). Curli can bind
a variety of host proteins, can mediate host cell adhesion and
invasion, and are involved in colonization and biofilm formation
(7–11). Bacterial biofilms constitute a protected mode of growth
against environmental stresses and immune defense, causing
high tolerance for antimicrobials, which results in persistent
Curli formation involves a complex molecular machinery that
is encoded by the divergently transcribed csgBA and csgDEFG
operons (7, 17). Curli fibers consist of two subunits: CsgA, which
constitutes the major portion of the fiber, and CsgB, the minor
component. Both CsgA and CsgB are secreted proteins with
similar molecular weights of ∼13 kDa, and their interaction
triggers wild-type curli formation in bacteria (5). The csgA gene
product (18) is a soluble, unstructured protein that requires the
presence of CsgB to assemble into fibers in vivo (4). In the ab-
sence of CsgB, CsgA is secreted from the cell as monomers, and
no fibrils are formed (4–6). The csgB gene product (19) is con-
sidered to be the nucleator for the assembly of curli (5). Unlike
CsgA, overexpressed CsgB can self-assemble into short polymers
on the bacterial surface when expressed alone (5). These data
suggest that CsgA and CsgB have different aggregation proper-
ties and play different roles in curli formation.
Previous studies have shown that CsgA and CsgB are highly
similar in terms of biochemical and biophysical properties; they
show high sequence homology, with about 50% similarity and
30% identity. The predicted structures of monomeric CsgA and
urli are highly aggregated, thin (2–6 nm diameter) amyloid
fibers expressed on the surface of Escherichia coli, Salmo-
CsgB are similar, consisting of five conserved, 18-residue tandem
strand-loop-strand motifs, each containing conserved glycine,
glutamine, and asparagine residues (7, 20, 21). In vitro, purified
CsgA is able to form fibers upon prolonged incubation that are
indistinguishable from wild-type curli as analyzed by EM (6).
CsgA aggregation shows a distinct lag, as detected by thioflavin T
(ThT) fluorescence changes, followed by a rapid increase in
fluorescence to a final plateau value (22). A C-terminal truncate
of CsgB, in which the C-terminal 19 amino acids have been re-
moved (CsgBtrunc), can also assemble into fibers in vitro that bind
to the amyloid-specific dyes Congo red and ThT (23). However,
CsgBtrunc is less efficient than full-length CsgB in mediating
CsgA nucleation in vivo (23). X-ray diffraction of fibrils formed
by purified CsgA and CsgB (WT, full length) showed the dis-
tinctive spacings (∼4.7 and ∼9 A), indicative of cross-β structure
that is commonly found in disease-associated amyloid (24). It
remains unclear how CsgA depends on the presence of the nu-
cleator protein CsgB to form functional amyloid fibrils in vivo.
In this study we compared the aggregation kinetics of both WT
CsgA and CsgB in vitro using multiple techniques. The results
reveal that CsgA and CsgB aggregate by different mechanisms
and suggest that the formation of curli fibers in vivo is regulated
by the ability of CsgB to rapidly form oligomeric structures that
alter the mechanism of aggregation of CsgA.
ThT Fluorescence Assay of Csg Aggregation. The aggregation of
CsgA and CsgBtrunchas previously been reported to show a lag
phase, as measured by ThT fluorescence change, followed by an
exponential rise in fluorescence to a final plateau value (22, 23,
25). Seeds made from CsgBtrunc(in which the C-terminal domain
has been truncated by 19 residues) can nucleate CsgA aggrega-
tion in vitro, but CsgBtruncis different from WT in nucleation of
curli formation in vivo (23). It is therefore important to examine
the properties of CsgB. Fig. 1A shows the time dependence for
CsgB aggregation as a function of CsgB concentration. Similar to
CsgBtrunc, CsgB exhibits a lag phase of ∼100 min, which is
shorter than that observed for CsgA (∼200 min) under the same
The effect of monomeric CsgB on the aggregation of CsgA was
investigated using the ThT assay. A 1:1 mixture of monomeric
CsgA and CsgB (4 μM each) was observed to have a lag phase
the lag phase typical of CsgA alone disappeared (Fig. 1B).
effect on the time course of the lag phase, suggesting that the
aggregation of CsgB was not affected by CsgA in the lag phase.
However, after 100 min, the presence of CsgB decreased the long
lag phase of CsgA. During this time, CsgB forms aggregates as
indicated by Fig. 1A. Seeds (oligomers) of CsgB abolished the lag
phase of CsgA and of CsgB (Fig. 2 A and B). The results suggest
that CsgB oligomers accelerate CsgA aggregation. Thus, the
Author contributions: Q.S., S.J.H., and C.F. designed research; Q.S., S.L.C., J.S.P., and B.F.
performed research; Q.S., B.F., S.J.H., and C.F. analyzed data; and Q.S., S.J.H., and C.F.
wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
| April 24, 2012
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absence of the CsgA lag phase when CsgA is mixed with CsgB in
Fig.1B is likely dueto theformation ofCsgBoligomersduring the
first 100 min that subsequently accelerate CsgA aggregation.
Circular Dichroism Changes. Far-UV CD changes were used to
follow the secondary structural changes of CsgA and CsgB dur-
ing aggregation (Fig. 3). Immediately after removal of Gdn and
initiating the aggregation process, the CD spectra of both CsgA
and CsgB are characteristic of a random coil with a minimum
close to 200 nm and little signal elsewhere. The two proteins,
however, show significant time-dependent CD differences at 216
nm, a wavelength that is characteristic for β-sheet formation
(Fig. 3 A and B). For CsgA during the first 24 h, the CD signal
exhibits little or no change. Over longer times, the CD signal at
200 nm decreases (from 2 d to 13 d) with only a slight change in
the signal at 216 nm (Fig. 3C). At very long times (9–13 d), the
CD spectrum of CsgA shows a weak minimum signal at ∼220 nm,
rather than at 216 nm, but probably characteristic of some β-sheet
structures (Fig. 3 C and D). The slow change of the CD signal at
200 nm after 1,000 min has a half-time of ∼4,800 min (Fig. 3E).
In contrast, the CD signal of CsgB rapidly increased at 200 nm
and decreased at 216 nm, indicating the appearance of β-sheet
structure (Fig. 3B). The CD change of CsgB at either 200 nm or
216 nm could be fit by a single exponential function with a half-
time of 180 min (Fig. 3E and Table 1).
Of interest is the comparison of CD and ThT over the first
1,000 min as shown in Fig. 4. For CsgB (Fig. 4A), the CD signal
change corresponds to the increase in ThT fluorescence, al-
though no lag phase is observed, which suggests that CsgB forms
aggregates with ordered β-sheet structure that can bind to ThT
and produce the characteristic ThT fluorescence. In contrast,
no correlation was observed between the CD signal change and
that monitored by ThT for CsgA (Fig. 4B). Although ThT fluo-
rescence increases for CsgA over time, there is no corresponding
change in CD, suggesting that at early times, CsgA forms
aggregates with little or no β-sheet structure. The difference be-
tween the behavior of CsgA and CsgB reflects a difference in the
mechanism of aggregation, which is discussed below.
Role of CsgB in Affecting CsgA Aggregation. Because CsgA and
CsgBalone showsignificantdifferencesin theCDsignalchange at
monitored by CD. Fig. 5 shows the CD change over incubation
time using concentrations of CsgA to CsgB at ratios of 20:1, 10:1,
and 5:1. In contrast to CsgA alone, the time-dependent spectra of
themixturesshowedapparentchangesat 216nm,characteristic of
β-sheet structure at all CsgA:CsgB ratios. At the concentrations
of CsgB used, from 0.39 to 1.12 μM, the CD216signal of all of the
mixtures was larger than that expected for CsgB alone (Table 1;
amplitudes). In these experiments the CD changes were fit by
a two-exponential function (Table 1). Interestingly, the rate con-
stant for the first phase is near that of CsgB alone (Table 1),
suggesting that this fast phase is mostly due to the rapid poly-
merization of CsgB. Thus, it is clear that CsgB rapidly forms
oligomers that change the aggregation mechanism of CsgA. Be-
cause CsgA alone shows little change at 216 nm, the large change
at 216 nm in the presence of substoichiometric amounts of CsgB
induced by oligomeric CsgB. Therefore, CsgB rapidly self-
assembles to oligomers/fibers with ordered β-sheet structure,
which serves as a nucleator for CsgA aggregates/fibers formation
with more-ordered β-sheet structures. At higher CsgB concen-
trations, the rate of the change at 216 nm increases, as would be
expected for more extensive CsgB aggregation. These results are
consistent with the in vivo observation that the formation of curli
requires CsgB serving as a nucleator (5).
Intrinsic Fluorescence Measurements. Changes in the intrinsic
fluorescence of a protein reflect changes in solvent accessibility
tored by ThT fluorescence. (A) The time course of CsgB (1–7 μM) aggregation
measured by fluorescence change at 482 nm. (B) Comparison of the time
course of ThT fluorescence change of CsgA (4 μM, dashed line), CsgB (4 μM,
solid line), and the mixture of CsgA and CsgB (4 μM each, circles). The asterisk
(*) and caret (^) highlight the differences in the lag phase of CsgB and CsgA,
respectively. Assays were performed using a plate reader. The standard con-
ditionsforthefibrillationswerein50mMKPO4(pH 7.2)at room temperature.
Aggregation/fibrillation of CsgB and its interaction with CsgA moni-
different seeds/oligomers made from preformed aggregates/fibers of either
CsgA or CsgB. Both CsgA and CsgB were 4 μM in 50 mM KPO4(pH 7.2) with or
without 1% seeds (by weight). Experiments were performed using a PTI
ThT fluorescence assay of (A) CsgA and (B) CsgB in the presence of
Shu et al. PNAS
| April 24, 2012
| vol. 109
| no. 17
of the side chains of aromatic amino acids. CsgA and CsgB differ
in tryptophan content. CsgA contains a single tryptophan
(W106), four tyrosines (Y26, 48, 50, and 151), and three phe-
nylalanines, whereas CsgB contains no tryptophan, six tyrosines
(Y24, 31, 88, 92, 109 and 129), and two phenylalanines.
For CsgA, using an excitation wavelength of 280 nm, the
protein exhibits two emission maxima: one at 303 nm due to the
four tyrosine residues and one at 350 nm due to the single
tryptophan (Fig. 6A). At an excitation wavelength of 295 nm,
only a single emission maximum, due to the tryptophan, is ob-
served (Fig. 6B). For CsgB, a single-emission maximum at 303
nm is observed using an excitation wavelength of 280 nm (Fig.
6A), whereas no fluorescence is observed when using an excita-
tion wavelength of 295 nm (Fig. 6B). Thus, using an excitation
wavelength of 280 nm, the fluorescence of the mixture (CsgA +
CsgB) at 350 nm is only due to CsgA, whereas that at 303 nm
measures both CsgA and CsgB (as shown in Fig. 6C). When
excited at 295 nm, the fluorescence of a CsgA/CsgB mixture
measures only that of CsgA, as shown in Fig. 6D.
The intrinsic fluorescence of CsgA alone, excited at 280 nm,
shows a small linear decrease over time, monitored by either
tyrosine or tryptophan signal (Fig. 6E), which is probably due to
the loss of soluble protein during the aggregation process.
However, the intrinsic fluorescence of CsgB alone, excited at 280
nm, shows an exponential decrease as a function of time (Fig.
6F) with a rate constant similar to the change in CD signal (Fig.
3E), confirming that the structure of CsgB rapidly changes with
a single exponential rate during its aggregation.
CD spectra of CsgA over 13 d. (D) CD spectrum of CsgA after 13 d, showing
a minimum at 220 nm. The time is shown in minutes (m), or hours (h). (E)
Comparison of CD signal change over time at 200 and 216 nm, for CsgA and
CsgB. All spectra were determined in a 0.1-cm path cell using 10 μM of
protein in 50 mM KPO4(pH 7.2). Similar CD behavior was observed with
a 1-cm cell using a protein concentration of 3 μM in 10 mM KPO4(pH 7.2).
Table 1 shows the kinetic parameters obtained from these data.
Far-UV CD spectra of (A) CsgA and (B) CsgB as a function of time. (C)
Table 1. Kinetic parameters of structural change during Csg protein aggregation
Protein CD, nmAmplitude1
−26.32 ± 0.29
8.71 ± 0.15
−1.47 ± 0.25
1.22 ± 0.42
−3.58 ± 0.49
1.70 ± 0.53
−4.98 ± 0.33
5.87 ± 1.72
0.0043 ± 0.00012
0.0042 ± 0.00018
0.0045 ± 0.0015
0.016 ± 0.01
0.002 ± 0.0006
0.0093 ± 0.0064
0.006 ± 0.00093
0.02 ± 0.0054
CsgA:CsgB = 20:1†
−12.53 ± 0.55
2.86 ± 0.55
−15.45 ± 2.82
2.78 ± 0.30
−11.92 ± 0.33
2.63 ± 0.15
0.00011 ± 0.000013
0.00012 ± 0.000048
0.00007 ± 0.00003
0.00021 ± 0.000077
0.00015 ± 0.000015
0.00051 ± 0.00008
CsgA:CsgB = 10:1‡
CsgA:CsgB = 5:1§
All experiments were carried out in 50 mM KPO4(pH 7.2). Amplitudes for CD are given as millidegrees (mdeg).
The parameters were obtained by fitting the CD data using a single-exponential function for CsgB, and a two-
exponential function for the mixture of CsgA and CsgB.
*The protein concentration of CsgB used was 10 μM.
†20:1 ratio of CsgA 7.8 μM to CsgB 0.39 μM.
‡10:1 ratio of CsgA 6.9 μM to CsgB 0.69 μM.
§5:1 ratio of CsgA 5.6 μM to CsgB 1.12 μM.
ing the aggregation of (A) CsgB and (B) CsgA. CsgA and CsgB were 4 μM for
the ThT assay and 10 μM for the CD measurements. Data obtained in 50 mM
KPO4(pH 7.2). For CsgB, the CD200was inverted from that shown in Fig. 3 for
comparison with the ThT change.
Comparison of normalized CD200and ThT fluorescence signals dur-
| www.pnas.org/cgi/doi/10.1073/pnas.1204161109 Shu et al.
The tyrosine fluorescence change [Fig. 6G; excitation (Ex) =
280 nm, emission (Em) = 303 nm] of the mixture of CsgA and
CsgB exhibits both the exponential decrease of CsgB and the
linear decrease of CsgA. The tryptophan fluorescence change of
the mixture is similar to that of CsgA with a linear decrease (Fig.
6G; Ex = 280 nm, Em = 350 nm). When excited at 295 nm (Fig.
6H), the mixture shows only the linear decrease seen for CsgA
alone. Thus, the intrinsic fluorescence results show that CsgB
undergoes a structural change either alone or when coincubated
Curli are examples of functional amyloid fibers that have been
observed in a wide variety of sources, including bacteria, fungi,
insects, invertebrate, and humans (6, 7, 26). Although Csg pro-
teins had been extensively studied in recent years, many ques-
tions remain, particularly with regard to the structures and
interactions that occur at early steps in the aggregation process
and lead to subsequent fibrillization.
CsgB Aggregation in Vitro. For CsgB, the CD signal at 216 nm
changes rapidly, as does its intrinsic fluorescence, suggesting
early formation of β-sheet structure. Fibril formation, as mea-
sured by ThT fluorescence change, is coincident with changes in
both CD200and CD216.
CsgA Aggregation in Vitro. The behavior of CsgA alone differs
dramatically from that of CsgB. Although CsgA forms visible
timeof∼200min,thereis nochangeintheCD signalat either200
or 216 nm. Instead, any CD signal change is very slow, occurring
over a period of days. Hence, any CsgA aggregate formed at early
times has little or no β-sheet structure. Atomic force microscopy
(AFM) data (Fig. 7) obtained after several days revealed amor-
phous aggregates of CsgA. Both normal fibers and amorphous
aggregates were found, as shown in Fig. 7A. Fibers could often be
seen growing from these amorphous aggregates (Fig. 7B).
CD data, we propose that CsgA does not undergo fibril formation
at early times of CsgA aggregation but rather forms amorphous
aggregates; thus, the ThT fluorescence at these times is a mea-
surement of the amorphous aggregates rather than fibrils. This
ratios. (A and B) 20:1 ratio of CsgA (7.8 μM) to CsgB (0.39 μM). (C and D) 10:1
ratio of CsgA (6.9 μM) to CsgB (0.69 μM). (E and F) 5:1 ratio of CsgA (5.6 μM)
to CsgB (1.12 μM). (A, C, and E) Comparison of CD spectra at different in-
cubation time for 3 d. (B, D, and F) Time course of CD signal change over 9 d
of incubation. The time is shown in minutes (m). Data obtained using
a 0.1-cm path cell in 50 mM KPO4(pH 7.2).
Far-UV CD change of the mixture of CsgA and CsgB at different
(A and B) Emission scans of CsgA alone and CsgB alone with the excitation
wavelength of (A) 280 nm and (B) 295 nm. (C and D) Emission scans of the
mixture of CsgA and CsgB with excitation at (C) 280 nm or (D) at 295 nm. (E–
H) The fluorescence intensity changes as a function of time. (E) CsgA alone
and (F) CsgB alone both using 280 nm excitation. (G and H) The mixture of
CsgA and CsgB excitation at (G) 280 nm and (H) 295 nm. The protein con-
centration for CsgA or CsgB alone was 1 μM, and 1 μM each for CsgA and
Intrinsic fluorescence change of CsgA and CsgB during aggregation.
Shu et al.PNAS
| April 24, 2012
| vol. 109
| no. 17
Also, when investigating the aggregation of β-lactoglobulin, Car-
rotta et al. (28) suggested that the structural motif recognized by
ThT can appear in protein aggregates. Some intrinsically disor-
dered proteins may initially form amorphous aggregates, and the
amyloid assemblies (fibers) emerge exclusively from within the
aggregates, as reported by others (29). Polymorphism of amyloid
has been widely observed (30, 31).
Over a long period (9–13 d), the CD spectrum of CsgA shows
a minimum ∼220 nm (Fig. 3 C and D), indicating CsgA even-
tually formed aggregates with β-strand–like structures. The slight
difference in the minimum of CD signal between CsgA (∼220
nm) and CsgB (∼216 nm) suggests that CsgA and CsgB aggre-
gates/fibers may differ somewhat structurally.
It has been previously noted that ThT fluorescence and CD
signal changes are not correlated for CsgA. For example, Wang
et al. (22) showed that the CD signal at 200 nm changed dra-
matically within 2 d, whereas the minimum at 220 nm increased
only slowly over a period of 2–15 d. In measurements over a period
of 6 h, Dueholm et al. (25) observed large CD changes at 197 nm
but little signal change at 216 nm, and that signal changes in CD
preceded those in ThT fluorescence. Differences in the kinetics of
aggregation between different studies may reflect differences in
protein preparation or experimental conditions.
CsgA Aggregation in the Presence of CsgB in Vitro. The aggregation
of CsgA in the presence of full-length CsgB has not previously
been reported. Our CD data show that, at substoichiometric
concentrations, CsgB changes the nature of the aggregation of
CsgA. Under conditions where both CsgA and CsgB are present,
CsgB rapidly self-assembles to oligomers with β-sheet structure,
which then serve as nucleators for CsgA aggregation. Our cur-
rent data, however, cannot determine the actual number of CsgB
monomers required to form a functional nucleator.
CsgA and CsgB Aggregation in Vivo. Wild-type curli are CsgA/CsgB
heteropolymers. The major subunit is CsgA, and CsgB is a minor
subunit. The fibers are ∼5–12 nm in diameter with irregular thin
branches. CsgA is unable to polymerize in the absence of CsgB;
thus, in the absence of either CsgA or CsgB, no surface struc-
tures are observed (5). However, when overexpressed in the
absence of CsgA, CsgB forms large quantities of cottony short
polymers lacking the filamentous structure of wild-type curli (5).
CsgB fused to maltose-binding protein also triggered the as-
sembly of curli that were distinctly curved and loosely aggre-
gated, 10–15 nm thick (5). These morphological differences can
be explained by the differences in structure and polymerization
kinetics that we observed between CsgA and CsgB. The differ-
ences in the length of the fibers should relate to the aggregation
rate and the relative concentration of subunits available. The fast
aggregation rate of CsgB limits the diffusion region of the mol-
ecules and results in shorter polymers, whereas CsgA with
a slower rate can form longer fibers. Moreover, different struc-
tural properties of the oligomers affect the packing mode of the
fibers, resulting in differences in shape and thickness.
amounts than CsgB. Our data show that CsgB rapidly forms
aggregates with β-sheet structure. These structures alter the for-
mation of CsgA aggregates. In vivo and in the absence of CsgB,
CsgA monomers diffuse away from the surface of the cells, which
we propose is a consequence of two factors: the relatively slow
aggregation of CsgA and the fact that CsgA intermediates are not
structured. We have shown that CsgA aggregation is accelerated
and that the aggregates formed are structurally changed in the
presence of CsgB. Thus, our data argue for a mechanism whereby
CsgB rapidly assembles to aggregates/fibers that nucleate the
formation of CsgA fibrils. Due to the rapid CsgB aggregation ki-
netics, CsgB is capable of forming short polymers condensed on
the cell surface when overexpressed, as described in a previous
report (5). In wild-type E. coli, the stoichiometry of CsgA to CsgB
precludes CsgB fiber formation. In sum, our data argue that the
formation of curli in vivo uses the different aggregation kinetics
and structural differences of CsgA and CsgB to form curli fibers.
Curli proteins CsgA and CsgB share many biochemical and
structural properties with disease-associated amyloids (6, 22, 23).
Wang et al. (22) observed that CsgA formed transient inter-
mediates that bind to A11, an antibody that specifically recog-
nizes intermediate oligomers, but not soluble monomers or
mature amyloid fibers of many disease-associated amyloids, such
as Aβ, islet amyloid polypeptide, polyglutamine, prion peptide,
and Sup35p (32–34). Therefore, curli biogenesis may provide an
excellent model for understanding the polymerization mecha-
nism of disease-associated amyloids. The structural and kinetic
mechanism observed in Csg proteins also provides insights into
the aggregation/fibrillization mechanism of other amyloids.
Curli biogenesis is a directed amyloid aggregation process re-
quiring specific molecular machinery to not only secrete subunits
to the extracellular surface but to also ensure that amyloid fibril
paper, we explored the aggregation of CsgA and CsgB both alone
andtogetherinvitro. WefoundthatCsgA andCsgBself-associate
to form aggregates/fibrils with different aggregation kinetics and
structural properties. For CsgA, the mechanism appears to be the
formation of amorphous aggregates from which fibrils appear af-
common path, with fibrils occurring early in the aggregation pro-
cess. Substoichiometric concentrations of CsgB change the ag-
gregation mechanism of CsgA to that characteristic of CsgB. Our
results indicate that the formation of wild-type curli requires
a combination of both CsgA and CsgB, with CsgB directing the
protein was 10 μM in 50 mM KPO4(pH 7.2) and incubated at 4 °C for 24 d
before imaging. The boxed region in A is shown magnified in B. (Scale bars:
A, 1 μm; B, 0.1 μM.)
Representative AFM tapping mode amplitude image of CsgA. The
| www.pnas.org/cgi/doi/10.1073/pnas.1204161109 Shu et al.
Materials and Methods Download full-text
Protein Expression and Purification. Wild-type CsgA and CsgB with 6 His-tag at
the C-terminal end (designated here as CsgA and CsgB) were cloned into
pET11d (New England Biolabs) and overexpressed in E. coli NDH471
(NEB3016 SlyD::aph). Both proteins were expressed in inclusion bodies. The
inclusion bodies (∼1 g) were suspended in 50-mL 8 M guanidine hydro-
chloride (Gdn), 50 mM KPO4(pH 8), stirred for 1 d, and then centrifuged. The
supernatant solution in 8 M Gdn was then passed through a 0.45-uM bottle-
top filter (Corning) and loaded onto a column packed with TALON metal
affinity resin (Clonetech). The protein was eluted with 200 mM imidazole in
6 M Gdn, 50 mM KPO4(pH 7.2) and stored at 4 °C. Immediately before
spectroscopy or other experiments, the protein was desalted and changed to
buffer of 10 or 50 mM KPO4(pH 7.2) using a Sephadex G25 Desalt column
(HiTrap; GE Healthcare). The start time of protein aggregation was consid-
ered to begin from the time that Gdn was removed. In the experiments
using a mixture of CsgA and CsgB, CsgA and CsgB were refolded for 5–15
min after removal of Gdn. The start time of the aggregation of the mixture
was considered to be the time that Gdn was removed (15 min).
Preparation of Csg Protein Seeds. Following removal of Gdn, CsgA or CsgB
(5–20 μM) was incubated in 50 mM KPO4(pH 7.2) in a glass tube at 4 °C. Csg
protein seeds were made from the samples incubated >30 d. The aggre-
gates/fibers were centrifuged, resuspended in water (1 mg/mL), and soni-
cated for 30 s. The visible aggregates/fibers in the samples disappeared and
the solution turned milky. This material was used as seeds at a 1:100 dilution.
ThT Assay. Aggregation was followed by the change in fluorescence of ThT
(35). When using the PTI fluorometer (Photon Technology International), the
excitation wavelength was 438 nm and the fluorescence emission was
recorded (from 450 to 550 nm) every 10 min after mixing the sample. The
measurements were made in 50 mM KPO4(pH 7.2) containing 25 μM ThT in
a total volume of 3 mL at 25 °C. When using the Tecan Infinite 200 plate
reader with the ThT assay, proteins were mixed with 25 μM ThT in a total of
125 μL in 96-well plates in duplicate and incubated at room temperature.
Samples were measured every 10 min using an excitation wavelength of 438
nm and emission >495 nm, using a 475-nm cutoff filter. Samples were
shaken for 10 s (Orbital shaker; amplitude 2 mm) before each reading.
Varying concentrations of protein (2–20 μM) were used in this study. Signal
intensities are in arbitrary units and differed between data collected on the
PTI fluorometer and the plate reader. All ThT assays were repeated 3×, and
representative data are shown. The lag phase for aggregation of CsgA was
similar to that of previous reports (22, 25). We observed, however, that at
high protein concentrations the solution turned cloudy at the end of the lag
phase, and the fluorescence signal became noisy, especially at protein con-
centrations >4 μM. Thus, to minimize this problem, a protein concentration
∼4 μM was used in most ThT assays.
Circular Dichroism. Far-UV CD spectra were recorded on a Jasco-J715 spec-
tropolarimeter with a 1-cm or 0.1-cm path cell, depending on the protein
concentration. Spectra were recorded at 25 °C as an average of 5 or 10 scans
from 190 to 240 nm. The scan rate was 50 nm/min with a response time of
1 s. All samples were mixed before every reading. Different CD experiments
were repeated with varying protein concentrations (2–15 μM).
Fluorescence Spectroscopy. The intrinsic fluorescence emission of CsgA and
CsgB were measured on a PTI fluorometer (Photon Technology International)
using an excitation wavelength of 280 or 295 nm at 25 °C. The protein
concentration was 1 μM in 50 mM KPO4(pH 7.2). Because CsgB does not
contain tryptophan, an excitation wavelength of 280 nm was used.
μM protein in 50 mM KPO4(pH 7.2) at 4 °C were diluted in 1:20 in Milli-Q
H2O and placed on a mica disk to adhere for 1 min. The sample was removed
by pipetting, and the mica was gently rinsed with Milli-Q H2O twice and
dried under a nitrogen stream. AFM images were acquired by tapping mode
using the Asylum Research MFP-3D-BIO AFM.
ACKNOWLEDGMENTS. We thank Dr. Ashley A. Nenninger for providing
constructs, Dr. Warren Lewis for helping with the ThT assay, and members of
the C.F. and S.J.H. laboratories for their helpful discussions and review of this
manuscript. This work was supported by National Institutes of Health Grant
AI48689 (to S.J.H.).
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Shu et al.PNAS
| April 24, 2012
| vol. 109
| no. 17