Intramolecular amide bonds stabilize pili on the surface of bacilli.
ABSTRACT Gram-positive bacteria elaborate pili and do so without the participation of folding chaperones or disulfide bond catalysts. Sortases, enzymes that cut pilin precursors, form covalent bonds that link pilin subunits and assemble pili on the bacterial surface. We determined the x-ray structure of BcpA, the major pilin subunit of Bacillus cereus. The BcpA precursor encompasses 2 Ig folds (CNA(2) and CNA(3)) and one jelly-roll domain (XNA) each of which synthesizes a single intramolecular amide bond. A fourth amide bond, derived from the Ig fold of CNA(1), is formed only after pilin subunits have been incorporated into pili. We report that the domains of pilin precursors have evolved to synthesize a discrete sequence of intramolecular amide bonds, thereby conferring structural stability and protease resistance to pili.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: 1. T antigens of group A hemolytic streptococci have been obtained in soluble form by digestion of the bacterial cells with pepsin or trypsin. Large quantities of this antigen were readily extracted from type 1 strains, whereas only small amounts could be obtained from strains of other types. 2. The T antigen, prepared in this way from a type 1 strain, was partially purified by chemical precipitation and further enzymatic digestion. An active fraction, apparently protein in nature, was separated electrophoretically at pH 7.00. The separated material, pooled and analyzed at the same pH, gave only a single peak. The isoelectric point of this substance was about pH 4.50. An elementary analysis was obtained. Although the T antigen was resistant to digestion with proteolytic enzymes and ribonuclease, it was readily inactivated by heat, especially in acid media and in strong salt solutions. The serological activity of this purified T substance was lost after exposure to ultraviolet radiation. 3. Analysis by means of the ultracentrifuge showed that the material was polydisperse and therefore probably impure. 4. The soluble form of the T substance was active in the precipitin reaction, in the fixation of complement, in inhibition of T agglutination, and as an antigen when injected into rabbits. The antibodies produced did not protect mice against infection with virulent strains of hemolytic streptococci containing the same T antigen. 5. The immunological specificity of T antigen in soluble form is the same as that of the T antigen in the intact streptococcus from which it was derived.Journal of Experimental Medicine 10/1946; 84(5):449-71. · 13.21 Impact Factor
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ABSTRACT: PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.Cell 07/2001; 105(6):733-43. · 31.96 Impact Factor
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ABSTRACT: The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.Biochimica et Biophysica Acta 12/2004; 1694(1-3):311-27. · 4.66 Impact Factor
Intramolecular amide bonds stabilize pili
on the surface of bacilli
Jonathan M. Budzika,1, Catherine B. Poorb,1, Kym F. Faullc, Julian P. Whiteleggec, Chuan Heb,2, and Olaf Schneewinda,2
aDepartments of Microbiology andbChemistry, University of Chicago, Chicago, IL 60637; andcPasarow Mass Spectrometry Laboratory, Neuropsychiatric
Institute, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, CA 90095
Communicated by Robert Haselkorn, University of Chicago, Chicago, IL, September 28, 2009 (received for review July 29, 2009)
Gram-positive bacteria elaborate pili and do so without the par-
ticipation of folding chaperones or disulfide bond catalysts. Sor-
tases, enzymes that cut pilin precursors, form covalent bonds that
link pilin subunits and assemble pili on the bacterial surface. We
determined the x-ray structure of BcpA, the major pilin subunit of
Bacillus cereus. The BcpA precursor encompasses 2 Ig folds (CNA2
and CNA3) and one jelly-roll domain (XNA) each of which synthe-
sizes a single intramolecular amide bond. A fourth amide bond,
derived from the Ig fold of CNA1, is formed only after pilin subunits
have been incorporated into pili. We report that the domains of
pilin precursors have evolved to synthesize a discrete sequence of
intramolecular amide bonds, thereby conferring structural stability
and protease resistance to pili.
CNA B domain ? jelly roll domain ? protease resistance ? sortase
or escape innate immune responses (1). In contrast to eukaryotic
cells and Gram-negative microbes, Gram-positive bacteria do not
Gram-positive bacteria, signal peptide carrying precursors of sur-
face proteins are secreted via the Sec pathway and require C-
terminal sorting signals for their incorporation into the envelope
(1). Sortases function as transpeptidases that cleave precursor
(3). For sortase A, B, or C, these intermediates are resolved by the
nucleophilic attack of the amino group of peptidoglycan cross-
cell wall envelope (1). In Bacillus cereus, a pilin-specific sortase
the YPKN pilin motif of the major pilus subunit, BcpA, as a
nucleophile (4). The product of this reaction is an intermolecular
D or A (5). The latter reaction prevents the incorporation of
additional pilin subunits and promotes anchoring of BcpA pili to
peptidoglycan crossbridges. BcpA pili contain a minor pilin, BcpB,
which is positioned at the tip of each fiber (6). Analogously to the
tip proteins of other pili, BcpB is thought to function as an adhesin
for host tissues (7, 8). The sorting signal of BcpB is cleaved by
sortase D, but not by sortase A. As the resulting acyl intermediate
between BcpB and sortase D can only be resolved by the nucleo-
philic attack of the YPKN motif, the minor subunit is deposited at
the tip of BcpA pili (6).
In previous studies, we engineered BcpA variants with strategi-
cally placed polyhistidine (His6) affinity tags and methionine (Met)
residues that were assembled into pili. Cyanogen bromide (CNBr)
treatment of purified pili generated homoserine-lactone breaks at
spectrometry and Edman degradation, thereby revealing the pres-
ence of 2 amide bonds, Lys162-Thr522, the sortase D-derived inter-
molecular linkage, as well as Lys37-Asn163, an intramolecular amide
he cell wall envelope of Gram-positive bacteria can be thought
of as a surface organelle that encompasses anchored proteins
bond positioned in the first CNA-B domain of BcpA (designated
CNA1) (4). Purification and mass spectrometry of the recombinant
major pilin, rBcpA, which lacks the N-terminal signal peptide and
C-terminal sorting signal, suggested the presence of intramolecular
Nevertheless, the contributions of intramolecular bonds toward
pilus structure and function have not yet been revealed.
Here we report the 3D x-ray structure of BcpA. The pilin
precursor encompasses 3 reverse-Ig (Ig) fold domains (CNA1–3)
and one jelly-roll domain (designated XNA). CNA2, CNA3and
only in mature, assembled pili. Informed by these results, we
examined the contribution of intramolecular amide bonds toward
pilus structure and function.
Intramolecular Amide Bonds in the BcpA Precursor. Recombinant
rBcpAHis-6, lacking the N-terminal signal peptide and the C-
terminal LPVTG sorting signal of the pilin precursor, was purified
by affinity chromatography from cleared lysates of Escherichia coli
(SI Appendix, Fig. S1). The predicted mass of the primary rBcpA-
His-6translational product is 56,697.27 (SI Appendix, Table S1). We
observed an average mass of 56,648.60, a measurement that is in
agreement with the formation of 3 intramolecular amide bonds,
each resulting in the loss of 17 Da (NH3) (SI Appendix, Table S1).
Analysis of the amino acid sequence of BcpA identified 3 CNA-B
domains (Pfam05738), Ig-like structures proposed to form a single
intramolecular amide bond (9). Each of the 3 CNA-B domains of
rBcpAHis-6is predicted to position a glutamic acid residue in close
proximity to lysine and asparagine residues; the glutamic acid is
thought to enable the formation of an intramolecular amide bond
between the side chains of lysine-asparagine (9). As expected,
alanine substitution of the conserved glutamic acid within CNA2
(Glu223Ala) or CNA3(Glu472Ala) abrogated the formation of the
corresponding amide bonds, as the average mass of each variant
rBcpAHis-6increased by 17 Da (4) (SI Appendix, Table S1). Simi-
larly, alanine substitution of residues engaged in the intramolecular
amide (Lys174or Asn256for CNA2, as well as Lys417or Asn512for
CNA3) abolished the formation of the corresponding amide bond;
these rBcpAHis-6mutants displayed only a mass difference of 34 Da
as compared to the predicted average mass of their primary
substitutions of Lys37or Asn163in CNA1did not affect the average
mass of purified rBcpAHis-6. Thus, although Lys37and Asn163
Author contributions: J.M.B., C.B.P., K.F.F., J.P.W., C.H., and O.S. designed research; J.M.B.,
C.B.P., K.F.F., and J.P.W. performed research; J.M.B., C.B.P., K.F.F., J.P.W., C.H., and O.S.
contributed new reagents/analytic tools; J.M.B., C.B.P., K.F.F., J.P.W., C.H., and O.S. ana-
lyzed data; and J.M.B., C.B.P., C.H., and O.S. wrote the paper.
The authors declare no conflict of interest.
1J.M.B. and C.B.P. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
November 24, 2009 ?
vol. 106 ?
engage in an intramolecular amide bond immediately adjacent to
the sortase D-derived intermolecular link of assembled pili (4), this
bond is not formed in rBcpA.
The CNA1 Domain of the BcpA Precursor Is Sensitive to Protease.
Purified rBcpAHis-6failed to produce diffracting crystals and was
subjected to limited protease cleavage. Treatment with trypsin
generated a protease resistant product, rBcpA*, that migrated
farther on Coomassie-stained SDS/PAGE than rBcpAHis-6without
protease treatment (SI Appendix, Fig. S1). Edman degradation
identified Asn163, of the YPKN pilin motif, as the N-terminal
residue of BcpA*, as all subsequently released residues agreed with
the predicted downstream sequence of BcpA (SI Appendix, Table
S2). Edman degradation released a single amino acid per sequenc-
ing cycle, confirming that the CNA1 domain within rBcpAHis-6,
unlike its counterpart within assembled pili (vide infra), lacks the
amide bond between Lys37and Asn163. BcpA*, when examined by
FTMS), generated an observed average mass of 40,377.2 (SI
Appendix, Fig. S1). The predicted average mass of BcpA* without
intramolecular amide bonds is 40,427.96. These data suggest that
BcpA* spans the remaining length of rBcpAHis-6 beginning at
Asn163and forms 3 intramolecular amide bonds. BcpA* was
expressed as a fusion to the C-terminal end of GST (GST, pJB171)
(SI Appendix, Fig. S1). GST-BcpA* was isolated by affinity chro-
matography and the GST tag removed with thrombin and BcpA*
average mass of 39,097.48 was observed for BcpA*. The predicted
presence of 3 intramolecular amide bonds (SI Appendix, Table S1).
Crystal Structure of BcpA*.BcpA*wascrystallized(SIAppendix,Fig.
S1) and its structure solved using multiple-wavelength anomalous
dispersion (MAD). BcpA* has an elongated, 3-domain structure
structures (Fig. 1A). CNA2and CNA3possess a reverse Ig-fold,
comprised of one 4-stranded ?-sheet and one 3-stranded ?-sheet
(Fig. 1B). XNA assumes a jelly-roll structure with 9 ?-strands (Fig.
1C). A single intramolecular amide bond was identified in CNA2
and CNA3(Fig. 1A). Continuous electron density demonstrated
the existence of amide bonds between the ?-amino group of lysine
and the carboxamide group of asparagine, i.e., Lys174-Asn265in
acid (Glu223or Glu472) is positioned in hydrogen bonding distance
of the amide bond.
A previously unrecognized amide bond between Lys273-Asn383
was identified in XNA (Fig. 1C and SI Appendix, Fig. S2). In
contrast to CNA2 and CNA3, where amide bond formation is
thought to be catalyzed by the side chain of glutamic acid, Asp312
is positioned in close proximity to Lys273and Asn383(SI Appendix,
Fig. S2). The side chain carboxylic acid of aspartic acid forms
of the intramolecular amide bond (SI Appendix, Fig. S2).
In the folded structure of BcpA*, the amide bonds of CNA2and
CNA3are located toward the C-terminal end of each domain, near
the interdomain connections between CNA2and XNA as well as
S2). The CNA2/CNA3amide bonds link the first and last ?-strands
of each domain, with participating lysine and asparagine residues
Appendix, Fig. S2). The XNA structure includes a ?-sandwich and
the intramolecular amide bond covalently joins the first and pen-
ultimate strands of the domain. The ?-strands on which the lysine
and asparagine residues reside assume antiparallel polarity, are
separated by one ?-strand, and are located on opposite sides of the
as the core structural unit of the XNA domain.
In mature polymerized BcpA, an intermolecular amide bond
between Lys162of the YPKN motif, positioned immediately adja-
cent to the N-terminal Asn163of BcpA*, is linked end-to-end with
the carboxyl group of Thr522, which is located 7 residues down-
stream of the C-terminal Ser515in Fig. 1A. Both termini of BcpA*
extend onto the surface of the structure. We presume that these
parts of the pilin protein are reorganized during pilus assembly via
the sortase D-catalyzed linkage as well as the folding of the CNA1
Catalytic Requirements for the Intramolecular Amide Bonds of BcpA.
rBcpAHis-6(primary translation product mass 56,697.27; observed
mass 56,644.26) or its variants were purified and their ability to
synthesize the intramolecular amide bonds in 3 domains—CNA2,
XNA and CNA3—was examined by a combination of molecular
biology and mass spectrometry. As reported above, alanine sub-
abrogated formation of the corresponding amide bond (SI Appen-
dix, Table S1). Similarly, alanine substitution of any one of the 6
residues engaged in the intramolecular amide bonds (Lys174-Asn265
for CNA2, Lys273-Asn383for XNA, Lys417-Asn512for CNA3) pre-
Table S1). Consistent with the proposed role of Asp312as the
catalytic residue for amide bond formation in XNA, alanine
substitution of this residue also abolished amide bond formation in
this domain (SI Appendix, Table S1). We constructed a triple
and Glu472(pJB217). This mutant rBcpAHis-6 did not form any
amide bonds, as its observed mass was within the experimental
Appendix, Table S1).
We constructed 3 plasmids (pJB150, pJB187 and pJB200) to
isolate branched peptides with intramolecular amide bonds in
CNA2, XNA, and CNA3from rBcpAHis-6(SI Appendix, Table S1).
XNA domains. (A) The crystal structure of BcpA* was solved using MAD. The
CNA2and CNA3domains are shown as blue ribbons and the XNA domain
purple. The Lys and Asn residues forming the intramolecular amide bonds in
each of the 3 crystallized domains are shown as red sticks. Topology diagrams
reverse Ig-fold (CNA) and jelly-roll fold (XNA). Both CNA2and CNA3share the
same overall topology. ?-strands are shown as arrows and ?-helices as cylin-
ders. Intramolecular amide bonds are shown as red lines.
Crystal structure of BcpA* and topology diagrams of the CNA and
Budzik et al.PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
etry and Edman degradation (SI Appendix, Fig. S3 and Tables
S3–S8). In summary, synthesis of intramolecular amide bonds
occurs at Lys-Asn residues that are positioned within electron
bonding distance of each folded domain of BcpA and must be in
mutations that altered catalytic or participating residues also pre-
vented amide bond formation, indicating that the contributions of
participating Asn or Lys residues, as well as their Glu or Asp
catalytic residues, are highly specific.
Intramolecular Amide Bonds in BcpA Pili. To identify intramolecular
amide bonds in pili, we used plasmid pJB230, encoding BcpA with
a Met-His6tag at Gly150, i.e., within the CNA1domain. To avoid
generation of complex mass spectra caused by multiple intramo-
lecular linkages, the BcpA variant of pJB230 carries an alanine
substitution at Asn163, which prevents synthesis of the CNA1amide
Met-His6tagged fragments were purified by affinity chromatogra-
phy and analyzed by Coomassie Blue stained SDS/PAGE (SI
Appendix, Fig. S4). Edman degradation identified compound A as
S4 and Table S9). Compound A harbors the intermolecular amide
bond of BcpA (Lys162-Thr522, SI Appendix, Fig. S4). Its observed
average mass (14,624.64) is in agreement with the intramolecular
amide of CNA2(SI Appendix, Fig. S4). To identify the residues
involved in the CNA2amide link, compound A was cleaved with
[NH2-GAVDLIKTGVNEK-(NNEEPTM*)-CO2H)], was charac-
terized by CAD fragmentation of the doubly charged ion at m/z
1065.54 (SI Appendix, Fig. S4). The a1? and b1? fragment ions
demonstrate the amide linkage between Lys174-Asn265(SI Appen-
dix, Table S10).
BcpA expressed from plasmid pJB232 also harbors the Met-His6
residues engaged in intramolecular amide bonds of the pilin pre-
cursor (Fig. 2A). Pili were isolated from B. anthracis srtA::ermC
(pJB232) (10), cleaved with CNBr, and purified by affinity chro-
matography (Fig. 2BC). The linear translational BcpA product of
pJB232, predicted mass 55,834, harbors 6 Met residues. CNBr
would be predicted to generate 7 daughter fragments with mass
14,178 (Leu382-Met510), 13,376 (Glu262-Met381), 13,119 [(His6)
Gly150-Met261], 7,681 (Asp26-Met96, the NH2-terminal fragment
following signal peptidase cleavage of BcpA), 5,955 [Gly97-Glu149
(Ala-Ser-Met)], 917 (Glu511-Met518), and 428.53 (Leu-Pro-Val-
Thr522)]. CNBr treatment of BcpA pili released a compound with
a mass of 49,728.86 as determined by ESI-FTMS (Fig. 2D). This
measurement can be explained by the presence of 4 intramolecular
amide bonds in BcpA pili and one intermolecular amide bond
The seventh CNBr fragment, m/z 5,955, is located between Gly97-
Glu149within CNA1[Gly97-Glu149(Ala-Ser-Met)]. Because Gly97-
Glu149is not linked by intra- or intermolecular amide bonds to the
remainder of the pilin, this compound does not copurify with the
of the 49,728.86 compound released 6 residues per cycle, confirm-
ing the presence of 5 amide bonds within each BcpA subunit of
assembled pili (Fig. 2E, SI Appendix, Table S11). Together, these
(Lys162-Thr522) as well as 4 intramolecular amide bonds involving
Lys37-Asn163, Lys174-Asn265, Lys273-Asn383, and Lys417-Asn512.
known to be resistant to proteases, a prerequisite for their func-
tional attributes as microbial adhesins on host tissues (11). Micro-
bial adhesion occurs on mucosal surfaces, a host milieu endowed
with innate defenses including proteases (11). BcpA pili were
purified from B. anthracis srtA::ermC harboring pJB12 and the
wild-type pilus operon bcpA-srtD-bcpB. Pili assembled with wild-
type BcpA were resistant to trypsin treatment, which is used here
as an indicator for the structural integrity of these fibers (Table 1).
at Asn163, Lys174, Asp312, or Glu472, thereby disrupting each of the
4 intramolecular amide bonds in CNA1, CNA2, XNA, or CNA3,
respectively. When expressed in bacilli and analyzed by immuno-
blotting, these variants assembled BcpA pili (SI Appendix, Fig. S5).
Purified pili were identified by labeling with immunogold particles
in BcpA pili. (A) Schematic to illustrate the structure or BcpA pili assembled
Asn) engaged in the formation of intramolecular amide bonds as well as the
intermolecular amide bond between Lys162(YPKN motif) and Thr522(in the
LPVT remnant of the cleaved sorting signal). BcpA pilin variants encoded by
pJB232 harbor 6 internal methionine residues that can be cleaved with cy-
anogen bromide (CNBr) to generate homoserine lactone breaks. (B) B. an-
thracis srtA::ermC (pJB232) was stained with ?-BcpA antibodies, 10 nm gold
with CNBr, purified by Ni-NTA chromatography and eluate analyzed by Coo-
massie stained SDS/PAGE. (D) The mass of compound B (arrow in C) was
determined by LC-ESI-FTMS. (E) The structure of compound B as derived by
Edman degradation and mass spectrometry. Diagram displays the peptide
sequence of 6 branched peptides, generated by CNBr cleavage at 6 internal
methionine (M) residues, and the intra- and intermolecular amide bonds that
connect all 6 peptides.
Four intramolecular and one intermolecular amide bond are formed
www.pnas.org?cgi?doi?10.1073?pnas.0910887106 Budzik et al.
and viewed by electron microscopy (Fig. 3). Alanine substitutions
that abrogated the intramolecular amide bonds of CNA1, CNA2, or
CNA3did not affect pilus assembly or length (Table 1). Alanine
substitution of Asp312, the catalytic residue of XNA, reduced the
length of pili. However, Lys273Ala, a mutation that also abrogates
formation of the XNA intramolecular amide, did not affect pilus
catalytic residue of CNA2, dramatically reduced pilus assembly and
length (SI Appendix, Fig. S5). Alanine substitution at Lys174, which
abrogates the intramolecular amide between Lys174-Asn265in the
polymerization (Table 1). Nevertheless, 2 catalytic residues for
amide bond formation, Glu223and Asp312, contribute in a discrete
manner to BcpA substrate recognition and sortase D-catalyzed
polymerization of pili.
To test whether BcpA mutant pili resist proteases, we incubated
isolated fibers with trypsin, an enzyme that cleaves polypeptides at
lysine, arginine, and histidine. Positively charged residues are
present in great abundance in BcpA—16% of all residues in the
mature pilin. When viewed by immunogold labeling and electron
microscopy, pili assembled from BcpA variants that cannot form
intramolecular amide bonds in CNA2 (Lys174Ala), XNA
(Lys273Ala), or CNA3(Glu472Ala) were all exquisitely sensitive to
trypsin, which cleaved these fibers into very small fragments (Fig.
3 and Table 1). Alanine substitution at Asn163, which abrogates
synthesis of the CNA1 derived intramolecular amide, caused a
moderate but significant increase in protease sensitivity. Taken
Table 1. Intramolecular amide bonds determine length and protease resistance of BcpA pili
Pili without trypsin
Pili after 2.2 ? 10?7M
Asn163Ala; Asp312Ala; Glu472Ala
Asn163Ala; Lys174Ala; Glu472Ala
— 1.41 (0.57)
1.3 ? 10?14
2.0 ? 10?11
3.9 ? 10?14
1.4 ? 10?9
2.1 ? 10?18
3.5 ? 10?14
3.6 ? 10?14
8.9 ? 10?15
8.9 ? 10?14
8.6 ? 10?13
1.2 ? 10?5
8.0 ? 10?6
2.7 ? 10?3
*Average pilus length measurement (n ? 20 immunoreactive particles) with the standard error of the mean in parentheses.
†Statistical significance was evaluated with the two-tailed homoscedastic Student’s t test, and P values were recorded.
intermolecular amide bonds. (B–K) Wild-type BcpA pili and their variants were purified from B. anthracis srtA::ermC. Isolated pili were either mock treated or
incubated with trypsin. The integrity of isolated pili was analyzed by immunogold labeling and transmission electron microscopy. See Table 1 for quantification
of pilus length.
Intramolecular amide bonds are required for the functional assembly of BcpA pili. (A) Diagram of polymerized BcpA pili with 4 intramolecular and one
Budzik et al. PNAS ?
November 24, 2009 ?
vol. 106 ?
no. 47 ?
together our data suggest that the intramolecular amide bonds of
CNA2and XNA display the largest contributions toward structural
stability of BcpA pili. Furthermore, pili assembled from Asp312Ala
variants appeared thinner (15 nm diameter) than those harboring
the intramolecular amide bond of XNA or the wild-type residue
Asp312, but lacking the Lys273-Asn383bond (30 nm diameter) (Fig.
3 B and H). Of note, our pilus detection method requires antibody
labeling and therefore cannot distinguish between the possibilities
that the diameter of the pilus structure may be diminished in XNA
variants or that the reactivity of pili with BcpA-specific antibodies
has been affected by the Asp312Ala substitution.
Pili, filamentous fibers often more than 1 ?m in length, are
comprised of protein subunits (pilins) and endowed with adhesive
subunits at their tips (12). Pili enable adherence and tissue invasion
of bacterial pathogens. Even a single proteolytic cut along the pilus
shaft, which may occur during assembly or in the mature structure,
would disable these adhesive factors and confer host resistance to
infection. If so, most bacterial pathogens are confronted with a
similar challenge—how to prevent cleavage of their pili by host
Assembly of protease resistant pili in the envelope of Gram-
negative bacteria involves folding catalysts as well as the oxidizing
environment of the periplasm (13, 14), a subcellular compartment
between the cytoplasmic and outer membranes of these microbes
(18). The location of disulfide bonds in these domains varies from
loops to a ?-strand in the same ?-sheet. Stabilizing disulfide bonds
products are subsequently assembled by an usher protein, the
central catalyst of pilus formation, which translocates nascent pili
across the outer membrane (20–22). Homologues of pilins, their
folding chaperones, and outer membrane ushers are found in the
genome sequences of many Gram-negative pathogens, implying
that pilus assembly occurs via a conserved pathway (23).
wall sacculi; however, these structures lack the folding catalysts and
oxidizing environment of the periplasm (1). Nevertheless, assembly
of pili in Gram-positive bacteria also proceeds by a universal
mechanism, involving major pilin subunits with YPKN pilin motifs
and LPXTG sorting signals, as well as pilin-specific sortases (24).
Previous work solved the crystal structures of 2 pilin proteins from
Gram-positive bacteria—the major subunit, Spy0128, of T1 pili
from Streptococcus pyogenes (9), and the minor subunit, GBS52,
from Streptococcus agalactiae (25). Both Spy0128 and GBS52
possess 2 intramolecular amide (isopeptide) bonds, and both struc-
tures encompass tandem Ig-like folds, i.e., 2 CNA-B domains.
Intramolecular amide (isopeptide) bonds exist in other cell-surface
Specifically, 2 domains, CNA-A and CNA-B, of the collagen-
binding adhesin CNA from S. aureus harbor intramolecular amide
bonds (26, 27).
The CNA2and CNA3domains in the current BcpA structure
display the same reverse IgG fold of CNA-B and intramolecular
amide bonds that reside on adjacent and parallel ?-strands, be-
longing to the same ?-sheet (27). Intra-sheet bonds covalently
connect the first and last strands of the domain and are buried
within the hydrophobic core of a ?-sandwich. XNA is the first
XNA assumes the same jelly-roll domain fold as Gram-negative
pilins, albeit that XNA generates an amide bond joining opposite
faces of the ?-sandwich domain. This unique feature suggests that
pili derived from XNA domains may be more stable to proteases
and resistant to thermal denaturing than pili assembled only from
CNA-B domains. The residue essential for forming the intramo-
lecular amide bond of XNA is Asp, in contrast to Glu from the
Most pilin subunits in Gram-positive bacteria appear to be built
(28). Further, most pilin subunits appear to be comprised of only 2
CNA-B domains (28). Lancefield and Dole reported Group A
streptococcal T antigens to be resistant to trypsin treatment (29). T
antigens are now appreciated as the major subunit of Group A
streptococcal pili that also include minor pilin subunits (30). The
recombinant precursor of the major pilin of T1 antigens harbors 2
CNA-B domains, each endowed with an intramolecular Asn-Lys
amide bond (9) of what appear to be the structural equivalents of
these intramolecular bonds are required for resistance of the
recombinant protein to protease (31). Nevertheless, the contribu-
tion of these bonds toward structural stability of T1 pili in the
streptococcal envelope is not yet known.
Here we examined BcpA pili of B. cereus with molecular biology
and structural analyses and derive a new model for the formation
of pilus fibers (Fig. 4). Assembly of fully functional BcpA pili
CNA2, XNA, and CNA3. Amide bonds in 3 BcpA domains, CNA2,
XNA, and CNA3, are found in the recombinant pilin precursor as
well as in assembled pili. We presume all 3 intramolecular amide
precursor, but it could also enable proper packing of BcpA inter-
mediates en route to the final pilus structure. A fourth intramo-
lecular amide, derived from the CNA1domain, is synthesized only
after the BcpA pilin precursor has been polymerized via the
Lys162-Thr522bond. In the absence of the CNA1amide bond, pili
remain partially sensitive to protease cleavage. As CNA1includes
the YPKN pilin motif, we presume the physiological role of its
amide bond may be to protect the peptide sequence surrounding
peptide removed. The CNA2, CNA3and XNA domains assume reverse Ig or
jelly-roll folds (2), form intramolecular amide bonds and enable presentation
of the YPKN amino group (4) for nucleophilic attack at the sortase D-acyl
intermediate with nascent pili, including the BcpB tip pilin (3). The product of
growing pilus. At this stage, the CNA1domain of the incoming BcpA subunit
is still unfolded (gray box with dashed borders) (5). Following folding and
intramolecular amide bond formation by the CNA1 domain, the mature,
protease resistant pilus is immobilized in the cell wall envelope of bacilli (6).
(B) Diagram displays the bcpA-srtD-bcpB gene cluster of B. cereus and the
relevant features of the encoded products.
Model for pilus assembly in Bacillus cereus. (A) The unfolded BcpA
www.pnas.org?cgi?doi?10.1073?pnas.0910887106Budzik et al.
the sortase D-catalyzed intermolecular amide bond by stabilizing
the reverse Ig-fold of this domain. Pili assembled without the XNA
amide bond, but not pili lacking the intramolecular linkages of
CNA1, CNA2, or CNA3, appear thinner than their wild-type
counterparts. If so, a second important function of the XNA
into the mature structure of the pilus fiber.
Materials and Methods
Bacterial Plasmids and Strains. Coding sequences of the bcpA-srtD-bcpB operon
mutations via Quick-Change mutagenesis.
BcpA for Crystallization. Purified BcpAHis-6was digested with trypsin and the
resistant core analyzed by mass spectrometry and Edman degradation. E. coli
to OD6000.6, 100 ml of cell culture was discarded, and amino acids were added
(lysine 0.1 mg/L, threonine 0.1 mg/L, phenylalanine 0.1 mg/L, leucine 0.05 mg/L,
isoleucine 0.05 mg/L, valine 0.05 mg/L, and selenomethionine 0.06 mg/L). GST-
BcpA was purified by GST-affinity chromatography, and the GST tag was re-
moved by on-column cleavage with 55 U of thrombin (GE). BcpA was concen-
trated (Amicon Ultra–15 centrifugal filter) and purified by gel filtration
chromatography (Superose 12 column). Selenomethionine-substituted BcpA
crystallized at room temperature with a reservoir solution of 20% PEG 3350 and
0.2 M calcium acetate hydrate. Crystals were frozen in N2(l) following cryopro-
at the Life Sciences Collaborative Access Team (LS-CAT) beamline 21-ID-D at the
Advanced Photon Source at Argonne National Laboratory (SI Appendix, Table
S13). The structure was determined by the multiple-wavelength anomalous
dispersion method with PHENIX (32).
Purification and Cleavage of Pili. B. anthracis srtA::ermC harboring plasmids
20 ?g/ml kanamycin. Bacteria and pili were scooped into water or 50 mM
ammonium bicarbonate, cells removed by centrifugation, and aliquots of pili
were digested with CNBr, mixed with loading buffer for SDS/PAGE and immu-
was purified by Ni-NTA affinity chromatography (4). Eluate was subjected to
SDS/PAGE and Coomassie Blue R-250 staining, transferred to PVDF membrane
and stained with amido black for Edman degradation, or analyzed by LC-ESI-
FTMS for molecular weight determination. RP-HPLC fractions from bacilli har-
boring pJB230 were digested with trypsin and analyzed by mass spectrometry.
Trypsin Cleavage of Pili. Bacilli were suspended in 1.75 ml of 50 mM ammonium
bicarbonate and OD600normalized to 1.1. Cells were removed by centrifugation
twice at 6,000 ? g for 5 min, and 0.5 ml of pili were incubated with 2.68 ?g of
sequencing grade modified trypsin (Promega) overnight at 37 °C for 19 h. Ali-
quots were stained with ?-BcpA antisera and goat anti-rabbit IgG 10 nm gold
conjugate before electron microscopy.
are described in SI Appendix.
ACKNOWLEDGMENTS. We thank Dr. Xiaojing Yang and staff at Argonne Na-
tional Laboratory-Advanced Photon Source for crystallography assistance. This
work was supported by US Public Health Service Grants AI38897 (to O.S.) and
AI074658 (to C.H.). J.M.B is a trainee of National Institutes of Health Medical
Scientist Training Program Grant GM07281. C.B.P. is a National Science Founda-
tion Graduate Research Fellow. O.S. and C.H. are members of and supported by
the Region V ‘‘Great Lakes’’ Regional Center of Excellence in Biodefense and
Emerging Infectious Diseases Consortium (NIAID, NIH Award 1-U54-AI-057153).
Data collection was performed at the Life Sciences Collaborative Access Team
the Advanced Photon Source at Argonne National Laboratory, which are sup-
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November 24, 2009 ?
vol. 106 ?
no. 47 ?
Corrections and Retraction
PSYCHOLOGICAL AND COGNITIVE SCIENCES
Correction for “Eye movement evidence that readers maintain and
act on uncertainty about past linguistic input,” by Roger Levy,
The authors note that panels A and B of Fig. 2 were transposed,
and that statistical significances for panels C and D were trans-
posed in the figure caption. The corrected figure and its legend
Correction for “Crystal structure analysis reveals Pseudomonas
PilY1 as an essential calcium-dependent regulator of bacterial
surface motility,” by Jillian Orans, Michael D. L. Johnson,
Kimberly A. Coggan, Justin R. Sperlazza, Ryan W. Heiniger,
MatthewC.Wolfgang, andMatthewR.Redinbo, which appeared
in issue 3, January 19, 2010, of Proc Natl Acad Sci USA
(107:1065–1070; first published December 28, 2009; 10.1073/
The authors note that due to a printer’s error, on page 1065,
right column, first full paragraph, seventh line, and page 1069,
right column, second paragraph, seventh line, the amino acids
6145–1163 should instead appear as 615–1163. This error does
not affect the conclusions of the article.
Correction for “Intramolecular amide bonds stabilize pili on
the surface of bacilli,” by Jonathan M. Budzik, Catherine B.
Poor, Kym F. Faull, Julian P. Whitelegge, Chuan He, and Olaf
Schneewind, which appeared in issue 47, November 24, 2009,
of Proc Natl Acad Sci USA (106:19992–19997; first published
November 10, 2009; 10.1073/pnas.0910887106).
The authors note that their manuscript was published without
a Protein Data Bank ID number to identify the crystal structure
of BcpA. The accession number for the structure is 3KPT.
i r t
i r t
sentences 2b and 3b) and overall sentence comprehension. (A) Proportion of trials with first-pass regression from critical word. (B) Go-past time from first
fixation on critical word to first fixation beyond it. (C) Proportion of trials with fixation on earlier preposition (at/toward) during go-past reading of critical
word. (D) Accuracy in comprehension-question answering. (E) First-pass time on critical word. In A, B, and D, interactions between preposition and critical-
word ambiguity are significant (all ANOVA P < 0.05); in C, the interaction is P = 0.087. In E, main effect of critical-word ambiguity is significant (ANOVA P <
0.05 by participants, P < 0.1 by items).
Means and standard errors of measures of processing difficulty associated with the critical word (e.g., tossed in sentences 2a and 3a; thrown in
| March 16, 2010
| vol. 107
| no. 11www.pnas.org
Retraction for “Triplex-forming oligonucleotide-orthophenan-
Fabio Cannata, Erika Brunet, Loïc Perrouault, Victoria Roig,
Slimane Ait-Si-Ali, Ulysse Asseline, Jean-Paul Concordet, and
Proc Natl Acad Sci USA (105:9576–9581; first published July 3,
2008;10.1073/pnas.0710433105); the undersigned authors wish to
note the following, “During efforts to extend this work, we have
been unable to reproduce the mutation data shown in this paper
admitted to an investigation committee having falsified the corre-
sponding sequencedata. Consequently, the conclusionconcerning
the induction of mutations by the orthophenanthroline-triplex
forming oligonucleotide conjugate (OP-19merTFO/LNA) in 10%
of cells is no longer supported by available evidence and the other
data concerning the cellular activity of OP-19merTFO/LNA con-
jugate should be reexamined. The undersigned authors therefore
retract the paper and the first author approves this retraction. We
apologize for any inconvenience this may have caused.”
| March 16, 2010
| vol. 107
| no. 11