Download The structure of secondary cell wall polymers: how

Microbiology (2005), 151, 643–651
Review
DOI 10.1099/mic.0.27749-0
The structure of secondary cell wall polymers:
how Gram-positive bacteria stick their cell walls
together
Christina Schäffer and Paul Messner
Correspondence
Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria
Paul Messner
[email protected]
The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the
past five decades. Outside the cytoplasmic membrane of these organisms the fundamental
polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and
osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic
acids are linked to some of the peptidoglycan chains. In this review these compounds are
considered as ‘classical’ cell wall polymers. In the course of recent investigations of bacterial
cell surface layers (S-layers) a different class of ‘non-classical’ secondary cell wall polymers
(SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell
surface. Comparative analyses have shown considerable differences in chemical composition,
overall structure and charge behaviour of these SCWPs. This review discusses the progress that
has been made in understanding the structural principles of SCWPs, which may have useful
applications in S-layer-based ‘supramolecular construction kits’ in nanobiotechnology.
Overview
The cell walls of Gram-positive bacteria usually contain a
variety of polysaccharides, a significant proportion of which
are covalently linked to peptidoglycan (PG), the major
scaffolding structure of the cell wall. As established in the
1980s, cell wall polysaccharides of Gram-positive organisms
can be classified on the basis of their structural characteristics into three distinct groups: (i) teichoic acids (Archibald
et al., 1968, 1993), (ii) teichuronic acids (Hancock &
Baddiley, 1985; Ward, 1981), and (iii) other neutral or
acidic polysaccharides which cannot be assigned to the two
former groups (Araki & Ito, 1989; Naumova & Shashkov,
1997). As all these compounds have long been attributed a
secondary role in cell wall function, they have been termed
‘secondary’ cell wall polymers (SCWPs). Biochemical and
genetic data accumulated over the past 30 years indicate
that the first two groups of these polysaccharides (‘classical’ SCWPs) play pivotal roles in normal cell function,
and consequently considerable energy is expended in their
biosynthesis (Archibald et al., 1993; Munson & Glaser, 1981;
Pooley & Karamata, 1994).
In the course of recent investigations of bacterial cell surface layer (S-layer) proteins from Bacillaceae (Messner &
Schäffer, 2003; Sleytr, 1978; Sleytr et al., 1996, 2002), novel
aspects of a group of ‘non-classical’ SCWPs have emerged
with regard to their structure and function. S-layers are
generally composed of identical (glyco)protein species
forming regular two-dimensional, lattices on bacterial cell
surfaces. The high metabolic load associated with the
0002-7749 G 2005 SGM
biosynthesis of S-layer proteins, together with their prominent cellular location, indicates that they provide a
positive selective advantage in the environment. It is
conceivable that the stable attachment of S-layer (glyco)proteins to the cell wall is important for the cell, and ‘nonclassical’ SCWPs have been identified as mediators for
non-covalent attachment of S-layers to the underlying PG
meshwork (Cava et al., 2004; Mesnage et al., 2000; Sára,
2001) (Fig. 1).
We review here the current knowledge on structural features, possible linkage types to the cell wall, and interactions
of ‘non-classical’ SCWPs in S-layer-carrying organisms
from the family Bacillaceae. Structural comparisons have
only recently started to be made, and nothing is as yet
known about the biosynthesis of the ‘non-classical’ SCWPs.
The structural information, however, has considerable
impact on the development of S-layer-based applications
in nanobiotechnology, for which these SCWPs may serve
as a basic building block in a ‘supramolecular construction
kit’ (Pum et al., 2004; Sampathkumar & Gilchrist, 2004;
Schäffer & Messner, 2004; Sleytr et al., 2002).
SCWPs of Gram-positive bacteria – a brief
retrospective
Teichoic and teichuronic acids are among the bestcharacterized SCWPs, with respect to both structure and
function. Although they are structurally diverse (Araki
& Ito, 1989; Naumova & Shashkov, 1997), the negative
charge of these anionic polymers mainly originates from
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643
C. Schäffer and P. Messner
of a barrier to prevent diffusion of nutrients and metabolites (Archibald et al., 1993; Baddiley, 1972; Fischer, 1994;
Hancock & Baddiley, 1985; Munson & Glaser, 1981; Navarre
& Schneewind, 1999).
Besides the considerable body of knowledge that has
accumulated about structural, biochemical and immunological features of teichoic and teichuronic acids from cell
walls of Gram-positive bacteria, it should be kept in mind
that further cell wall polysaccharides may be present in these
organisms. These will be discussed in the next section.
Fig. 1. Schematic representation of the cell wall profile of (A)
S-layer-protein- and (B) S-layer-glycoprotein-carrying Grampositive bacilli to indicate the cellular location of SCWP. The
dotted ovals serve to highlight the SCWP. The SCWP is covalently bound to muramic acid residues of PG but attached noncovalently, presumably by a lectin-type interaction (Sára, 2001),
to the S-layer protein. CM, cytoplasmic membrane; MP, membrane protein; PG, peptidoglycan; SP, S-layer protein; SLG, Slayer glycoprotein glycan; SCWP, secondary cell wall polymer.
phosphate (teichoic acids) or carboxyl (teichuronic acids)
groups, and they may also elaborate acidic side chains
containing glycerol, phosphate, organic acids (e.g. pyruvic
and succinic acid), or sulphate. While the overall structure
of the different types of teichoic acids is well documented
(Archibald et al., 1993; Fischer, 1988; Hancock & Baddiley,
1985; Munson & Glaser, 1981), the structural features of
teichuronic acids are less well understood and only a few
have been subjected to full chemical analyses (Munson
& Glaser, 1981; Ward, 1981). These anionic polymers
account for 10–60 % (by weight) of the bacterial cell wall,
with the relative amount depending on the culture conditions (Archibald et al., 1993; Ellwood & Tempest, 1969;
Rogers et al., 1980). Although the exact biological function (or functions) of these negatively charged SCWPs is
not fully understood, several general functions have been
attributed to them. These include (i) binding of divalent
cations, (ii) role in the balance of metal ions for membrane
functionality, (iii) binding of proteins, (iv) role in folding
of extracellular metallo-proteins, (v) providing a source
of phosphate under phosphate starvation conditions, (vi)
interaction with cell wall lytic enzymes, and (vii) formation
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The ‘non-classical’ group of SCWPs
The discovery of ‘non-classical’ SCWPs
Our research has focused on glycosylated S-layer proteins,
which are regarded as ideal model systems for studying the
glycosylation of prokaryotic proteins (for recent reviews
see Messner & Schäffer, 2003; Novotny et al., 2004; Schäffer
& Messner, 2004). In the course of the purification of
S-layer glycoproteins, which constitute up to 15 % of the
cell walls of Bacillaceae (Schäffer et al., 2001), minor
amounts of a second glycoconjugate are frequently coisolated (Altman et al., 1990, 1996; Messner et al., 1987;
Schäffer et al., 1999, 2000, 2004; Steindl et al., 2002).
Considering the available data about multi-glycosylated
S-layer proteins from archaea (Sumper & Wieland, 1995),
these ‘additional carbohydrate structures’ were originally
interpreted as being a second set of S-layer glycoprotein
glycan chains of low abundance. Improved purification
and separation methods, however, eventually led to the
clear assignment of these compounds as a class of SCWPs,
which accounts for a substantial amount (7–15 % by
weight) of the PG of the investigated organisms. The
investigated SCWP–PG complexes comprise the intact
glycan moieties and portions of PG of variable size, due
to random degradation during the preparation procedure.
Based on compositional and structural data, we suggest
that classification of the ‘non-classical’ SCWPs from S-layercarrying Bacillaceae into group (iii) of Araki & Ito (1989)
is most appropriate.
Common and variable features of ‘non-classical’
SCWPs from Bacillaceae
Although the structural analysis of SCWPs from S-layer
carrying Bacillaceae is still in its infancy, comparison of
those SCWPs for which data are available has revealed a
number of common features (Table 1; see also Fig. 2). These
can be summarized as follows.
Group I. The structure of the glycan portion of the
SCWP–PG complex of Paenibacillus alvei CCM 2051 was
elucidated to be [(Pyr4,6)-b-D-ManpNAc-(1R4)-b-DGlcpNAc-(1R3)]n~11-(Pyr4,6)-b-D-ManpNAc-(1R4)-aD-GlcpNAc-(1R (Schäffer et al., 2000). Each repeating
unit disaccharide of this SCWP is substituted with 4,6linked pyruvic acid residues, conferring the overall anionic
character of the SCWP. Upon prolonged exposure of the
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Secondary cell wall polymers
Table 1. Features of ‘non-classical’ SCWPs from S-layer-carrying Bacillaceae
Features
Glycan composition
Sugar constituents
Possible non-carbohydrate modifications
Charge
Repeating units
Number of glycoses per repeating unit
Average molecular mass
Backbone structures
Group I SCWP
Group II SCWP
Heteropolysaccharide
GlcpNAc, ManpNAc, GalpNAc, Manp-2,3-diNAcA, Glcp, Ribf
Pyruvate, phosphate, acetate
Negative or neutral
2–15; linear or branched (chain-length variation possible)
2–5
~4–6 kDa
Group III SCWP
Antennary structure
Content in PG
Substitution of muramic acid residues
Type of linkage to muramic acid
Number of different SCWPs per organism
[R3)-b-D-ManpNAc-(1R4)-b-D-GlcpNAc-(1R]
[R4)-b-D-Manp-2,3-diNAcA-(1R6)-a-D-Glcp-(1R4)-b-D-Manp-2,3-diNAcA(1R3)-a-D-GlcpNAc(1R]
No common motif
Possible
7–15 % (by weight)
20–25 %
Under investigation; phosphodiester or pyrophosphate linkages may exist
1
Fig. 2. Structures of SCWPs of S-layer carrying Bacillaceae. Open symbols, gluco-configuration (%, N-acetylglucosamine;
#, D-glucose; , N-acetylmuramic acid); black symbols, manno-configuration (&, N-acetylmannosamine; X, 2,3-di-Nacetylmannosaminuronic acid, R1=COOH, R2=CONH2, R3=CONHCOCH3, R4=CON(COCH3)2); light grey symbols,
galacto-configuration ( , N-acetylgalactosamine); dark grey symbol, ribo-configuration ( , D-ribofuranose); , phosphatecontaining linker group.
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C. Schäffer and P. Messner
polymer sample to acidic conditions, as obtained upon
solvation in D2O during NMR experiments, considerable
loss of pyruvate residues can occur. In a first set of experiments, 31P-NMR spectroscopy revealed two phosphorus
signals with a markedly different integral ratio, which was
interpreted as a possible pyrophosphate linkage between
SCWP and PG. Indirect support for this assumption came
from treatment with EDTA, SDS, and sonication of the
SCWP–PG complex immediately prior to the NMR measurement. The 31P spectrum then revealed two phosphorus signals with an integral ratio of almost 1 : 1. Thus,
{
the presence of a a-D-GlcpNAc-(1RO)-PO{
2 -O-PO2 (OR6)-MurNAc linkage unit was inferred from that set
of biochemical and NMR data.
The ManNAc-GlcNAc backbone disaccharide motif corresponds to that frequently observed in other cell wall polysaccharides, albeit with an inversion of the anomeric
configuration of the D-GlcNAc residue at the reducing
end (Fig. 2). It has been suggested that inversion of the
anomeric linkage plays a role in the initial polymerization
event of the carbohydrate chain during glycoconjugate
biosynthesis, because the attachment of the first repeating
unit is a critical step for the complete and correct elongation
of the polysaccharide chain (Olsthoorn et al., 2000).
The ManNAc-GlcNAc backbone disaccharide motif is also
reminiscent of the linkage unit of certain teichoic acids
(Araki & Ito, 1989). However, in the ‘non-classical’ SCWPs
this motif is repeated several times, thus constituting the
entire glycan moiety of those SCWPs.
Pyruvic-acid-containing SCWPs have also been reported
for the S-layer carrying organisms Bacillus sphaericus CCM
2177 (Ilk et al., 1999) and Bacillus anthracis (Mesnage et al.,
2000); however, information is not available on either their
full structures or their linkage to the PG layer.
In contrast to anionic polymers, neutral polysaccharides
possessing the identical backbone motif are found in the
PG of other Bacillaceae. The SCWPs of Thermoanaerobacterium thermosaccharolyticum strains D120-70 (Altman
et al., 1990; C. Schäffer, H. Kählig & P. Messner, unpublished results) and E207-71 (Altman et al., 1996) display
the commonly encountered R3)-b-D-ManpNAc-(1R4)-bD-GlcpNAc-(1R4)-b-D-ManpNAc-(1R3)-b-D-GlcpNAc(1R motif. Although previously it was thought that in
strain D120-70 this motif is extended by galactose residues
(Altman et al., 1990), the most recent results show that
alternating ManNAc residues are substituted by ribofuranose side chains (C. Schäffer, H. Kählig, R. Christian
& P. Messner, unpublished results), as also demonstrated for
the SCWP of strain E207-71 (Altman et al., 1996).
Group II. The first SCWP–PG complex of an S-layer-
carrying organism for which the structure was completely
elucidated was purified from Geobacillus stearothermophilus NRS 2004/3a (Messner et al., 1987; Schäffer et al.,
1999). The anionic polymer, comprising on average six
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tetrasaccharide repeating units with the structure R4)-bD-Manp-2,3-diNAcA-(1R6)-a-D-Glcp-(1R4)-b-D-Manp2,3-diNAcA-(1R3)-a-D-GlcpNAc(1R, represents the
SCWP of many G. stearothermophilus wild-type strains.
Preliminary analysis of that SCWP indicated a mass range
of 4000 to 6000. To identify the linkage region between
the repeating units and the PG, 1H spectra were recorded
with simultaneous 13P decoupling to detect signals of
phosphorylated sugars. Recent NMR data suggest that,
instead of a phosphodiester bond which serves as linker
for teichoic and teichuronic acids, this SCWP is linked
to C-6 of muramic acid of PG via a pyrophosphate
bridge, with about 20–25 % of muramyl residues being
substituted by SCWP glycans (Schäffer et al., 1999).
Interestingly, preliminary analyses of the SCWP of Geobacillus tepidamans GS5-97T indicate that its backbone
structure is reminiscent of that of G. stearothermophilus
NRS 2004/3a, with additional modifications of the carboxyl groups of the Manp-2,3-diNAcA residues, turning
the anionic character of the glycan into a neutral one. A
recent investigation has demonstrated that, in this organism, the SCWP is linked to muramic acid by a common
phosphodiester linkage. The definitive structure of the
SCWP of G. tepidamans GS5-97 is currently being elucidated (C. Steindl, C. Schäffer, P. Messner & N. Müller,
unpublished).
Group III. The charge-neutral SCWP isolated from Aneurini-
bacillus thermoaerophilus DSM 10155 represents a hitherto
unique bacterial glycan structure (Steindl et al., 2002;
Wugeditsch, 1998). Salient features of that SCWP, as
deduced from NMR spectroscopy, are: (i) the biantennary
oligosaccharide structure, and (ii) the chemical homogeneity of the oligosaccharide part (defined glycan chain
length) (Fig. 2). It is important to note that a biantennary
SCWP has, so far, not been reported for a member of the
domain Bacteria. Thus, this SCWP resembles the glycoprotein glycans of eukaryotic rather than polysaccharides of
prokaryotic organisms (Varki et al., 1999).
General considerations about ‘non-classical’ SCWP
structure
Comparison of the overall composition of all ‘non-classical’
SCWPs analysed so far reveals that the more complex group
II glycans exhibit the same alternating order of gluco and
manno sugars as the group I glycans (Fig. 2). The glycan
chain starts with a GlcNAc residue at the reducing end
and ends with Manp-2,3-diNAcA. It is possible that the
group II structures have evolved from the simpler group I
structures by the introduction of residues such as glucose
or Manp-2,3-diNAcA through the action of strain-specific
enzymes. Preliminary data on the biosynthetic origin of
the Manp-2,3-diNAcA residues of the lipopolysaccharide
of Bordetella pertussis (Wing et al., 2004) have established
that the relevant nucleotide-activated intermediate is
synthesized from UDP-GlcNAc via several reaction steps
including oxidation at C-6, amination, transacetylation,
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Secondary cell wall polymers
and epimerization at C-3, prior to its insertion into the
repeating unit of the SCWP. Likewise, deacetylation and
deamination of GlcNAc to Glc is conceivable.
Concerning the linkage type between SCWPs and PG, our
studies suggest that phosphodiester and pyrophosphate
linkages may exist in different organisms. However, it
might well be that the pyrophosphate-linked glycans
identified in SCWP preparations represent biosynthetic
intermediates, which under certain conditions can be
detected as dominating molecular species in 31P-NMR
spectra (N. Müller, C. Schäffer & P. Messner, unpublished
data). It is known, for instance, from Bacillus cereus that
sugar-pyrophosphoryllipid intermediates can be present
in membrane preparations (Yamamori et al., 1978). Under
in vivo conditions, the pyrophosphate bridge might represent a predetermined cleavage site with possible relevance
for glycan biosynthesis. In general, pyrophosphate bridges
are commonly formed in intermediates involved in the
biosynthesis of different prokaryotic cell wall polymers.
Currently the linkages between SCWPs and PG are being
investigated in detail. Interestingly, for all investigated
SCWPs, the glycose residue linked to the bridging phosphate residue is in the a-configuration. Whether this has
any impact on the biosynthesis of the SCWPs remains to
be established.
Interactions of SCWPs and S-layers from Bacillaceae
In addition to the previously mentioned general features,
novel properties for SCWPs have emerged from research on
S-layers. S-layers are two-dimensional crystalline protein
lattices on the outermost surface layer of bacteria from
almost all phylogenetic branches (Sleytr, 1978; Sleytr et al.,
1996). S-layer proteins are of interest because of their
involvement in important physiological processes which
include, in the case of pathogenic bacteria, the infection
mechanism (Navarre & Schneewind, 1999). If present,
S-layer (glyco)proteins are the most abundant cellular
proteins. Provision of any kind of selection advantage by
the S-layer to a bacterium in its natural environment
requires the stable attachment of the S-layer to the cell
surface in vivo. During evolution, Gram-positive bacteria
have developed various strategies for displaying proteins
on their surface. These strategies include predominantly
covalent binding of LPXTG-carrying proteins to PG, but a
number of non-covalent binding strategies have also been
developed (Navarre & Schneewind, 1999). Reattachment
experiments of isolated S-layer glycoproteins from thermophilic clostridia revealed the non-covalent character of the
interaction between S-layer and PG (Sleytr, 1976). For
Lactobacillus buchneri it was shown very early that hydroxyl
groups of a neutral cell wall polysaccharide are responsible
for the attachment of the S-layer protein to the cell wall
(Masuda & Kawata, 1985).
Possible cell-wall-targeting mechanisms. Recently, the
cell-wall-targeting mechanism of S-layer proteins has been
investigated in more detail (Cava et al., 2004; Mesnage
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et al., 2000; Sára, 2001). These studies suggest that, in
general, S-layer proteins have two functional regions: a
cell-wall-targeting domain, which in most of the organisms investigated thus far is located at the N-terminus,
and a C-terminal self-assembly domain. The existence of
a cell-wall-targeting domain in S-layer proteins was substantiated by Fujino et al. (1993) and Lupas et al. (1994),
who identified motifs of approximately 55 amino acids,
containing 10–15 conserved residues, which were designated SLH (S-layer homology) domains. SLH domains,
usually composed of one to three modules, are frequently
used means for targeting proteins to the cell surface. They
are found not only in various S-layer proteins but also in
many other surface-associated proteins, e.g. the cellulosomes (Bayer et al., 2004) or other surface-associated
enzymes (Liu et al., 1996). In the case of Bacillus anthracis, the aetiological agent of anthrax, the molecular basis
of the interaction between SLH domains and SCWPs
was established by elucidating their binding properties
(Mesnage et al., 2000). Both S-layer proteins of B. anthracis
(EA1 and Sap) possess SLH domains that bind the
S-layer proteins directly to PG (Mesnage et al., 1997,
1999). A different binding mechanism was proposed for
the S-layer protein of Geobacillus stearothermophilus strain
PV72/p2 (Ries et al., 1997; Sára et al., 1998a). In this
organism two different binding domains were identified
in the N-terminal region of the S-layer protein SbsB, one
for SCWP and another for PG (Sára, 2001; Sára et al.,
1998b). In Thermus thermophilus a similar binding
mechanism was identified between an S-layer–outer membrane complex and the cell wall. There is a strong interaction of the SLH domain of the S-layer protein with a
pyruvylated component of a highly immunogenic SCWP
(Cava et al., 2004).
Originally the involvement of pyruvyl groups in S-layer
binding was inferred from observations in B. anthracis
(Mesnage et al., 2000). It was demonstrated that CsaB is
involved in the addition of pyruvate to a PG-associated
polysaccharide fraction, and that this modification is
necessary for binding of the S-layers via SLH domains.
Interestingly, the csaAB operon was found to be present in
several bacterial species (Mesnage et al., 2000). The amount
of pyruvate in cell walls of slh+ csaB+ strains was in the
range of ~2 mg pyruvate per mg cell wall. Pyruvate was
also identified in the SCWP repeating units of Bacillus
sphaericus CCM 2177 (Ilk et al., 1999) and Paenibacillus
alvei CCM 2051 (Schäffer et al., 2000) (compare with
Fig. 2), which may be taken as an indication that pyruvate,
or more generally speaking, negative charges of SCWPs,
constitute a widespread mechanism for anchoring S-layer
proteins containing SLH domains to the bacterial cell wall.
In this context it should be mentioned that, according to
recent investigations with B. sphaericus CCM 2177, SCWP–
S-layer protein interaction occurs via four SLH domains
(Huber et al., 2005), which is different from the accepted
paradigm of the involvement of one to three SLH motifs
for S-layer protein binding.
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C. Schäffer and P. Messner
Besides the anchoring mechanism involving SLH domains,
another mechanism that possibly utilizes basic amino acids,
present in the cell-wall-targeting region and known for
their direct interaction with carbohydrates, may apply for
S-layer proteins devoid of SLH domains (Table 2). In the
genus Geobacillus, SLH domains have only been identified
on the S-layer protein SbsB of G. stearothermophilus PV72/
p2 (Kuen et al., 1997; GenBank accession no. X98095). All
other investigated strains, such as G. stearothermophilus
PV72/p6 (SbsA; Kuen et al., 1994; X71092), G. stearothermophilus ATCC 12980 (SbsC; Jarosch et al., 2000; AF055578), a
variant strain of G. stearothermophilus ATCC 12980 (SbsD;
Egelseer et al., 2001; AF228338), and G. stearothermophilus
NRS 2004/3a (SgsE; Schäffer et al., 2002; AF328862) possess
S-layer proteins devoid of SLH domains. For the S-layer
protein SbsC it was shown experimentally that a positively
charged N-terminal fragment (amino acids 31–257) is responsible for anchoring the S-layer subunits. Using several
truncated forms of SbsC it was demonstrated that this
region is not required either for self-assembly or for generating the oblique lattice structure (Jarosch et al., 2001).
Interestingly, none of the SCWPs of the organisms that
possess S-layer proteins without SLH domains are modified
with pyruvyl groups and some of them have a net-neutral
charge (see Fig. 2 and Table 2). These observations support the notion that, in addition to the previously discussed involvement of pyruvate, other mechanisms can be
involved in the binding of S-layer proteins to the PG (Cava
et al., 2004; Mesnage et al., 2000; Sára, 2001). In addition,
the non-conserved character of S-layer binding mechanisms
is shown by the observation that the cell-wall-targeting
domain is not necessarily located in the N-terminal region
of the S-layer protein. Well-documented examples of Cterminal anchoring are the S-layer proteins of Lactobacillus
acidophilus ATCC 4556 (Smit et al., 2001) and Lactobacillus
crispatus (Antikainen et al., 2002). Recently, for an S-layer
Table 2. ‘Non-classical’ SCWPs as linker structures for S-layer proteins to the cell wall
Organism
S-layer protein
SCWP
Site of interaction between
SCWP and S-layer protein
N-terminus (aa 33–204), three
SLH motifs
Geobacillus stearothermophilus PV72/p2
SbsB (X98095)
Bacillus sphaericus CCM
2177
SbpA (AF211170)
Negatively charged
(structure under
investigation)
Negatively charged
Paenibacillus alvei CCM
2051
Bacillus anthracis
Sequence unknown
Negatively charged
N-terminus (aa 33–202), four
SLH motifs; involvement of
pyruvyl residues of SCWP
proposed
Unknown
Sap (P49051), EA1
(P94217)
G. stearothermophilus
NRS 2004/3a
G. stearothermophilus
PV72/p6
G. stearothermophilus
ATCC 12980
Geobacillus tepidamans
GS5-97
Aneurinibacillus thermoaerophilus DSM 10155
SgsE (AF328864)
Negatively charged
(structure
unknown)
Negatively charged
N-terminus, three SLH motifs;
involvement of pyruvyl
residues of SCWP
N-terminus, no SLH motifs
SbsA (X71092)
Negatively charged
SbsC (AF055578)
Negatively charged
SgtA (AY883421)
Neutral
SatB (AY395579)
Neutral
N-terminus (aa
SLH motifs
N-terminus (aa
SLH motifs
N-terminus, no
(proposed site
C-terminus, no
(proposed site
Thermoanaerobacterium
thermosaccharolyticum
D120-70
Unknown
Neutral
Unknown
T. thermosaccharolyticum
E207-71
Unknown
Neutral
Unknown
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References
Kuen et al. (1997);
Ries et al. (1997);
Sára et al. (1998a, b)
Huber et al. (2005);
Ilk et al. (1999)
Schäffer et al. (2000)
Mesnage et al. (1999, 2000)
31–257), no
Messner et al. (1987);
Schäffer et al. (1999)
Kuen et al. (1994)
31–257), no
Jarosch et al. (2000, 2001)
SLH motifs
of interaction)
SLH motifs
of interaction)
C. Steindl and others,
in preparation
Wugeditsch (1998);
Steindl et al. (2002);
S. Zayni, P. Messner &
C. Schäffer, unpublished
Altman et al. (1990);
C. Schäffer, H. Kählig,
R. Christian & P. Messner,
unpublished
Altman et al. (1996);
C. Schäffer, H. Kählig,
R. Christian & P. Messner,
unpublished
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Secondary cell wall polymers
protein of another member of the Bacillaceae (Aneurinibacillus thermoaerophilus DSM 10155), C-terminal cell wall
anchoring was proposed (S. Zayni, P. Messner & C. Schäffer,
unpublished data).
The current hypothesis is that the binding between
S-layer proteins and SCWPs is based on a lectin-type
interaction (Sára, 2001). Although, generally, lectins display
low affinities for carbohydrates, their interactions should
be highly specific (Lee & Lee, 2000). Indeed, high binding
specificity was demonstrated for the S-layer protein
SbsB of G. stearothermophilus PV72/p2 and the negatively
charged SCWP of that organism by real-time surface
plasmon resonance biosensor technology (Mader et al.,
2004). That study revealed the presence of at least
two binding sites on a single SCWP molecule with a
separation of about 14 nm and an overall Kd of
7?761027 M. This poses the question of which of the
motifs on the structurally defined SCWPs (compare with
Fig. 2) would be available for the proposed lectintype binding. In group I SCWPs the substitution of the
ManNAc residues and, in particular, the ManNAc at the
non-reducing end, occurs mainly at C-4 (and C-6 in the case
of pyruvate substituents). This implies that only the
hydroxyl groups at C-3 of ManNAc would be available
for the proposed lectin-like linkage. Consequently, for the
non-covalent interaction between SCWP and S-layer
protein, a specificity similar to that of a mannose/
mannosamine-specific lectin may be predicted. In the case
of the group II SCWPs of G. stearothermophilus only the
hydroxyl groups at C-4 would be available for lectin binding.
Consequently, as with group I SCWPs, mannose-like
specificity is predicted for resulting lectin-like interaction.
In contrast, for the group III SCWP of A. thermoaerophilus
DSM 10155, in which the glycan chains are terminated
with GlcNAc residues, a lectin-like interaction with glucose/
glucosamine-specificity is predicted with additional doubling of the binding motifs due to the presence of two
antennae.
Additional properties of S-layers. In the course of the
investigation of the SCWP of Aneurinibacillus thermoaerophilus DSM 10155, an interesting observation was made,
which let us propose a novel property for this SCWP:
mediation of water-solubility of the organism’s S-layer
glycoprotein under in vitro conditions. Indeed the S-layer
glycoprotein of that organism is, so far, the only reported
completely water-soluble S-layer glycoprotein (Wugeditsch,
1998). In vivo, the S-layer subunits of A. thermoaerophilus
DSM 10155 assemble into a square S-layer glycoprotein
lattice, whereas isolated S-layer glycoprotein subunits do
not self-assemble; this property can, however, be restored
in vitro after addition of polyethylene glycols as crystallizing agents to a glycoprotein preparation. Detailed analysis
of the purified S-layer self-assembly products indicated
that the self-assembly process is preceded by the dissociation of the co-purified SCWP from the S-layer glycoprotein
(Steindl et al., 2002). This suggests the following scenario
http://mic.sgmjournals.org
for the in vivo biological role of the SCWP of A. thermoaerophilus DSM 10155. On the intact bacterial cell, the
SCWP holds the S-layer subunits in close proximity,
enabling inter- and intermolecular interaction, and thus
permitting S-layer glycoprotein lattice formation to occur.
When the supporting PG layer is removed during S-layer
glycoprotein preparation, the covalent bonds between
SCWP and the PG backbone are cleaved, releasing the
S-layer glycoprotein with the polymer attached. It is
conceivable that polyethylene glycols can replace SCWP
at the lectin-like binding site on the S-layer protein,
thereby functioning as macromolecular crowding agents.
The resulting situation might be seen as mimicry of the
excluded volume effect thought to prevail on the bacterial
cell surface in vivo, which enables the glycosylated S-layer
subunits of A. thermoaerophilus DSM 10155 to interact
with each other through hydrophobic interactions, resulting in S-layer lattice formation (Steindl et al., 2002). This
interpretation supports the assumption of a lectin-type
binding between S-layer (glyco)proteins and SCWP (Sára,
2001). The biantennary structure of the SCWP obviously
results in a doubling of binding motifs for the S-layer
glycoprotein, allowing a biologically relevant binding
strength to be attained.
Conclusions and outlook
The diversity observed among different SCWP structures of
Bacillaceae follows a general theme which is well known
from other cell surface structures, such as the serotypes of
lipopolysaccharides (Raetz & Whitfield, 2002) and capsular
polysaccharides (Sutherland, 1999). Presumably, this diversity is responsible for creating microenvironments in which
different organisms can survive under unfavourable conditions. Current data indicate that ‘non-classical’ SCWPs
function as mediators for anchoring S-layer (glyco)proteins
from Bacillaceae to the bacterial cell wall.
One interesting aspect of future research will concern the
coordination of S-layer (glyco)protein biosynthesis and
SCWP biosynthesis. It is known that the biosynthesis of
S-layer proteins is a very complex, finely tuned process in
which the amount of the protein component, its translocation through the cell wall, and its incorporation into
the existing S-layer has to be coordinated with the growth
rate of the bacterium and the production of other cell wall
components, such as PG and SCWP. By analogy with the
biosynthesis mechanisms of other glycoconjugates, it seems
reasonable to assume that the biosynthesis of SCWP and
aglycone (PG) occur at different cellular locations and that
a later ligation step results in formation of the covalent
linkage between the SCWP and PG.
Overall, the complete elucidation of the structure and
biosynthesis of several ‘non-classical’ SCWPs will contribute
to our general understanding of the various mechanisms
underlying the tethering of S-layer (glyco)proteins to the
cell surface of Gram-positive bacteria.
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649
C. Schäffer and P. Messner
Acknowledgements
We thank Dr Iain B. Wilson for critical reading of the manuscript and
Sonja Zayni for help with the preparation of the figures. This work
was supported by the Austrian Science Fund, projects P15612-B10
and P15840-B10 (to P. M.).
Huber, C., Ilk, N., Rünzler, D., Egelseer, E. M., Weigert, S., Sleytr,
U. B. & Sára, M. (2005). The three S-layer-like homology motifs of
the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not
sufficient for binding to the pyruvylated secondary cell wall polymer.
Mol Microbiol 55, 197–205.
Ilk, N., Kosma, P., Puchberger, M., Egelseer, E. M., Mayer, H. F.,
Sleytr, U. B. & Sára, M. (1999). Structural and functional analyses of
the secondary cell wall polymer of Bacillus sphaericus CCM 2177 that
serves as an S-layer-specific anchor. J Bacteriol 181, 7643–7646.
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