Article

Lactobacillus surface layer proteins: Structure, function and applications

Department of Veterinary Biosciences, Division of Microbiology and Epidemiology, University of Helsinki, P.O. Box 66, 00014, Helsinki, Finland.
Applied Microbiology and Biotechnology (Impact Factor: 3.34). 05/2013; 97(12). DOI: 10.1007/s00253-013-4962-2
Source: PubMed

ABSTRACT

Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea. They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted pI. The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.

Full-text

Available from: Ulla Hynönen
MINI-REVIEW
Lactobacillus surface layer proteins: structure, function
and applications
Ulla Hynönen & Airi Palva
Received: 18 March 2013 /Revised: 26 April 2013 /Accepted: 27 April 2013 /Published online: 16 May 2013
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Bacterial surface (S) layers are the outermost
proteinaceous cell envelope structures found on members
of nearly all taxonomic groups o f bacteria and Archaea.
They are composed of numerous identical subunits forming
a symmetric, porous, lattice-like layer that completely
covers the cell surface. The subunits are held together and
attached to cell wall carbohydrates by non-covalent interac-
tions, and they spontaneously reassemble in vitro by an
entropy-driven process. Due to the low amino acid sequence
similarity among S-layer proteins in general, verification of
the presence of an S-layer on the bacterial cell surface
usually requires electron microscopy. In lactobacilli,
S-layer proteins have been detected on many but not all
species. Lactobacillus S-layer proteins differ from those of
other bacteria in their smaller size and high predicted pI. The
positive charge in Lactobacillus S-layer proteins is concen-
trated in the more conserved cell wall binding domain,
which can be either N- or C-terminal depending on the
species. The more variable domain is responsible for the
self-assembly of the monomers to a periodic structure. The
biological functions of Lactobacillus S-layer proteins are
poorly understoo d, b ut in s ome sp ecies S-layer proteins
mediate bacterial adherence to host cells or extracel lular
matrix proteins or have protective or enzymatic functions.
Lactobacillus S-layer proteins show potential for use as
antigen carriers in live oral vaccine desig n because of their
adhesive and immunomodulatory properties and the general
non-pathogenicity of the species.
Keywords Surface layer protein
.
S-layer protein
.
Lactobacillus
.
Adhesion
Introduction
Bacterial surface (S) layers are proteinaceous cell envelope
structures ubiquitously found in Gram-positive and Gram-
negative bacterial species and in Archaea (Sára and Sleytr
2000). When present, they form the outermost layer of the
cell, being occasionally covered only by capsules (Fouet et al.
1999). S-layers are composed of numerous identical
(glyco)protein subunits, 40200 kDa in molecular weight,
which form a two-dimensional, regular and highly porous
array with oblique (p1, p2), square (p4) or hexagonal (p3,
p6) symmetry. The subunits are held together and attached to
the underlying cell surface by non-covalent interactions and
have an intrinsic, entropy-driven tendency to form regular
structures either in solution or on a solid support in vitro.
The subunit proteins are typically rich in acidic and hydro-
phobic amino acids but low in sulphur-containing amino acids
and have a low predicted overall pI value (Sára and Sleytr
2000). S-layer protein genes are highly expressed. Several
S-layer protein genes in the genome of a single strain have
been describe d, but all of the genes are not necessarily
expressed at the same time; silent genes, antigenic variation
based on S-layer gene expression (reviewed by Boot and
Pouwels 1996;SáraandSleytr2000;Thompson2002), alter-
native expression of S-layer protein genes in or ex vivo
(reviewed b y Fouet 2009), sequential expression during
growth (Mignot et al. 2004) and, rarely, superimposed S-
layers (Stewart and Murray 1982; Cerquetti et al. 2000)or
S-layers composed of two different S-layer proteins (Rothfuss
et al. 2006; Fagan et al. 2009; Goh et al. 2009; Sekot et al.
2012) have been described. Due to the low overall sequence
similarity among S-layer protein genes and the lack of a
universal signature sequence, confirmation of the presence
of an S-layer still relies largely on electron microscopy.
In recent decades, information about the biological func-
tions of S-layer proteins has accumulated, but no common
function for all S-layers ha s eme rged. The f unctions
U. Hynönen
:
A. Palva (*)
Department of Veterinary Biosciences, Division of Microbiology
and Epidemiology, University of Helsinki, P.O. Box 66,
00014 Helsinki, Finland
e-mail: Airi.Palva@helsinki.fi
Appl Microbiol Biotechnol (2013) 97:52255243
DOI 10.1007/s00253-013-4962-2
Page 1
characterized thus far include, e.g., the determination or
maintenance of cell shape (Mescher and Strominger 1976;
Engelhardt 2007a ) and functions as a molecular sieve (Sára
and Sleytr 1987; Sára et al. 1990), as a binding site for large
molecules (Kay et al. 1985; Phipps and Kay 1988;
Matuschek et al. 1994; Egelseer et al. 1995, 1996; Peters
et al. 1995), ions (Schultze-Lam et al. 1992 ; Pollmann et al.
2006; Klingl et al. 2011) or phages (Howard and Tipper
1973; Ishiguro et al. 1984; Fouet 2009) and as a mediator of
bacterial adhesion (Doig et al. 1992; Toba et al. 1995;
Noonan and Trust 1997; Hynönen et al. 2002; Buck et al.
2005; Sakakibara et al. 2007; Poppin ga et al. 2012). In
pathogenic bacteria, S-layers may contribute to virulence
by several mechanisms, including adhesion, coaggregation
(Shimotahira et al. 2013), a ntigenic variation (Thompson
2002; Spigaglia et al. 2011), protection from complement
or from phagocytosis (Doig et al. 1992; Thompson 2002;
Shimotahira et al. 2013) or modulation of T-cell or cytokine
responses (Wang et al. 2000; Ausiello et al. 2006; Sekot et
al. 2011; Settem et al. 2013). Further, S-layer proteins may
protect the bacterial cell from vario us environmental factors
such as mechanical and osmotic stresses (Engelhardt 2007a,
b), antimicrobial peptides (de la Fuente-Núñez et al. 2012),
radiation (Kotiranta et al. 1999), changes in environmental
pH (Gilmour et al. 2000), bacteriophage s (H oward a nd
Tipper 1973), ba cterial or eukaryotic microbial predators
(Koval an d Hynes 1991; Tarao et al.
2009) or bacteriolytic
enzymes (Lortal et al. 1992). Some S-layer proteins have the
potential to act as degradative enzymes (Calabi et al. 2001;
Ahn et al. 2006; Prado Acosta et al. 2008), and the S-layer
protein of a marine Synechococcus strain is involved in
motility (Brahamsha 1996; McCarren et al. 2005).
Due to the self-assembly properties and the highly ordered,
regular structure down to the nanometer scale, S-layers
have a vast application potential in (nano)biotechnology.
Applications of S-layers can be roughly divided into two
groups. The first comprises applications utilizing (genetically
engineered) S-layered bacterial cells, S-layer (fusion) proteins
or only the expression and/or secretion signals of S-layer
protein genes in various biological systems, including vaccine
development, heterologous protein production and surface
display. The second group utilizes isolated, usually recombi-
nant, S-layer proteins for non-life (nano) technological appli-
cations (see the comprehensive recent reviews by Schuster et
al. 2006; Sleytr et al. 2007; Schuster and Sleytr 2009; Ilk et al.
2011; Pum et al. 2013).
Lactic acid bacteria are Gram-positive, non-pathogenic
micro-organisms characterized by the production of lactic
acid as the main end-product of carbohydrate metabolism.
Within lactic acid bacteria, the genus Lactobacillus forms a
large, heter ogeneous group consisting of non-sporulating,
anaerobic or microaerophilic, catalase-negative, fermenta-
tive organisms with a low G+C content (3253 %) and
complex nutritional requirements. Lactobacilli have been
isolated from various environments, including plants, food-
stuffs, silage and sewage, and they have been found in the
gastrointestinal and genital tracts of humans and animals,
where they form part of the normal flora (Ka ndler and Weiss
1986; Axelsson 19 98; Hay ashi e t al. 2005; Felis and
Dellaglio 2007). Bes ides having a long history of use in
food and feed fermentations, lactic acid bacteria have
aroused interest owing to the health beneficial (probiotic)
properties of some strains. They have proved promising also
as potential vehicles for the delivery of therapeu tic and
prophylactic molecules, such as vaccine antigens in humans
(Seegers 2002; Wells and Mercenier 2008).
Occurrence and general properties of Lactobacillus
S-layer proteins
In the genus Lactobacillus, S-layers have been found in sev-
eral but not all species. Biochemical or functional data have
been published about the S-layer proteins of Lactobacillus
brevis, Lactobacillus buchneri, Lactobacillus helveticus and
Lactobacillus hilgardii and organisms of the former
Lactobacillus acidophilus group (Johnson et al. 1980), includ-
ing L. acidophilus, Lactobacillus amylovorus, Lactobacillus
crispatus and Lactobacillus gallinarum. In addition, strains of
Lactobacillus amylolyticus, Lactobacillus gigeriorum,
Lactobacillus kefiranofaciens, Lactobacillus pasteurii and
Lactobacillus ultunensis carry predicted S-layer protein genes
in their completely or partially sequenced genomes (see
Table 1). Lactobacillus kefir and Lactobacillus parakefir have
been shown to possess an S-layer (Garrote et al. 2004), al-
though the genes have not been sequenced. In earlier studies,
S-layers have been demonstrated by electron microscopy on
Lactobacillus fermentum and Lactobacillus delbrueckii sub-
species bulgaricus (Kawata et al. 1974; Masuda and Kawata
1983), but species identification of these strains has subse-
quently been questioned (Boot et al. 1996c). Supporting this is
thefactthatnoL. fermentum S-layer protein gene sequence
can be found in public databases, and the whole genome
sequencing of L. delbrueckii subspecies bulgaricus did not
reveal any S-layer protein gene (Hao et al. 2011;Makarovaet
al. 2006); thus, at present these species can be considered as
non-S-layer producers. Likewise, in an earlier study, a regular
layer was observed on Lactobacillus casei (Barker and Thorne
1970), but according to Boot et al. (1996b), no S-layer
protein-encoding gene is present in this species, and the isolate
probably would now be reclassified to another species.
Moreover, as the demonstration of S-layer proteins on the
surface of L. casei, Lactobacillus paracasei subsp ecies
paracasei and Lactobacillus rhamnosus by Zhang et al.
(2010b) and Guo et al. (2012) is not yet confirmed, L. casei
is also currently considered as a non-S-layer producer.
5226 Appl Microbiol Biotechnol (2013) 97:52255243
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Table 1 Lactobacillus strains carrying S-layer protein genes with sequences in public databases
Strain Slp reference
a
Genome
Status Reference
L. acidophilus ATCC 4356 Boot et al. (1995)
JCM 1038 AAF65561
Unspecified AEW12794
NCFM Buck et al. (2005) Completed Altermann et al. (2005)
30SC Annotation Completed Oh et al. (2011)
ATCC 4796 Annotation Ongoing
L. amylolyticus DSM 11664 Annotation Ongoing
L. amylovorus GRL 1112 Jakava-Viljanen and Palva (2007) Completed Kant et al. (2011a)
GRL 1118 Jakava-Viljanen and Palva (2007) Completed Kant et al. (2011b)
GRL 1115 Jakava-Viljanen and Palva (2007) Ongoing
DSM 16698 Palva et al. unpublished Ongoing
L. brevis ATCC 8287 Vidgren et al. (1992) Ongoing
ATCC 14869 Jakava-Viljanen et al. (2002)
KB290 BAK78870
M8 AFD33419
ATCC 367 Åvall-Jääskeläinen et al. (2008) Completed Makarova et al. (2006)
DSM 20054 ABBC45 Annotation Ongoing
L. brevis ssp gravesensis ATCC 27305 Annotation Ongoing
L. buchneri CD034 Annotation Completed Heinl et al. (2012)
ATCC 11577 Annotation Ongoing
L. crispatus JCM 5810 Sillanpää et al. (2000)
LMG 12003 Sillanpää et al. (2000)
F5.7 Mota et al. (2006)
ZJ001 Chen et al. (2009)
K2-4-3 Hu et al. (2011)
K313 Sun et al. (2012)
M247 AJ007839
MH315 AB110090, AB11091
Unspecified AAY41912
Unspecified AAY41916
ST1 Hurmalainen et al. 2007 Completed Ojala et al. (2010)
125-2-CHN CTV-05 MV-1A-US
MV-3A-US 2141 JV-V01
Annotation Ongoing
L. gallinarum D109, D195-2, D256, D109,
D255, ATCC 33199, D42 D44-2
Hagen et al. (2005)
DSMZ 10532 AAS83409
L. gigeriorum CRBIP 24.85 Annotation Ongoing
L. helveticus /suntoryeus CNRZ 892 Callegari et al. (1998)
CNRZ 1269 CAA63409
ATCC 12046 Lortal et al. (1992) CAB46984
GCL 1001
b
BAB72065
JCM 1003
b
BAB72066
JCM 1007 BAB72067
JCM 1008 BAB72068
IMPC M696 CNRZ 35 IMPC HLM1
IMPC I60 CNRZ 303 ATCC 15009
Ventura et al. (2000)
K1/R0052 ADK74769
Y10 M4 Cachat and Priest (2005)
Appl Microbiol Biotechnol (2013) 97:52255243 5227
Page 3
All of the Lactobacillus S-layer proteins characterized thus
far are preceded by a 2532-amino-acid signal peptide, indi-
cating secretion through the general secretory pathway. The
deduced amino acid sequences of mature Lactobacillus S-layer
prote ins vary considerably (Åvall-Jääskeläinen and Palva
2005), and even the S-layer proteins of the same strain, when
present, may be markedly different in sequence (Jakava-
Viljanen et al. 2002; Hagen et al. 2005). As in the case of
S-layers in general, a remarkable similarity between the de-
duced amino acid sequences can only be found between related
species, e.g. between the S-layer proteins of the former
L. acidophilus group organisms and some L. helveticus strains
(Antikainen et al. 2002; Hagen et al. 2005). Methods for the
identification of L. crispatus (Horie et al. 2002b)and
L. helveticus (Ventura et al. 2000) based on the presence of
the S-layer protein genes have been developed, but the appli-
cability for the latter species has subsequently been questioned
due to the observed heterogeneity within L. helveticus S-layer
protein gene sequences. Moreover, the relationship between
L. helveticus cell surface proteinase and surface layer proteins
is currently not clear (Gatti et al. 2005). The comparison of
phylogenetic trees based on 22 deduced Lactobacillus S-layer
protein (or the S-layer-like Apf protein) sequences and the 16S
rRNA gene sequences of the corresponding Lactobacillus
species revealed a similar overall clustering of strains (Åvall-
Jääskeläinen and Palva 2005). However , when the phyloge-
netic trees constructed on the basis of the S-layer protein genes
of a set of L. acidophilus-related organisms, including strains
of the novel Lactobacillus suntoryeus species [later reclassified
as L. helveticus (Naser et al. 2006)], were compared with trees
constructed on the basis of 16S rRNA or elongation factor Tu
(tuf) gene sequences of the same species, the novel strains
grouped together in the latter tree, but not in the former tree
based on the S-layer protein genes. This indicates a strong
selective pressure driving the diversification of S-layer protein
genes within at least some L. acidophilus-related organisms as
well (Cachat and Priest 2005). Nevertheless, the remarkable
similarities on the amino acid level between the S-layer pro-
teins of L. acidophilus-related organisms have led to the pro-
posal of using LC-MS/MS analysis of S-layer proteins for
typing strains within this group (Podlesny et al. 2011). A
phylogenetic tree based on those Lactobacillus S-layer protein
sequences for which gene expression data are currently avail-
ableisshowninFig.1. It clearly separates the S-layer proteins
of L. brevis and L. hilgardii from the S-layer proteins of
L. helveticus and the
L. acidophilus group or ganisms but in-
dicates great variability within the S-layer proteins of L. aci-
dophilus group as, e.g., the S-layer proteins of different strains
of L. gallinarum may be very distantly related (see, e.g., LgsF
of L. gallinarum D109).
The S-layer protein Slp A of L. acidophilus NCFM is
identical in sequence with SA of L. acidophilus ATCC
4356, although the strains are clearly distinquishable by
pulse field g el electrophoresis of chromosomal DNA
(Sanders and Klaenhammer 2001 ). L. acidophilus NCFM
harbours a larger diversity of mobi le genetic elements than
other probiotic lactic acid bacteria (Altermann et al. 2005).
Although the elements do not show similarity to currently
known integrative and conjugative elements (Wozniak and
Waldor 2010), the possibility of horizontal gene transfer in
Table 1 (continued)
Strain Slp reference
a
Genome
Status Reference
Slh02 AGD98690
MIMLh5 Taverniti et al. (2012)
Unspecified AAL36968
DPC4571 Annotation Completed Callanan et al. (2008)
H10 Annotation Completed Zhao et al. (2011)
MTCC5463 Annotation Completed Prajapati et al. (2011)
R0052 Annotation Completed Tompkins et al. (2012)
DSM20075 Annotation Ongoing
L. hilgardii B706 Dohm et al. (2011)
ATCC 8290 Annotation Completed
L. kefiranofaciens ZW3 Annotation Completed Wang et al. (2011)
L. pasteurii CRBIP 24.76 Annotation Ongoing
L. ultunensis DSM 16047 Annotation Ongoing
a
GenBank reference number is indicated if no publication about the S-layer protein is available. Annotation; identification based on genomic
annotation only
b
Proteinase (PrtY)-like (Gatti et al. 2005; Yamamoto et al. 2000); see text
5228 Appl Microbiol Biotechnol (2013) 97:52255243
Page 4
the acquisition of slpA gene in L. acidophilus cannot be
ruled out. A more likely explanation for the presence of an
identical slp gene in two genetically different strains is,
however, that the strains have a common origin as their
genomic organization is predominantly the same (Sanders
and Klaenhammer 2001).
S-layer proteins of lactobacilli differ from S-layer proteins
in general in their smaller size (2571 kDa) and high predicted
overall pI value (9.4 10.4). The lattice symmetry of
Lactobacillus S-layer proteins, when known, is of oblique or
hexagonal type (reviewed by Åvall-Jääskeläinen and Palva
2005). A glycan structure of a Lactobacillus S-layer protein
has to date been determined only for L. buchneri (Messner et
al. 2008), whereas glycosylated S-layer proteins have been
described in L. kefir (Mobili et al. 2009a). Secondary structure
predictions for S-layer proteins are of limited value thus far as
the prediction algorithms are based on the available structures
of very dissimilar types of proteins. A prediction performed
for the amino acid sequences of the unprocessed forms of six
Lactobacillus S-layer proteins suggested on average 14 %
α-helices, 39 % extended strands and 47 % random coils in
these proteins (Åvall-Jääskeläinen and Palva 2005). Physical
measurements revealing secondary structures have been
conducted f or a few Lactobacillus species. A Fourier
Fig. 1 A neighbour-joining
phylogenetic tree based on
Lactobacillus S-layer protein
sequences for which gene
expression data are currently
available. The scale bar
indicates the phylogenetic
distances expressed as the
number of amino acid
substitutions per sequence.
Bootstrap values are indicated
at the nodes for 500 replicates.
The partial S-layer protein
sequence of L. helveticus
MIMLh5 was excluded from
the analysis. Asterisk
corresponding gene is known to
be silent
Appl Microbiol Biotechnol (2013) 97:52255243 5229
Page 5
transform infrared spectroscopy study performed for the
S-layer proteins of L. kefir and L. brevis indicated α-helix
contents of 021 %, β-sheet contents of 2350 % and other
structure contents, including β-turns and random coils, of
3763 % in these proteins. For example, the proportions of
α-helix, β-sheet, and other structures in SlpA of L. brevis
ATCC 8287 were 0, 50 and 50 %, respectively (Mobili et al.
2009b). Atomic force microscopy studies of the S-layer pro-
tein CbsA of L. crispatus and its N- and C-terminal fragments
suggested the presence of at least four α-helical structures of
variable sizes, rather than β-sheets, in the N-terminal part of
CbsA (Verbelen et al. 2007). Elucidation of the tertiary struc-
ture of S-layer proteins has been hindered by their molecular
weights mostly not being in the suitable range (<40 kDa) for
nuclear magnetic resonance studies and by their low solubil-
ity, i.e., their tendency to form two-dimensional lattices rather
than three-dimensional crystals in solution. Consequently, no
three-dimensional structures of Lactobacillus S-layer proteins
at atomic resolution are currently available.
In addition to the verified S-layer proteins presented earlier,
surface proteins described as S-layer like have been identi-
fied on th e surface of severa l Lactobacillus gasseri and
Lactobacillus johnsonii strains. These aggregation promoting
factor (Apf) proteins share several characteristics with
Lactobacillus S-layer proteins, such as their relative abundance
on the cell surface, extractability by lithium chloride (LiCl),
amino acid composition, predicted physical properties like
high pI and indispensability for growth (Ventura et al. 2002).
The Apf proteins of L. gasseri 4B2 are critical for cell shape
maintenance, but they are not the sole components responsible
for the aggregative phenotype (Jankovic et al. 2003). Despite
the similarities with surface layer proteins, the S-layer nature of
Apf proteins has not yet been fully confirmed. Each L. gasseri
and L. johnsonii strain carries two apf genes in tandem orien-
tation, both of which are expressed at the same time as
monocistr onic units (Ventura et al. 2002; Jankovic et al.
2003), a feature not very common among Lactobacillus slp
genes (see "Expression of Lactobacillus Slayer protein genes).
A three-layered cell envelope structure similar to that observed
on S-layered Lactobacillus strains and modifiable by LiCl has
been observed in thin-section samples of L. johnsonii cells by
electron microscopy (Ventura et al. 2002). However, no mi-
crographs obtained by freeze-fracturing techniques or by other
methods revealing the symmetric organization of the proteins
have been published, and no reports are available about the
recrystallization of the proteins in vitro. Proteins designated as
Apf, with C-terminal similarity to Apf proteins of L. gasseri
and L. johnsonii, have subsequently been described for several
Lactobacillus and other bacterial species (Goh and
Klaenhammer 2010), like L. crispatus M247 (Marcotte et al.
2004) and L. acidophilus NCFM (Goh and Klaenhammer
2010). L. acidophilus NCFM has also a true S-layer
(Konstantinov et al. 2008), and L. crispatus M247 at least
carries an S-layer gene (Table 1). Thus, based on the current
data, the designation of L. gasseri and L. johnsonii Apf pro-
teins as belonging to an S-layer like family (Ventura et al.
2002), but not as true S-layer proteins, is justifiable.
Expression of Lactobacillus S-layer protein genes
The very efficient synthesis of S-layer proteins in lactobacilli is
achieved by several means: (1) The half lives of the S-layer
protein gene transcripts have been determined to be exception-
ally long (14 and 15 min for the S-layer protein genes of
L. brevis ATCC 8287 and L. acidophilus ATCC 4356, respec-
tively) (Boot et al. 1996b;Kahalaetal.1997). At least in the
case of L. acid ophilus ATCC 4356 (Narita et al. 2006)andL.
crispatus K313 (Sun et al. 2012), this is due to the long 5
untranslated region (UTR) of the transcript forming a stabilizing
secondary structure, as originally suggested by Boot et al.
(1 996b). However , the 5 UTR of L. br evis ATCC 8287 slpA
transcript is not exceptionally long (Vidgren et al. 1992); (2) A
biased codon usage, correlating with efficient gene expression in
lactobacilli (Pouwels and Leunissen 1994), has been observed
for the S-layer protein genes of L. brevis ATCC 8287 (V idgren
et al. 1992)andL. acidophilus ATCC 4356 (Boot et al. 1995);
(3) The promoters of S-layer protein genes are efficient, even to
the extent that they have been used in heterologous expression
and protein production systems (see "Applications of
Lactobacillus Slayer proteins); (4) The presence of two pro-
moters, which may act to enhance and/or regulate gene expres-
sion, has been identified upstream of the slpA gene of L. brevis
ATCC 8287 (V idgren et al. 1992; Hynönen et al. 2010), slpB of
L. brevis ATCC 14869 (Jakava-Viljanen et al. 2002)andslpA of
L. acidophilus ATCC 4356 (Boot et al. 1996b). In L. brevis
ATCC 8287, both slp promoters were shown to be active
(Kahala et al. 1997; Hynönen et al. 2010), whereas in L.
acidophilus ATCC 4356, only the downstream promoter is
functional under the conditions tested (Boot et al. 1996b).
The presence of multiple S-layer protein genes in the same
strain is common in lactobacilli (Boot et al. 1996c;Hagenet
al. 2005), but so far only the S-layer protein genes slpB and
slpD of L. brevis ATCC 14869 (Jakava-Viljanen et al. 2002),
slpB and slpC of L. crispatus K313 (Sun et al. 2012)andslpA
and slpX of L. acidophilus NCFM (or slpB and slpX of the
slpA knock-out mutant of L. acidophilus NCFM) (Goh et al.
2009) have been shown to be expressed simultaneously. Thus,
silent S-layer protein genes, under the conditions tested, are
common and represented, e.g., by the slpB genes of L. aci-
dophilus ATCC 4356, NCIMB 8607, LMG 11428, LMG
11469 (Boot et al. 1995) and NCFM (Konstantinov et al.
2008), cbsB of L. crispatus JCM 5810 (Sillanpää et al.
2000) and ZJ001 (Chen et al. 2009), SlpNB of L. crispatus
LMG 12003 (personal communication; Sillanpää et al. 2000),
slpA of L. crispatus K313 (Sun et al. 2012), slpC of L. brevis
5230 Appl Microbiol Biotechnol (2013) 97:52255243
Page 6
ATCC 14869 (Jakava-Viljanen et al. 2002), several lgs genes
of L. gallinarum (Hagen et al. 2005), and probably also by one
of the two S-layer protein genes identified in L. amylovorus by
DNA hybridization (Boot et al. 1996c), although the presence
of two identical-sized S-layer proteins on the bacterial surface
cannot be excluded. According to a preliminary SDS-PAGE
analysis of seven porcine L. amylovorus isolates, only one
isolate was suggested to express two S-layer protein genes at
the same time, while for the remaining strains, only one S-
layer protein was present (Jakava-Viljanen and Palva 2007).
The genomes of L. gallinarum strains have two genes
encoding S-layer proteins: acommononeandastrain-
specific one, but each strain produces only a single S-layer
protein, which is always encoded by the strain-specific gene
(Hagen et al. 2005). In the sequenced genomes of L. brevis
ATCC 367 (Makarova et al. 2006)andL. buchneri CD034
(Heinl et al. 2012), two or several complete genes, re-
spectively, have been identified by sequence homology
(Åvall-Jääskeläinen et al. 2008; Hein l et al. 2012), but
the expression of these genes is unknown.
The mechanism of the differential expression of slp genes
has been well documented in L. acidophilus 4356, in which an
inversion of a chromosomal segment leads to the placement of
the silent gene in front of the active slp promoter (Boot et al.
1996a). This event seems to be unfavoured under laboratory
conditions as the silent gene is at the expression site only in
0.3 % of the chromosomes of a broth culture of L. acidophilus
4356. No condition favouring the expression of the silent gene
has thus far been characterized (Boot et al. 1996a). A similar
chromosomal inversion mechanism has subsequently been
shown to operate in L. acidophilus NCFM, where the inacti-
vation of the S-layer protein gene slpA by homologous recom-
bination led to the appearance of an alternate S-layer protein,
SlpB, in the mutant strain NCK1377-CI (Buck et al. 2005;
Konstantinov et al. 2008).
Information about adaptive changes in Lactobacillus S-
layer gene expression, not known to involve chromosomal
rearrangements, is scarce. In L. brevis ATCC 14869, the dif-
ferential expression of the slpB and slpD genes is related to the
oxygen content of the growth medium and the growth stage:
slpB is expressed irrespective of oxygen content and equally in
different growth phases, while slpD is predominantly
expressed in aerated cultures and mainly in the exponential
phase. The onset of
slpD expression is most likely mediated by
a soluble cytoplasmic factor, and it was surmised to be part of a
stress response; a concomitant change in colony morphology,
presumably not directly linked to the S-layer protein type, was
also observed. Neither the nature/mechanism of action of the
soluble regulator nor the reason for the silence of the slpC gene
in this strain is known (Jakava-Viljanen et al. 2002). Stress-
mediated regulation has been suggested also on other occa-
sions. The expression of the S-layer protein gene of L. aci-
dophilus NCC 2628 was induced whe n the strain was
cultivated under conditions of limited protein supply (Schär-
Zammaretti et al. 2005). An effect of bile salts was observed in
L. acidophilus ATCC 4356, where concentrations of 0.01
0.05 % were shown to increase slpA gene expression, while
the expression was decreased in 0.1 % bile; concom itant
changes were observed in colony morphology and cell surface
hydrophobicity (Khaleghi et al. 2010). In the same strain,
sublethal concentrations of penicillin G were shown to increase
the amount of SlpA on the bacterial surface, but the results
were not in accordance with slpA gene expression (Khaleghi et
al. 2011). The expression of the S-layer protein gene of
L. acidophilus NCFM, in contrast, was not significantly in-
duced during the passage through an in vitro gastrointestinal
tract model (Weiss and Jespersen 2010). In L. brevis ATCC
8287, a slight repression effect of bile on slpA promoter activ-
ity was observed, but neither bile, pancreatin nor an uncom-
mon carbon source had an effect on the amount of SlpA
expressed on the bacterial cell surface (Hynönen et al. 2010).
Similarly, in L. hilgar dii, wine-related stress factors, like the
presence of ethanol, copper sulphate or p-coumaric acid, did
not affect S-layer protein production measurable by SDS-
PAGE (Dohm et al. 2011).
Completely S-layer-negative Lactobacillus mutants are dif-
ficult or impossible to create (Palva et al. unpublished data;
Boot et al. 1996a;Martinezetal.2000;Bucketal.2005),
indicating that at least one functional S-layer protein gene is
essential for S-layered lactobacilli, and expression of S-layer
protein genes thus could be anticipated to be constitutive.
However, some of the examples earlier indicate that variation
and regulation at the transcriptional and/or translational level
also exist. Recently, genes encoding alternative sigma factors
have been identified in the sequenced genomes of several
Lactobacillus species, and numerous potential transcription
factor genes are also present (Azcarat e-Peril et al. 2008 ),
indicating potential for the regulated expression of slp genes
under special conditions in these organisms. However, cur-
rently the transcriptional and translational regulation mecha-
nisms of Lactobacillus S-layer protein genes on a molecular
level are almost completely unexplored.
Cell wall binding an d self-assembly regions
of Lactobacillus S-layer proteins
One or both of the two structural regions generally present in
S-layer proteins, i.e., the region involved in the attachment of
the S-layer subunit to the cell envelope and the region involved
in S-layer assembly, have so far been characterized in the
following S-layer proteins: in the SA protein of L. acidophilus
ATCC 43 56 (Smit e t al.
2001), in CbsA of L. crispatus JCM
5810 (Antikainen et al. 2002), in SlpB of L. crispatus strains
K313 and K2-4-3 (Hu et al. 2011; Sun et al. 2012), in SlpA of
L. crispatus ZJ001 (Chen et al. 2009), in SlpA of L. brevis
Appl Microbiol Biotechnol (2013) 97:52255243 5231
Page 7
ATCC 8287 vall-äskeläinen et al. 2008) and in the S-layer
protein of L. hilgardii B706 (Dohm et al. 2011).
The first five strains listed above belong to the former
L. acidophilus group (Johnson et al. 1980), and the amino
acid sequences of their S-layer proteins show extensive sim-
ilarity in the C-terminal region (Hu et al. 2011; Sun et al.
2012). Extending the alignment to the S-layer proteins of
organisms of the closely related L. helveticus (Collins et al.
1991; Felis and Dellaglio 2007) also indicates a remarkable
conservation of the C-terminal region (Antikainen et al. 2002),
suggesting a conserved function for this region. Indeed in SA
(Smit et al. 2001), CbsA (Antikainen et al. 2002) and in the
SlpB proteins of K313 (Sun et al. 2012) and K2-4-3 (Hu et al.
2011), the C-terminal part of the S-layer protein, approximate-
ly 125 amino acids in length, is responsible for binding to the
cell envelope. In SA, only one of the two 65-amino-acid
repeats of the cell wall binding region is necessary for binding,
and an enhancing role for the other repeat has been suggested
(Smit and Pouwels 2002). In contrast, in the S-layer proteins
of L. brevis ATCC 8287 and L. hilgardii B706, organisms
completely unrelated to L. acidophilus, the N-terminal parts of
the proteins comprise the cell wall binding region (Åvall-
Jääskeläinen et al. 2008;Dohmetal.2011). Nevertheless,
all of the S-layer proteins described earlier have a similar
charge distribution with a high predicted pI in the cell wall
binding part (Smit et al. 2001;Antikainenetal.2002vall-
Jääskeläinen et al. 2008; Sun et al. 2012;Dohmetal.2011).
Thus, an electrostatic interaction occurring between the cell
wall binding regions and the negatively charged cell wall
polymers has been proposed (Antikainen et al. 2002).
Lactobacillus S-layer proteins do not possess surface layer
homology domains (Lupas et al. 1994), repeated motifs 5060
amino acids in length, which are known to be involved in the
binding of many S-layer proteins to the cell wall, for instance,
those of Bacillus anthracis (Mesnage et al. 1999), Geobacillus
stearothermophilus PV72/p2 (Ries et al. 1997; Sára et al.
1998), Lysinibacillus sphaericus
CCM 2177 (Ilk et al. 1999)
and Clostridium thermocellum NCIMB 10682 (Lemaire et al.
1998). Instead two repeated amino acid sequences with ho-
mology to the tyrosine/phenylalanine containing carbohydrate-
binding motifs of clostridia l toxins and streptococcal
glucosyltransferases (Wren 1991; von Eichel-Streiber et al.
1992) are present in the cell wall binding regions of the
above-mentioned SA, CbsA and SlpA of L. acidophilus,
L. crispatus and L. brevis, respectively. These motifs are also
found in the C-terminal parts of the silent S-layer protein SB of
L. acidophilus ATCC 4356, the S-layer protein of L. helveticus
CNRZ 892 and the non-S-layer proteins of lactic acid bacteria
known to be associated with the cell envelope (Smit et al.
2001; Åvall-Jääskeläinen et al. 2008).Thecellwallreceptors
of the S-layer proteins of L. acidophilus and L. crispatus have
indeed been shown to be carbohydrates: SA of L. acidophilus
ATCC 4356 and CbsA and SlpB of L. crispatus JCM 5810 and
K313, respectively, interact with teichoic acids (Antikainen et
al. 2002; Smit and Pouwels 2002; Sun et al. 2012); CbsA also
binds to lipoteichoic acids isolated from Staphylococcus aure-
us and Streptococcus faecalis, but not to the teichuronic
acid/polysaccharide fraction of the cell wall of L. crispatus
JCM 5810 (Antikainen et al. 2002). On the contrary, the cell
wall components interacting with the S-layer proteins of
L. brevis ATCC 8287 and L. hilgardii apparently are non-
teichoic acid polysaccharides as trichloroacetic acid (TCA)
treatment of the cell walls had no effect on the interaction
(Åvall-Jääskeläinen et al. 2008; Dohm et al. 2011); TCA
treatment at +4 °C has been reported to selectively remove
teichoic acids (Hancock and Poxton 1988). Supporting this is
the fact that, in earlier studies, the cell walls of L. brevis and
L. buchneri were shown to contain neutral polysaccharides that
were suggested to be involved in the anchoring of the S-layer
protein via hydrogen bonding (Masuda and Kawata 1980,
1985). In contrast to the well-characterized exopolysaccharides
of lactic acid bacteria (De V uyst and Degeest 1999; Welman
and M addox 2003), the cell wall polysac charides of
lactobacilli other than teichoic acids are poorly known. The
detailed structure of a neutral wall polysaccharide of L. casei
has been determined (Nagaoka et al. 1990), but no precise
structures for such polysaccharides of L. buchneri or L. brevis
strains are available.
In SA of L. acidophilus,CbsAofL. crispatus and SlpA of
L. crispatus ZJ001 and L. brevis, the more variable part of the
protein (N-terminal in the S-layer proteins of L. acidophilus or
L. crispatus, C-terminal in SlpA of L. brevis) is responsible for
the self-assembly of the S-layer protein monomers to a peri-
odic S-layer lattice, as shown by the mapping of the self-
assembly properties of truncated recombinant S-layer proteins
by transmission electron microscopy (Sillanpää et al. 2000;
Smit et al. 2001, 2002; Antikainen et al. 2002vall-
Jääskeläinen et al. 2008;Chenetal.2009); these regions thus
most likely represent the surface-exposed parts of the proteins.
SA, CbsA and SlpA of L. brevis, and apparently also the other
S-layer proteins described earlier, can thus be viewed as two-
domain proteins with a cell wall binding domain and a self-
assembly domain facing the extracellular environment, the
former being not or less exposed to the environment. In SA,
this view is supported by extensive proteolytic and chemical
breakdown experiments (Smit et al. 2001); moreover, in the
S-layer proteins of L. brevis and L. hilgardii,theC-terminal
parts were found to be trypsin resistant (Åvall-Jääskeläinen et
al. 2008;Dohmetal.2011).
More detailed information is available about the structures
of the self-assembly domains of SA of L. acidophilus ATCC
4356, CbsA of L. crispatus JCM 5810 and SlpA of L. brevis
ATCC 8287. According to insertion and deletion mutagenesis
and proteolytic studies of SA, the N-terminal self-assembly
domain is probably organized into two subdomains of approx-
imately 12 and 18 kDa, linked by a surface-exposed loop. The
5232 Appl Microbiol Biotechnol (2013) 97:52255243
Page 8
very N-terminus of SA is not critical for crystallization and is
probably buried inside the domain or facing the cell wall or
S-layer pore. Conserved regions and regions predicted to form
secondary structures in SA are necessary for the formation of a
regular lattice (Smit et al. 2002). The lack of necessity of the
very N-terminal end and the importance of the conserved
regions for self-assembly have also been demonstrated for
CbsA, where the conserved, valine-rich flanking regions of
the self-assembly domain are especially important for the
formation of the S-lay er lattice and may have a role in
directing the formation of a regular polymer; changes in the
morphology of the self-assembly products of CbsA fragments
were seen accompanying a mutation of even a single residue
in these conserved border regions as well as with the stepwise
truncation of the self-assembly region. The C-terminal cell
wall binding domain has a stabilizing role in the recrystalliza-
tion of CbsA monomers by allowing a more efficient sheet
formation (Antikainen et al. 2002). The locations of a set of
defined amino acids in SlpA of L. brevis have been mapped by
cysteine-scanning mutagenesis combined with sulfhydryl
modification. The analysis, based on measuring the surface
accessibilities of the residues when the protein is in a mono-
meric or self-assembled form, grouped the residues according
to their locations within the polymerized S-layer structure: to
those located in the interior of the subunit, to those on the
outer surface of the polymerized protein layer, to those on the
inner surface of the layer and to those likely located in the
subunitsubunit interface and pore or inner surface of the
layer. The results c onfirmed the two-domain structure of
SlpA and revealed several sites of high surface accessibility
(Vilen et al. 2009).
Functions of Lactobacillus S-layer proteins
Adhesive functions
The most often proposed function for Lactobacillus S-layers
is the mediation of bacterial adherence to various targets. In a
number of studies, the loss of the S-layer protein from the
bacterial surface by chemical means (Kos et al. 2003; Garrote
et al. 2004; Frece et al. 2005; Chen et al. 2007; Jakava-
Viljanen and Palva 2007; Tallon et al. 2007) or the covering
of the layer by other molecules during prolonged cultivation
(Schneitz et al. 1993) has been shown to decrease adhesion to
different targets, but the role of the S-layer protein in adher-
ence in these studies has not been directly demonstrated. The
haemagglutinating activity of L. acidophilus JCM 1034 and
the mucin binding activities of related strains were shown to
be linked to their S-layer proteins, although the involvement
of other guanidine hydrochloride-extractable components of
the cell wall in this lectin-like activity could not be excluded,
and/or the effect of aggregation of the S-layer proteins
possibly causing unspecific effects could not be completely
ruled out (Yamada et al. 1994; Takahashi et al. 1996).
Likewise, in the study of Golowczyc et al. (2009), where the
carbohydrate-dependent co-aggregation of L. kefir with yeast
or red blood ce lls was suggested to be S-layer-mediated,
conclusions were drawn from the effects of LiCl and SDS
treatments of L. kefir cells, and the solubility of the S-layer
proteins in the LiCl extracts of L. kefir used in the aggregation
assays was not demonstrated. Also, in the study of Uchida et
al. (2006), which showed an interaction between the S-layer
protein of L. brevis OLL 2772 and human blood group A
antigen by a surface plasmon resonance assay, a dialysed
guanidine hydrochloride extract of bacterial cells was used
as an analyte, leaving the effects of the levels of purity and
solubility of the protein debatable.
The role of a Lactobacillus S-layer protein in bacterial
adherence has been unequivocally shown in a few instances,
where recombinant S-layer proteins (Toba et al. 1995; Sun
et al. 2012), S-layer-negative mutants (Konstantinov et al.
2008), highly purified monomeric proteins (de Leeuw et al.
2006) or a surface display system for the S-layer protein
(Hynönen et al. 2002) was used.
Recombinant forms of CbsA of L. crispatus JCM 5810
(Toba et al. 1995; Sillanpää et al. 2000) and SlpB of
L. crispatus K313 (Sun et al. 2012) both bind collagen types
I and IV. In contrast, the recombinant form of the non-
expressed SlpB protein of L. crispatus JCM 5810, which
showed 43 % sequence identity to CbsA at the amino acid
level, does not bind these collagens (Sillanpää et al. 2000).
L. crispatus JCM 5810 cells also bind to collagen-rich regions
of chicken colon in vitro, while guanidine hydrochloride-
treated cells are unable to bind, suggesting biological rele-
vance for the observed collagen binding of CbsA (Sillanpää et
al. 2000). The N-terminal amino acid residues at position
31274 of mature CbsA are needed for collagen binding,
and mostly the same residues (32271) are needed for the
reassembly of CbsA monomers to an S-layer, suggesting the
dependence of collagen binding on the periodic structure
(Sillanpää et al. 2000). The display of CbsA on the surface
of a non-S-layered L. casei strain through a PrtP cell wall
anchor rendered the recombinant cells able to bind collagens,
although the anchoring system probably does not allow the
monomers to form a true S-layer (Martinez et al. 2000). While
the sequence similarity between CbsA and the S-layer protein
SlpB of L. crispatus K313 is restricted to the C-terminal cell
wall binding region, the N-terminal part of SlpB also binds
collagen. More than 341 N-terminal amino acid residues are
needed for binding (Sun et al. 2012), but no data are available
if the collagen binding and polymerization require the same
amino acid residues as demonstrated for CbsA. The recombi-
nant protein comprising the N-terminal part of the S-layer
protein of L. crispatus strain ZJ001, in turn, binds to detached
HeLa cells (Chen et al. 2009).
Appl Microbiol Biotechnol (2013) 97:52255243 5233
Page 9
A further well-characterized adhesive Lactobacillus S-
layer protein is SlpA on L. acidophilus NCFM cells, which
binds to the dendritic cell-specific ICAM-3-grabbing
nonintegrin (DC-SIGN) receptor on human immature dendrit-
ic cells, leading to cytokine production and modulation of the
immune response. The slpA knock-out mutant expressing
SlpB and SlpX is significantly reduced in binding to
DC-SIGN, and the interaction leads to the induction of differ-
ent cytokines (Konstantinov et al. 2008). Initially, a role for
SlpA of L. acidophilus NCFM was demonstrated in binding to
Caco-2 cells as the binding of the knock-out mutant of the
gene in locus LBA 1377 was decreased by 84 % compared
with the wild type (Buck et al. 2005). However, the gene in
locus LBA 1377 was subsequently annotated as a putative
mucus binding protein, and SlpA was localized in locus LBA
0169. Nevertheless, SlpA encoded by the gene in LBA 0169
has later been detected on the surface of several Caco-2 cell
binding L. acidophilus isolates (Ashida et al. 2011). It is
identical in sequence with the SA protein of L. acidophilus
ATCC 4356, suggesting that these strains might have similar
adhesive and immunomodulatory properties as well as surface
layer-associated murein hydrolase activity (see Protective,
enzymatic and other functions).
Finally, the S-layer protein SlpA of L. brevis ATCC 8287
mediates the binding of the bacterial cells to several human
epithelial cell lines and fibronectin, as revealed by expressing
fragments of slpA in a surface display system based on the H7
flagella of Escherichia coli. Eighty-one amino acids from the
N-terminal part of SlpA were sufficient to confer binding to
epithelial cells (Hynönen et al. 2002). The binding functions of
SlpA were verified using a non-adhesive Lactococcus strain, in
which a nicin-inducible surface display system with a cell wall-
anchoring peptide from lactococcal AcmA was used to display
the binding region of SlpA on the cell surface (Åvall-
Jääskeläinen et al. 2003). Khang et al. (2009), in turn, used
purified SlpA green fluorescent protein (GFP) fusion proteins
to show the binding of SlpA to undifferentiated human HT-29
cells, although more attention could have been focused on
controlling the specificity of the interaction in this study. The
binding of SlpA to extracellular matrix proteins has been
further confirmed by de Leeuw et al. (2006), who demonstrated
a direct interaction between the chromatographically purified,
monomeric form of SlpA and soluble fibronectin or laminin by
surface plasmon resonance assays. The binding mechanisms to
fibronectin and laminin were found to be different and pro-
posed to be mediated by different regions of SlpA.
In addition to the above-mentioned examples of specific
binding, the S-layers of lactobacilli may have a non-specific
enhancing effect on binding to surfaces, like those encountered
in the gastrointestinal or urogenital tract, as they are generally
hydrophobic and may thus enhance adhesion to hydrophobic
surfaces (van der Mei et al. 2003). This effect is, however ,
dependent on the ionic strength of the environment (Vadillo-
Rodríguez et al. 2005). Some Lactobacillus S-layers, but not
all, have even been found to change their surface hydropho-
bicity in response to environmental ionic strength, thus possi-
bly offering different binding capacities. In the case of the SA
protein of L. acidophilus ATCC 4356, the decrease in hydro-
phobicity associated with higher environmental ionic strength
is hypothesized to be due to the shrinkage of the S-layer and
the consequent partial exposure of the inner, more hydrophilic
C-terminal domain (Vadillo-Rodriguez et al. 2004).
Protective, enzymatic and other functions
To date, a couple of functions other than adhesion have been
shown or proposed for Lactobacillus S-layer proteins. The
presence of the S-layer protein decreases the susceptibility of
L. helveticus ATCC 12046 to mutanolysin (Lortal et al. 1992),
the susceptibility of L. acidophilus M92 to gastric and pan-
creatic juice (Frece et al. 2005) and the susceptibility of
L. hilgardii wine isolate B706 to wine-related conditions like
the presence of copper sulphate or tannic acid (Dohm et al.
2011). On the other hand, the S-layer proteins of brewery
isolates of L. brevis were deduced not to act as barriers for
the hop bittering substance isohumulone (Yasui et al. 1995).
The auxiliary S-layer c omponent SlpX of L. acidophilus
NCFM probably affects the permeability of the S-layer as
the slpX-negative mutant is more susceptible to SDS and more
resistant to bile than the wild type (Goh et al. 2009). The
C-terminal part of the S-layer protein SA of L. acidophilus
ATCC 4356 has been shown to have murein hydrolase
(endopeptidase) activity against the cell wall of, e.g.,
Salmonella enterica (Prado Acosta et al. 2008), but the bio-
logical relevance of this finding was not investigated. A role
as a phage receptor has been suggested for the S-layer protein
of L. helveticus CNRZ 892 (Callegari et al. 1998).
Applications of Lactobacillus S-layer proteins
Vaccine development
During the recent years, the number of applications devel-
oped or suggested for Lactobacillus S-layer proteins has
gradually increased. One of the fields currently extensively
studied is the construction of S-layer fusion proteins for use
in imm unization in man or animals. Especially the develop-
ment of live Lactoba cillus strains carrying S-layers com-
posed of hybrid proteins on their surface is of interest as
such strains have potential for use as live mucosal vaccines.
Several findings support this approach: (1) The non-
pathogenicity of lactobacilli and their ability to survive the
passage through the gastrointestinal tract enables a simple,
safe and efficient route of oral antigen delivery; (2) A clear
relationship exists between antigen expression levels and
5234 Appl Microbiol Biotechnol (2013) 97:52255243
Page 10
immune response (Grangette et al. 2001; Seegers 2002), and
surface displ ay with an S-layer protein as a carrier results in
the simultaneous expression of the foreign peptide as
hundreds of thousands of regularly arranged copies on the
cell; (3) Lactobacillus cells as well as surface layer arrays
have intrinsi c adjuvant properti es (Smith et al. 1993;
Miettinen et al. 1996; Maassen et al. 2000; Seegers 2002;
Bega nović et al. 2011), and the simultaneous display of
immunomodulating molecules in the S-layer could further
enhance or direct the immune response; (4) As antigen
carrier systems can be significantly improved by the co-
display of adhesins (Cano et al. 1999; Liljeqvist et al.
1999), the various binding functions described earlier might
prove advantageous in the targeted delivery of antigenic
molecules. For instance, the identification of the S-layer
protein of L. acidophilus NCFM as the binding ligand for
the dendritic cell-specific antigen DC-SIGN (Konstantinov
et al. 2008) makes this probiotic stra in or its S-layer an
attractive tool for oral vaccine design. So far, only a system
utilizing L. acidophilus NCFM cells, not yet its S-layer, as a
carrier for an antigen with a small dendritic cell-targeting
peptide has been developed (Mohamadzadeh et al. 2009).
The develo pment of Lactobacillus vaccine carriers based
on hybrid S-layers is at an early stage. Small model peptides
have been displayed in each monomer of the S-layer of
L. brevis ATCC 8287 (Åvall-Jääskeläinen et al. 2002) and
L. acidophilus ATCC 4356 (Smit et al. 2002) by chromo-
somal integration based on homologous recombination.
Similarly, surface display of GFP in the S-layer proteins
on chicken Lactobacillus isolates has been achieved by
utiliz ing the gene fragment encompa ssing the e xpression
and secretion signals and the region encoding the cell wall
binding domain of the S-layer protein of L. crispatus (Mota
et al. 2006). As a prerequisite for hybrid S-layer-based
vaccine development, a systematic mapping of surface-
accessible amino acids has been performed for the S-layer
protein of L. brevis ATCC 8287 (Vilen et al. 2009). Apart from
hybrid S-layer proteins, non-adhesive antigen delivery vehi-
cles like lactococci have been rendered adhesive by the sur-
face display of adhesive S-layer proteins or S-layer-derived
peptides such as those of L. crispatus JCM 5810 (Martinez et
al. 2000)orL. brevis ATCC 8287 (Åvall-Jääskeläinen et al.
2003). Preliminary experiments have also been performed in
the field of passive immunization by utilizing the epithelial
cell binding S-layer protein of L. brevis KCTC 3102 (ATCC
8287) as a purified, immunoglobulin binding fusion protein to
target antibodies to the intestinal surfaces of calves in order to
prevent neonatal diarrhoea (Khang et al. 2009). In this small
field study, a higher recovery of calves from diarrhoea was
obtained by administering antiviral and antibacterial anti-
bodies in combination with the fusion protein than by admin-
istering the an tibodies alone, although the mech anism of
protection remained speculative.
Applications based on anti-adhesive and anti-infectious
effects
Another potential application is t he use of S-layers or
S-layered lactobacilli as anti-adhesive agents or as other ther-
apeutic or preventative measures against infectious diseases.
In many studies, however, the anti-adhesive effects observed
for S-layer proteins against different pathogens, as described,
e.g., for the S-layer proteins of L. crispatus (Horie et al. 2002a;
Chen et al. 2007), L. helveticus (Sherman et al. 2005;Johnson-
Henry et al. 2007)andL. kefir (Golowczyc et al. 2007), cannot
unequivocally be attributed to the surface layer proteins per se,
as the dialysed extracts used in the inhibition studies appar-
ently contained also other LiCl or guanidium hydrochloride-
extractable cell surface components as well as aggregates of
the S-layer protein, and thus the specificities of the inhibitions
were compromised. The same holds true for the study of
Martínez et al. (2012), which demonstrated the inhibition of
JUNV infection by the surface protein extract of L. acidoph-
ilus ATCC 4356 in a DC-SIGN expressing cell culture model,
although the interaction between SlpA of ATCC 4356/NCFM
and DC-SIGN has previously been demonstrated
(Konstantinov et al. 2008). Similarly, the results of the study
of Carasi et al. (2011), which showed the potential of L. kefir
S-layer proteins for decreasing the cytopathic effect of
Clostridium difficile culture supernatants or toxins to Vero
cells, were obtained using unpurified LiCl extracts of L. kefir
cells and thus cannot be considered as fully conclusive.
The identification of the S-layer protein of L. acidophilus
NCFM as the binding ligand for the dendritic cell-specific
antigen DC-SIGN and the different cytokine responses
elicited by SlpA and the alternative S-layer protein SlpB
(Konstantinov et al. 2008) have raised interest in studying
the contribution of the S-layer protein of NCFM to its probi-
otic action. There is an association between DC-SIGN poly-
morphisms and allergic sensitization, and the colonization of
1-month-old infants by L. acidophilus slightly decreases the
risk of allergic dermatitis (Penders et al. 2010), but still the
role of the SlpADC-SIGN interaction in immun ological
tolerance and its biological significance is far from clear. On
the other hand, in the cellular mechanisms of inflammatory
bowel disease, lipoteichoic acids of L. acidophilus NCFM
seem to have a major role, as pre-treatment of mice by LTA-
negative L. acidophilus NCFM ameliorated dextran sulphate
sodium-induced inflammatory colitis (Mohamadzadeh et al.
2011). Interestingly, the presence of SlpA on an slpB
-
slpX
-
mutant actually increases the pro-inflammatory action of LTA
compared with the LTA-expressing parental strain (Zadeh et
al. 2012). Thus, a role for SlpB and SlpX in regulating LTA-
induced inflammation has been suggested (Lightfoot and
Mohamadzadeh 2013). A mutant lacking SlpB and SlpX but
carrying SlpA also tends to be cleared from the mouse gas-
trointestinal tract more rapidly than the wild type, but the
Appl Microbiol Biotechnol (2013) 97:52255243 5235
Page 11
mechanism is not known (Zadeh et al. 2012). Some indica-
tions about the contribution of the S-layer protein of L.
acidophilus ATCC 4356/NCFM to probiotic action were
obtained in the study of Li et al. (2011b), in which the
chromatographically purified S-layer protein SlpA was shown
to counteract a Salmonella-induced transepithelial electric
resistance decrease and IL-8 secretion as well as to inhibit
Salmonella-induced F-actin rearrangements and JNK and p38
activation in Caco-2 cells. In another study, the same protein
was shown to activate the ERK1/2 signaling pathway and to
inhibit caspase-3 activity in Salmonella-infected Caco-2 cells,
thereby decreasing Salmonella-induced Caco-2 cell apoptosis
and cell damage (Li et al. 2011a). Interestingly, apart from
probiotics, even the S-layer protein of a dairy strain of
L. helveticus was found to reduce NF-κB activation in Caco-
2 cells while triggering the expression of TLR2-mediated pro-
inflammatory factors in human and mouse macrophages, thus
showing stimulatory effects on innate immunity. In this study,
special effort was exerted to demonstrate the purity of the
S-layer preparation used (Taverniti et al. 2012).
A novel application has been suggested for the S-layer
protein SA of L. acidophilus ATCC 4356. The murein hydro-
lase activity o f the C-terminal part of SA, shown by the
degradation of cell wall preparations of Gram-negative path-
ogens (Prado Acosta et al. 2008), acts synergistically with the
well-documented antibacterial agent, nisin, against bacterial
pathogens. The combination of these two inhibits the growth
of both Gram-positive and Gram-negative pathogens, as ex-
emplified by Salmonella enterica, Staphylococcus aureus and
Bacillus cereus, through a mechanism that involves the dissi-
pation of the transmembrane proton motive force (Prado-
Acosta et al. 2010).
Chemical conjugates and liposomes
Some biochemical and physical studies of isolated Lactobacillus
S-layer proteins aiming at biotechnological or clinical applica-
tions have also been initiated. Small molecular probes like biotin
or fluorescein isothiocyanate have been conjugated to purified
S-layer proteins of L. brevis using amine-based coupling chem-
istry. The S-layer protein bioconjugates formed, purified by
affinity chromatography, were capable of self-assembling into
regular layers, where the surface coverage of the conjugated
molecules is homogeneous and the density controllable. The
method offers a way to display several different and high
molecular weight molecules at an interface (Sampathkumar
and Gilchrist 2004). Further , positively charged liposomes have
been coated by the S-layer proteins of L. br evis and L. kefir
(Hollmann et al. 2007, 2010a). Importantly for future vaccine
applications, the S-layer proteins markedly increased the stabil-
ity of the liposomes under unfavourable conditions, e.g. at low
pH, at high temperature or in the presence of bile salts or
pancreatic extract, as measured by the rele ase o f a fluorescent
marker compound, and the effect could be further enhanced by
cross-linking the proteins with glutaraldehyde (Hollmann et al.
2007). The stabilizing effect was shown to be based on the
neutralization of the charge repulsion between stearylamine
molecules in the liposome, leading to increased acyl chain
packing and membrane rigidity. The glycosylated S-layer pro-
tein of L. kefir had higher affinity to the liposomes than the non-
glycosylated one of
L. brevis (Hollmann et al. 2010a), but no
striking differences were found between the liposome-
stabilizing effects of the two proteins (Hollmann et al. 2007).
The kinetics of the interaction between the S-layer pro-
tein and a lipid monolayer was found to be dependent on
the composition of the membrane and could be modulat-
ed by components t hat modify the hydration state of the
lipid i nterface (Hollmann et al. 2010b).
Expression/secretion signals in heterologous gene
expression
The expre ssion and/or secretion signals of Lactobacillus
S-layer protein genes have also been utilized in biotechnolog-
ical applications (Savijoki et al. 1997; Kahala and Palva 1999;
Novotny et al. 2005; Lizier et al. 2010; Zhang et al. 2010a).
The region encompassing the double promoter and the
ribosome-binding sequence up to the start of the slpA gene of
L. brevis ATCC 8287 (Kahala and Palva 1999), or the region
containing additionally the slpA signal peptide gene sequence
(Savijoki et al. 1997), have been used in Lactobacillus and
Lactococcus hosts for intracellular or extracellular protein
production. Using slpA expression and secretion signals, se-
cretion levels of beta-lactamase up to 80 mg/l have been
achieved. Differences exist between the recognition efficiency
of the signals in different hosts; high-level extracellular protein
production with slpA signals was achieved in Lactococcus
lactis and Lactobacillus plantarum and moderate production
in L. gasseri,whileinL. casei the expression signals were not
recognized (Savijoki et al. 1997). On the other hand, the
promoter region of L. acidophilus ATCC 4356 S-layer protein
gene was highly active in L. casei (Boot et al. 1996b) but
functioned poorly in L. reuteri (Lizier et al. 2010). The
transcriptional activity in heterologous hosts could be signifi-
cantly improved or decreased by the modification of native slp
gene promoter, and both strain- and context-dependent effects
of the introduced sequences have been detected (McCracken
and Timms 1999). Adding merely the signal peptide encoding
sequence of slpA from L. brevis ATCC 8287 upstream of the 5
end of the human interferon alpha gene increased the secretion
efficiency of interferon alpha in L. lactis threefold compared to
the signal peptide encoding sequence of lactococcal Usp45,
but the total interferon production was lower in the strain with
the slpA signal peptide encoding sequence (Zhang et al.
2010a). The recent development of a counterselective gene
replacement system for the chromosomal integration of genes
5236 Appl Microbiol Biotechnol (2013) 97:52255243
Page 12
in L. acidophilus (Goh et al. 2009) has enabled protein pro-
duction using chromosomally located S-layer protein pro-
moters in lactobacilli. The promoter of the S-layer protein
gene slpA of L. acidophilus NCFM was found to direct the
expression of the reporter gene gusA3, leading to a higher
expression level than that obtained from a plasmid, when the
reporter gene was placed between the stop codon and the
transcriptional terminator of slpA (Douglas and Klaenhammer
201 1).
Concluding remarks
Present knowledge about Lactobacillus S-layer proteins
supports the view of Gram-positive S-layer proteins as
two-domain entities, where one domain is responsible for
cell wall binding and the other for the self-assembly of the
regular surface layer. The common theme of carbohydrates
as the binding sites for S-layer proteins in the cell walls of
Gram-positive bacteria is also supported, although the de-
tailed anchoring molecules and mechanisms vary among
different lactobacilli. Biophysical methods are increasingly
utilized in the structural studies of S-layers, and together
with computer model ling-based methods they will probably
allow for high-resolution structure s of Lactobacillus S-layer
proteins, which currently are scarce owing to difficulties in
obtaining high-quality crystals for X-ray crystallography.
As food-grade and potentially probiotic organisms,
lactobacilli are excellent candidates for health-related appli-
cations like live oral vaccines, where their ability to survive
in the gastrointestinal tract could be utilized and their
S-layer proteins could be used as carriers of antigens or
other medically important molecules, possibly in combina-
tion with immunostimulatory or adhesive molecules. In this
approach, the polymeric nature and inherent adjuvant prop-
erties of S-layers are apparently an advantage. Further, im-
mobilization of recom binant S-layer proteins combined with
the display of foreign molecules in the S-layer forms the
basis for the development of different solid-phase reagents,
such as biocatalysts, diagnostic devices, biose nsors and
biosorbents, where the typical positive charge of the cell
wall-binding domain of Lactobacillus S-layer proteins could
augment the immobilization. While most of the biotechno-
logical applications of S-layer proteins so fa r have been
designed for the S-layer proteins of thermophilic bacilli,
the increasing knowledge about the structure and biology
of Lactobacillus S-layer proteins, as well as the developing
tools to genetically manipulate these organisms, will pave
the way to applications utilizing the S-layer proteins of these
beneficial and easily cultivable bacteria.
Acknowledgments This work was performed in the Centre of
Excellence on Microbial Food Safety Research, Academy of Finland.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
References
Ahn JS, Chandramohan L, Liou LE, Bayles KW (2006) Characterization
of CidR-mediated regulation in Bacillus anthracis reveals a previ-
ously undetected role of S-layer proteins as murein hydrolases. Mol
Microbiol 62:11581169
Altermann E, Russell WM, Azcarate-Peril A, Barrangou R, Buck BL,
McAuliffe O, Souther N, Dobson A, Duong T, Callanan M, Lick
S, Hamrick A, Cano R, Klaenhammer TR (2005) Complete
genome sequence of the probiotic lactic acid bacterium
Lactobacillus acidophilus NCFM. PNAS 102:39063912
Antikainen J, Anton L, Sillanpää J, Korhonen TK (2002) Domains in
the S-layer protein CbsA of Lactobacillus crispatus involved in
adherence to collagens, laminin and lipoteichoic acids and in self-
assembly. Mol Microbiol 2:381394
Ashida N, Yanagihara S, Shinoda T, Yamamoto N (2011)
Characterization of adhesive molecule with affinity to Caco-2
cells in Lactobacillus acidophilus by proteome analysis. J Biosci
Bioeng 112:333337
Ausiello CM, Cerquetti M, Fedele G, Spensieri F, Palazzo R, Nasso M,
Frezza S, Mastrantonio P (2006) Surface layer proteins from
Clostridium difficile induce inflammatory and regulatory cyto-
kines in human monocytes and dendritic cells. Microbes Infect
8:26402646
Åvall-Jääskeläinen S, Palva A (2005) Lactobacillus surface layers and
their applications. FEMS Microbiol Rev 29:511529
Åvall-Jääskeläinen S, Kylä-Nikkilä K, Kahala M, Miikkulainen-Lahti T,
Palva A (2002) Surface display of foreign epitopes on the
Lactobacillus brevis S-layer . Appl Environ Microbiol 68:59435951
Åvall-Jääskeläinen S, Lindholm A, Palva A (2003) Surface display of
the receptor-binding region of the Lactobacillus brevis S-layer
protein in Lactococcus lactis provides nonadhesive lactococci
with the ability to adhere to intestinal epithelial cells. Appl
Environ Microbiol 69:22302236
Åvall-Jääskeläinen S, Hynönen U, Ilk N, Pum D, Sleytr UB, Palva A
(2008) Identification and characterization of domains responsible
for self-assembly and cell wall binding of the surface layer protein
of Lactobacillus brevis ATCC 8287. BMC Microbiol 8:165
Axelsson L (1998) Lactic acid bacteria: Classification and phys-
iology. Lactic acid bacteria: microbiology and functional as-
pects In: Salmi nen S, von Wright A (eds), pp. 173. Marcel
Dekker, New York.
Azcarate-Peril MA, Altermann E, Goh YJ, Sanozky-Dawes RB, Pfeiler
EA, O'Flaherty S, Buck BL, Dobson A, Duong T, Miller MJ,
Barrangou R, Klaenhammer TR (2008) Analysis of the genome
sequence of Lactobacillus gasseri ATCC 33323 reveals the mo-
lecular basis of an autochthonous intestinal organism. Appl
Environ Microbiol 74:46104625
Barker DC, Thorne KJ (1970) Spheroplasts of Lactobacillus casei and
the cellular distribution of bactoprenol. J Cell Sci 7:755785
Beganović J, Frece J, Kos B, Leboš Pavunc A, Habjanič K, Sušković J
(2011) Functionality o f the S-layer protein from the probiotic
strain Lactobacillus helveticus M92. Antonie Van Leeuwenhoek
100:4353
Boot HJ, Pouwels PH (1996) Expression, secretion and antigenic varia-
tion of bacterial S-layer proteins. Mol Microbiol 21:11171123
Boot HJ, Kolen CP, Pouwels PH (1995) Identific ation, cl oning,
and nucleotide s equence of a silent S-layer protein gene of
Lactobacillus acidophilus ATCC 4356 which has extensive
Appl Microbiol Biotechnol (2013) 97:52255243 5237
Page 13
similarity w ith the S -laye r protei n gene o f this species. J
Bacteriol 177:72227230
Boot HJ, Kolen CP, Pouwels PH (1996a) Interchange of the
active and silent S-layer protein genes of Lactobacillus aci-
dophilus by inversion of the chromosomal slp segment. Mol
Microbiol 21:799809
Boot HJ, Kolen CP, Andreadaki FJ, Leer RJ, Pouwels PH (1996b) The
Lactobacillus acidophilus S-layer protein gene expression site
comprises two consensus promoter sequences, one of which di-
rects transcription of stable mRNA. J Bacteriol 178:53885394
Boot HJ, Kolen CP, Pot B, Kersters K, Pouwels PH (1996c) The presence
of two S-layer-protein-encoding genes is conserved among species
related to Lactobacillus acidophilus. Microbiology 142:23752384
Brahamsha B (1996) An abundant cell-surface polypeptide is required
for swimming by the nonflagellated marine cyanobacterium
Synechococcus. Proc Natl Acad Sci U S A 93:65046509
Buck BL, Altermann E, Svingerud T, Klaenhammer TR (2005)
Functional analysis of putative adhesion factors in Lactobacillus
acidophilus NCFM. Appl Environ Microbiol 71:83448351
Cachat E, Priest FG (2005) Lactobacillus suntoryeus sp. nov., isolated
from malt whisky distilleries. Int J Syst Evol Microbiol 55:3134
Calabi E, Ward S, Wren B, Paxton T, Panico M, Morris H, Dell A,
Dougan G, Fairweather N (2001) Molecular characterization of
the surface layer p roteins from Clostr idium di fficile.Mol
Microbiol 40:11871199
Callanan M, Kaleta P, O'Callaghan J, O'Sullivan O, Jordan K,
McAuliffe O, Sangrador-Vegas A, Slattery L, Fitzgerald GF,
Beresford T, Ross RP (2008) Genome sequence of Lactobacillus
helveticus, an organism distinguished by selective gene loss and
insertion sequence element expansion. J Bacteriol 190:727735
Callegari ML, Riboli B, Sanders JW, Cocconcelli PS, Kok J, Venema
G, Morelli L (1998) The S-layer gene of Lactobacillus helveticus
CNRZ 892: cloning, sequence and heterologous expression.
Microbiology 144(Pt 3):719726
Cano F, Liljeqvist S, Nguyen TN, Samuelson P, Bonnefoy JY, Ståhl S,
Robert A (1999) A surface-display ed cholera toxin B peptide
improves antibody responses using food-grade staphylococci for
mucosal subunit vaccine delivery. FEMS Immunol Med
Microbiol 25:289298
Carasi P, Trejo FM, Pérez PF, De Antoni GL, Serradell Mde L (2011)
Surface proteins from Lactobacillus kefir antagonize in vitro cyto-
toxic effect of Clostridium difficile toxins. Anaerobe 18:135142
Cerquetti M, Molinari A, Sebastianelli A, Diociaiuti M, Petruzzelli R,
Capo C, Mastrantonio P (2000) Characterization of surface layer
proteins from different Clostridium difficile clinical isolates.
Microb Pathog 28:363372
Chen X, Xu J, Shuai J, Chen J, Zhang Z, Fang W (2007) The S-layer
proteins of Lactobacillus crispatus strain ZJ001 is responsible for
competitive exclusi on against Escherichia coli O157:H7 and
Salmonella typhimurium. Int J Food Microbiol 115:307
312
Chen X, Chen Y, Li X, Chen N, Fang W (2009) Characterization of
surface layer proteins in Lactobacillus crispatus isolate ZJ001. J
Microbiol Biotechnol 19:11761183
Collins MD, Rodrigues U, Ash C, Aguirre M, Farrow JAE, Martinez-
Murcia A, Phillips BA, Williams AM, Wallbanks S (1991)
Phylogenetic analysis of the genu s Lactobacillus and related
lactic acid bacteria as determined by reverse trans criptase se-
quencing of 16S rRNA. FEMS Microbiol Lett 77:512
de la Fuente-Núñez C, Mertens J, Smit J, Hancock RE (2012) The
bacterial surface layer provides protection against antimicrobial
peptides. Appl Environ Microbiol 78:54525456
de Leeuw E, Li X, Lu W (2006) Binding characteristics of the
Lactobacillus brevis ATCC 8287 surface layer to extracellular
matrix proteins. FEMS Microbiol Lett 260:210215
De Vuyst L, Degeest B (1999) Heteropolysaccharides from lactic acid
bacteria. FEMS Microbiol Rev 23:153177
Dohm N, Petri A, Schlander M, Schlott B, König H, Claus H (2011)
Molecular and biochemical properties of the S-layer protein from
the wine bacterium Lactobacillus hilgardii B706. Arch Microbiol
193:251261
Doig P, Emödy L, Trust TJ (1992) Binding of laminin and fibronectin
by the trypsin-resistant major structural domain of the crystalline
virulence surface array protein of Aeromonas salmonicida. J Biol
Chem 267:4349
Douglas GL, Klaenhammer TR (2011) Directed chromosomal integra-
tion and expression of the reporter gene gusA3 in Lactobacillus
acidophilus NCFM. Appl Environ Microbiol 77:73657371
Egelseer E, Schocher I, Sára M, Sleytr UB (1995) The S-layer
from Bacillus stearothermophilus DSM 2358 functions as an
adhesion site for a high-molecular-weight amylase. J
Bacteriol 177:14441451
Egelseer EM, Schocher I, Sleytr UB, Sára M (1996) Evidence that an
N-terminal S-layer protein fragment triggers the release of a cell-
associated high-molecular-weight amylase in Bacillus
stearothermophilus ATCC 12980. J Bacteriol 178:56025609
Engelhardt H (2007a) Are S-layers exoskeletons? The basic function of
protein surface layers revisited. J Struct Biol 160:115124
Engelhardt H (2007b) Mechanism of osmoprotection by archaeal S-
layers: a theoretical study. J Struct Biol 160:190199
Fagan RP, Albesa-Jove D, Qazi O, Svergun DI, Brown KA,
Fairweather NF (2009) Structural insights into the molecular
organization of the S-layer from Clostridium difficile.Mol
Microbiol 71:13081322
Felis GE, Dellaglio F (2007) Taxonomy of Lactobaci lli a nd
Bifidobacteria. Curr Issues Intest Microbiol 8:4461
Fouet A (2009) The surface of Bacillus anthracis. Mol Aspects Med
30:374385
Fouet A, Mesnage S, Tosi-Couture E, Gounon P, Mock M (1999)
Bacillus anthracis s urface: capsule and S-layer. J Appl
Microbiol 87:251255
Frece J, Kos B, Svetec IK, Zgaga Z, Mrsa V, Suskovic J (2005)
Importance of S-layer proteins in probiotic activity of
Lactobacillus acidophilus M92. J Appl Microbiol 98:285292
Garrote GL, Delfederico L, Bibiloni R, Abraham AG, Perez PF,
Semorile L, De Antoni GL (2004) Lactobacilli isolated from kefir
grains: evidence of the presence of S-layer proteins. J Dairy Res
71:222230
Gatti M, Rossetti L, Fornasari ME, Lazzi C, Giraffa G, Neviani E
(2005) Heterogeneity of putative surface layer proteins in
Lactobacillus helveticus. Appl Environ Microbiol 71:75827588
Gilmour R, Messner P, Guffanti AA, Kent R, Scheberl A, Kendrick N,
Krulwich TA (2000) Two-dimensional gel electrophoresis analy-
ses of pH-dependent protein expression in facultatively
alkaliphilic Bacillus pseudofirmus OF4 lead to characterization
of an S-layer protein with a role in alkaliphi ly. J B acteri ol
182:59695981
Goh YJ, Klaenhammer TR (2010) Functional roles of aggregation-
promoting-l ike factor in stress tolerance and adherence of
Lactobacillus acidophilus NCFM. Appl Environ Microbiol
76:50055012
Goh YJ, Azcarate-Peril MA, O'Flaherty S, Durmaz E, Valence F,
Jardin J, Lortal S, Klaenhammer TR (2009) Development and
application of a upp-based counterselective gene replacement
system for study of the S-layer protein SlpX in Lactobacillus
acidophilus NCFM. Appl Environ Microbiol 75:30933105
Golowczyc MA, Mobili P, Garrote GL, Abraham AG, De Antoni GL
(2007) Protective action of Lactobacillus kefir carrying S-layer
protein against Salmonella enterica serovar Enteritidis. Int J Food
Microbiol 118:264273
Golowczyc MA, Mobili P, Garrote GL, de Los Angeles Serradell M,
Abraham AG, De Antoni G L (2009) Interaction between
Lactobacillus kefir and Saccharomyces lipolytica isolated from
5238 Appl Microbiol Biotechnol (2013) 97:52255243
Page 14
kefir grains: evidence for lectin-like activity of bacterial surface
proteins. J Dairy Res 76:111116
Grangette C, Müller-Alouf H, Goudercourt D, Geoffroy MC, Turneer
M, Mercenier A (2001) Mucosal immune responses and protec-
tion against tetanus toxin after intranasal immunization with re-
combinant Lactobacillus plantarum. Infect Immun 69:15471553
Guo CF, Zhang LW, Han X, Yi HX, Li JY, Tuo YF, Zhang YC, Du M,
Shan YJ, Yang L (2012) Screening for cholesterol-lowering pro-
biotic based on deoxycholic acid removal pathway and studying
its functional mechanisms in vitro. Anaerobe 18:516522
Hagen KE, Guan LL, Tannock G W, Korver DR, Allison GE
(2005) Detect ion, characterization,andinvitroandinvivo
expression of genes encoding S-proteins i n Lactobacil lus
gallinarum strains isolated from chicken crops. Appl
Environ Microbiol 71:66336643
Hancock IC, Poxton IR (1988) Bacterial cell surface techniques. Wiley,
New York
Hao P, Zheng H, Yu Y, Ding G, Gu W, Chen S, Yu Z, Ren S, Oda M,
Konno T, Wang S, Li X, Ji ZS, Zhao G (2011) Complete sequenc-
ing and pan-genomic analysis of Lactobacillus delbrueckii subsp.
bulgaricus reveal its genetic basis for industrial yogurt produc-
tion. PLoS One 17:e15964
Hayashi H, Takahashi R, Nishi T, Sakamoto M, Benno Y (2005)
Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal
human colonic microbiota using 16S rRNA gene libraries and
terminal restriction fragment length polymorphism. J Med
Microbiol 54:10931101
Heinl S, Wibberg D, Eikmeyer F, Szczepanowski R, Blom J, Linke B,
Goesmann A , Gr ab herr R, Schwab H, Pühler A, Schlüter A
(2012) Insights into the completely annotated genome of
Lactobacillus buchneri CD034, a strain isolated from stable grass
silage. J Biotechnol 161:153166
Hollmann A, Delfederico L, G likmann G, De Antoni G, Semorile
L, Disalvo EA (2007) Characterization of li posomes coated
with S-layer proteins from l actobacilli. Biochim Biophys
Acta 1768:393400
Hollmann A, Delfederico L, De Antoni G, Semorile L, Disalvo EA
(2010a) Interaction of bacterial surface layer proteins with lipid
membranes: synergysm between surface charge density and chain
packing. Colloids Surf B: Biointerfaces 79:191197
Hollmann A, Delfederico L, De Antoni G, Semorile L, Disalvo EA
(2010b) Relaxation processes in the adsorption of surface layer
proteins to lipid membranes. J Phys Chem B 114:1661816624
Horie M, Ishiyama A, Fujihira-Ueki Y, Sillanpää J, Korhonen TK,
Toba T (2002a) Inhibition of the adherence of Escherichia coli
strains to basement membrane by Lactobacillus crispatus ex-
pressing an S-layer. J Appl Microbiol 92:396403
Horie M, Kajikawa HS, Toba T (2002b) Identification of Lactobacillus
crispatus by polymerase chain reaction targeting S-layer protein
gene. Lett Appl Microbiol 35:5761
Howard L, Tipper DJ (1973) A polypeptide bacteriophage receptor:
modified cell wall protein subunits in bacteriophage-resistant mu-
tants of Bacillus sphaericus strain P-1. J Bacteriol 113:14911504
Hu S, Kong J, Sun Z, Han L, Kong W, Yang P (2011) Heterologous
protein display on the cell surface of lactic acid bacteria mediated
by the S-layer protein. Microb Cell Fact 10:86
Hurmalainen V, Edelman S, Antikainen J, Baumann M, Lähteenmäki
K, Korhonen TK (2007) Extracellular proteins of Lactobacillus
crispatus enhance activation of human plasminogen.
Microbiology 153:11121122
Hynönen U, Westerlund-Wikström B, Palva A, Korhonen TK (2002)
Identification by flagellum display of an epithelial cell- and
fibronectin-binding function in the SlpA surface protein of
Lactobacillus brevis. J Bacteriol 184:33603367
Hynönen U, Åvall-Jääskeläinen S, Palva A (2010) Characterization
and separate activities of the two promoters of the Lactobacillus
brevis S-layer protein gene. Appl Microbiol Biotechnol 87:657
668
Ilk N, Kosma P, Puchberger M, Egelseer EM, Mayer HF, Sleytr UB,
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 181:76437646
Ilk N, Egelseer EM, Sleyt r UB (2011) S-layer fusion proteins
construction principles and applications. Curr Opin Biotechnol
22:824831
Ishiguro EE, Ainsworth T, Harkness RE, Kay WW, Trust TJ (1984) A
temperate bacteriophage specific for strains of Aero monas
salmonicida possessing A-layer, a cell surface virulence factor.
Curr Microbiol 10:199202
Jakava-Viljanen M, Palva A (2007) Is olation of surface (S) layer
protein carrying Lactobacillus species from porcine intestine and
faeces and characterization of their adhesion properties to differ-
ent host tissues. Vet Microbiol 124:264273
Jakava-Viljanen M, Åvall-Jääskeläinen S, Messner P, Sleytr UB, Palva
A (2002) Isolation of three new surface layer protein genes (slp)
from Lactobacillus brevis ATCC 14869 and characterization of
the change in their expression under aerated and anaerobic con-
ditions. J Bacteriol 184:67866795
Jankovic I, Ventura M, Meylan V, Rouvet M, Elli M, Zink R (2003)
Contribution of aggregation-promoting factor to maintenance of cell
shape in Lactobacillus gasseri 4B2. J Bacteriol 185:32883296
Johnson JL, Phelps CF, Cummins CS, London J, Gasser F (1980)
Taxonomy of the Lactobacillus acidophilus group. Int J Syst
Bacteriol 30:5368
Johnson-Henry KC, Hagen KE, Gordonpour M, Tompkins TA, Sherman
PM (2007) Surface-layer protein extracts from Lactobacillus
helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7
adhesion to epithelial cells. Cell Microbiol 9:356367
Kahala M, Palva A (1999) The expression signals of the Lactobacillus
brevis slpA gene direct efficient heterologous protein production
in lactic acid bacteria. Appl Microbiol Biotechnol 51:7178
Kahala M, Savijoki K, Palva A (1997) In vivo expression of the
Lactobacillus brevis S-layer gene. J Bacteriol 179:284286
Kandler O, Weiss N (1986) In: Sneath PHA, Mair NS, Sharpe ME,
Holt JG (eds) Regular, nonsporing Gram-positive rods. Bergey's
manual of systematic bacteriology. Williams and Wi lkins,
Baltimore, pp 12081234
Kant R, Paulin L, Alatalo E, de Vos WM, Palva A (2011a) Genome
sequence of Lactobacillus amylovorus GRL1112. J Bacteriol
193:789790
Kant R, Paulin L, Alatalo E, de Vos WM, Palva A (2011b) Genome
sequence of Lactobacillus amylovorus GRL1118, isolated from
pig ileum. J Bacteriol 193:3147
3148
Kawata T, Masuda K, Yoshino K, Fujimoto M (1974) Regular array in
thecellwallofLactobacillus fermenti as revealed by freeze-
etching and negative staining. Jpn J Microbiol 18:469476
Kay WW, Phipps BM, Ishiguro EE, Trust TJ (1985) Porphyrin binding
by the surface array virulence protein of Aeromonas salmonicida .
J Bacteriol 164:13321336
Khaleghi M, Kermanshahi RK, Yaghoobi MM, Zarkesh-Esfahani SH,
Baghizadeh A (2010) Assessment of bile salt effects on S-layer
production, slp gene expression and some physicochemical prop-
erties of Lactobacillus acidophilus ATCC 4356. J Microbiol
Biotechnol 20:749756
Khaleghi M, Kasra Kermanshahi R, Zarkesh-Esfahani SH (2011)
Effects of penicillin G on morphology and certain physiological
parameters of Lactobacillus acidophilus ATCC 4356. J Microbiol
Biotechnol 21:822829
Khang YH, Park HY, Jeong YS, Kim JA, Kim YH (2009)
Recombinant S-layer proteins of Lactobacillus brevis mediating
antibody adhesion to calf intestine alleviated neonatal diarrhea
syndrome. J Microbiol Biotechnol 19:511519
Appl Microbiol Biotechnol (2013) 97:52255243 5239
Page 15
Klingl A, Moissl-Eichinger C, Wanner G, Zweck J, Huber H, Thomm
M, Rachel R (2011) Analysis of the surface proteins of
Acidithiobacillus ferrooxidans strain SP5/1 and the new, pyrite-
oxidizing Acidithiobacillus isolate HV2/2, and their possible in-
volvement in pyrite oxidation. Arch Microbiol 193:867882
Konstantin ov SR, Smidt H, de Vos WM, Bruijns SC , Si ng h SK,
Valence F, Molle D, Lortal S, Altermann E, Klaenhammer TR,
van Kooyk Y (2008) S layer protein A of Lactobacillus acidoph-
ilus NCFM regulates immature dendritic cell and T cell functions.
Proc Natl Acad Sci U S A 105:1947419479
Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003)
Adhesion and aggregation ability of probiotic strain Lactobacillus
acidophilus M92. J Appl Microbiol 94:981987
Kotiranta AK, Ito H, Haapasalo MP, Lounatmaa K (1999) Radiation
sensitivity of Bacillus cereus with and without a crystalline sur-
face protein layer. FEMS Microbiol Lett 179:275280
Koval SF, Hynes SH (1991) Effect of paracrystalline protein surface
layers on predation by Bdellovibrio bacteriovorus. J Bacteriol
173:22442249
Lemaire M, Miras I, Gounon P, Beguin P (1998) Identification of a region
responsible for binding to the cell wall within the S-layer protein of
Clostridium thermocellum. Microbiology 144(Pt 1):211217
LiP,YinY,YuQ,YangQ(2011a)Lactobacillus acidophi lus S-
layer protein-mediated inhibition of Salmonella-induced apo-
ptosis i n Caco-2 cells. Biochem Biophys Res Commun
409:142147
Li P, Yu Q, Ye X, Wang Z, Yang Q (2011b) Lactobacillus S-layer
protein inhibition of Salmonella-induced reorganiz ation of the
cytoskeleton and activation of MAPK signalling pathways in
Caco-2 cells. Microbiology 157(Pt 9):26392646
Lightfoot YL, Mohamadzadeh M (2013) Tailoring gut immune re-
sponses with lipoteichoic acid-deficient Lactobacillus acidophi-
lus. Front Immunol 4:25
Liljeqvist S, Cano F, Nguyen TN, Uhlen M, Robert A, Ståhl S (1999)
Surface display of fun ctional fibronectin-binding domains on
Staphylococcus carnosus. FEBS Lett 446:299304
Lizier M, Sarra PG, Cauda R, Lucchini F (2010 ) Comparison of
expression vectors in La ctobacillus reuteri strains. FEMS
Microbiol Lett 308:815
Lortal S, van Heijenoort J, Gruber K, Sleytr UB (1992) S-layer of
Lactobacillus helveticus ATCC 12046: isolation, chemical char-
acterization and reformation after extraction with lithium chloride.
J Gen Microbiol 138:611618
Lupas A, Engelhardt H, Peters J, Santarius U, Volker S, Baumeister W
(1994) Domain structure of the Acetogenium kivui surface layer
revealed by el ectron crystallography and sequence analysis. J
Bacteriol 176:12241233
Maassen CB, van Holten-Neelen C, Balk F, den Bak-Glashouwer
MJ, Leer RJ, Laman JD, Boersma WJ, Claassen E (2000) Strain-
dependent induction of cytokine profiles in the gut by orally admin-
istered
Lactobacillus strains. Vaccine 18:26132623
Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E,
Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V,
Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein
DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y,
Benson A, Baldwin K, Lee JH, Díaz-Muñiz I, Dosti B, Smeianov
V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou R,
Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F,
Broadbent J, Hutkins R, O'Sullivan D, Steele J, Unlu G, Saier M,
Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D
(2006) Comparative genomics of the lactic acid bacteria. Proc
Natl Acad Sci U S A 103:1561115616
Marcotte H, Ferrari S, Cesena C, Hammarström L, Morelli L, Pozzi G,
Oggioni MR (2004) The aggregation-promoting factor of
Lactobacillus crispatus M247 and its genetic l ocus. J Appl
Microbiol 97:749756
Martinez B, Sillanpää J, Smit E, Korhonen TK, Pouwels PH (2000)
Expression of cbsA encoding the collagen-binding S-protein of
Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC
393(T). J Bacteriol 182:68576861
Martínez MG, Prado Acosta M, Candurra NA, Ruzal SM (2012) S-
layer proteins of Lactobacillus acidophilus inhibits JUNV infec-
tion. Biochem Biophys Res Commun 422:590595
Masuda K, Kawata T (1980) Reassembly of the regularly arranged
subunits in the cell wall of Lactob acillus brevis and t heir
reattachment to cell walls. Microbiol Immunol 24:299308
Masuda K, Kawata T (1983) Distribution and chemical characteriza-
tion of regular arrays in the cell walls of strains of the genus
Lactobacillus. FEMS Microbiol Lett 20:145150
Masuda K, Kawata T ( 1985) Reassembly o f a regularly arranged
protein in the cell wall of Lactobacillus buchneri and its
reattachment to cell walls: chemical modification studies .
Microbiol Immunol 29:927938
Matuschek M, Burchhardt G, Sahm K, Bahl H (1994) Pullulanase of
Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium
thermosulfurogenes): molecular analysis of the gene, composite
structure of the enzyme, and a common model for its attachment
to the cell surface. J Bacteriol 176:32953302
McCarren J, Heuser J, Roth R, Yamada N, Martone M, Brahamsha B
(2005) Inactivation of swmA results in the loss of an outer cell layer
in a swimming Synechococcus strain. J Bacteriol 187:224230
McCracken A, Timms P (1999) Efficiency of transcription from pro-
moter sequence variants in Lactobacillus is both strain and con-
text dependent. J Bacteriol 181:65696572
Mescher MF, Strominger JL (1976) Structural (shape-maintaining) role
of the cell surface glycopr otein of Halobact erium salinarium.
Proc Natl Acad Sci U S A 73:26872691
Mesnage S, Tosi-Couture E, F ouet A (1999) Production and cell
surface anchoring of functional fusions between the SLH motifs
of the Bacillus anthracis S-layer proteins and the Bacillus subtilis
levansucrase. Mol Microbiol 31:927936
Messner P, Steiner K, Z arschler K, Schäffer C (2008) S-layer
nanoglycobiology of bacteria. Carbohydr Res 343:1934
1951
Miettinen M, Vuopio-Varkila J, Varkila K (1996) Production
of human tumo r necrosis factor alp ha, interleukin-6, and
interleukin-10 is induced by lactic acid bacteria. Infect Immun
64:54035405
Mignot T, Couture-Tosi E, Mesnage S, Mock M, Fouet A (2004) In
vivo Bacillus anthracis gene expression requires PagR as an
intermediate effector of the AtxA signalling cascade. Int J Med
Microbiol 293:619624
Mobili P, Serradell Mde L, Trejo SA, Aviles Puigvert FX, Abraham
AG, De Antoni GL (2009a) Heterogeneity of S-layer proteins
from aggregating and non-aggregating Lactobacillus kefir strains.
Antonie Van Leeuwenhoek 95:363372
Mobili P, Londero A, Maria TMR, Eusébio MES, De Antoni GL,
Fausto R, Gómez-Zavaglia A (2009b) Characterization of S-
layer proteins of Lactobacillus by FTIR spectroscopy and differ-
ential scanning calorimetry. Vib Spectrosc 50:6877
Mohamadzadeh M, Duong T, Sandwick SJ, Hoover T, Klaenhammer
TR (2009) Dendritic cell targeting of Bacillus anthracis protective
antigen expressed by Lactobacillus acidophilus protects mice
from lethal challenge. Proc Natl Acad Sci USA 106:43314336
Mohamadzadeh M, Pfeiler EA, Brown JB, Zadeh M, Gramarossa M,
Managlia E, Bere P, Sarraj B, Khan MW, Pakanati KC, Ansari
MJ, O'Flaherty S, Barrett T, Klaenhammer TR (2011) Regulation
of induced colonic inflammation by Lactobacillus acidophilus
deficient in lipoteichoic acid. Proc Natl Acad Sci U S A
108(Suppl 1):46234630
Mota RM, Moreira JL, Souza MR, Horta MF, Teixeira SM, Neumann
E, Nicoli JR, Nunes AC (2006) Genetic transformation of novel
isolates of chicken Lactobacillus bearing probiotic features for
5240 Appl Microbiol Biotechnol (2013) 97:52255243
Page 16
expression of heterologous proteins: a tool to develop live oral
vaccines. BMC Biotechnol 6:2
Nagaoka M, Muto M, Nomoto K, Matuzaki T, Watanabe T, Yokokura
T (1990) Structure of polysaccharidepeptidoglycan c omplex
from the cell wall of Lactobacillus casei YIT9018. J Biochem
108:568571
Narita J, Ishida S, Okano K, Kimura S, Fukuda H, Kondo A (2006)
Improvement of protein production in lactic acid bacteria using 5-
untranslated leader sequence of slpA from Lactobacillus acidoph-
ilus. Improvement in protein production using UTLS. Appl
Microbiol Biotechnol 73:366373
Naser SM, Hagen KE, Vancanneyt M, Cleenwerck I, Swings J,
Tompkins TA (2006) Lactobacillus suntoryeus Cachat and Priest
2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen
1919) Bergey et al. 1925 (Approved Lists 1980). Int J Syst Evol
Microbiol 56(Pt 2):355360
Noonan B, Trust TJ (1997) The synthesis, secretion and role in viru-
lence of the paracrystalline surface protein layers of Aeromonas
salmonicida and A. hydrophila. FEMS Microbiol Lett 154:17
Novotny R, Scheberl A, Giry-Laterriere M, Messner P, Schaffer C
(2005) Gene cloning, functional expression and secretion of the
S-layer protein SgsE from Geobacillus stearothermophilus NRS
2004/3a in Lactococcus lactis . FEMS Microbiol Lett 242:2735
Oh S, Roh H, Ko HJ, Kim S, Kim KH, Lee SE, Chang IS, Kim S, Choi
IG (2011) Complete genome sequencing of Lactobacillus aci-
dophilus 30SC, isolated from swine intestine. J Bact eriol
193:28822883
Ojala T, Kuparinen V, Koskinen JP, Alatalo E, Holm L, Auvinen P,
Edelman S, Westerlund-Wikström B, Korhonen TK, Paulin L,
Kankainen M (2010) Genome sequence of Lactobacillus
crispatus ST1. J Bacteriol 192:35473548
Penders J, Thijs C, Mommers M, Stobberingh EE, Dompeling E,
Reijmerink NE, van den Brandt PA, Kerkhof M, Koppelman GH,
Postma DS (2010) Intestinal lactobacilli and the DC-SIGN gene for
their recognition by dendritic cells play a role in the aetiology of
allergic manifestations. Microbiology 156:32983305
Peters J , Nitsc h M, Kuhlmorgen B, Golbik R, Lupas A,
KellermannJ,EngelhardtH,PfanderJP,MüllerS,Goldie
K (1995) Tetrabrachion: a filamentous archaebacterial surface
protein assembly of unusual structure and extreme stability. J
Mol Biol 245:385401
Phipps BM, Kay WW (1988) Immunoglobulin binding by the regular
surface array of Aeromonas salmonicida. J Biol Chem 263:9298
9303
Podlesny M, Jarocki P, Komon E, Glibowska A, Targonski Z (2011)
LC-MS/MS analysis of surface layer proteins as a useful method
for the identification of lactobacilli from the Lactobacillus aci-
dophilus group. J Microbiol Biotechnol 21:421429
Pollmann K, Raff J, Merroun M, Fahmy K, Selenska-Pobell S (2006)
Metal binding by bacteria from uranium mining waste piles and
its technological applications. Biotechnol Adv 24:58
68
Poppinga L, Janesch B, Fünfhaus A, Sekot G, Garcia-Gonzalez E,
Hertlein G, Hedtke K, Schäffer C, Genersch E (2012)
Identification and functional analysis of the S-layer protein SplA
of Paenibacillus larvae, the causative agent of American foul-
brood of honey bees. PLoS Pathog 8:e1002716
Pouwels PH, Leunissen JA (1994) Divergence in codon usage o f
Lactobacillus species. Nucleic Acids Res 22:929936
Prado Acosta M, Mercedes Palomino M, Allievi MC, Sanchez Rivas
C, Ruzal SM (2008) Murein hydrolase activity in the surface layer
of Lactobacillus acidophilus ATCC 4356. Appl Environ
Microbiol 74:78247827
Prado-Acosta M, Ruzal SM, Allievi MC, Palomino MM, Sanchez
Rivas C (2010) Synergistic effects of the Lactobacillus acidoph-
ilus surface layer and nisin on bacterial growth. Appl Environ
Microbiol 76:974977
Prajapati JB, Khedkar CD, Chitra J, Suja S, Mishra V, Sreeja V, Patel
RK, Ahir VB, Bhatt VD, Sajnani MR, Jakhesara SJ, Koringa PG,
Joshi CG (2011) Whole-genome shotgun sequencing of an
Indian-origin Lactobacillus helveticus strain, MTCC 5463, with
probiotic potential. J Bacteriol 193:42824283
Pum D, Toca-Herrera JL, Sleytr UB (2013) S-layer protein self-
assembly. Int J Mol Sci 14:24842501
Ries W, Hotzy C, Schocher I, Sleytr UB, Sára M (1997) Evidence that
theN-terminalpartofthe S-layer protein from Bacillus
stearothermophilus PV72/p2 recognizes a secondary cell wall
polymer. J Bacteriol 179:38923898
Rothfuss H, Lara JC, Schmid AK, Lidström ME (2006) Involvement
of the S-layer proteins Hpi and SlpA in the maintenance of cell
envelope integrity in Deinococcus radiodurans R1. Microbiology
152:27792787