Estructura y Arquitectura Del Peptidoglucano

REVIEW ARTICLE
Peptidoglycan structure and architecture

Waldemar Vollmer1, Didier Blanot2 & Miguel A. de Pedro3
1
Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2Enveloppes Bactériennes
et Antibiotiques, IBBMC, UMR 8619 CNRS, Univ Paris-Sud, Orsay, France; and 3Centro de Biologı́a Molecular Severo Ochoa, Consejo Superior de
Investigaciones Cientı́ficas, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain
Correspondence: Waldemar Vollmer, Abstract

Institute for Cell and Molecular Biosciences,
Medical School, University of Newcastle upon
The peptidoglycan (murein) sacculus is a unique and essential structural element
Tyne, Catherine Cookson Building, in the cell wall of most bacteria. Made of glycan strands cross-linked by short
Framlington Place, Newcastle upon Tyne, NE2 peptides, the sacculus forms a closed, bag-shaped structure surrounding the
4HH, UK. Tel.: 144 0 191 222 6295; fax: 144 cytoplasmic membrane. There is a high diversity in the composition and sequence
0 191 222 7424; e-mail: w.vollmer@ncl.ac.uk of the peptides in the peptidoglycan from different species. Furthermore, in several
species examined, the fine structure of the peptidoglycan significantly varies with
Received 12 July 2007; revised 21 October the growth conditions. Limited number of biophysical data on the thickness,
2007; accepted 22 October 2007.
elasticity and porosity of peptidoglycan are available. The different models for the
First published online 8 January 2008.
architecture of peptidoglycan are discussed with respect to structural and physical
DOI:10.1111/j.1574-6976.2007.00094.x
parameters.
Editor: Arie van der Ende
Keywords
peptidoglycan; murein; bacterial cell wall.
genes (but no peptidoglycan) have been found in the green

Introduction plant Arabidopsis thaliana (five genes) and the moss Physco-
Peptidoglycan (murein) is an essential and specific compo- mitrella patens (nine genes); these genes would be involved
nent of the bacterial cell wall found on the outside of the in chloroplast division (Machida et al., 2006).
cytoplasmic membrane of almost all bacteria (Rogers et al., This review provides a brief overview on the diversity and
1980; Park, 1996; Nanninga, 1998; Mengin-Lecreulx & variability of the chemical structure of peptidoglycan in
Lemaitre, 2005). Its main function is to preserve cell different bacteria, and summarizes the available data on the
integrity by withstanding the turgor. Indeed, any inhibition biophysical properties of the cell wall. Finally, structural
of its biosynthesis (mutation, antibiotic) or its specific aspects required for modelling the architecture of the
degradation (e.g. by lysozyme) during cell growth will result peptidoglycan sacculus are discussed.
in cell lysis. Peptidoglycan also contributes to the mainte-
nance of a defined cell shape and serves as a scaffold for
anchoring other cell envelope components such as proteins Chemical structure of peptidoglycan
(Dramsi et al., 2008) and teichoic acids (Neuhaus &
Baddiley, 2003). It is intimately involved in the processes of General structure and characteristic features
cell growth and cell division. Peptidoglycan (and the genetic The main structural features of peptidoglycan are linear
arsenal necessary to its biosynthesis) is absent in Mycoplas- glycan strands cross-linked by short peptides (Rogers et al.,
mas, Planctomyces and the scrub typhus agent Orientia 1980) (Fig. 1). The glycan strands are made up of alternating
(Rickettsia) tsutsugamushi (Moulder, 1993; Tamura et al., N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid
1995). It has never been detected in Chlamidiae although (MurNAc) residues linked by b-1 ! 4 bonds. The D-lactoyl
most biosynthetic genes exist (Chopra et al., 1998; Ghuysen group of each MurNAc residue is substituted by a peptide
& Goffin, 1999). Conversely, peptidoglycan is present in stem whose composition is most often L-Ala-g-D-Glu-meso-
the photosynthetic organelles (‘cyanelles’) of the glaucocys- A2pm (or L-Lys)-D-Ala-D-Ala (A2pm, 2,6-diaminopimelic
tophyte algae (Aitken & Stanier, 1979). A few biosynthetic acid) in nascent peptidoglycan, the last D-Ala residue being
FEMS Microbiol Rev 32 (2008) 149–167

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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150 W. Vollmer et al.
Fig. 1. Structure of the peptidoglycan of Escherichia coli. The glycan strands consist of alternating, b-1 ! 4-linked GlcNAc and MurNAc residues, and
are terminated by a 1,6-anhydroMurNAc residue. The yellowish labelled part represents the basic disaccharide tetrapeptide subunit (monomer), which is
also written with the conventional amino acid and hexosamine abbreviations on the left-hand side. The middle part shows a cross-linked peptide, with
the amide group connecting both peptide stems drawn in red. [Reproduced with permission from Mengin-Lecreulx D & Lemaitre B (2005), Copyright
(r SAGE Publications, 2005), by permission of Sage Publications Ltd].
lost in the mature macromolecule. Cross-linking of the tion reactions. Secondary modifications in the glycan
glycan strands generally occurs between the carboxyl group strands such as N-deacetylation, O-acetylation and N-gly-
of D-Ala at position 4 and the amino group of the diamino colylation are frequently found and are the topic of another
acid at position 3, either directly or through a short peptide review in this issue (Vollmer, 2008). In the Gram-positive
bridge. Therefore, the chemical traits of this heteropolymer Staphylococcus aureus, the glycan strands may contain either
involve the presence of an unusual sugar (MurNAc), of a MurNAc or a GlcNAc residue at the reducing end; the
g-bonded D-Glu, of L–D (and even D–D) bonds and of latter residue indicates that cleavage of the strand by an
nonprotein amino acids (e.g. A2pm). N-acetylglucosaminidase had occurred (Boneca et al., 2000).
The structural features outlined in the preceding para- In all Gram-negative and some Gram-positive species (e.g.
graph are retrieved in all bacterial species known to date. Bacillus subtilis), the glycan strands do not have a reducing
However, a certain degree of variation exists either in the (MurNAc or GlcNAc) end but terminate with a 1,6-anhy-
peptide stem, in the glycan strands or in the position or droMurNAc residue, which has an intramolecular ring from
composition of the interpeptide bridge. Interspecies varia- C1 to C6. It is not known whether the 1,6-anhydroMurNAc
tion is the general case; it has been the subject of the residues present in the sacculi have been formed during
monumental work of Schleifer & Kandler (1972) 35 years termination of glycan strand synthesis, or whether they are
ago and has served in the establishment of the tri-digital the result of degradation by lytic transglycosylases, or both.
system of peptidoglycan classification used universally. In species with high activity of glycan strand-cleaving
However, in the same species, there can be variations in the enzymes (glucosaminidases and muramidases), the pepti-
fine structure according to the growth conditions (growth doglycan may contain glycan strands with all possible
phase, medium composition, intra/extracellular growth, combinations of GlcNAc and MurNAc residues at the ends.
presence of antibiotics). In the next sections, this study will These hydrolytic enzymes must be inactivated rapidly and
present an overview of the different types of variations removed quantitatively when peptidoglycan is prepared for
encountered. glycan strand length analysis to avoid cleavage of the strands
after peptidoglycan isolation (Ward, 1973). Different meth-
ods have been applied to determine the average length of the
The glycan strands in peptidoglycan
glycan strands and the length distribution: (1) quantifica-
The glycan strands are formed by oligomerization of mono- tion of the fraction of the reducing hexosamine residues
meric disaccharide peptide units (lipid II) by transglycosyla- after chemical reduction (Rogers, 1970; Ward, 1973), (2)

c 2008 Federation of European Microbiological Societies FEMS Microbiol Rev 32 (2008) 149–167

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Structure and architecture of peptidoglycan 151
enzymatic addition of galactosamine residues to the GlcNAc length was 45.1 disaccharide units. The average chain length
end and their quantification (Schindler et al., 1976), (3) of all glycan strands was estimated as 21 disaccharide units,
quantification of the fraction of the 1,6-anhydroMurNAc- which is slightly less than the average chain length of 25–35
containing disaccharide peptide subunits that are the hall- disaccharide units calculated from the fraction of 1,6-
mark of the glycan strand ends in some species (Glauner anhydroMurNAc residues (Gmeiner et al., 1982; Glauner,
et al., 1988; Quintela et al., 1995a) and (4) release of the 1988). The average glycan chain length is greater (ca. 50
glycan strands by an amidase and their purification by an disaccharide units) in the peptidoglycan from P. morganii, as
anion exchange column, followed by their separation by calculated from the fraction of 1,6-anhydroMurNAc con-
reverse-phase HPLC (Harz et al., 1990). The latter method is taining muropeptides (Quintela et al., 1995a). The glycan
restricted to the separation of glycans from 1 to 30 dis- strands from H. pylori are particularly short, with an average
accharide units. Glycans that are longer than 30 disaccharide chain length of o 10 disaccharide units (Costa et al., 1999),
units elute together in one peak. and they become even shorter during transition from a
There are only limited data on the average chain length spiral to a coccoid cell shape in the stationary growth phase
and the chain length distribution of the glycan strands in (Costa et al., 1999; Chaput et al., 2007). It might be
different species. Remarkably, the average chain length of the important for the integrity of the peptidoglycan net that
glycan strands does not correlate with the thickness of the the glycan strand ends are hyper-cross-linked in H. pylori
peptidoglycan layer, as there are Gram-positive species with (Costa et al., 1999).
a thick cell wall with either short (S. aureus) or long (B.
subtilis) glycan strands. Similarly, there are Gram-negative
Variation in the peptide stem
species with either short (Helicobacter pylori) or long
(Proteus morganii) glycan strands. The variations of the peptide stem can be divided into two
The glycan strands in the peptidoglycan of Bacilli (B. categories: (1) those due to the specificity of the Mur ligases,
subtilis, Bacillus licheniformis and Bacillus cereus) have an the enzymes responsible for its biosynthesis, and (2) those
average chain length between 50 and 250 disaccharide units occurring at a later step of the biosynthesis [see accompany-
(Hughes, 1971; Warth & Strominger, 1971; Ward, 1973). In ing chapters in this issue (Barreteau et al., 2008; Bouhss
contrast, the glycan strands of S. aureus are much shorter, et al., 2008)]. These variations are enumerated in Table 1.
with an average chain length of about 18 disaccharide units The first amino acid of the peptide stem is added by the
(Tipper et al., 1967; Ward, 1973). Separation of the staphy- MurC ligase. In most bacterial species, this amino acid is
lococcal glycan strands by HPLC revealed that the predomi- L-Ala; in rare cases, Gly or L-Ser is added instead (Table 1).
nant chain length was between 3 and 10 disaccharide units. Two interesting cases deserve to be mentioned. First, the
Longer glycan strands with more than 26 disaccharide units enzymes from Mycobacterium tuberculosis and Mycobacter-
represented about 10–15% of the total glycan material ium leprae have the same in vitro specificity pattern towards
(Boneca et al., 2000). The L-ornithine-containing peptido- L-Ala and Gly; however, the amino acid found at the first
glycan of Deinococcus radiodurans Sark, a Gram-positive position of the peptide stem is different (L-Ala for the
bacterium that is extremely resistant to ionizing radiations, former and Gly for the latter). This appears to be due to
had glycan strands terminated by 1,6-anhydroMurNAc the growth conditions (Mahapatra et al., 2000). The second
residues with an average chain length of about 20 disacchar- case is that of Chlamydia trachomatis: the MurC activity
ide units (Quintela et al., 1999a). adds L-Ala, L-Ser and Gly with similar in vitro efficiencies.
The average chain length of the glycan strands in Gram- This absence of specificity prevents from deducing the
negative bacteria can be calculated from the fraction of 1,6- nature of the first amino acid of the putative chlamydial
anhydroMurNAc residues containing disaccharide peptide peptidoglycan (Hesse et al., 2003).
subunits. Different species differ in the average chain length The amino acid at the second position is added by the
of the glycan strands but the normal range lies between 20 MurD ligase. In all species this enzyme adds D-Glu, the
and 40 disaccharide units (Tuomanen et al., 1989; Quintela modifications encountered (Table 1) occurring at a later
et al., 1995a, b). As shown for Escherichia coli, the average step.
chain length of the glycan strands also varies to some extent The greatest variation is found at position 3. The addition
with the strain and growth conditions (Glauner, 1988). of the third amino acid is catalyzed by the MurE ligase. This
Escherichia coli glycan strands of up to 30 disaccharide units amino acid is generally a diamino acid, either meso-A2pm
have been separated by HPLC (Harz et al., 1990). The (most Gram-negative bacteria, Mycobacteria, Bacilli) or
average chain length of the glycan strands from 1 to 30 L-Lys (most Gram-positive bacteria); in certain species,
disaccharide units was 8.9 disaccharide units in strain W7. other diamino acids (L-Orn, LL-A2pm, meso-lanthionine,
Glycan strands longer than 30 disaccharides represented L-2,4-diaminobutyric acid, D-Lys) or monoamino acids
about 25–30% of the total material, and their average chain (L-homoserine, L-Ala, L-Glu) are encountered (Table 1). As

c2008 Federation of European Microbiological Societies

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Table 1. Amino acid variations in the peptide stem

Position Residue encountered Examples
1 L-Ala Most species
Gly Mycobacterium leprae, Brevibacterium imperiale
L-Ser Butyribacterium rettgeri
2 D-Isoglutamate Most Gram-negative species
D-Isoglutamine
Most Gram-positive species, Mycobacteria
threo-3-Hydroxyglutamate Microbacterium lacticum
3 meso-A2pm Most Gram-negative species, Bacilli, Mycobacteria
L-Lys Most Gram-positive species
L-Orn Spirochetes, Thermus thermophilus
L-Lys/L-Orn Bifidobacterium globosum
L-Lys/D-Lys Thermotoga maritima
LL-A2pm Streptomyces albus, Propionibacterium petersonii
meso-Lanthionine Fusobacterium nucleatum
L-2,4-Diaminobutyrate Corynebacterium aquaticum
L-Homoserine Corynebacterium poinsettiae
L-Ala Erysipelothrix rhusiopathiae
L-Glu Arthrobacter J. 39
Amidated meso-A2pm Bacillus subtilis
2,6-Diamino-3-hydroxypimelatew Ampuraliella regularis
w
L-5-Hydroxylysine Streptococcus pyogenesz
N -Acetyl-L-2,4-diaminobutyrate
g
Corynebacterium insidiosum
4 D-Ala All bacteria
5 D-Ala Most bacteria
D-Ser Enterococcus gallinarum
D-Lac Lactobacillus casei, Enterococci with acquired
resistance to vancomycin
These residues result from reactions occurring posterior to the action of Mur ligases.
w
The process of formation of these residues (direct incorporation by MurE or subsequent hydroxylation of the nonhydroxylated residue) is unclear
(Muñoz et al., 1966; Perkins, 1969).
z
In this organism, a 10 : 1 ratio of lysine to hydroxylysine was found Muñoz et al. (1966).
for the second position, further modifications of the third mers of lysine, but no meso-A2pm (Huber et al., 1986), is
amino acid occur posterior to MurE action (Table 1). capable of adding in vitro L-Lys, D-Lys and meso-A2pm with
In most cases, the MurE enzyme is highly specific for the comparable efficiencies (Boniface et al., 2006). In the L-Lys-
relevant amino acid; this has been demonstrated for the containing UDP-MurNAc-tripeptide product, the D-Glu-L-
meso-A2pm-adding and L-Lys-adding enzymes from E. coli Lys bond has the conventional g ! a arrangement; how-
and S. aureus, respectively (Boniface, 2007). In Corynebac- ever, in the D-Lys-containing product, D-Lys is acylated by
terium pointsettiae, where L-homoserine (L-Hse) is found at the g-carboxylate of D-Glu on its e-amino function. This
position 3, MurE is specific both for the amino acid and the leads to the synthesis of two peptide stems: the conventional
nucleotide precursor UDP-MurNAc-Gly-D-Glu, the ‘wrong’ L-Ala-D-Glu (g ! a) L-Lys-D-Ala-D-Ala and the unusual
nucleotide with L-Ala at position 1 being a poor substrate; L-Ala-D-Glu (g ! e) D-Lys. The absence of meso-A2pm in
this ensures a correct synthesis of the ‘right’ peptide stem T. maritima peptidoglycan is explained by its very low
Gly-g-D-Glu-L-Hse-D-Ala-D-Ala (Wyke & Perkins, 1975). intracellular pool with respect to those of L- and D-Lys
However, the MurE enzyme sometimes seems to be devoid (Boniface et al., 2006).
of strict specificity, and this affects the final composition of Amino acids at positions 4 and 5 are added as a dipeptide,
peptidoglycan. In certain species of the genus Bifidobacter- in most cases D-Ala-D-Ala. The synthesis of the dipeptide is
ium, two diamino acids, L-Lys and L-Orn, are retrieved at the carried out by the Ddl enzyme and its incorporation into the
third position of the peptide stem (Schleifer & Kandler, peptide stem by the MurF ligase. It has been established that
1972). As a matter of fact, it has been shown that MurE from the latter has a high degree of specificity for the C-terminal
Bifidobacterium globosum can incorporate both diamino amino acid (Duncan et al., 1990; Bugg et al., 1991). This is
acids indifferently (Hammes et al., 1977). MurE from complementary to the specificity of the former, which
Thermotoga maritima, a Gram-negative species whose pep- resides mainly on the N-terminal amino acid, and this
tidoglycan contains similar proportions of both enantio- constitutes a ‘double-sieving’ mechanism ensuring the


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synthesis of a pentapeptide stem ending mainly in D-Ala-D- a-carboxyl group of meso-A2pm through the e-amino func-
Ala (Neuhaus & Struve, 1965; Duncan et al., 1990). D-Ala is tion of its C-terminal lysine residue: in this transpeptidation
predominantly found at position 4 in all species. D-Lactate reaction, the tetrapeptide moiety L-Ala-g-D-Glu-meso-
(D-Lac) or D-Ser is found at position 5 in strains endowed A2pm-D-Ala is the acyl donor while the side chain amine of
with natural or acquired resistance to vancomycin (Table 1), the C-terminal residue of the lipoprotein is the acyl acceptor.
the affinity of the D-Ala-D-Lac and D-Ala-D-Ser moieties for Lately, it has been demonstrated that in E. coli, enzymes
the antibiotic being much lower than that of the conven- homologous to the L,D-transpeptidase of Enterococcus
tional D-Ala-D-Ala moiety (Healy et al., 2000). A certain faecium are responsible for the attachment of Braun’s
proportion of Gly, presumably escaping from the double- lipoprotein to the peptide stem (Magnet et al., 2007b).
sieving mechanism, is often found at position 4 or 5 in lieu Gram-positive bacteria contain many surface proteins
of D-Ala. The proportion is low (ca. 1%) in E. coli, but (e.g. protein A, fibrinonectin-binding proteins, collagen
reaches 19% in Caulobacter crescentus (Markiewicz et al., adhesin) anchored to peptidoglycan and involved in
1983; Glauner et al., 1988). pathogenic processes. The anchoring reaction is catalyzed
Other variations in peptidoglycan composition (amida- by a membrane protein called sortase A (Marraffini et al.,
tion, hydroxylation, acetylation, attachment of amino acids 2006). Sortase A from S. aureus cleaves the Thr–Gly
or other groups, attachment of proteins) occur after the peptide bond of the sorting signal (consensus sequence
action of the Mur ligases, often at the level of lipid II. These Leu–Pro–Xaa–Thr–Gly, where Xaa is any amino acid) pre-
modifications concern essentially positions 2 and 3. It sent in the surface protein and links the a-carboxyl group of
should be mentioned that most enzymes responsible for Thr to the side-chain amine at the third position of a
these modifications are still unknown. peptide stem. In this case, the surface protein is the acyl
Amidation of the a-carboxyl of glutamic acid (yielding D- donor whereas the peptide moiety L-Ala-g-(a-D-Glu-NH2)-
e
isoglutamine) and the e-carboxyl group of meso-A2pm is L-Lys(N -Gly5)-D-Ala is the acyl acceptor. It has been
very frequent. It has been shown in Mycobacterium smegma- demonstrated that the acceptor substrate of sortase A is
tis that lipid II is the substrate for amidation reactions; in lipid II.
fact, lipid II in this species appears as a mixture of non-,
mono- and di-amidated molecules (Mahapatra et al., 2005).
Variation in cross-linking
The hydrocarbon chain of D-Glu, meso-A2pm or L-Lys is
hydroxylated in some species. In the case of D-Glu, it was Most variations of the peptide moiety of peptidoglycan
demonstrated that hydroxylation occurs after the cytoplas- occur in its mode of cross-linkage and in the composition
mic steps (Schleifer et al., 1967); it depends on the oxygen of the interpeptide bridge (Fig. 2 and Table 2). There are two
supply during growth, cells grown under microaerophilic main groups of cross-linkage (Schleifer & Kandler, 1972). In
conditions displaying almost no hydroxylated glutamic acid the first group (3–4 cross-linkage), the cross-linkage extends
(Schleifer et al., 1968). In Corynebacterium insidiosum, from the amino group of the side-chain of the residue
where unsubstituted L-2,4-diaminobutyric acid is the sub- at position 3 of one peptide subunit (acyl acceptor) to the
strate for MurE, the g-amino group of the diamino acid is carboxyl group of D-Ala at position 4 of another (acyl
acetylated in the UDP-MurNAc-pentapeptide precursor and donor). As mentioned above, this is the most common kind
in peptidoglycan (Perkins, 1971; Wyke & Perkins, 1975). In of cross-linkage. It can be either direct (most Gram-negative
certain organisms, an amino acid or another amine-con- bacteria) or through an interpeptide bridge (most Gram-
taining moiety, such as glycine (Micrococcus luteus, Arthro- positive bacteria). In the second group (2–4 cross-linkage),
bacter tumescens), glycine amide (Arthrobacter athro- found only among coryneform bacteria, especially the
cyaneus), D-alanine amide (Arthrobacter sp. NCIB9423), phytopathogenic corynebacteria, the cross-linkage extends
cadaverine (Selenomonas ruminantium) or N-acetylputres- between the a-carboxyl group of D-Glu at position 2 of one
cine (Cyanophora paradoxa), is added to the a-carboxyl peptide subunit (acyl acceptor) and the carboxyl group of
group at position 2 (Schleifer & Kandler, 1972; Kamio D-Ala at position 4 of another (acyl donor). In this case, for
et al., 1981; Pfanzagl et al., 1996). the first subunit to be an acceptor, an interpeptide bridge
The peptide stem constitutes the point of covalent containing a diamino acid must be present. The cross-
anchoring of cell envelope proteins to peptidoglycan linking reactions are catalyzed by the transpeptidase domain
(Dramsi et al., 2008). In E. coli and other related Gram- of penicillin-binding proteins, enzymes that have been
negative bacteria, Braun’s lipoprotein is the only protein studied extensively, in particular in human pathogenic
covalently linked to peptidoglycan known to date (Braun & bacteria (Sauvage et al., 2008).
Rehn, 1969; Braun & Sieglin, 1970). It is a 58-amino acid The size of the interpeptide bridge ranges from one to
protein whose N-terminal glyceryl-cysteine residue is mod- seven amino acid residues. Various amino acids are encoun-
ified by the addition of three fatty acids. It is attached to the tered: Gly, L-Ala, L- or D-Ser, D-Asx, L- or D-Glu, etc. As


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(a) (b) (c)

-G–M- -G–M- -G–M-
1 L-Ala 1 L-Ala
1 Gly
2 D-Glu 2 D-Glu NH δ
γ 2 D-Glu D-Orn D-Ala 4
ε γ ε
3 m-A pm D-Ala 4 3 L-Lys (Gly) D-Ala 4 γ
3 L-Hse L-Hse 3
4 D-Ala m-A pm 3 4 D-Ala L-Lys 3 γ
γ γ
4 D-Ala D-Glu 2
D-Glu 2 D-Glu NH 2
L-Ala
Gly 1
L-Ala 1 1
-G–M- -G–M- -G–M-
(d)
-G–M-
1 L-Ala
2 D-Glu Gly
γ
ε γ
3 L-Lys D-Ala L-Lys D-Glu L-Ala D-Ala 4
4 D-Ala Gly L-Lys 3

γ
D-Glu Gly 2
L-Ala 1
-G–M-
Fig. 2. Examples of cross-linkage and interpeptide bridge. (a), Escherichia coli (direct 3–4 cross-link); (b), Staphylococcus aureus (3–4 cross-link with a
pentaglycine bridge); (c), Corynebacterium pointsettiae (2–4 cross-link with a D-ornithine bridge); (d), Micrococcus luteus (3–4 cross-link with a bridge
consisting of a peptide stem). G, N-acetylglucosamine; M, N-acetylmuramic acid.
Table 2. Characterized branching enzymes, and nature of the interpeptide bridges synthesized
Enzyme(s) Family Bridge Species Reference
FmhB, FemA, FemB Fem transferase (Gly)5 Staphylococcus aureus Schneider et al. (2004)
BppA1, BppA2 Fem transferase L-Ala-L-Ala Enterococcus faecalis Bouhss et al. (2002)
FemX Fem transferase L-Ala-L-Ser or Weissella viridescens Biarrotte-Sorin et al. (2004)
L-Ala-L-Ser-L-Ala
MurM, MurN Fem transferase L-Ser-L-Ala or Streptococcus pneumoniae Fiser et al. (2003)
L-Ala-L-Ala
w
Aslfm ATP-grasp D-Asx Enterococcus faecium Bellais et al. (2006)
FemX catalyses the addition of the first amino acid residue (L-Ala) of the side chain; the second (L-Ser) and third (L-Ala) residues are added by unknown
Fem transfereases (Villet et al., 2007).
w
The substrate of Aslfm is D-Asp, which is engaged through its b-carboxyl group; in mature peptidoglycan, the a-carboxyl group is partially amidated.
already mentioned, the interpeptide bridges of 2–4 cross- the enzymes responsible for their biosynthesis (‘branching
links contain necessarily (but not exclusively) a diamino enzymes’). In fact, it is only recently that some branching
acid (L- or D-Lys, D-Orn, D-2,4-diaminobutyrate). For many enzymes have been purified and characterized (Table 2).
years, the important diversity of composition of the inter- They can be divided into two groups according to the
peptide bridges has contrasted with the poor knowledge of nature of the amino acid incorporated: (1) Glycine and


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L-amino acids are activated as aminoacyl-tRNAs and trans- percentage of monomers is low (o10%), most peptidogly-
ferred to the precursors by a family of nonribosomal peptide can units being present as oligomers (lengths up to non-
bond-forming enzymes called Fem transferases (Mainardi amers were detected by HPLC and up to eicosamers deduced
et al., 2008). (2) D-Amino acids are activated as acyl from calculation) (Snowden & Perkins, 1990).
phosphates by proteins belonging to the ATP-grasp family,
which is composed of highly diverse enzymes that catalyze Classification of peptidoglycans according to
the ATP-dependent ligation of a carboxyl group to an amino their structure
or imino nitrogen, a hydroxyl oxygen or a thiol sulpfur
Considering that the variations in peptidoglycan structure
(Galperin & Koonin, 1997). The precursor substrate of the
have taxonomic implications, Schleifer and Kandler estab-
branching enzymes varies among species: lipid II for S.
lished a tri-digital system of classification of peptidoglycans.
aureus enzymes, UDP-MurNAc-pentapeptide for Weissella
The first digit, a Roman capital letter, represents the mode of
viridescens FemX or both for Enterococcus faecalis enzymes.
cross-linkage (A and B for the 3–4 and 2–4 cross-linkages,
An interesting case deserves to be mentioned. In M. luteus
respectively). The second digit, a number, refers to the type
(lysodeikticus), a large proportion of the peptide stems
of interpeptide bridge, or lack of it, involved in the cross-
[L-Ala-g-(a-D-Glu-Gly)-L-Lys-D-Ala] becomes detached
linkage. The third digit, a Greek letter, indicates the amino
from the MurNAc residues by an amidase reaction and
acid found at position 3 of the peptide stem. As a conse-
forms peptide cross-bridges between positions 3 and 4
quence, the examples of peptidoglycan depicted in Fig. 2
(Fig. 2). These bridges often contain several pentapeptides
have the following types: A1g (a), A3a (b), B2b (c) and A2a
joined ‘head-to-tail’ (Ghuysen et al., 1968). The nature of
(d). For further details about this classification, the reader is
the enzyme catalyzing the unusual transpeptidation reaction
invited to refer to the review of Schleifer & Kandler (1972)
between D-Ala (acyl donor) and the N-terminal L-Ala (acyl
or to the book of Rogers et al. (1980).
acceptor) is unknown.
D-Ala at position 4 is not the only possible acyl donor: the
Variations in peptidoglycan fine structure
carboxyl group of the amino acid at position 3 can also play
this role. This gives rise to the appearance of 3–3 cross-links, The fine structure of the bacterial sacculi is reflected in the
which were originally discovered in Mycobacteria (Wietzer- detailed muropeptide composition of peptidoglycan as
bin et al., 1974). In M. smegmatis, 3–3 cross-links represents determined by means of high-resolution techniques. Infor-
one third of all cross-links. In other bacteria, their propor- mation on the abundance and peculiarities of families of
tion is low but increases during the stationary phase or in b- muropeptides with specific structural functions is crucial to
lactam-resistant strains (Pisabarro et al., 1985; Mainardi understand the architecture and physiology of the sacculus
et al., 2000). Their formation is catalyzed by penicillin- itself. In a first approximation, the most relevant muropep-
insensitive L,D-transpeptidases (Mainardi et al., 2005). tide families are: cross-linked muropeptides, as their abun-
As for the main peptide chain, the interpeptide bridge can dance is related to the mesh size and strength of the sacculus;
be further modified after its assembly. In Enterococcus 1,6-anhydroMurNAc muropeptides (if present), which re-
faecium, where the Aslfm enzyme ligates the b-carboxyl late to the average length of peptidoglycan strands, and
group of D-Asp to the UDP-MurNAc-pentapeptide muropeptides acting as links for other molecules such as
(Table 2), part of the a-carboxyl groups are subsequently Braun’s lipoprotein in Gram-negative and wall teichoic acids
amidated, thereby leading to a peptidoglycan composed of or polysaccharides in Gram-positive species (Glauner &
b-D-aspartyl and D-isoasparaginyl residues in similar Höltje, 1990; Höltje & Glauner, 1990; Höltje, 1998; de Pedro,
amounts (Bellais et al., 2006). In Thermus thermophilus, 2004).
where the amino acid at position 3 is L-Orn and the bridge Systematic studies in the model bacterium E. coli
consists of a diglycyl residue between positions 3 and 4, a revealed that sacculi are endowed with a large metabolic
significant proportion of glycyl residues not engaged in the plasticity, which is indicated by the variability of peptido-
bridge with the donor peptide stem are acylated with glycan muropeptide composition and surface density
phenylacetic acid (Quintela et al., 1995b). (amount of peptidoglycan per unit of cell surface area) in
Besides the diversity in the nature of cross-linkage, there response to the growth and environmental conditions
is a considerable variation in the degree of cross-linkage, (Mengin-Lecreulx & van Heijenoort, 1985; Prats & de Pedro,
which varies from ca. 20% in E. coli to over 93% in 1989; Höltje, 1998).
S. aureus (Rogers et al., 1980). Translated in terms of Investigations of the mechanisms of precursor incorpora-
muropeptide content, these figures mean that in E. coli, tion into the sacculus indicated that newly inserted and old
most peptidoglycan units appear as monomers peptidoglycans had different muropeptide compositions,
(ca. 50%) or dimers (ca. 40%), higher oligomers being a and that the macromolecule suffers a maturation, or aging,
minority (Glauner et al., 1988); conversely, in S. aureus, the before new and old materials become indistinguishable


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(de Pedro & Schwarz, 1981; Burman & Park, 1983; Pisabarro Only a few Gram-positive bacteria have lent themselves to
et al., 1985; Glauner & Höltje, 1990). In E. coli, new analysis by HPLC and in most cases their composition could
peptidoglycan is less cross-linked, and has little covalently be only partially solved (Garcia-Bustos et al., 1987; Laitinen
bound lipoprotein, but is richer in muropeptides with a & Tomasz, 1990; de Jonge et al., 1992; Atrih et al., 1996,
pentapeptide side chain (potential donors in DD-transpepti- 1999a, b; Quintela et al., 1999a, b; Severin et al., 2004;
dation reactions) and is made up of longer glycan strands. Boneca et al., 2007). A large variability in fine structure is
Aging brings with it a progressive variation of the indicated evident, as expected from their heterogeneity in chemical
parameters in a process that apparently requires about one composition and cross-linking. Even among the more
mass doubling time to complete. Interestingly, most of the homogeneous Gram-negative organisms, large differences
difference in cross-linkage between new and old peptidogly- in fine structure have been clearly shown in spite of the
cans can be accounted for by de novo synthesis of LD-A2pm- limited number of well-studied organisms (Folkening et al.,
A2pm (3–3) cross-linked muropeptides (de Pedro & 1987; Höltje & Glauner, 1990; Quintela et al., 1995a, b,
Schwarz, 1981; Glauner & Höltje, 1990). 1999b; Costa et al., 1999). Therefore, it seems that there is
Peptidoglycan fine structure is also subjected to global no optimal or standard value for parameters as cross-linkage
variations when the state of growth changes (Pisabarro et al., or glycan strand length, but rather each species selects the
1985; Glauner et al., 1988). The transition of E. coli from values (or range of values) appropriate for its particular life
exponential growth into a resting phase brings with it a conditions.
drastic modification in the composition, and likely struc- Variations in peptidoglycan fine structure have also been
ture, of sacculi. Most remarkable alterations affect the degree associated with bacterial pathogenesis in a number of cases.
of cross-linkage, which increases by 30–40% (from 27–30 up Well-documented examples are the changes exhibited by
to 36–42% of cross-linked muropeptides), the mean glycan peptidoglycan in Neisseria gonorrhoeae peptidoglycan re-
chain length, which declines to about one half (from 30–35 lated to secretion of cytotoxin (Cloud & Dillard, 2002); in
down to 15–18 disaccharides chain1), and the lipoprotein H. pylori associated with morphological transitions (Costa
content, which increases by about 50% (from 8–10 up to et al., 1999); and in Salmonella typhimurium associated with
13–15% of lipoprotein-bound muropeptides) (Pisabarro intracellular colonization of epithelial cells (Quintela et al.,
et al., 1985; Blasco et al., 1988; Glauner et al., 1988). As for 1997).
peptidoglycan aging, most of the variation in cross-linkage To conclude, it seems that bacteria are remarkably able to
could be accounted for by a dramatic increase in the relative ‘fine tune’ the structure of the cell wall to best suit their
abundance of LD-A2pm-A2pm cross-linked muropeptides, individual needs, and to better adapt to changing, and often
from 5–6% to 11–13% of the total muropeptides (Pisabarro challenging, environmental conditions. The nature of the
et al., 1985). Of course, the inverse transition also takes place profits bacteria derived from these adaptations is still
when cells resume active growth from a resting condition. unknown, but is likely relevant for their survival.
Although data are quite limited, recovery of the muropep-
tide composition characteristic for actively growing cells
Variation upon amino acid supplementation or
might involve active modification of total peptidoglycan in
in genetically engineered bacteria
addition to the expected variation due to mixing of old
(resting phase) and new (growth phase) peptidoglycans (de When present at a high concentration in the growth
la Rosa et al., 1985; Pisabarro et al., 1985). medium, analogues of peptidoglycan amino acids can be
A rather surprising ability of E. coli is its aptitude to incorporated into the macromolecule and modify its com-
modify peptidoglycan surface density. Studies conducted position. The most-known example is that of glycine, which
under conditions limiting supply of precursors showed that can replace alanine at position 1, 4 or 5 in several bacterial
E. coli can reduce the amount of peptidoglycan per surface species (Hammes et al., 1973). Similarly, D-amino acids such
unit down to one half the normal value, maintaining a as D-Met, D-Trp or D-Phe can replace D-Ala at position 4 in
typical morphology and growth parameters (Prats & de E. coli (Caparros et al., 1992). The fact that several A2pm
Pedro, 1989; Caparros et al., 1992). Cells with reduced analogues are able to complement A2pm auxotrophy
peptidoglycan content were nevertheless more sensitive to in E. coli dap mutants suggests their incorporation at
penicillin and other damaging agents. It has been proposed position 3 in peptidoglycan (see references in Mengin-
that the ability to reduce the content of peptidoglycan could Lecreulx et al. 1994). Interestingly, the presence of hydro-
help cells to deal with transient inhibitions of cell wall xylysine, which is often considered to be a natural constitu-
biosynthesis and therefore increase their chances of survival ent of the peptidoglycan of certain species, is in most cases
(Prats & de Pedro, 1989). the result of particular growth conditions, namely lysine
A comprehensive survey of peptidoglycan fine structure deprivation and hydroxylysine supplementation (see e.g.
in different bacterial species is simply nonexistent at present. Smith & Henderson, 1964; Shockman et al., 1965).


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Peptidoglycan composition varies in mutants or geneti- Peptidoglycan composition and structure can also evolve
cally engineered cells with respect to wild type. This is well under the selective pressure of antibiotics. This aspect is
documented in E. coli for the amino acid at position 3 developed in the present issue by Mainardi et al. (2008).
of the peptide stem. A dapF mutant lacking A2pm epimerase
was shown to contain a huge pool of LL-A2pm that was Biophysical properties of peptidoglycan
incorporated into peptidoglycan (Mengin-Lecreulx et al.,
Peptidoglycan sacculi are bag-shaped molecules with unique
1988). Similarly, in a dapA strain in which genes of the
biophysical properties. On the one hand, sacculi have the
methionine pathway are either deleted or overexpressed,
strength to withstand the cell’s turgor of up to 25 atmo-
A2pm was totally replaced by meso-lanthionine and/or L-
spheres present in Gram-positive bacteria. On the other
allo-cystathionine (Richaud et al., 1993; Mengin-Lecreulx
hand, the sacculi are not rigid walls but are flexible, allowing
et al., 1994). Recenty, the peptidoglycan of E. coli cells
reversible expansion under pressure, and they have relatively
overexpressing the murE gene of S. aureus or T. maritima
wide pores, enabling diffusion of large molecules such as
was shown to contain high amounts of lysine (Mengin-
proteins. Because the peptidoglycan completely surrounds
Lecreulx et al., 1999; Boniface et al., 2006). The replacement
the cytoplasmic membrane, the sacculus has a similar size
of meso-A2pm at position 3 by an analogue resulted in a
and shape as the bacterial cells from which it was isolated.
decrease of the proportion of dimer. For cells overexpressing
the S. aureus murE gene, this defect in the degree of cross-
Thickness of the cell wall peptidoglycan
linking even led to a lytic phenotype.
There kinds of experiments were also applied to genes Upon staining with a heavy metal, the thin sacculi from
coding for Fem transferases. The heterospecific expression Gram-negative bacteria appear in electron microscopy (EM)
of the femhB, femA and femB genes of S. aureus in Enter- pictures as flat, empty cell envelopes (Fig. 3). The thickness
ococcus faecalis led to the production of peptide stems of peptidoglycan has been determined from electron micro-
substituted by mosaic side chains that were effectively graphs of thin sections of E. coli cells. However, the results
engaged in cross-bridges (Arbeloa et al., 2004). Similar should be considered with caution when chemical fixation
results were obtained when the bppA1 gene of Enterococcus and dehydration are used in combination with certain
faecalis was expressed in Enterococcus faecium (Magnet et al., staining techniques. Depending on the procedure applied,
2007a). the peptidoglycan layer has a thickness between 1.5 and
Fig. 3. EM of purified sacculi. Sacculi purified from Escherichia coli (a, b) and from the extreme thermophile Thermus thermophilus (c) by repeated
incubations in boiling 4% sodium dodecyl sulfate were spread onto copper grids, washed, stained with 1% uranyl acetate and observed under a
transmission electron microscope. Sample A was subjected to carbon platinum shadowing at an angle of 151 to emphasize the surface features and
thickness. Scale bars represent 0.5 mm. Note the folds at the polar regions as the cylindrical structure collapses onto a flat surface.


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15 nm (Murray et al., 1965; de Petris, 1967; Hobot et al., 50–60 nm at the older regions of the cell surface (Touhami
1984; Leduc et al., 1985, 1989; Beveridge, 1999). The main et al., 2004). Upon mechanical removal of patches of outer
concerns with these methods are (1) that the dehydration wall, AFM produced images of regularly arranged, about
and fixation might change the thickness of the peptidogly- 26 nm thick strands running perpendicular to the long axis
can and (2) that the measurements determine the thickness of rod-shaped cells of Lactobacillus helveticus. Although the
of the line formed by the contrasting metal, which is not nature of these structures is unknown, it has been proposed
necessarily identical to the thickness of the peptidoglycan that they are made of bundles of twisted peptidoglycan
layer (de Petris, 1967; Wientjes et al., 1991). The introduc- strands (Firtel et al., 2004).
tion of cryo-transmission electron microscopy (cryo-TEM)
of frozen hydrated sections proved to be a major improve- Elasticity of sacculi
ment because this technique omits the chemical fixation and
Low-angle laser light scattering was used to determine the
staining procedures. With this method, the peptidoglycan
change of the mean surface of E. coli sacculi following
can be seen as a thin line beneath the outer membrane in
an alteration of the net charge, either by changing to a low
thin sections of Gram-negative bacteria. In E. coli and
or a high pH, or by chemical modification. From these
Pseudomonas aeruginosa, the peptidoglycan layer is
studies, it was concluded that the sacculi are elastic and can
6.35 0.53 and 2.41 0.54 nm thick, respectively (Matias
reversibly expand and shrink threefold without rupture
et al., 2003). These data are in agreement with measure-
(Koch & Woeste, 1992). Osmotic challenge of growing
ments of the thickness of isolated sacculi from E. coli with
E. coli cells results in a maximum shrinkage in the surface
two different methods. Small-angle neutron scattering of
area of 33%, or a maximum swelling of 23% (Koch, 1984;
hydrated, isolated sacculi (without bound lipoprotein)
Baldwin et al., 1988). Similarly, living cells as well as isolated
yielded a thickness of 2.5 nm of 75–80% of the surface,
cell walls of Bacillus megaterium contract when they are
corresponding to the thickness of a single layer (Labischinski
transferred from water to salt solution (Marquis, 1968).
et al., 1991). The remaining 20–25% of the surface
When the cytoplasmic membrane of filamentous grown
had a maximum thickness of 7 nm, which would correspond
E. coli cells was destroyed with a detergent, there was a
to a triple layer. By atomic force microscopy (AFM), a
sudden decrease in the cell surface area of about 45% due to
thickness of 3.0 0.5 nm of air-dried sacculi from
the relaxation of the peptidoglycan (Koch et al., 1987).
E. coli was determined, and this value increased to
Apparently, the peptidoglycan is under dynamic stress in
6.0 0.5 nm upon rehydration of the sacculi (Yao et al.,
the living cell due to the cell’s turgor and there is
1999). AFM showed, too, that sacculi from P. aeruginosa
a limit to which the peptidoglycan can be maximally
were thinner, 1.5 0.5 nm in the air-dried form and
stretched.
3.0 0.5 nm when rehydrated, as compared with sacculi
Interestingly, isolated E. coli sacculi are significantly
from E. coli.
more deformable in the direction of the long axis of the
Application of the cryo-TEM technique to Gram-positive
cell (elastic modulus, 1.5 107–3 107 N m2; average,
bacteria revealed a bipartite organization of the cell wall
2.5 107 N m2) than in the direction perpendicular to the
(Matias & Beveridge, 2005, 2006, 2007; Zuber et al., 2006).
long axis (elastic modulus, 3.5 107–6 107 N m2; aver-
In all species analyzed, there is a zone of low density
age, 4.5 107 N m2) (Yao et al., 1999). The elastic modulus
presumably lacking polymeric wall structure next to the
is lower for material with greater elasticity. This is consistent
plasma membrane. This ‘inner wall zone (IWZ)’ or ‘peri-
with the observation that the changes in the volume
plasmic space’ has a thickness between 16 nm (in S. aureus)
of osmotically shocked E. coli cells are mainly due
and 22 nm (in B. subtilis). The ‘outer wall zone’ (OWZ) of
to changes in the cell length, whereas the cell diameter is
higher density is likely to represent the polymeric peptido-
virtually constant (van den Bogaart et al., 2007). It was
glycan–teichoic acid complex (with attached surface pro-
suggested that the anisotropy in elasticity is the consequence
teins). The thickness of the OWZ most likely varies with the
of the predominant alignment of the (more flexible) pep-
species, growth phase of the cells and growth conditions,
tides in the direction of the long axis of the cell and of the
and was determined to be in the range of 15–30 nm (in
(more rigid) glycan strands perpendicular to the direction of
S. aureus, B. subtilis, Streptococcus gordonii and Enterococcus
the long axis. Such a network has been modelled, and the
gallinarum). Interestingly, the septal cell wall region in S.
theoretical calculations of the elastic moduli are in good
aureus has five layers, with two layers of high density
agreement with the measured values (Boulbitch et al., 2000).
(OWZs) sandwiched between three layers of lower density
(IWZs) (Matias & Beveridge, 2007). AFM allowed visualiza-
Porosity of sacculi
tion of concentric rings surrounding a central depression on
a new hemisphere after daughter cell separation, as well as a Fluorescence-labelled dextrans of different sizes have been
network of fibers and large holes with diameters of used to determine the diameters of holes in the peptidoglycan


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network in E. coli and B. subtilis (Demchick & Koch, 1996). As commented above the sacculus is a covalently
Interestingly, the pores were of similar average sizes in closed structure built up from glycan strands that are cross-
peptidoglycans from Gram-negative and Gram-positive linked to each other through peptide bridges. In E. coli, and
species and they were relatively homogenous in size: the in general in Gram-negative bacteria, the sacculus is very
mean radius of the pores was 2.06 nm for E. coli peptidogly- thin, in particular compared with the thick, dense appear-
can and 2.12 nm for B. subtilis peptidoglycan, which is a ance of cell walls from Gram-positive species. These
similar value as the pore radius of at maximum 2.5 nm for B. basic properties, defined long ago, naturally lead to the
subtilis and B. megaterium cell walls described in a previous concept of the sacculus as static, regular and planar net-like
study (Hughes et al., 1975). From these values it was polymeric macromolecule, a concept which can still be
calculated that globular, uncharged proteins with molecular traced down to present-day textbooks. However, this some-
weights of up 22–24 kDa should be able to penetrate the what simplistic vision seems to be far from reality and
isolated, relaxed peptidoglycan. Globular proteins of up to the bacterial sacculus is proving itself to be a particularly
50 kDa or more should be able to diffuse through stretched intractable subject for structural studies. Application of even
peptidoglycan layer in the cell. Indeed, disruption of the the more powerful tools in structural analysis, as X-ray
outer membrane of E. coli in combination with a hyper- diffraction, EM, AFM, low angle neutron scattering, and
osmotic shock released a subset of proteins from the cell others have provided only limited information (Formanek
which were similar to those proteins which were able et al., 1976; Labischinski et al., 1979, 1985, 1991; Yao et al.,
to pass through a 100-kDa cut-off filter. Perhaps this 1999; Matias et al., 2003). Perhaps the most important
value is determined by the molecular sieving properties concept to derive from these studies is that the distribution
of the stretched peptidoglycan layer (Vazquez-Laslop of subunits in the sacculus is far from the highly regular
et al., 2001). ‘quasi crystalline’ schemes proposed in the past and so often
used to represent the cell wall.
From a structural point of view, the basic problem is to
Modelling the structure of the bacterial
define how individual glycan strands are arranged relative
sacculus to each other and to the cell axes. The interactions among
To understand how a biological structure grows, a detailed neighbouring glycan strands are in turn conditioned by
knowledge of how the individual components are organized three parameters; thickness, cross-linkage and length dis-
and arrayed with respect to each other is of prime relevance. tribution of the glycan strands. These three parameters
Unravelling the molecular architecture of the bacterial determine the number of chemical bonds per unit of surface
sacculus has been a constant aspiration for many microbiol- area opposing the turgor, and define the basic constrains in
ogists, but it is proving to be a frustrating topic. In model making.
particular, the architecture of the cell wall of Gram-positive
bacteria is far from being understood. Gram-positive species
Are sacculi from E. coli mono- or multilayered?
not only have a thick, multi-layered peptidoglycan but other
major surface polymers linked to it (Vollmer, 2008). Whereas the multilayered structure of sacculi from Gram-
The anionic poly(ribitol-phosphate) or poly(glycerol-phos- positive bacteria is generally accepted from EM and
phate) teichoic acids may account for up to 50% of the mass biochemical data (Beveridge & Matias, 2006; Matias &
of the cell wall. Many species also have capsular polysacchar- Beveridge, 2007), the nature of the Gram-negative sacculi is
ides which are often covalently linked to the peptidoglycan. still uncertain. The extreme thinness (3–4 nm) assigned to
In addition, there are many surface proteins either linked the E. coli sacculus in early EM work (Murray et al., 1965; de
covalently to peptidoglycan or bound noncovalently to cell Petris, 1967) was consistent with the sacculus being a planar
wall polymers. To decipher the architecture of this three- monolayer of peptidoglycan. However, later evidence from
dimensional assembly of different polymeric components different fields suggests a more complex situation. Neutron
and its enlargement during bacterial growth will be a major scattering studies on purified cell walls indicated that sacculi
challenge for the future. With respect to the architecture of consist of a mixture of mono (75%) and trilayered (25%)
the sacculus of Gram-negative bacteria, E. coli has been the regions (Labischinski et al., 1991), although no data is
best-studied subject. Therefore, the structural aspects of available about the distribution of the mono and trilayered
sacculi from this organism will be concentrated on. The idea areas. That the sacculus is ‘more than a monolayer’ finds
of the following comments is not so much to criticize additional support on the experimental determination of
existing models, but to point out aspects of cell wall biology the amount of peptidoglycan per cell which was close to
and biochemistry which are overlooked, but must be theoretical estimations for a single layered sacculus, but
accounted for, by present day models to help improve future could still accommodate sizeable areas with a trilayered
developments. structure (Wientjes et al., 1991), on the ability of E. coli to


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keep proper shape and growth rate with a severely reduced permitted an accurate analysis of the size distribution of
amount of peptidoglycan per unit of cell surface area (Prats glycan strands (Harz et al., 1990). However the method still
& de Pedro, 1989; Caparros et al., 1992), and on the suffers from a key limitation in that only glycan strands
identification of cross-linked trimers and tetramers in between 1 and 30 disaccharide units can be individually
sacculi (Glauner et al., 1988) which also favours a (partially) resolved. Longer strands cannot be separated from each
multi-layered structure. Application of AFM (Yao et al., other but at least the proportion of muropeptides in strands
1999) and advanced sample preparation techniques for longer than 30 disaccharides can be evaluated, and an
cryo-TEM (Matias et al., 2003) indicated that E. coli sacculi average value can be calculated from the proportion of 1,6-
are considerably thicker (6.0 nm by AFM, 6.3 nm by cryo- anhydroMurNAc-containing muropeptides. Information
TEM) than first thought. Perhaps more convincing than gathered by this method (Harz et al., 1990; Obermann &
absolute thickness measurements is the fact that sacculi Höltje, 1994; Höltje, 1998; Ishidate et al., 1998), showed
from another typical Gram-negative organism, P. aerugino- some unexpected, but crucial features of sacculi, that can be
sa, had only one half the thickness of those from E. coli summarized as: (1) about two thirds of total muropeptides
(3.0 and 2.5 nm for AFM and cryo-TEM, respectively). As are assembled in strands shorter than 30 disaccharide units;
sacculi from both species are made of identical subunits (2) the distribution is continuous with strands of every
(Quintela et al., 1995a), the more likely explanation is that length between 1 and 30 subunits; (3) the mean and modal
sacculi from E. coli, and probably other Gram-negative values for the distribution of short strands (o 30 dis-
bacteria, indeed include sizable regions of multilayered accharides chain1) are about 7–8 and 9–10 disaccharide
peptidoglycan. units per strand, respectively, with a large positive skewness;
(4) the ca. 40% of total material in long strands (4 30 dis-
accharides chain1) is recovered as a pool with a mean GCL
The length distribution of glycan strands
of 45 disaccharides chain1.
The sacculus is made up of glycan strands cross-linked to Therefore, sacculi of E. coli contain relatively short glycan
each other through peptide bridges. As the physico chemical strands with a broad length distribution and with a strong
properties of the glycan and peptide moieties are very tendency to cross-link to each other through the glycan
different, in particular the ability of each to change con- strand terminating muropeptides.
formation under stress (Barnickel et al., 1979; Labischinski
et al., 1985; Labischinski & Maidhof, 1994; Koch, 2000b), an
Cross-linking peptidoglycan strands
important parameter for the understanding of the macro-
molecular structure of the sacculus is the length distribution In Gram-negative bacteria peptidoglycan strands are
of the glycan strands. The peptide stems are of a fixed length, bound (cross-linked) to each other through the peptide
and in principle distributed regularly along the glycan back- side chains of the monomeric disaccharide penta- (tetra)-
bone. Therefore the number of possible interstrand connec- peptide subunits (Schleifer & Kandler, 1972). Cross-linking
tions is also a direct function of glycan chain length (GCL). in Gram-negative species, in particular in E. coli, is relatively
For a long time the only way to determine GCL was based on low and mediated by peptide bridges consisting of the
quantification of glycan strand terminal muropeptides heptapeptide L-Ala ! D-Glu ! meso-A2pm ! D-Ala !
(Schindler et al., 1976; Beachey et al., 1981; Glauner et al., meso-A2pm’D-Glu’L-Ala (DD-Ala-meso-A2pm, or 3–4,
1988). This permits calculation of a mean value for GCL bridges), the result of DD-transpeptidation reactions respon-
(mGCL). Reported results (Pisabarro et al., 1985, 1987; sible for incorporation of new precursors into the cell wall
Glauner, 1988; Glauner & Höltje, 1990) show four structu- (Schleifer & Kandler, 1972; Quintela et al., 1995a; Höltje,
rally relevant features: (1) wild-type E. coli strains in 1998).
exponential growth have mGCL from 25 to 35 disaccharide Application of HPLC methods demonstrated the pre-
units; (2) the mGCL changes widely in response to the state sence of a second kind of cross-linking in E. coli (Glauner
of growth of the cells: in E. coli mGCL drops to ca. 15 et al., 1988) and other Gram-negative bacteria (Quintela
disaccharides in resting cells; (3) more than 80% of 1,6- et al., 1995a), the so-called (LD)-A2pm-A2pm (or 3–3)
anhydroMurNAc terminal muropeptides are cross-linked bridges. These bridges are shorter by one amino acid
and there are indications of a similar situation for the (a hexapeptide instead of the normal heptapeptide) and
GlcNAc termini as deduced from peptidoglycan labelled are, apparently, the product of penicillin-insensitive LD-
with galactosyl transferase (Schindler et al., 1976; Beachey transpeptidase activity (Höltje, 1998). Sacculi from growing,
et al., 1981; C. Altmutter, unpublished data). wild type E. coli strains usually exhibit a degree of cross-
Application of a new method based on enzymatic clip- linkage (molar proportion of cross-linked muropeptides
ping of peptide stems with human serum amidase followed relative to total muropeptides) of about 25–35%. That
by HPLC separation of the resulting linear polysaccharides means that on average every third to second disaccharide in


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a peptidoglycan strand would be cross-linked to another 1998; Koch, 1998a, b, 2000a, b; Dmitriev et al., 1999, 2003;
adjacent strand. Similar values seem to be common among Yao et al., 1999; Pink et al., 2000; Vollmer & Höltje, 2004;
Gram-negative species, although data are still limited Meroueh et al., 2006). Most available evidence favoured
(Quintela et al., 1995a; Costa et al., 1999). LD-A2pm-A2pm models postulating glycan strands oriented parallel to the
bridges account for 7–16% of total cross-linked muropep- cell surface, and in most cases with the glycan backbones
tides (Pisabarro et al., 1985; Glauner, 1988; Glauner & transversal to the cell longitudinal axis. More recently an
Höltje, 1990), a minor but certainly significant fraction. alternative model based on glycan strands oriented perpen-
However the function and distribution of A2pm-A2pm dicular to the surface of the sacculus has been proposed
bridges in the sacculus remains unknown. The positive (Dmitriev et al., 1999, 2003; Meroueh et al., 2006). As
identification of cross-linked trimers and tetramers by indicated above, it is not intended here to enter into a
means of HPLC techniques in E. coli sacculi demonstrated discussion of models so far proposed, but rather point out
that a particular subunit in one glycan strand could be some aspects, in the authors’ opinion, overlooked in those
linked to two, or even three, other glycan strands (Glauner, models.
1988; Glauner & Höltje, 1990). Although trimers account A good starting point is to refer to a recent comment
for only about 3–4% of total muropeptides (dimers add up (Young, 2006) on the same topic which emphasizes what a
to 30–40%), the fact that they represent connection nodes weak point for most models is: the requirement for rather
for multiple glycan strands suggest a relevant structural role restrictive structural and morphological parameters. All the
for this minor class of muropeptides (Glauner et al., 1988; main models (Höltje, 1996, 1998; Koch, 2000b; Dmitriev
Höltje, 1998). Tetramers are barely detectable with reported et al., 2003) do require a high degree of order in the array of
abundances about 0.2% of total muropeptides, a propor- both glycan strands and cross-links, a requirement opposed
tionally low value, which nevertheless corresponds to some by experimental evidence (Labischinski et al., 1979, 1985;
thousands of muropeptides per sacculus. Following the Labischinski & Maidhof, 1994). As commented in the
same argument as above scarce tetramers could still be preceding sections the main structural parameters in the
structurally significant (Glauner et al., 1988). As commented sacculus are subjected to drastic changes on the course of
above the degree of cross-linkage is a variable parameter normal growth. This calls for dynamic rather than static
influenced by the state of growth of the cell, and aging of models because the sacculus as such is in a continuous state
newly inserted muropeptides (de Pedro & Schwarz, 1981; of change. Furthermore, any credible model should have an
Pisabarro et al., 1985; Glauner & Höltje, 1990). intrinsic ability to accommodate the size and shape changes
Because disaccharide subunits in peptidoglycan strands (in particular in cell diameter, but also more dramatic ones
are rotated with respect to each other due to the influence as round and branched shapes) that cells can exhibit under
of the lactyl group in MurNAc, consecutive peptide stems specific conditions.
point out in different directions (Labischinski et al., 1979, Sacculi are made of glycan strands with a very wide
1985). Early estimations indicated rotation angles close to distribution of sizes, with a substantial proportion of total
901 between consecutive disaccharides, and therefore a peptidoglycan in strands too short to be connected to
periodicity of four muropeptides, that is every fourth nearby strands by more than two cross-links. Such strands
peptide stem would roughly point in the same direction could structurally be assimilated to a long range cross-link,
(Labischinski et al., 1985). However, recent calculations with connecting longer and relatively distant strands (Costa et al.,
synthetic peptidoglycan fragments indicate a rotation angle 1999; de Pedro, 2004). Even if it is generally assumed, there
close to 1201 (Meroueh et al., 2006), consistent with a is no evidence at all that cross-linkage happens regularly
periodicity of three muropeptides. The periodicity of pepti- alongside the glycan strands. Indeed, the tendency of glycan
doglycan conditions the cross-linking between adjacent strand termini to be highly cross-linked means that cross-
strands, as only those peptide stems with the correct relative linkage in internal positions is lower than the mean value for
orientation can be proficient substrates for transpeptida- total peptidoglycan. Therefore, it is likely that long glycan
tion. An immediate consequence of these facts is that strands might have relatively long uncross-linked stretches.
adjacent peptidoglycan strands are unlikely to be cross- A final comment on the organization of glycan strands
linked to each other by consecutive muropeptides (Koch, comes from the very existence of cross-linked trimers and
1998a, b). tetramers. These groups of muropeptides might not be very
abundant, but still enough to be significant elements of the
sacculus with about (1–3) 104 and 103 trimers and tetra-
The orientation of the glycan strands
mers per sacculus, respectively. The interesting point about
The more controversial aspect related to the structure of the these two families of muropeptides is that they represent
sacculus is the orientation and distribution of the glycan connecting hubs for several crossing glycan strands.
strands (Formanek et al., 1976; Verwer et al., 1978; Höltje, An attractive idea is that trimers and tetramers represent


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linking points of short-to-long glycan strands. A normal research can lead to the discovery of peptidoglycans whose
cross-link bridging two nearby, long glycan strands could act structure diverge from the overall structure defined at the
as the attachment point for a short glycan strand acting as a beginning of this review. A recent example is represented by
long range connection to another relatively remote long T. maritima, in which a D-Lys residue with an ‘upside-down’
glycan strand. Interestingly, about 30% of cross-linked 1,6- arrangement has been identified (Boniface et al., 2006). It is
anhydroMurNac muropeptides are trimers, whilst only likely that such an unusual motif, which coexists with the
10% of cross-linked nonterminal muropeptides are trimers conventional L-Lys-containing motif, is at the origin of a
as calculated from data in Glauner et al. (1988). particular type of cross-link. Another example is that of
That is, glycan strand termini seem to have a high tendency Chlamydiae, for which no muramic acid-containing pepti-
to cross-link to a dimeric muropeptide to form a trimer. doglycan was detected to date. However, the biosynthetic
Whether this tendency is more marked in short or long activities characterized so far (MurA, MurC, Ddl) are
glycan strands cannot be decided at present, and of course functional either in vitro or in a heterologous context
trimers could show no preference at all relative to glycan (McCoy & Maurelli, 2006). It is difficult to imagine that
strand length. these enzymes do not participate in the synthesis of a
As for the remaining, major fraction of cross-linked specific macromolecule, the structure of which presumably
trimers and tetramers geometrical arguments require that greatly differs from that of usual peptidoglycan.
three, or four, peptidoglycan strands cross over (Glauner In the last two decades, the improvement of analytical
et al., 1988). There are no data available on the angles methods (HPLC, MS) has allowed to show that, within a
between multiple glycan strands linked together at these particular species, variations in peptidoglycan fine structure
positions. In the extreme cases, they could run parallel, occur as a function of aging, medium composition, patho-
antiparallel, or perpendicular to each other. Regions of genesis, or presence of antibiotics. This type of research has
multiple glycan strand crossings could be related with the implications not only in the field of bacterial physiology, but
postulated regions of multilayered peptidoglycan. Whether also in those of innate immunity, pathogenicity, and anti-
or not simple crossing of strands could be enough to explain bacterial therapy.
the neutron diffraction results (Labischinski et al., 1991) has One major task for the future is to determine the
not been addressed yet. molecular architecture of peptidoglycan in Gram-positive
Finally, another aspect models should be able to account and Gram-negative species, which is not possible with
for is the ability of E. coli cells, and likely others, to today’s techniques. This includes determination of the
accommodate variations in the amount of peptidoglycan orientation of the glycan strands and peptides with respect
per unit of cell surface. Under normal growth conditions to the cell’s axes and the distribution pattern of particular
results suggest that E. coli has about two times as much structures (LD-cross-links, dimeric and trimeric peptides,
peptidoglycan as would be strictly required to preserve glycan strand ends, attachment sites for other polymers) on
physical integrity, normal shape and growth parameters the surface of the cell wall. Knowing the architecture of
(Prats & de Pedro, 1989; Caparros et al., 1992). It is peptidoglycan is a prerequisite for solving the mechanism(s)
interesting to note that recent measurements of cell wall of cell wall growth. As outlined in this review, models of
thickness in E. coli and P. aeruginosa indicate that the former peptidoglycan architecture should be based on the length
is twice as thick as the latter (Yao et al., 1999; Matias et al., distribution of the glycan strands, the degree of cross-
2003). If indeed E. coli does have an ‘excess’ of peptidoglycan linkage, the thickness of the macromolecule, the number of
relative to other species as P. aeruginosa, then Gram-negative subunits per cell surface, as well as on biophysical data on
bacteria should have to be considered a more structurally the porosity and elasticity of the sacculus.
heterogeneous group than formerly thought.
Concluding remarks Acknowledgements

Many data on the chemical structure and the biophysics of This work was supported by the European Commission
peptidoglycan have been gathered over the last decades. through the EUR-INTAFAR project (LSHM-CT-2004-
However, knowledge of this fascinating molecule is still very 512138) (to W.V. and D.B.), the Deutsche Forschungsge-
limited. For example, the analysis of peptidoglycan compo- meinschaft (DFG) within the Forschergruppe Bakterielle
sition with high-resolution techniques has been performed Zellhülle (FOR 449) (to W.V.), the Centre National de la
so far only for a few bacterial species. A significantly Recherche Scientifique (UMR 8619) (to D.B.), grant
enlarged data set on peptidoglycan fine structures will be of BFU2006-04574 from Ministerio de Educación y Ciencia,
interest for different research fields including bacterial Spain (to M.A.P) and an institutional grant from Fundación
taxonomy, physiology, and pathogenesis. Moreover, such Ramón Areces (to M.A.P).


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References Boneca IG, Dussurget O, Cabanes D et al. (2007) A critical role

for peptidoglycan N-deacetylation in Listeria evasion from the
Aitken A & Stanier RY (1979) Characetrization of peptidoglycan
host innate immune system. Proc Natl Acad Sci USA 104:
from cyanelles of Cyanophora paradoxa. J Gen Microbiol 112:
997–1002.
219–223.
Boniface A (2007) Etude des relations structure-activité au sein
Arbeloa A, Hugonnet JE, Sentilhes AC, Josseaume N, Dubost L,
de la famille des Mur synthétases, enzymes de la voie de
Monsempes C, Blanot D, Brouard JP & Arthur M (2004)
biosynthèse du peptidoglycane. Thesis, Universite Paris-Sud,
Synthesis of mosaic peptidoglycan cross-bridges by hybrid
Orsay, France.
peptidoglycan assembly pathways in gram-positive bacteria.
Boniface A, Bouhss A, Mengin-Lecreulx D & Blanot D (2006) The
J Biol Chem 279: 41546–41556.
MurE synthetase from Thermotoga maritima is endowed with
Atrih A, Zollner P, Allmaier G & Foster SJ (1996) Structural
an unusual D-lysine adding activity. J Biol Chem 281:
analysis of Bacillus subtilis 168 endospore peptidoglycan and
15680–15686.
its role during differentiation. J Bacteriol 178: 6173–6183.
Bouhss A, Josseaume N, Severin A et al. (2002) Synthesis of the
Atrih A, Bacher G, Allmaier G, Williamson MP & Foster SJ
L-alanyl-L-alanine cross-bridge of Enterococcus faecalis
(1999a) Analysis of peptidoglycan structure from vegetative
peptidoglycan. J Biol Chem 277: 45935–45941.
cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan
Bouhss A, Trunkfield AE, Bugg TDH & Mengin-Lecreulx D
maturation. J Bacteriol 181: 3956–3966.
(2008) The biosynthesis of peptidoglycan lipid-linked
Atrih A, Bacher G, Korner R, Allmaier G & Foster SJ (1999b)
intermediates. FEMS Microbiol Rev, in press.
Structural analysis of Bacillus megaterium KM spore Boulbitch A, Quinn B & Pink D (2000) Elasticity of the rod-
peptidoglycan and its dynamics during germination. shaped gram-negative eubacteria. Phys Rev Lett 85: 5246–5249.
Microbiology 145: 1033–1041. Braun V & Rehn K (1969) Chemical characterization, spatial
Baldwin WW, Sheu MJ, Bankston PW & Woldringh CL (1988) distribution and function of a lipoprotein (murein-
Changes in buoyant density and cell size of Escherichia coli in lipoprotein) of the E. coli cell wall. The specific effect of trypsin
response to osmotic shocks. J Bacteriol 170: 452–455. on the membrane structure. Eur J Biochem 10: 426–438.
Barnickel G, Labischinski H, Bradaczek H & Giesbrecht P (1979) Braun V & Sieglin U (1970) The covalent murein-lipoprotein
Conformational energy calculation on the peptide part of structure of the Escherichia coli cell wall. The attachment site of
murein. Eur J Biochem 95: 157–165. the lipoprotein on the murein. Eur J Biochem 13: 336–346.
Barreteau H, Kovač A, Boniface A, Sova M, Gobec S & Blanot D Bugg TD, Wright GD, Dutka-Malen S, Arthur M, Courvalin P &
(2008) Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Walsh CT (1991) Molecular basis for vancomycin resistance in
Microbiol Rev, in press. Enterococcus faecium BM4147: biosynthesis of a depsipeptide
Beachey EH, Keck W, de Pedro MA & Schwarz U (1981) peptidoglycan precursor by vancomycin resistance proteins
Exoenzymatic activity of transglycosylase isolated from VanH and VanA. Biochemistry 30: 10408–10415.
Escherichia coli. Eur J Biochem 116: 355–358. Burman LG & Park JT (1983) Changes in the composition of
Bellais S, Arthur M, Dubost L et al. (2006) Aslfm, the D-aspartate Escherichia coli murein as it ages during exponential growth.
ligase responsible for the addition of D-aspartic acid onto the J Bacteriol 155: 447–453.
peptidoglycan precursor of Enterococcus faecium. J Biol Chem Caparros M, Pisabarro AG & de Pedro MA (1992) Effect of
281: 11586–11594. D-amino acids on structure and synthesis of peptidoglycan in
Beveridge TJ (1999) Structures of gram-negative cell walls and Escherichia coli. J Bacteriol 174: 5549–5559.
their derived membrane vesicles. J Bacteriol 181: 4725–4733. Chaput C, Labigne A & Boneca IG (2007) Characterization of
Beveridge TJ & Matias VR (2006) Ultrastructure of gram-positive Helicobacter pylori lytic transglycosylases Slt and MltD.
cell walls. Gram-Positive Pathogens (Fischetti VA, Novick RP, J Bacteriol 189: 422–429.
Ferretti JJ, Portnoy DA & Rood JI, eds), pp. 3–11. American Chopra I, Storey C, Falla TJ & Pearce JH (1998) Antibiotics,
Society for Microbiology, Washington, DC. peptidoglycan synthesis and genomics: the chlamydial
Biarrotte-Sorin S, Maillard AP, Delettré J, Sougakoff W, Arthur M anomaly revisited. Microbiology 144: 2673–2678.
& Mayer C (2004) Crystal structures of Weissella viridescens Cloud KA & Dillard JP (2002) A lytic transglycosylase of Neisseria
FemX and its complex with UDP-MurNAc-pentapeptide: gonorrhoeae is involved in peptidoglycan-derived cytotoxin
insights into FemABX family substrates recognition. Structure production. Infect Immun 70: 2752–2757.
12: 257–267. Costa K, Bacher G, Allmaier G et al. (1999) The morphological
Blasco B, Pisabarro AG & de Pedro MA (1988) Peptidoglycan transition of Helicobacter pylori cells from spiral to coccoid
biosynthesis in stationary-phase cells of Escherichia coli. is preceded by a substantial modification of the cell wall.
J Bacteriol 170: 5224–5228. J Bacteriol 181: 3710–3715.
Boneca IG, Huang ZH, Gage DA & Tomasz A (2000) de Jonge BL, Chang YS, Gage D & Tomasz A (1992)
Characterization of Staphylococcus aureus cell wall glycan Peptidoglycan composition of a highly methicillin-resistant
strands, evidence for a new beta-N-acetylglucosaminidase Staphylococcus aureus strain. The role of penicillin binding
activity. J Biol Chem 275: 9910–9918. protein 2A. J Biol Chem 267: 11248–11254.


by guest
on 23 August 2018
de la Rosa EJ, de Pedro MA &Vazquez D (1985) Penicillin binding Isolation of a new peptide dimer, N-a-[L-alanyl-g-(a-D-
proteins: role in initiation of murein synthesis in Escherichia glutamylglycine)]-L-lysyl-D-alanyl-N-a-[L-alanyl-g-(a-D-
coli. Proc Natl Acad Sci USA 82: 5632–5635. glutamylglycine)]-L-lysyl-D-alanine. Biochemistry 7:
Demchick P & Koch AL (1996) The permeability of the wall 1450–1460.
fabric of Escherichia coli and Bacillus subtilis. J Bacteriol 178: Glauner B (1988) Separation and quantification of muropeptides
768–773. with high-performance liquid chromatography. Anal Biochem
de Pedro MA (2004) Topological domains in the cell wall of 172: 451–464.
Escherichia coli. Molecules in Time and Space: Bacterial Shape, Glauner B & Höltje J-V (1990) Growth pattern of the murein
Division and Phylogeny (Vicente M, Tamames J, Valencia A & sacculus of Escherichia coli. J Biol Chem 265: 18988–18996.
Mingorance J, eds), pp. 27–58. Kluwer Academic/Plenum Glauner B, Höltje J-V & Schwarz U (1988) The composition of
Publishers, New York. the murein of Escherichia coli. J Biol Chem 263: 10088–10095.
de Pedro MA & Schwarz U (1981) Heterogeneity of newly Gmeiner J, Essig P & Martin HH (1982) Characterization of
inserted and preexisting murein in the sacculus of Escherichia
minor fragments after digestion of Escherichia coli murein with
coli. Proc Natl Acad Sci USA 78: 5856–5860.
endo-N,O-diacetylmuramidase from Chalaropsis, and
de Petris S (1967) Ultrastructure of the cell wall of Escherichia coli
determination of glycan chain length. FEBS Lett 138: 109–112.
and chemical nature of its constituent layers. J Ultrastruct Res
Hammes W, Schleifer KH & Kandler O (1973) Mode of action of
19: 45–83.
glycine on the biosynthesis of peptidoglycan. J Bacteriol 116:
Dmitriev BA, Ehlers S & Rietschel ET (1999) Layered murein
revisited: a fundamentally new concept of bacterial cell wall 1029–1053.
Hammes WP, Neukam R & Kandler O (1977) On the specificity
structure, biogenesis and function. Med Microbiol Immunol
(Berlin) 187: 173–181. of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic
Dmitriev BA, Toukach FV, Schaper KJ, Holst O, Rietschel ET & acid: diamino acid ligase of Bifidobacterium globosum. Arch
Ehlers S (2003) Tertiary structure of bacterial murein: the Microbiol 115: 95–102.
scaffold model. J Bacteriol 185: 3458–3468. Harz H, Burgdorf K & Höltje J-V (1990) Isolation and separation
Dramsi S, Davison S, Magnet S & Arthur M (2008) Surface of the glycan strands from murein of Escherichia coli by
proteins covalently attached to peptidoglycan: examples from reversed-phase high-performance liquid chromatography.
both gram-positive and gram-negative bacteria. FEMS Anal Biochem 190: 120–128.
Microbiol Rev, in press. Healy VL, Lessard IA, Roper DI, Knox JR & Walsh CT (2000)
Duncan K, van Heijenoort J & Walsh CT (1990) Purification and Vancomycin resistance in enterococci: reprogramming of the
characterization of the D-alanyl-D-alanine-adding enzyme D-ala-D-Ala ligases in bacterial peptidoglycan biosynthesis.
from Escherichia coli. Biochemistry 29: 2379–2386. Chem Biol 7: R109–R119.
Firtel M, Henderson G & Sokolov I (2004) Nanosurgery: Hesse L, Bostock J, Dementin S, Blanot D, Mengin-Lecreulx D &
observation of peptidoglycan strands in Lactobacillus helveticus Chopra I (2003) Functional and biochemical analysis of
cell walls. Ultramicroscopy 101: 105–109. Chlamydia trachomatis MurC, an enzyme displaying UDP-N-
Fiser A, Filipe SR & Tomasz A (2003) Cell wall branches, acetylmuramate: amino acid ligase activity. J Bacteriol 185:
penicillin resistance and the secrets of the MurM protein. 6507–6512.
Trends Microbiol 11: 547–553. Hobot JA, Carlemalm E, Villiger W & Kellenberger E (1984)
Folkening WJ, Nogami W, Martin SA & Rosenthal RS (1987)
Periplasmic gel: new concept resulting from the reinvestigation
Structure of Bordetella pertussis peptidoglycan. J Bacteriol 169:
of bacterial cell envelope ultrastructure by new methods. J
4223–4227.
Bacteriol 160: 143–152.
Formanek H, Schleifer KH, Seidl HP, Lindemann R & Zundel G
Höltje J-V (1996) A hypothetical holoenzyme involved in the
(1976) Three-dimensional structure of peptidoglycan of
replication of the murein sacculus of Escherichia coli.
bacterial cell walls: infra red investigations. FEBS Lett 70:
150–154. Microbiology 142(Part 8): 1911–1918.
Höltje J-V (1998) Growth of the stress-bearing and shape-
Galperin MY & Koonin EV (1997) A diverse superfamily of
enzymes with ATP-dependent carboxylate-amine/thiol ligase maintaining murein sacculus of Escherichia coli. Microbiol Mol
activity. Protein Sci 6: 2639–2643. Biol Rev 62: 181–203.
Garcia-Bustos JF, Chait BT & Tomasz A (1987) Structure of the Höltje J-V & Glauner B (1990) Structure and metabolism of the
peptide network of pneumococcal peptidoglycan. J Biol Chem murein sacculus. Res Microbiol 141: 75–89.
262: 15400–15405. Huber R, Langworthy TA, Konig H, Thomm M, Woese CR, Sleytr
Ghuysen JM & Goffin C (1999) Lack of cell wall peptidoglycan UB & Stetter KO (1986) Thermatoga maritima sp. nov.
versus penicillin sensitivity: new insights into the chlamydial represents a new genus of unique extremely thermophilic
anomaly. Antimicrob Agents Chemother 43: 2339–2344. eubacteria growing up to 90 1C. Arch Microbiol 144: 324–333.
Ghuysen JM, Bricas E, Lache M & Leyh-Bouille M (1968) Hughes RC (1971) Autolysis of Bacillus cereus cell walls and
Structure of the cell walls of Micrococcus lysodeikticus. 3. isolation of structural components. Biochem J 121: 791–802.


by guest
on 23 August 2018
Hughes RC, Thurman PF & Stokes E (1975) Estimates of the Magnet S, Arbeloa A, Mainardi JL et al. (2007a) Specificity of L,D-
porosity of Bacillus licheniformis and Bacillus subtilis cell walls. transpeptidases from gram-positive bacteria producing
Z Immunitatsforsch Exp Klin Immunol 149: 126–135. different peptidoglycan chemotypes. J Biol Chem 282:
Ishidate K, Ursinus A, Höltje J-V & Rothfield L (1998) Analysis of 13151–13159.
the length distribution of murein glycan strands in ftsZ and ftsI Magnet S, Bellais S, Dubost L, Fourgeaud M, Mainardi JL, Petit-
mutants of E. coli. FEMS Microbiol Lett 168: 71–75. Frère S, Marie A, Mengin-Lecreulx D, Arthur M & Gutmann L
Kamio Y, Itoh Y & Terawaki Y (1981) Chemical structure of (2007b) Identification of the L,D-transpeptidases responsible
peptidoglycan in Selenomonas ruminantium: cadaverine links for attachment of the Braun lipoprotein to Escherichia coli
covalently to the D-glutamic acid residue of peptidoglycan. peptidoglycan. J Bacteriol 189: 3927–3931.
J Bacteriol 146: 49–53. Mahapatra S, Crick DC & Brennan PJ (2000) Comparison of the
Koch AL (1984) Shrinkage of growing Escherichia coli cells by
UDP-N-acetylmuramate: L-alanine ligase enzymes from
osmotic challenge. J Bacteriol 159: 919–924.
Mycobacterium tuberculosis and Mycobacterium leprae.
Koch AL (1998a) The three-for-one model for gram-negative wall
J Bacteriol 182: 6827–6830.
growth: a problem and a possible solution. FEMS Microbiol
Mahapatra S, Yagi T, Belisle JT, Espinosa BJ, Hill PJ, McNeil MR,
Lett 162: 127–134.
Brennan PJ & Crick DC (2005) Mycobacterial lipid II is
Koch AL (1998b) Orientation of the peptidoglycan chains in the
sacculus of Escherichia coli. Res Microbiol 149: 689–701. composed of a complex mixture of modified muramyl and
Koch AL (2000a) Simulation of the conformation of the murein peptide moieties linked to decaprenyl phosphate. J Bacteriol
fabric: the oligoglycan, penta-muropeptide, and cross-linked 187: 2747–2757.
nona-muropeptide. Arch Microbiol 174: 429–439. Mainardi JL, Legrand R, Arthur M, Schoot B, van Heijenoort J &
Koch AL (2000b) Length distribution of the peptidoglycan chains Gutmann L (2000) Novel mechanism of beta-lactam resistance
in the sacculus of Escherichia coli. J Theor Biol 204: 533–541. due to bypass of DD-transpeptidation in Enterococcus faecium. J
Koch AL & Woeste S (1992) Elasticity of the sacculus of Biol Chem 275: 16490–16496.
Escherichia coli. J Bacteriol 174: 4811–4819. Mainardi JL, Fourgeaud M, Hugonnet JE et al. (2005) A novel
Koch AL, Lane SL, Miller JA & Nickens DG (1987) Contraction of peptidoglycan cross-linking enzyme for a beta-lactam-
filaments of Escherichia coli after disruption of cell membrane resistant transpeptidation pathway. J Biol Chem 280:
by detergent. J Bacteriol 169: 1979–1984. 38146–38152.
Labischinski H & Maidhof H (1994) Bacterial peptidoglycan: Mainardi JL, Villet R, Bugg TDH, Mayer C & Arthur M (2008)
overview and evolving concepts. Bacterial Cell Wall (Ghuysen Evolution of peptidoglycan biosynthesis under the selective
JM & Hakenbeck R, eds), pp. 23–38. Elsevier, Amsterdam. pressure of antibiotics in gram-positive bacteria. FEMS
Labischinski H, Barnickel G, Bradaczek H & Giesbrecht P (1979) Microbiol Rev, in press.
On the secondary and tertiary structure of murein. Low and Markiewicz Z, Glauner B & Schwarz U (1983) Murein structure
medium-angle X-ray evidence against chitin-based and lack of DD- and LD-carboxypeptidase activities in
conformations of bacterial peptidoglycan. Eur J Biochem 95: Caulobacter crescentus. J Bacteriol 156: 649–655.
147–155. Marquis RE (1968) Salt-induced contraction of bacterial cell
Labischinski H, Barnickel G, Naumann D & Keller P (1985) walls. J Bacteriol 95: 775–781.
Conformational and topological aspects of the three- Marraffini LA, Dedent AC & Schneewind O (2006) Sortases and
dimensional architecture of bacterial peptidoglycan. Ann Inst
the art of anchoring proteins to the envelopes of gram-positive
Pasteur Microbiol 136A: 45–50.
bacteria. Microbiol Mol Biol Rev 70: 192–221.
Labischinski H, Goodell EW, Goodell A & Hochberg ML (1991)
Matias VR & Beveridge TJ (2005) Cryo-electron microscopy
Direct proof of a ‘more-than-single-layered’ peptidoglycan
reveals native polymeric cell wall structure in Bacillus subtilis
architecture of Escherichia coli W7: a neutron small-angle
168 and the existence of a periplasmic space. Mol Microbiol 56:
scattering study. J Bacteriol 173: 751–756.
240–251.
Laitinen H & Tomasz A (1990) Changes in composition of
Matias VR & Beveridge TJ (2006) Native cell wall organization
peptidoglycan during maturation of the cell wall in
pneumococci. J Bacteriol 172: 5961–5967. shown by cryo-electron microscopy confirms the existence of a
Leduc M, Frehel C & van Heijenoort J (1985) Correlation periplasmic space in Staphylococcus aureus. J Bacteriol 188:
between degradation and ultrastructure of peptidoglycan 1011–1021.
during autolysis of Escherichia coli. J Bacteriol 161: 627–635. Matias VR & Beveridge TJ (2007) Cryo-electron microscopy of
Leduc M, Frehel C, Siegel E & van Heijenoort J (1989) cell division in Staphylococcus aureus reveals a mid-zone
Multilayered distribution of peptidoglycan in the periplasmic between nascent cross walls. Mol Microbiol 64: 195–206.
space of Escherichia coli. J Gen Microbiol 135: 1243–1254. Matias VR, Al-Amoudi A, Dubochet J & Beveridge TJ (2003)
Machida M, Takechi K, Sato H et al. (2006) Genes for the Cryo-transmission electron microscopy of frozen-hydrated
peptidoglycan synthesis pathway are essential for chloroplast sections of Escherichia coli and Pseudomonas aeruginosa.
division in moss. Proc Natl Acad Sci USA 103: 6753–6758. J Bacteriol 185: 6112–6118.


by guest
on 23 August 2018
McCoy AJ & Maurelli AT (2006) Building the invisible wall: Perkins HR (1969) The configuration of 2,6-diamino-3-
updating the chlamydial peptidoglycan anomaly. Trends hydroxypimelic acid in microbial cell walls. Biochem J 115:
Microbiol 14: 70–77. 797–805.
Mengin-Lecreulx D & Lemaitre B (2005) Structure and Perkins HR (1971) Homoserine and diaminobutyric acid in the
metabolism of peptidoglycan and molecular requirements mucopeptide-precursor-nucleotides and cell walls of some
allowing its detection by the Drosophila innate immune plant-pathogenic corynebacteria. Biochem J 121: 417–423.
system. J Endotoxin Res 11: 105–111. Pfanzagl B, Allmaier G, Schmid ER, de Pedro MA & Löffelhardt
Mengin-Lecreulx D & van Heijenoort J (1985) Effect of growth W (1996) N-acetylputrescine as a characteristic constituent of
conditions on peptidoglycan content and cytoplasmic steps of cyanelle peptidoglycan in glaucocystophyte algae. J Bacteriol
its biosynthesis in Escherichia coli. J Bacteriol 163: 208–212. 178: 6994–6997.
Mengin-Lecreulx D, Michaud C, Richaud C, Blanot D & van Pink D, Moeller J, Quinn B, Jericho M & Beveridge T (2000) On
Heijenoort J (1988) Incorporation of LL-diaminopimelic acid the architecture of the gram-negative bacterial murein
into peptidoglycan of Escherichia coli mutants lacking sacculus. J Bacteriol 182: 5925–5930.
diaminopimelate epimerase encoded by dapF. J Bacteriol 170: Pisabarro AG, de Pedro MA & Vazquez D (1985) Structural
2031–2039. modifications in the peptidoglycan of Escherichia coli
Mengin-Lecreulx D, Blanot D & van Heijenoort J (1994) associated with changes in the state of growth of the culture. J
Replacement of diaminopimelic acid by cystathionine or Bacteriol 161: 238–242.
lanthionine in the peptidoglycan of Escherichia coli. J Bacteriol Pisabarro AG, Garcia del Portillo F, de la Rosa EJ & de Pedro MA
176: 4321–4327. (1987) Initiation of murein biosynthesis in E. coli cells treated
Mengin-Lecreulx D, Falla T, Blanot D, van Heijenoort J, Adams with b-lactam antibiotics. FEMS Microbiol Lett 42: 81–84.
DJ & Chopra I (1999) Expression of the Staphylococcus aureus Prats R & de Pedro MA (1989) Normal growth and division of
UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase Escherichia coli with a reduced amount of murein. J Bacteriol
in Escherichia coli and effects on peptidoglycan biosynthesis 171: 3740–3745.
and cell growth. J Bacteriol 181: 5909–5914. Quintela JC, Caparros M & de Pedro MA (1995a) Variability of
Meroueh SO, Bencze KZ, Hesek D, Lee M, Fisher JF, Stemmler TL peptidoglycan structural parameters in gram-negative
& Mobashery S (2006) Three-dimensional structure of the bacteria. FEMS Microbiol Lett 125: 95–100.
bacterial cell wall peptidoglycan. Proc Natl Acad Sci USA 103: Quintela JC, Pittenauer E, Allmaier G, Aran V & de Pedro MA
4404–4409. (1995b) Structure of peptidoglycan from Thermus
Moulder JW (1993) Why is Chlamydia sensitive to penicillin in thermophilus HB8. J Bacteriol 177: 4947–4962.
the absence of peptidoglycan? Infect Agents Dis 2: 87–99. Quintela JC, de Pedro MA, Zollner P, Allmaier G & Garcia-del
Muñoz E, Ghuysen JM, Leyh-Bouille M, Petit JF, Heymann H, Portillo F (1997) Peptidoglycan structure of Salmonella
Bricas E & Lefrancier P (1966) The peptide subunit Na-(L- typhimurium growing within cultured mammalian cells. Mol
alanyl-D-isoglutaminyl)-L-lysyl-D-alanine in cell wall Microbiol 23: 693–704.
peptidoglycans of Staphylococcus aureus strain Copenhagen, Quintela JC, Garcia-del Portillo F, Pittenauer E, Allmaier G & de
Micrococcus roseus R 27, and Streptococcus pyogenes group A, Pedro MA (1999a) Peptidoglycan fine structure of the
type 14. Biochemistry 5: 3748–3764. radiotolerant bacterium Deinococcus radiodurans Sark. J
Murray RGE, Steed P & Elson HE (1965) The location of the Bacteriol 181: 334–337.
mucopeptide in sections of cell wall of Escherichia coli and Quintela JC, Zollner P, Garcia del Portillo F, Allmaier G & de
other gram-negative bacteria. Can J Microbiol 11: 547–560. Pedro MA (1999b) Cell wall structural divergence among
Nanninga N (1998) Morphogenesis of Escherichia coli. Microbiol Thermus spp. FEMS Microbiol Lett 172: 223–229.
Mol Biol Rev 62: 110–129. Richaud C, Mengin-Lecreulx D, Pochet S, Johnson EJ, Cohen GN
Neuhaus FC & Baddiley J (2003) A continuum of anionic charge: & Marlière P (1993) Directed evolution of biosynthetic
structures and functions of D-alanyl-teichoic acids in gram- pathways. Recruitment of cysteine thioethers for constructing
positive bacteria. Microbiol Mol Biol Rev 67: 686–723. the cell wall of Escherichia coli. J Biol Chem 268: 26827–26835.
Neuhaus FC & Struve WG (1965) Enzymatic synthesis of analogs Rogers HJ (1970) Bacterial growth and the cell envelope. Bacteriol
of the cell-wall precursor. I. Kinetics and specificity of uridine Rev 34: 194–214.
diphospho-N-acetylmuramyl-L-alanyl-D-glutamyl-L-lysine: D- Rogers HJ, Perkins HR & Ward JB (1980) Microbial Cell Walls and
alanyl-D-alanine ligase (adenosine diphosphate) from Membranes. Chapman and Hall, London.
Streptococcus faecalis R. Biochemistry 4: 120–131. Sauvage E, Charlier P, Terrak M & Ayala JA (2008) The penicillin-
Obermann W & Höltje J-V (1994) Alterations of murein binding proteins: structure and role in peptidoglycan
structure and of penicillin-binding proteins in minicells from biosynthesis. FEMS Microbiol Rev, in press.
Escherichia coli. Microbiology 140: 79–87. Schindler M, Mirelman D & Schwarz U (1976) Quantitative
Park JT (1996) The murein sacculus. Escherichia coli and determination of N-acetylglucosamine residues at the non-
Salmonella (Neidhardt FC, ed), pp. 48–57. ASM Press, reducing ends of peptidoglycan chains by enzymic attachment
Washington, DC. of [14C]-D-galactose. Eur J Biochem 71: 131–134.


by guest
on 23 August 2018
Schleifer KH & Kandler O (1972) Peptidoglycan types of bacterial coli: consequences of osmotic stress. Mol Microbiol 64:
cell walls and their taxonomic implications. Bacteriol Rev 36: 858–871.
407–477. Vazquez-Laslop N, Lee H, Hu R & Neyfakh AA (2001) Molecular
Schleifer KH, Plapp R & Kandler O (1967) Identification of sieve mechanism of selective release of cytoplasmic proteins by
threo-3-hydroxyglutamic acid in the cell wall of osmotically shocked Escherichia coli. J Bacteriol 183:
Microbacterium lacticum. Biochem Biophys Res Commun 28: 2399–2404.
566–570. Verwer RW, Nanninga N, Keck W & Schwarz U (1978)
Schleifer KH, Plapp R & Kandler O (1968) Die Arrangement of glycan chains in the sacculus of Escherichia
Aminosäuresequenz des Mureins von Microbacterium coli. J Bacteriol 136: 723–729.
lacticum. Biochim Biophys Acta 154: 573–582. Villet R, Fonvielle M, Busca P et al. (2007) Idiosyncratic features
Schneider T, Senn MM, Berger-Bächi B, Tossi A, Sahl HG & in tRNAs participating in bacterial cell wall synthesis. Nucleic
Wiedemann I (2004) In vitro assembly of a complete, Acids Res 35: 6870–6883.
pentaglycine interpeptide bridge containing cell wall precursor Vollmer W (2008) Structural variation in the glycan strands of
(lipid II-Gly5) of Staphylococcus aureus. Mol Microbiol 53: bacterial peptidoglycan. FEMS Microbiol Rev, in press.
675–685. Vollmer W & Höltje J-V (2004) The architecture of the murein
Severin A, Tabei K & Tomasz A (2004) The structure of the cell (peptidoglycan) in gram-negative bacteria: vertical scaffold or
horizontal layer(s)? J Bacteriol 186: 5978–5987.
wall peptidoglycan of Bacillus cereus RSVF1, a strain closely
Ward JB (1973) The chain length of the glycans in bacterial cell
related to Bacillus anthracis. Microb Drug Resist 10: 77–82.
walls. Biochem J 133: 395–398.
Shockman GD, Thompson JS & Conover MJ (1965) Replacement
Warth AD & Strominger JL (1971) Structure of the peptidoglycan
of lysine by hydroxylysine and its effects on cell lysis in
from vegetative cell walls of Bacillus subtilis. Biochemistry 10:
Streptococcus faecalis. J Bacteriol 90: 575–588.
4349–4358.
Smith WG & Henderson LM (1964) Relationships of lysine and
Wientjes FB, Woldringh CL & Nanninga N (1991) Amount
hydroxylysine in Streptococcus faecalis and Leuconostoc
of peptidoglycan in cell walls of gram-negative bacteria.
mesenteroides. J Biol Chem 239: 1867–1871.
J Bacteriol 173: 7684–7691.
Snowden MA & Perkins HR (1990) Peptidoglycan cross-linking
Wietzerbin J, Das BC, Petit JF, Lederer E, Leyh-Bouille M &
in Staphylococcus aureus. An apparent random polymerisation
Ghuysen JM (1974) Occurrence of D-alanyl-(D)-meso-
process. Eur J Biochem 191: 373–377. diaminopimelic acid and meso-diaminopimelyl-meso-
Tamura A, Ohashi N, Urakami H & Miyamura S (1995) diaminopimelic acid interpeptide linkages in the
Classification of Rickettsia tsutsugamushi in a new genus, peptidoglycan of Mycobacteria. Biochemistry 13: 3471–3476.
Orientia gen. nov., as Orientia tsutsugamushi comb. nov. Int J Wyke AW & Perkins HR (1975) The specificity of enzymes adding
Syst Bacteriol 45: 589–591. amino acids in the synthesis of the peptidoglycan precursors of
Tipper DJ, Strominger JL & Ensign JC (1967) Structure of the cell Corynebacterium poinsettiae and Corynebacterium insidiosum.
wall of Staphylococcus aureus, strain Copenhagen. VII. Mode of J Gen Microbiol 88: 159–168.
action of the bacteriolytic peptidase from Myxobacter and the Yao X, Jericho M, Pink D & Beveridge T (1999) Thickness and
isolation of intact cell wall polysaccharides. Biochemistry 6: elasticity of gram-negative murein sacculi measured by atomic
906–920. force microscopy. J Bacteriol 181: 6865–6875.
Touhami A, Jericho MH & Beveridge TJ (2004) Atomic force Young KD (2006) Too many strictures on structure. Trends
microscopy of cell growth and division in Staphylococcus Microbiol 14: 155–156.
aureus. J Bacteriol 186: 3286–3295. Zuber B, Haenni M, Ribeiro T, Minnig K, Lopes F, Moreillon P &
Tuomanen E, Schwartz J, Sande S, Light K & Gage D (1989) Dubochet J (2006) Granular layer in the periplasmic space of
Unusual composition of peptidoglycan in Bordetella pertussis. gram-positive bacteria and fine structures of Enterococcus
J Biol Chem 264: 11093–11098. gallinarum and Streptococcus gordonii septa revealed by cryo-
van den Bogaart G, Hermans N, Krasnikov V & Poolman B electron microscopy of vitreous sections. J Bacteriol 188:
(2007) Protein mobility and diffusive barriers in Escherichia 6652–6660.


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on 23 August 2018

Estructura y Arquitectura Del Peptidoglucano

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Estructura y Arquitectura Del Peptidoglucano

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REVIEW ARTICLE

Peptidoglycan structure and architecture

Correspondence: Waldemar Vollmer, Abstract

Editor: Arie van der Ende

genes (but no peptidoglycan) have been found in the green

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Table 1. Amino acid variations in the peptide stem

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(a) (b) (c)

-G–M- -G–M- -G–M-

4 D-Ala Gly L-Lys 3

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Concluding remarks Acknowledgements

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References Boneca IG, Dussurget O, Cabanes D et al. (2007) A critical role

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