Reviews

Regulation of peptidoglycan synthesis

and remodelling
Alexander J. F. Egan, Jeff Errington    and Waldemar Vollmer    ✉
Abstract | Bacteria surround their cell membrane with a net-like peptidoglycan layer, called
sacculus, to protect the cell from bursting and maintain its cell shape. Sacculus growth during
elongation and cell division is mediated by dynamic and transient multiprotein complexes, the
elongasome and divisome, respectively. In this Review we present our current understanding
of how peptidoglycan synthases are regulated by multiple and specific interactions with cell
morphogenesis proteins that are linked to a dynamic cytoskeletal protein, either the actin-like
MreB or the tubulin-like FtsZ. Several peptidoglycan synthases and hydrolases require activation
by outer-membrane-anchored lipoproteins. We also discuss how bacteria achieve robust cell wall
growth under different conditions and stresses by maintaining multiple peptidoglycan enzymes
and regulators as well as different peptidoglycan growth mechanisms, and we present the
emerging role of ld-transpeptidases in peptidoglycan remodelling.

Turgor
Force pushing the cytoplasmic
membrane against the cell wall,
caused by the osmotic flow of
water into the cytoplasm.
Centre for Bacterial Cell
Biology, Biosciences Institute,
Newcastle University,
Newcastle upon Tyne,
United Kingdom.
✉e-mail: w.vollmer@ncl.ac.uk
https://doi.org/10.1038/
s41579-020-0366-3
Bacteria are capable of propagating in diverse, often
changing and adverse environments, which enables
them to colonize almost any site on Earth. Growing
bacteria maintain their species-specific cell shape and
pass it to their progeny, but they may change their size
and cell shape depending on the environmental condi-
tions1. How bacteria grow and divide whilst maintaining
their cell shape is a fundamental unanswered question
in microbiology.
The shape of a bacterial cell is maintained by the pep-
tidoglycan layer, also called the ‘sacculus’, that encases the
cytoplasmic membrane to protect the cell from bursting
due to turgor2. A bacterial cell must preserve the integ-
rity of its sacculus at all times to prevent lysis and death.
The sacculus is a net-like layer composed of glycan
chains that are connected by short peptides. It is mainly
single-layered in diderm (Gram-negative) bacteria such
as Escherichia coli, which have an outer membrane, and
multilayered in monoderm (Gram-positive) species
such as Bacillus subtilis. Growth of the sacculus requires
the synthesis of new peptidoglycan and its incorpora-
tion into the existing layer or layers and is accompanied
by the release of old peptidoglycan material from the
sacculus3.
The peptidoglycan precursor lipid II is synthesized
at the inner leaflet of the cytoplasmic membrane and is
transported to the periplasmic side, where it is utilized
by peptidoglycan synthases (Fig. 1). Peptidoglycan glyco-
syltransferases (GTases) polymerize the glycan chains,
and transpeptidases (TPases) form peptide crosslinks.
Penicillin-binding proteins (PBPs) of class A (also called
aPBPs) catalyse both reactions, by virtue of their GT51
type GTase and Ser-type TPase domains4. Class B PBPs
(bPBPs) are monofunctional TPases with an N-terminal
non-catalytic ‘pedestal’ domain that places the TPase
domain away from the membrane and interacts with
other proteins. The two types of monofunctional GTases
are the SEDS (shape, elongation, division and sporula-
tion) proteins and monofunctional GT51 GTases4,5.
Peptidoglycan hydrolases are present in multiple vari-
ants in all bacteria, where they have major roles — for
example, in peptidoglycan turnover during growth and
cell division, cell shape maintenance or bacterial inter­
actions. The major classes of peptidoglycan hydrolases
are N-acetylmuramidases (lysozymes and lytic trans-
glycosylases), N-acetylglucosaminidases, amidases,
endopeptidases and carboxypeptidases6.
During the past decade it has emerged that peptido­
glycan synthases and hydrolases are spatiotemporally
regulated at multiple levels, presumably to ensure that
sacculus growth is synchronized with the cell cycle and
aligned with the growth rate of the cell. Our knowledge
about the mechanisms underlying the regulation of sac-
culus growth and cell morphology is limited to a handful
of model bacteria, mostly from the phyla Proteobacteria
and Firmicutes. This Review expands on a previous
article from 2012 (ref.3) and focuses on key players in
peptidoglycan synthesis and its regulation, presenting
recent discoveries and new developments in the molec-
ular mechanisms of sacculus growth regulation and
bacterial morphology. Where other species are not men-
tioned, we present the current knowledge of peptido­
glycan synthesis in E. coli, expanding to other species for
important aspects. We refer the reader to other articles

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Spore cortex
A specialized spore cell wall
layer uniquely deposited
inward from the enveloping
mother cell compartment.
Proton motive force
(PMF). Force that promotes the
movement of protons across
membranes down the electro-
chemical potential gradient,
in most cells generated by an
electron transport chain that
acts as a proton pump.
Elongasome
Dynamic multiprotein complex
responsible for peptidoglycan
synthesis in the lateral wall of
many rod-shaped bacteria.
Actin
Family of cytoskeletal proteins
that dynamically form micro­
filaments to perform a range
of important cellular processes
in both eukaryotes and
prokaryotes. The prokaryotic
actin-like MreB is part of
the elongasome in many
rod-shaped bacteria.
NAture Reviews | MICRoBIoLogy
about specific features of peptidoglycan growth and
cell division in coccal-shaped and ovococcal-shaped
Firmicutes, like Staphylococcus aureus and Streptococcus
pneumoniae, respectively7,8; in ε-proteobacteria, like
Caulobacter crescentus and Helicobacter pylori9–11; in
the predatory bacterium Bdellovibrio bacteriovorus12;
in Mycobacterium spp.13; and in intracellular pathogens
like Chlamydia spp.14,15. Recent advances in this area
have been made possible by the development and use of
fluorescent d-amino acids (FDAAs) that are specifically
incorporated into peptidoglycan by transpeptidases16,
by novel high-resolution microscopy techniques17 and by
in vitro reconstruction of biochemical processes18–20.
Molecular mechanisms
The majority of proteins essential for peptidoglycan
synthesis, from the enzymes catalysing crucial reactions
to morphogenesis proteins, have been known for some
time (Table 1), but our understanding of how these pro-
teins function at the molecular level remains limited.
Owing to recent technological advances, new features
of many previously described proteins have been discov-
ered; for example, high-resolution structural informa-
tion about peptidoglycan synthases and their associated
proteins has been crucial in determining the molecu-
lar details of the regulation of peptidoglycan synthesis.
A recent review provides an excellent overview of our
current structural insights21.
Transporting lipid II across the cytoplasmic membrane.
The identity of the protein or proteins responsible for
transporting, or ‘flipping’, lipid II from the inner to the
outer leaflet of the cytoplasmic membrane for incorpo-
ration into peptidoglycan remained elusive for decades.
Before the SEDS proteins were found to have GTase
activity, they were considered the most likely candidates
for carrying out this function. The model rod-shaped
bacteria E. coli and B. subtilis each possess two SEDS
proteins, RodA and FtsW, that are essential for saccu-
lus growth during elongation and division, respectively
(B. subtilis has a third SEDS protein, SpoVE, which is
involved in spore cortex synthesis22). SEDS proteins are
integral membrane proteins with ten transmembrane
helices23 and, based on indirect cellular evidence 24,
were hypothesized to flip lipid II across the cytoplas-
mic membrane. Indeed, the SEDS protein FtsW was
the first, and so far the only, protein shown to transport
lipid II across a membrane when reconstituted in pro-
teoliposomes25, which suggests, by extension, that the
paralogous RodA is also a flippase. However, neither
RodA nor FtsW was implicated in lipid II flipping in
a cellular assay in which colicin M was added to cells
in order to digest only the flipped lipid II facing the
periplasm of E. coli26,27. This assay identified MurJ (also
known as MviN) as the lone lipid II flippase in E. coli.
MurJ is an integral membrane protein, it is essential for
peptidoglycan synthesis, and its depletion leads to the
accumulation of peptidoglycan precursors in the cell28,29.
These observations — coupled with the fact that MurJ
is a member of the MOP (multidrug, oligosaccharide-
lipid, polysaccaride) transporter superfamily of pro-
teins, a family previously implicated in transporting
Reviews
undecaprenyl pyrophosphate-linked oligosaccharides
— were indicative of a role in lipid II synthesis and/or
transport30. Indeed, purified MurJ binds lipid II, and
lipid II binding is abrogated in non-functional or inacti­
vated versions of MurJ31. However, the same study that
revealed the in vitro flippase activity of FtsW showed
none for MurJ25. The subsequent high-resolution struc-
ture of MurJ revealed an inward-open central cavity,
presumably the site for substrate binding, and that the
protein functions by an alternate-access or rocker-switch
mechanism. The proton motive force (PMF) and binding
and release of a sodium ion seem to drive the neces-
sary conformational changes and move the MurJ cargo
across the membrane32–35 (Fig. 2). In E. coli and H. pylori,
the O-antigen flippase Wzk can substitute for the loss
of MurJ36, and B. subtilis has an alternative presumed
lipid II flippase, called Amj, which is essential in cells
lacking MurJ26.
Whether MurJ or the SEDS proteins, or both of these
together, flip lipid II is still uncertain, as key caveats
remain for each set of studies. For the in vitro experi-
ments, the lack of a PMF across the artificial membrane
may explain the lack of observed flippase activity of
MurJ25. For the in vivo experiments, the absence of an
apparent contribution from FtsW and/or RodA could
be explained if the lipid II flipped by these proteins was
immediately incorporated into the sacculus, a possibility
the experiments did not explore27. Another considera-
tion is that colicin M rapidly inhibits peptidoglycan syn-
thesis by disrupting carrier lipid recycling37 and hence
is not a neutral tool for the process being analysed.
One possible solution that may resolve the conflicting
strands of evidence is that MurJ and the SEDS proteins
work together in the flipping of lipid II. The localization
of MurJ to mid-cell during cell division, where it pre-
sumably flips lipid II for septal peptidoglycan synthesis,
requires both lipid II synthesis and a functional FtsW38.
It is estimated that E. coli requires that ~5,000 molecules
of lipid II be flipped per second in order to maintain
the rate of peptidoglycan synthesis39. Perhaps MurJ
and the SEDS proteins fulfil this rapid flipping require-
ment together; however, future studies will be required
in order to investigate this hypothesis.
Controlling the location of peptidoglycan synthesis.
The many protein–protein interactions detected in the
cell and with purified peptidoglycan enzymes and cell
morphogenesis proteins, together with their cellular
localization dependencies and dynamics, all support
the existence of dynamic and possibly transient multi­
protein complexes for sacculus growth. These com-
plexes presumably comprise peptidoglycan synthases,
their regulators and peptidoglycan hydrolases essential
for breaking bonds within the wall to accommodate the
insertion of new material. Meanwhile, overall cell wall
morphogenesis is ultimately governed by cytoskeletal
proteins. The exact nature of these complexes, as well
as how they facilitate peptidoglycan growth, remains to
be resolved. In rod-shaped bacteria, at least two distinct
machineries exist (Fig. 2). The elongasome (also referred
to as the Rod complex), organized by the actin homo-
logue MreB, facilitates the insertion of new material
volume 18 | August 2020 | 447
Reviews

Diderm sp. Monoderm sp.

40–60
ManNAc
GroP
Poly-RboP or GroP
40–60

Outer membrane OmpA

P
Lpp LD-TPase

L-Lys-NH2

Amidase LD-EPase
Peptidoglycan NH2 LD-TPase NH2 LD-TPase
DL-CPase exo-LT
sacculus
40–60
DD-EPase
endo-LT 2HN
NH2
DD-TPase LD-CPase DD-TPase
Acceptor Donor
peptide peptide
HO Lipid II
Nascent OH
peptidoglycan
GTase LCP
DD-CPase
Periplasm P P P enzymes P
P P P P P
? BacA, PgpB, YbjG MurJ, FtsW MurG MraY
Cytoplasmic membrane
P P P P
Cytoplasm P P
U P P
U P P
GlcNAc
MurNAc
anhMurNAc
Lipid II Lipid I
L-Ala
D-Ala
MurF
Meso-Dap
L-Glu MurA MurB MurC MurD MurE
U P P U P P U P P U P P U P P
D-Glu
D-amino acid
P Phosphate DdlA
MurI
U Uridine Alr or DadX

Undecaprenol

Divisome
Transient and dynamic
multiprotein complex that
divides a bacterial cell.
Tubulin
Family of cytoskeletal proteins
that form microtubules in
eukaryotes. The tubulin-like
FtsZ forms dynamic filaments
to organize cell division
in bacteria.
during growth along the lateral part of the cell cylinder.
The divisome, under the ultimate control of the tubulin
homologue FtsZ, facilitates peptidoglycan synthesis dur-
ing cell division. The core and accessory components of
the E. coli divisome and elongasome are illustrated in
Fig. 2 and listed in Table 1; how the cytoskeletal elements
MreB and FtsZ may control peptidoglycan synthesis
and so maintain cell morphology will be discussed in
detail below. The remainder of this section focuses on
our current understanding of the molecular details of
how proteins within these complexes control peptid­
oglycan synthases. The reader is directed to recent com-
prehensive reviews on the divisome for more detailed
information40–42.
Controlling peptidoglycan synthesis activities through
protein interactions. Spatial and temporal control
of peptidoglycan synthase function is exerted by
protein-binding partners. One of the best-studied pep-
tidoglycan synthases is class A PBP1B from E. coli, owing
to its tractability for in vitro biochemistry. PBP1B con-
tributes to a substantial proportion of peptidoglycan
synthesis in the cell43,44. Class A PBP1A and PBP1B are
referred to as semi or partially redundant, as the cell only
needs one of these enzymes for growth45. However, each
enzyme seems to have a preferred role: PBP1B localizes
at mid-cell during cell division and interacts with several
essential division proteins (such as ZipA, FtsW, FtsN,
FtsQ–FtsL–FtsB and the class B PBP3)46–50; by contrast,
PBP1A localizes predominantly to the cell periphery
during growth and interacts with the class B PBP2,
which is essential for elongation51. The fact that PBP1A
and PBP1B can substitute for each other suggests that
they can form similar interactions, but this remains to be
explored. In all cases, the interaction partners listed exert
some order of control on the PBPs, from ensuring proper

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◀ Fig. 1 | Peptidoglycan synthesis, cleavage and modification. Shown are the necessary
reactions and enzymes responsible for the synthesis and hydrolysis of peptidoglycan
within the cell envelopes of monoderm (Gram-positive) and diderm (Gram-negative)
bacteria. Cytosolic and membrane-associated steps lead to assembly of the lipid II
precursor. MurA and MurB synthesize UDP-MurNAc, and the collective activities of the
specific amino acid ligases MurC, MurD, MurE, MurF and Ddl, as well as of the racemases
Alr or Dadx and MurI, is needed to synthesize the stem peptide at UDP-MurNAc. MraY
and MurG produce lipid I and lipid II, respectively. For simplicity , the pentapeptide from
Escherichia coli is shown; amino acids other than d-Glu and meso-Dap, at positions 2
and 3, respectively , are present in many other species. Undecaprenol phosphate (C55-P)
is the carrier lipid for peptidoglycan and teichoic acid precursors. Lipid II is flipped across
the cytoplasmic or inner membrane, mediated by MurJ and perhaps FtsW, where it is the
substrate for glycosyltransferases (GTases) that polymerize the nascent peptidoglycan
chains. These are attached to the existing sacculus and/or to other nascent chains by
dd-transpeptidases (dd-TPases). Peptides are trimmed by dd-carboxypeptidases,
ld-carboxypeptidases and dl-carboxypeptidases (CPases), and crosslinks are cleaved
by dd-endopeptidases and ld-endopeptidases (EPases). Amidases remove the peptide
from glycan chains, and lytic transglycosylases (LT) cleave the glycan chains, producing
1,6-anhydro-N-acetylmuramic acid (anhMurNAc) residues at the released products.
ld-TPases are responsible for ld-crosslinks, and for the covalent attachment of the
outer-membrane-anchored lipoprotein Lpp in species that have it. Diderm species
also feature proteins, such as the widely conserved outer membrane protein A (OmpA),
that non-covalently bind to the peptidoglycan sacculus, which is important for envelope
stability. ld-TPases and dd-TPases can both incorporate non-canonical d-amino acids,
such as the fluorescent d-amino acid (FDAA) probes used to label regions of peptidoglycan
growth (not shown). The diphosphate form of the carrier lipid, C55-PP (undecaprenol
pyrophosphate), is synthesized by UppS and also released in GTase reactions and
subsequently is dephosphorylated to the monophosphate form, C55-P, by the phosphatases
BacA (also called UppS), PgpB and YbjG. Alr, Ala racemase, biosynthetic; DadX, Ala
racemase, catabolic; DdlA , d-Ala-d-Ala ligase A; GlcNAc, N-acetylglucosamine; GroP,
glycerol phosphate; ManNAc, N-acetyl-d-mannosamine; meso-Dap, meso-diaminopimelic
acid; MraY, phospho-N-acetylmuramoyl-pentapeptide transferase; MurA , UDP-GlcNAc
enolpyruvyl transferase; MurB, UDP-MurNAc dehydrogenase; MurC, UDP-MurNAc l-Ala
ligase; MurD, UDP-MurNAc-l-Ala-d-Glu ligase; MurE, UDP-MurNAc-l-Ala-d-Glu-meso-
Dap ligase; MurF, UDP-MurNAc-tripeptide-d-alanyl-d-Ala ligase; MurG, undecaprenyldiphospho-
muramoylpentapeptide beta-N-acetylglucosaminyltransferase; MurI, Glu racemase;
MurJ, MOP (multidrug, oligosaccharide-lipid, polysaccharide) transporter (lipid II flippase);
MurNAc, N-acetylmuramic acid; Poly-RboP/GroP, poly-ribitol/glycerol phosphate.
localization through to directly affecting catalysis (see
below). Despite their semi-redundancy, recent evidence
suggests that PBP1B has a more important role in the
cell under certain conditions. PBP1B, and not PBP1A,
is required for recovery from a cell wall free spheroplast
state52, growth in the presence of β-lactam antibiotics
through bypassing dd-TPases53, peptidoglycan repair
during stress response54 and growth at low pH55.
Strikingly, several of the class A PBPs require activ­
ation by a cognate outer-membrane-anchored Lpo lipo-
protein. In E. coli, the function of PBP1A and PBP1B in
the cell is absolutely reliant on the binding of LpoA and
LpoB, respectively56,57, presumably to regulate peptid­
oglycan synthesis in response to the porous properties
of the sacculus3. Each Lpo protein is anchored to the
outer membrane and spans the periplasm58–60 to inter-
act with its cognate, inner-membrane-anchored class
A PBP through a small, non-catalytic domain: ODD in
PBP1A and UB2H in PBP1B57. The structure of PBP1A
from E. coli and how LpoA interacts with and stimulates
this synthase are not known. The UB2H domain is sit-
uated between the two catalytic domains in the PBP1B
structure, and the binding interface with LpoB has been
mapped58,61,62. The binding of LpoA or LpoB to their
cognate PBP stimulates enzymatic activity in different
ways. LpoA binding to PBP1A mainly activates TPase,
NAture Reviews | MICRoBIoLogy
Reviews
whereas LpoB binding to PBP1B activates both GTase
and TPase57,58,63,64. Moreover, Pseudomonas aeruginosa
has a PBP1B homologue with a UB2H domain but no
LpoB, and the function of PBP1B in this organism is reli-
ant on a different Lpo protein, LpoP65. A combination of
genetic, biochemical and structural work has shown that
LpoB, and presumably LpoP, exerts control over PBP1B
function by inducing conformational changes that affect
the dynamics of crucial motifs within the enzyme to
stimulate catalysis63,65,66 (Fig. 3).
Another recently emerging example of an absolute
requirement for protein binding for function is the
relationship between the SEDS proteins and their cog-
nate class B PBPs67. Purified FtsW from S. aureus and
Streptococcus thermophilus only showed GTase activ-
ity in the presence of their cognate class B PBPs (PBP1
and PBP2x, respectively)67. E. coli RodA was shown to
require PBP2 for activity68, but B. subtilis RodA, which
is essential in the absence of class A PBPs69, showed weak
GTase activity without its cognate class B PBP5. Like class
A PBPs, SEDS proteins elongate the reducing end of the
growing glycan chain70. The nature of this activity needs
to be further explored, because the crystal structure of
RodA lacks obvious binding sites for lipid II and the
growing glycan chain23. This might imply that the bind-
ing of the membrane-proximal portion of a class B PBP
alters the structure so as to create such substrate-binding
sites, or perhaps it constitutes part of the active site itself.
The link between E. coli FtsW and PBPs seems to be
more complex48. FtsW interacts directly with PBP1B and
inhibits its GTase activity by binding to and sequestering
lipid II. Addition of PBP3 to the reaction between PBP1B
and FtsW restored GTase activity, presumably by allowing
PBP1B to access lipid II48. Hence, FtsW could function as
a ‘delivery system’ for lipid II (or nascent glycan chains
produced by FtsW), only allowing the initiation of new
peptidoglycan synthesis at the right moment, in the right
place during cell division, controlled by PBP3 (ref.48).
Essential cell division proteins also affect PBPs
directly. In E. coli, the divisome is assembled in two steps,
whereby early division proteins — FtsZ, FtsA, ZipA and
a small amount of FtsN — localize to the future division
site early in the cell cycle71, to eventually recruit PBPs
to drive a pre-septal phase of peptidoglycan synthesis.
Pre-septal peptidoglycan synthesis takes place at future
cell division sites independent of the septum peptidogly-
can synthase PBP3 and most of the essential cell division
proteins. PBP1A and PBP1B are recruited to pre-septal
sites via interactions with ZipA and FtsN50. These link
the PBPs to FtsZ polymers either directly (ZipA) or indi-
rectly via FtsA (FtsN). Both ZipA and FtsN interact with
the class A PBPs, presumably via their transmembrane
and perhaps part of their cytosolic regions, and stimulate
their GTase activity19,49,50. The divisome matures through
the arrival of late cell division proteins. FtsQ, FtsL and
FtsB form a stable complex and seem to function as a
key checkpoint for divisome activation, thus directly
affecting PBP1B and PBP3 function47. In this model,
FtsQ–FtsL–FtsB inhibits PBP1B and PBP3 until enough
FtsN accumulates at mid-cell, driven by the binding of
its C-terminal SPOR domain to septal peptidoglycan72,73.
Above a crucial threshold, FtsN triggers septation by
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Table 1 | Cell morphogenesis proteins and peptidoglycan synthase regulators

Protein Role Mechanism of action (if known) In vitro Essentialb Conservation
dataa
Cell division — Z-ring assembly and regulation
FtsZ Cytoskeletal protein, tublin structural GTPase, forms dynamic polymers that Yes Yes Widely conserved
homologue and master regulator of treadmill around the cell circumference
cell division; principal component of
the Z-ring
FtsA A principal membrane tether for FtsZ; Interacts with FtsZ and the cytoplasmic Yes Yes Widely conserved
required for the initiation of division leaflet of the membrane
once the divisome complex is formed
ZipA Promotes Z-ring assembly and Membrane-anchored protein, interacts Yes Yes γ-Proteobacteria
membrane attachment; coordinates with FtsZ and also with essential division
peptidoglycan synthases during peptidoglycan synthases
pre-septal peptidoglycan synthesis
ZapA Promotes Z-ring assembly and Interacts with FtsZ, promoting Yes No Widely conserved
stability filament bundling
ZapB Redundant role in ensuring Z-ring Forms filaments that interact with ZapA , Yes No Some
assembly localizes within the Z-ring γ-proteobacteria
ZapC Z-ring stabilization Interacts with FtsZ, promoting filament Yes No Some
bundling and suppressing GTPase activity γ-proteobacteria
ZapD Redundant role in promoting Z-ring Interacts with FtsZ, stimulating protofilament Yes No Some
assembly and stability , analogous to association β-proteobacteria
ZapA and
γ-proteobacteria
ZapE Negative regulator of Z-ring Interacts with FtsZ late in cell division, Yes No Gram-negative
structure disrupts filament formation bacteria
FtsE and Regulate FtsA dynamics, promoting FtsE–FtsX together form an ATP-binding cassette Yes No Widely conserved
FtsX proper divisome assembly; regulates transporter-like complex; ATP hydrolysis-induced
peptidoglycan autolysin activity conformational changes control EnvC, which in
during division turn controls AmiA and AmiB
EzrA Negative regulator of Z-ring May scaffold the divisome complex, bridging Yes No Gram-positive
assembly , ensuring proper dynamics Z-ring and associated proteins with ‘late’ bacteria with low
and complex structure division proteins, including peptidoglycan GC content
synthases
SepF Redundant role with FtsA in ensuring Interacts with FtsZ suppressing GTPase activity , Yes No Gram-positive
Z-ring assembly , as well as regulation promoting filament stability and bundling bacteria and
of septal morphology cyanobacteria
GpsB Adaptor protein that functions as a Interacts with class A PBPs and shuttles them Yes Essential in Firmicutes
scaffold for peptidoglycan synthesis to the correct cellular location, depending on S. aureus; not
complexes and Z-ring dynamics cell cycle; also interacts with EzrA , MreC and essential in
(Staphylococcus aureus only) (indirectly) RodZ Bacillus subtilis
Cell division – ‘late’-associating proteins of the divisome
FtsK Bridge between the ‘early’ Z-ring Membrane-anchored protein, interacts with Limited Yes Widely conserved
division proteins and the ‘late’ numerous divisome components (FtsZ, FtsQ,
divisome proteins involved FtsL and PBP3); nonessential N-terminal
in peptidoglycan synthesis; domain involved in DNA translocation
decatenation of sister chromosomes
FtsQ FtsQ, FtsL and FtsB together form FtsQLB interacts with FtsW, PBP3, PBP1B and Yes Yes Widely conserved
(DivIB) a complex within the membrane FtsN; regulates the peptidoglycan synthesis
that constitutes a key structural activities of PBP3 (reducing TPase function)
FtsL Yes Yes Widely conserved
element of the divisome; FtsQ– and PBP1B (reducing GTase function)
FtsB FtsL–FtsB recruits and regulates the antagonistically with FtsN Yes Yes Widely conserved
(DivIC) peptidoglycan synthetic portion of
the complex FtsW–PBP3 (with or
without PBP1B)
FtsW Peptidoglycan polymerizing GTase Integral membrane protein of the SEDS family. Yes Yes Widely conserved
and lipid II transport across the FtsW has been shown to possess GTase activity
membrane when bound by its cognate class B PBP, to flip
lipid II across a membrane and to be required
for localization of MurJ in the cell (both its
presence and function)
PBP3 Class B PBP (dd-TPase) required for Essential for peptidoglycan synthesis during Yes Yes Widely conserved
peptidoglycan crosslinking during cell division, interacts with FtsW, PBP1B and
division FtsN; required for FtsW function

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Table 1 (cont.) | Cell morphogenesis proteins and peptidoglycan synthase regulators

Protein Role Mechanism of action (if known) In vitro Essentialb Conservation
dataa
Cell division – ‘late’-associating proteins of the divisome (cont.)
FtsN Its arrival provides signal of Binds to septal peptidoglycan; interacts Yes Yes γ-Proteobacteria
divisome completion to FtsA , with PBP3, FtsW and PBP1B, stimulating the and some
initiating constriction; regulates peptidoglycan synthesis activities of the latter; β- proteobacteria
peptidoglycan synthesis also interacts with FtsA
MurJ Lipid II transport across the Localizes to division site concurrently with Noc Yes Widely conserved
(also membrane PG synthesis enzymes, depending on lipid II
known as synthesis and also the activities of PBP3 and
MviN) FtsW. Protein structure shows that transport
likely occurs by alternate access mechanism,
using the PMF for energy.
Tol–Pal Outer membrane constriction during A trans-envelope complex comprising the inner Limited Essential in Proteobacteria
cell division, coordination with membrane proteins TolQ, TolR , TolA , periplasmic Pseudomonas
peptidoglycan synthesis TolB and outer-membrane-anchored Pal; binds aerguginosa,
to peptidoglycan (TolR and Pal); interacts with Caulobacter
PBP1B from Escherichia coli and modulates crescentus, not
activity in coordination with outer-membrane essential in
constriction E. coli
Cell elongation — elongasome or rod complex
MreB Cytoskeletal protein, actin structural ATPase, forms dynamic membrane-associated Yes Yes Widely conserved
(MreBH, homologue required for the generation polymers that rotate around the circumference in rod-shaped
Mbl) and maintenance of cylindrical rod of the rod, enriched at areas of negative mean bacteria
shape; MreBH and Mbl are redundant curvature; MreB polymers have an intrinsic
paralogues of MreB in B. subtilis bend and are able to deform membranes in vitro
MreC Maintenance of rod shape, regulation Membrane-anchored protein, interacts with No Yes Widely conserved
of peptidoglycan synthesis by the MreB, MreD and PBP2, inducing a structural
elongasome change in the non-catalytic domain of the
latter, presumably regulating peptidoglycan
crosslinking activity
MreD Maintenance of rod shape The precise role of MreD in cell elongation is No Yes γ-Proteobacteria
unknown and some
β- proteobacteria
RodZ Regulator of MreB polymerization Membrane-anchored protein, interacts No Yes Proteobacteria,
and localization dynamics; functions with MreB, RodA , PBP2 and PBP1A (and some
as an adaptor between MreB and PBP1B), thereby providing a scaffold for the Firmicutes and
peptidoglycan synthases of the elongasome Actinobacteria
elongasome
RodA Peptidoglycan polymerizing GTase Integral membrane protein of the SEDS family; Yesd Yes Widely conserved
and (putatively) lipid II transport shown to possess weak GTase activity alone,
across the membrane increased by the addition of PBP2; assumed to
share the ability of FtsW to flip lipid II (on the
basis of homology)
PBP2 Class B PBP (dd-TPase) required for Essential for peptidoglycan synthesis during Yes Yes Widely conserved
peptidoglycan crosslinking during cell elongation; interacts with RodA , PBP1A
elongation and MreC; stimulates the GTase activity of
PBP1A and RodA
Peptidoglycan synthase regulators
LpoA Essential for PBP1A function in E. coli Outer-membrane-anchored lipoprotein, Yes Essential in γ-Proteobacteria
interacts with PBP1A and stimulates TPase Haemophilus
activity influenzae, not
essential in E. coli
LpoB Essential for PBP1B function in E. coli
Outer-membrane-anchored lipoprotein, Yes No Some
interacts with PBP1B from E. coli, stimulating γ-proteobacteria
both GTase and TPase activities by inducing
conformational changes in the synthase
CpoB Modulates the function of PBP1B–LpoB Accessory protein of Tol–Pal, interacting with Yes No Proteobacteria
in coordination with outer-membrane TolA; periplasmic; interacts with PBP1B–LpoB,
constriction during cell division modulating the stimulation of TPase activity
LpoP Essential for PBP1B function in Outer-membrane-anchored lipoprotein, Limited No Some
P. aeruginosa interacts with PBP1B from P. aeruginosa, γ-proteobacteria
inducing enzyme activation by a mechanism
similar to that of LpoB–PBP1B in E. coli
GTase, glycosyltransferase; PBP, penicillin-binding protein; PMF, proton motive force; SEDS, shape, elongation, division and sporulation; TPase, transpeptidases.
a
In vitro data pertaining to protein function. bUnder standard laboratory conditions. cMurJ did not transport lipid II across a membrane in vitro; one study shows
lipid II binding, while another shows none. dFlipping of lipid II by RodA was not shown in vitro.

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Reviews

Outer membrane

Division Elongation
Peptidoglycan LpoA

PBP 1B

2
PBP

1A
PBP Interaction
3

PBP
LpoB

RodZ
FtsN

MreC
Periplasm
MurJ
FtsQLB

MreD
PMF PMF ?
FtsEX
FtsK

Inner membrane
FtsW MraY
ZipA MurG
FtsA MreB
Cytoplasm
FtsZ RodA
ZapA–E Lipid II

Fig. 2 | Lipid II flipping and peptidoglycan synthesis during elongation and division. The schematic shows the
multiprotein complexes for peptidoglycan synthesis during cell division and elongation in Escherichia coli, including
the preferred penicillin-binding protein (PBP)–Lpo proteins, according to known localization patterns and interactions in the
cell (PBP1B with PBP3 and FtsW, and PBP1A with PBP2). However, it is known in E. coli that PBP1A and PBP1B can substitute
for each other in the cell in standard laboratory conditions. The arrow on the right indicates the direct interaction between
PBP1A and PBP2 (ref.51), although PBP1A and LpoA function semi-autonomously from the rest of the elongation complex43.
Essential members of the divisome and elongasome complexes are shown. Loss of any of the components leads to perturbed
or arrested complex function and a drastic alteration of cell morphology , with the exception of the Zap proteins of the
divisome. MraY and MurG produce lipid II at the inner membrane and co-localize with both the division and elongation
complexes in E. coli. The flippase MurJ requires the proton motive force (PMF) in order to drive conformational changes for
its alternating-access transport mechanism. MurJ co-localization with the divisome requires an active FtsW, which suggests
that it could deliver lipid II to the complex via FtsW. In vitro evidence suggests that FtsW and PBP3 regulate the access of
PBP1B to lipid II. The precise molecular architecture of the divisome and elongasome and how they insert new material into
the existing peptidoglycan layer are not yet known. For simplicity , peptidoglycan hydrolases known to associate with each
complex, and to be required for their proper function, are not shown. MraY, phospho-N-acetylmuramoyl-pentapeptide
transferase; MurG, undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase; MurJ, MOP
(multidrug, oligosaccharide-lipid, polysaccharide) transporter (lipid II flippase).

unblocking the inhibition of PBP1B and PBP3–FtsW
by FtsQ–FtsL–FtsB74. How the various effectors affect
PBP1B is currently unknown, but it is possible that
they restrict (FtsQ–FtsL–FtsB) or support (FtsN and/or
ZipA) the dynamics of important structural motifs that
are also affected by LpoB63. Indeed, it was recently shown
that another protein, CpoB, can directly modulate the
LpoB-mediated activation of TPase by affecting con-
formational changes within PBP1B63,75. It remains to
be established which of these interactions and regula-
tory mechanisms are conserved in other bacteria and
which are specific to E. coli. In all, the emerging picture
suggests that the divisome is regulated by a network
of functional interactions and signalling between key
regulators and peptidoglycan synthases that initiate
constriction and regulate its progression, to ensure that
peptidoglycan synthesis happens at the right moment
and place with an optimal rate (Fig. 3).
Our understanding of regulatory interactions within
the elongasome is more limited, especially biochemically,
but themes similar to those discussed for the divisome
have recently emerged. MreC is a widely conserved elon-
gasome protein, essential for rod shape, and it interacts
with MreB and PBP2. MreC has been implicated in acti-
vating peptidoglycan synthesis by the elongasome and is
thought to induce conformational changes in PBP2 that
favour not only its TPase activity, but the GTase activity
of RodA68. Preliminary in vivo fluorescence resonance
energy transfer (FRET) data suggest that MreC and
MreD affect the activity of a RodA–PBP2 complex dif-
ferently, and that this regulation is important for proper
cell shape76. Indeed, the co-structure of an MreC–PBP2
complex from H. pylori reveals an extended interface
between the two proteins that was shown to be essential
to maintaining cell morphology and growth77. Hence,
MreC and MreD may coordinate the peptidoglycan syn-
thesis activities of the elongasome with the morphogene-
sis function of MreB. PBP2 was also previously shown to
form a cooperative interaction with PBP1A. Binding of
PBP2 stimulates PBP1A GTase activity in vitro, and this
interaction also improves PBP2 TPase activity, boost-
ing the attachment of newly synthesized material to a
peptid­oglycan PG sacculus in vitro. These data suggest
that the two synthases function together to synthesize
and attach new peptidoglycan51.
Morphogenic control
As we mentioned above, the key proteins that control cell
wall morphogenesis, FtsZ and MreB, are homologues
of the well-known eukaryotic cytoskeletal proteins

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 Reviews

Bactofilins
Bacterial cytoskeletal proteins
that form sheet-like structures
or filaments near the cell
membrane to guide
morphogentic processes.
tubulin and actin, respectively. Like their eukaryotic
counterparts, FtsZ and MreB polymerize dynamically
in a manner that is influenced by nucleotide binding and
hydrolysis. Each protein interacts directly or indirectly
with many members of the divisome and elongasome
machineries. The roles and interactions of these pro-
teins have been reviewed in detail previously42,78, and
here we focus mainly on recent results aimed at eluci-
dating how the protein dynamics influence the shape
and form of cell wall synthesis. Although we focus on
MreB and FtsZ, some bacteria use additional cytoskeletal
elements, such as intermediate filaments and bactofilins,
to maintain a curved or helical cell shape or to position
cell appendages79.
Early immunofluorescence experiments with FtsZ
revealed a ring-like localization at prospective or ongo-
ing cell division sites80, prompting the notion that divi-
sion proteins might form a localized ring-like machine
defining the site of constriction. It emerged that FtsZ was
a master regulator of division nucleating the recruitment
of other division proteins. The localization experiments
were confirmed and extended by subsequent fluorescent
fusion imaging experiments that not only confirmed
the ring-like structure of FtsZ81,82 but also showed that the
protein is dynamic, with turnover of subunits in the ring
occurring with a half time of about 8–9 s (Ref.83).
In the past few years, the advent of super-resolution
imaging and fast and sensitive cameras, together with

A
Aa TP Ab Ac Ad TP Ae
FtsQLB
α
PBP1B

3
CpoB

PBP
LpoB
UB2H Lipid II
α

TolA
FtsN
GT
Inner
C55-PP FtsW
membrane
PgpB

B PBP1A
Ba TP Bb Bc Bd
PBP2
LpoA

MreC
?
Inner RodA
membrane

Fig. 3 | Emerging themes in peptidoglycan synthase regulation. In each
part, our current understanding of how protein interaction partners affect
peptidoglycan synthesis activities is illustrated. A | The emerging themes for
the relatively well-characterized Escherichia coli penicillin-binding protein 1B
(PBP1B). B | The themes for the less well-characterized E. coli proteins RodA ,
PBP2 and PBP1A. The effects of binding partners are displayed across the
different parts Aa–Ae and Ba–Bd, but this is done for illustrative simplicity and
is not indicative of temporal separation or mutual exclusivity , as these aspects
are not yet fully understood. Aa | PBP1B is thought to exist in either active or
inactive states, depending on the state of dynamic structural motifs. Two
α-helices (α) within the glycosyltransferase (GT) and transpeptidase (TP)
catalytic domains that contact residues in the regulatory UB2H domain are
shown in the inactive conformation (indicated by the white stars). The
diphosphate form of the carrier lipid, undecaprenol pyrophosphate (C55-PP),
inhibits further GTase reactions unless it is processed to undecaprenol
phosphate (C55-P)20. Ab | Binding of LpoB to the UB2H domain induces
conformational changes (indicated by arrows) that affect the dynamics of
the α-helices and promote the active conformation of PBP1B (indicated by the
yellow stars). The C55-PP phosphatase PgpB binds to PBP1B in the inner
membrane and boosts GTase activity by removing the inhibitory product,
C55-PP, which enables its recycling20. Ac | Aside from its own GTase activity ,
FtsW inhibits PBP1B by preventing access to lipid II. Ad | The addition of PBP3
to in vitro reactions between PBP1B and FtsW restored the GTase activity
of PBP1B by altering lipid II binding to FtsW, which then presumably enables
PBP1B to access the substrate. However, the presence of FtsQ–FtsL–FtsB
(FtsQLB) inhibits both PBP1B GTase and PBP3 TPase activity. Whether the
FtsQLB-mediated inhibitory effect on PBP1B GTase activity is due to changes
in the identified dynamic structural motifs is currently unknown. The Tol-
associated protein CpoB binds to PBP1B–LpoB and negatively modulates
the TPase regulatory dynamics without affecting the GTase activity. Ae | The
recruit­ment of FtsN during cell division is a signal to the early divisome
proteins to initiate constriction while simultaneously signalling FtsQLB to
relieve its inhibitory effect on PBP1B and PBP3 (and perhaps FtsW). FtsN
binding also stimulates PBP1B GTase activity synergistically with LpoB.
Binding of the core Tol protein TolA to PBP1B relieves the inhibitory effect of
CpoB on the PBP1B TPase and coordinates synthase function with outer-
membrane constriction during cell division. TolA binding also stimulates
PBP1B GTase activity synergistically with LpoB and FtsN. The contribution of
FtsW to glycan synthesis is omitted from this illustration, for simplicity. Ba | The
structure of E. coli RodA has no obvious binding site for lipid II or the growing
glycan chain. E. coli PBP2 features two domains external to the trans­membrane
helix, forming a regulatory ‘pedestal’ atop which is the TPase domain.
Bb | Recent bio­chemical work has shown that most SEDS (shape, elongation,
division and sporulation) proteins, such as RodA and FtsW, are inactive
without their cognate class B PBP, which suggests that class B PBP binding
opens up, or constitutes part of, the active site. Bc | Binding of MreC to PBP2–
RodA is hypothesized to induce a conformational change that promotes an
active conformation of the complex. Bd | PBP2 interacts with PBP1A , and their
binding affects the catalysis of both proteins, stimulating PBP1A GTase activity
and PBP2 TPase activity , which enables the two proteins to more efficiently
attach new peptidoglycan to existing sacculi in vitro. PBP1A activity relies on
LpoA binding in the cell, which presumably induces stimulatory
conformational changes that produce an effect similar to the observed effect
of LpoB on PBP1B, but those changes are not yet characterized.

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Reviews
3D-struc tured illumination
microscopy
(3D-SIM). An imaging
technique based on the
use of spatially structured
excitation illumination;
it allows reconstruction of
super-resolution images with
approximately twice the
resolution of regular, diffraction-
limited microscopy (down
to ~110 nm).
Photoactivated localization
microscopy
(PALM). Super-resolution
microscopy technique based
on repetitive imaging of
stochastically photoactivatable
or photoswitchable fluorescent
proteins; it can achieve a
resolution of 10–20 nm.
Total internal reflection
fluorescence microscopy
(TIRF). Uses an evanescent
wave (a very thin
electromagnetic field) to
selectively excite and image
fluorophores within a 100-nm
to 200-nm distance from the
coverslip–specimen interface.
454 | August 2020 | volume 18 www.nature.com/nrmicro
new labelling methods for nascent peptidoglycan, has
provided new insights into the connections between
filament dynamics, localization and peptidoglycan
synthesis17. Studies based on 3D-structured illumination
microscopy (3D-SIM) and photoactivated localization
micro­scopy (PALM) have revealed that the FtsZ ring is
not continuous but patchy in E. coli84, C. crescentus85 and
S. pneumoniae86. A recent study used a combination of
total internal reflection fluorescence microscopy (TIRF)
microscopy, and a novel method to stand B. subtilis
cells on end, to visualize FtsZ filament dynamics in
parallel with cell wall synthesis87. By pulse-labelling
cells with fluorescent d-amino acids, the authors
showed that the peptidoglycan material in the clos-
ing septum is synthesized at discrete sites that trans-
locate circumferentially around the cell. They went
on to show that these translocating sites of synthesis
coincide with the movement of a division-site-specific
synthetic enzyme, PBP2B. Crucially, they found that
FtsZ formed discrete, discontinuous foci or short fil-
aments that also moved circumferentially around the
cell division site, with similar kinetics. The FtsZ motion
was shown to be due to treadmilling, and manipula-
tion of the treadmilling velocity affected not only the
motion of FtsZ but also an FtsZ-associated cytoplasmic
division protein, FtsA, as well as PBP2B and peptido-
glycan synthesis, whereas FtsZ motion was unaffected
by the inhibition of peptidoglycan synthesis. Thus,
the treadmilling motion of FtsZ controls the location
and rate of peptidoglycan synthesis during annular
invagination of the B. subtilis division site. A similar
treadmilling behaviour was found for E. coli FtsZ88.
Preliminary data suggest that in E. coli, PBP3 follows
treadmilling FtsZ filaments via a Brownian ratchet
mechanism, and the speed of treadmilling affects
the processivity of the endtracking of PBP3 (ref.89).
In addition, early findings showed that the treadmilling
of FtsZ also drives the fast movement of a popula-
tion of presumably inactive FtsW molecules in order
to distribute these along the septum; a population of
slow FtsW molecules moves independent of FtsZ but
depending on septal peptidoglycan synthesis 90. In
S. aureus, cytokinesis is initially slow and depends
on FtsZ treadmilling, but it speeds up and becomes
independent of FtsZ once MurJ localizes at the sep-
tum and septal peptidoglycan synthesis begins 91.
Treadmilling of purified FtsZ–FtsA co-polymers has
previously been reconstituted on supported bilayers92.
The same experimental system has recently been used
to show that FtsN and FtsQ mol­e cules, whilst not
showing any directed movement themselves, enrich
at treadmilling FtsZ–FtsA filaments owing to tran-
sient interactions in a diffusion-and-capture mecha-
nism18. Curiously, however, in this case perturbations
in the rate of treadmilling did not affect the rate of
peptidoglycan synthesis or septal closure, and in
S. pneumoniae, movement dynamics of the septal pepti-
doglycan synthases FtsW and PBP2x depended on ongo-
ing peptidoglycan synthesis and not on the rate of FtsZ
treadmilling93. The significance of the different coupling
behaviours between FtsZ and synthetic enzymes in
E. coli, B. subtilis and S. pneumoniae is not yet clear, but
they might conceivably reflect differences in, for exam-
ple, septal morphology, peptidoglycan thickness and/or
the presence of an outer membrane.
The mreB gene was originally identified via shape-
defective mutants of E. coli94, and it has been suggested
that that protein belongs to the actin superfamily95.
It then emerged that MreB, as well as a paralogous pro-
tein in B. subtilis called Mbl, formed filamentous struc-
tures located in the cylindrical part of the cell, and that
the presence of an MreB homologue correlated with a
rod shape across a wide range of bacteria96,97. Labelling
of nascent peptidoglycan synthesis revealed a discontin-
uous pattern that resembled that of the MreB or Mbl
filaments, which suggests a direct involvement of MreB
proteins in cell wall morphogenesis96. As for FtsZ, vari­
ous synthetic and degradative proteins involved in cell
wall elongation were then shown to associate with MreB
proteins, which is consistent with the notion that fila-
ments of MreB are responsible for organizing cell wall
synthesis and dictating a cylindrical cell shape (reviewed
in ref.78). MreB filaments were then described as rotat-
ing circumferentially98,99, but, as for FtsZ, our view of
the detailed localization dynamics of MreB proteins has
become more complicated as imaging methods have
improved (reviewed in ref.100). Three groups found
that MreB filaments in actively growing B. subtilis or
E. coli cells are relatively short, often visible only as
diffraction-limited foci (<200 nm), and that these rotate
more or less circumferentially around the cell. Unlike
with FtsZ, the motion of which is thought to be driven
by treadmilling (see above), MreB motion is dependent
on active peptidoglycan synthesis101–103, and prelimi-
nary data suggest that MreB motion is also affected by
membrane fluidity104. These experiments suggested that
the role of MreB is to orientate peptidoglycan synthesis
in order to generate or maintain a smooth cylindrical
shape, but how this orientation is governed remained
unclear. The most recent publications have focused on
the size and arrangement of MreB filaments and explo-
ration of the idea that orientation is governed by inter-
actions between curvature in MreB filaments and the
cell membrane.
The length of MreB filaments seems to depend greatly
on growth rate and is prone to experimental artefacts
or changes in growth conditions. One study described
the filament length in B. subtilis as often being >1 μm
long and thus capable of traversing nearly half of the
cell circumference105; another study reported that dur-
ing exponential growth, most MreB filaments are less
than 200 nm long106. In E. coli, most recent estimates
put the length at typically 500 nm107. There is general
agreement that MreB filaments migrate in the direction
of their long axis. They tend to orientate and guide the
movement of peptidoglycan synthesis approximately in
the direction of principal membrane curvature — which
is usually perpendicular to the long axis of the cell cyl-
inder108. This directionality is maintained as cell width
is artificially increased until the cell becomes more or
less spherical, at which point motion becomes isotropic.
A similar effect on the orientation of MreB filaments
was seen in in vitro experiments with purified MreB and
liposomes of various shapes. Another study also showed

that the recovery of shape from sphere to rod was mani­
fested by oriented movement of MreB filaments at sites
of bulging, which then grew out into nascent rods108.
E. coli differs from B. subtilis in at least two impor-
tant ways. First, the width of E. coli varies systematically
according to the growth rate, whereas that of B. subtilis is
almost constant. Second, E. coli has a thin peptidoglycan
layer, presumably requiring much tighter coordination
of synthetic and autolytic enzymes. Variations in cell
diameter correlate with changes in the helical pitch of
MreB filaments, which suggests that in this organism,
unlike B. subtilis, MreB filament orientation, and there-
fore the orientation of newly synthesized glycan strands,
is influenced by membrane curvature107. It seems pos-
sible that these discrepancies reflect differences in the
ways that E. coli and B. subtilis MreB proteins associate
with the cytoplasmic membrane: E. coli MreB has an
N-terminal amphipathic region that enables direct inter-
action with the membrane109, a region that the B. subtilis
protein lacks.
Presently, progress in better understanding how these
cytoskeletal proteins contribute to cell morphogenesis
is probably being limited by our poor understanding
of the fine structure of peptidoglycan. The degree of
vari­ation in the tracks made by MreB filaments suggests
that they might not follow template strands in the exist­
ing pep­tidoglycan, in which case connections to the
exist­ing structure must be opportunistic, depending on
proximity to available acceptor side chains.
Finally, we need to acknowledge that many organ-
isms, especially cocci, such as Staphylococcus species;
ovococci, such as Streptococcus species; and tip-growing
organisms, do not have MreB proteins and must there-
fore have developed other mechanisms to control
peptidoglycan synthesis and cell shape.
Cell envelope stability
Many bacteria — for example, E. coli and B. subtilis — are
capable of propagating under various growth conditions
that differ in the composition and physico-chemical
properties of the surrounding milieu. The bacterial
cell surface is particularly exposed to environmental
conditions. Whereas the site of peptidoglycan syn­
thesis is to some degree protected by the thick cell wall
in monoderm bacteria, and by the outer membrane in
diderm bacteria, peptidoglycan synthesis is likely to
be affected by external factors such as temperature,
the pH value of the growth medium or the presence of
metal ions and small chemicals that can penetrate the
cell envelope. Recent work has shed some initial light
on the mechanisms by which bacteria achieve robust-
ness in peptidoglycan synthesis. Here we focus on two
emerging aspects. First, many bacteria maintain large
sets of peptid­oglycan enzymes and their regulators,
whereby individual enzymes are active and are regulated
differently, but collectively they cover the full range of
growth conditions, which ensures robust peptidoglycan
synthesis over a wide range of environments110. Second,
it has recently emerged that bacteria can repair defec-
tive peptidoglycan in order to survive otherwise lethal
insults and toxic chemicals that cause defects in the
peptidoglycan layer54.
NAture Reviews | MICRoBIoLogy
Reviews
Robustness by multiple enzymes, regulators and mecha-
nisms. E. coli has ~40 different enzymes that synthesize
and hydrolyse peptidoglycan in the periplasm3; hence,
on average each of the eight periplasmic peptidoglycan
reactions is catalysed by ~5 enzymes. Little is known
about the specific functions of the seemingly redundant
peptidoglycan synthases. However, of the two main
bifunctional peptidoglycan synthases, PBP1A and its
activator LpoA are more important for growth at alka-
line pH values, whereas PBP1B and LpoB are more
important for growth under acidic conditions, as has
been shown by phenotypic analysis of mutant strains
and is consistent with the activity profile of the purified
enzymes55. Hence, E. coli seems to maintain these two,
semi-redundant enzymes, which can both function in
lateral growth and cell division, in order to grow over
a wider range of pH values. Salmonella enterica subsp.
enterica serovar Typhimurium has the canonical PBP3
TPase required for cell division under standard growth
conditions, but this species has a second PBP3 paral-
ogue, called PBP3SAL, that enables the cell to divide
within acidic phagosomes of the eukaryotic host cell111.
Of all peptidoglycan enzymes, the peptidoglycan
hydrolases seem to be most redundant. The cell uses
some of the hydrolases only in certain conditions. PBP5
is the main dd-carboxypeptidase in E. coli required for
maintaining proper cell shape112. However, in acidic con-
ditions, a dd-carboxypeptidase of unknown function,
PBP6B, that was previously considered ‘minor’ becomes
important113. PBP6B is more active and stable at acidic
than at neutral and alkaline pHs and was shown to pro-
vide dd-carboxypeptidase activity in cells growing at a
pH of ~5. Another example in E. coli is the lytic trans-
glycosylase MltA, which is more active at 30 °C than at
37 °C, both in vivo and in vitro114. Vibrio cholerae has
three semi-redundant dd-endopeptidases — ShyA, ShyB
and ShyC — of which ShyB is upregulated and required
in order to support cell growth under conditions of zinc
starvation115.
Three amidases — AmiA, AmiB and AmiC —
contribute most to septum cleavage during cell division
(lytic transglycosylases and dd-endopeptidases have
a smaller contribution) in E. coli, consistent with the
cell-chaining phenotype of mutants lacking multiple
amidases116. The amidases are activated differently, either
from inside the cell (AmiA and AmiB) or from the outer
membrane (AmiC). AmiA and AmiB are acti­vated by
EnvC, a catalytically inactive member of the M23 pep-
tidase family117,118. The EnvC-mediated activation of
amidases requires the interaction of EnvC with the ABC
transporter-type membrane proteins FtsE–FtsX, which
presumably couple the hydrolysis of cytosolic ATP with
the progression of cell division119. By contrast, AmiC is
activated by the outer-membrane-anchored lipoprotein
NlpD118,120, which contains a LysM peptidoglycan-binding
domain121. Interestingly, both of these amidase regulators
are involved in other processes: FtsE–FtsX also partici-
pates in divisome assembly by coupling peptidoglycan
synthesis and hydrolysis via interactions with divisome
proteins122, and NlpD couples peptidoglycan hydrolysis
and outer-membrane invagination during cell division123.
FtsE–FtsX-activated peptidoglycan hydrolases are also
volume 18 | August 2020 | 455
Reviews

RpoS
Alternative sigma factor for
stationary-phase gene
expression.
found in other bacteria — for example, B. subtilis and
S. pneumoniae124,125. B. subtilis requires at least one of two
peptidoglycan hydrolases, the FtsE–FtsX-activated CwlO
or the cell-wall-anchored LytE, for cell elongation126,127.
In S. pneumoniae, FtsE–FtsX activates the peptidoglycan
hydrolase PcsB, which cleaves the septal peptidoglycan for
daughter cell separation125,128.
Three out of the six dd-endopeptidases (which
hydrolyse dd-crosslinks) — MepS, MepM and MepH
— are redundantly essential for cell elongation in
E. coli, and depletion of all three results in lysis, presum-
ably because cells cannot insert new glycan strands into
the peptidoglycan layer129. MepS is unique among these
enzymes, in that it is subject to high turnover by the
periplasmic protease Prc; cells lacking Prc have a ten-
fold increased level of MepS130. The degradation of MepS
depends on the outer-membrane-anchored protein NlpI,
which binds MepS and delivers it to Prc131. NlpI recently
emerged as an adaptor protein that also interacts with
other dd-endopeptidases, including PBP4, PBP7 and
MepM, without substantially affecting their cellular
abundance132. Because some of the endopeptidases also
interact directly with the peptidoglycan synthase PBP1A
and/or its regulator LpoA, it is possible that NlpI func-
tions to localize the endopeptidases near the outer mem-
brane and at active peptidoglycan synthesis complexes,
to ensure that the hydrolysis of peptidoglycan is spatially
coupled to sites of peptidoglycan growth129,132,133.
Other bacteria use different sets of peptidoglycan
hydrolases for growth or to maintain their particular
cell shape. V. cholerae uses lytic transglycosylases (rather
than amidases) for daughter cell separation134. H. pylori
and Campylobacter jejuni use sets of carboxypeptida­
ses and endopeptidases to grow with helical cell shape,
which is important for host colonization and patho-
genicity in both species, and some of the hydrolases are
active when cells acquire a coccal cell shape during the
transition to stationary phase135–140.
Robustness by ld-transpeptidases and peptidoglycan
repair. The essential PBPs produce the majority of pep-
tide crosslinks in peptidoglycan, using pentapeptides
as donors in dd-transpeptidation reactions. The struc-
turally unrelated ld-transpeptidases (LDTs) use tetra-
peptides as donors and can have different functions.
In E. coli, LDTs catalyse two nonessential reactions.
Three of the LDTs (LdtA, LdtB and LdtC) attach the
outer-membrane-anchored Braun’s lipoprotein (Lpp)
to peptidoglycan, providing a firm connection between
peptidoglycan and the outer membrane that stabilizes
the cell envelope141. Moreover, three other LDTs of the
same enzyme family (LdtD, LdtE and LdtF) are involved
in the formation of ld-crosslinks in peptidoglycan,
which connect the amino acids at position 3 of two stem
peptides54,142.
ld-crosslinks are of low abundance (~5–15%) in
E. coli but are the most abundant crosslink type in cer-
tain bacteria, such as mycobacteria or Clostridiales.
Interestingly, in tip-growing mycobacteria, the activities
of PBPs and LDTs are spatially and temporally separated.
PBPs are involved in cell wall growth at the tip of the cell,
whereas LDTs participate in the maturation of the
peptidoglycan at the lateral wall143. Agrobacterium tume-
faciens has 14 LDT-encoding genes, and some of the gene
products may contribute to polar growth144,145.
LDTs are not inhibited by most β-lactam antibiotics,
with the notable exception of carbapenems146, and some
strains of Enterococcus faecium use LDTs to bypass the
need for otherwise essential PBPs, producing exclusively
ld-crosslinks when growing in the presence of β-lactam
antibiotics147 (Fig. 4). This PBP bypass mechanism also
involves dd-carboxypeptidases, which produce the tetra-
peptide donors for the LDTs, and confers resistance to
most β-lactams. Copper ions inactivate LDTs at a low
concentration that does not prevent cell growth (MIC),
impairing cell envelope stability in E. coli and preventing
PBP bypass in E. faecium148.
Recent work has revealed another role of an LDT in
the pathogen S. enterica subsp. enterica serovar Typhi, the
causative agent of typhoid fever. Intracellular S. Typhi
cells secrete a large, multisubunit toxin that is assem-
bled in the periplasm. Toxin secretion occurs at sites
where the peptidoglycan has been edited by the LDT
YcbB (homologue of E. coli LdtD) and requires a peptid­
oglycan hydrolase, the muramidase TtsA, which specif-
ically cleaves in the ld-crosslinked peptidoglycan in
order to enable the passage of the toxin through the
peptidoglycan layer149.
In E. coli, the ldtD gene is part of the Cpx-mediated
cell envelope stress response, whereas ldtE and ldtF show
RpoS-dependent expression in the stationary phase54,150.
All three corresponding LDTs become essential in cells
with disturbed outer-membrane biogenesis — that is,
upon inhibition of lipopolysaccharide (LPS) transport
to the outer membrane, owing to depletion of the essen-
tial lptC gene or by treatment of cells with LPC-058,
an inhibitor of the LPS biosynthesis enzyme LpxC54.
These cells produce an unusually high proportion
of ld-crosslinks and also require the GTase activity of
PBP1B, its activator LpoB and the dd-carboxypeptidase
PBP6B in order to survive the severe cell envelope stress
conditions. Presumably, these proteins and LdtD repair
defects in the peptidoglycan layer that arise from dis-
rupted LPS transport to the outer membrane54,151 (Fig. 4).
Interestingly, an E. coli mutant with increased levels of
LdtD and the alarmones guanosine tetraphosphate and
guanosine pentaphosphate (collectively referred to as
(p)ppGpp) can grow in the presence of otherwise lethal
concentrations of ampicillin, producing exclusively
ld-crosslinked peptidoglycan, similar to strains of
E. faecium that use the PBP bypass mechanism53. Hence,
under antibiotic stress, when dd-transpeptidases are
inhibited, E. coli switches its mode of peptidoglycan
synthesis and uses its peptidoglycan repair system to
grow with a fully functional, ld-crosslinked peptido-
glycan (Fig. 4), which demonstrates the versatility and
robustness of the peptidoglycan synthesis process.
Concluding remarks
Recent years have brought substantial progress in our
knowledge about the regulation of bacterial cell wall syn-
thesis and morphology, with particular emphasis, first, on
the role of protein–protein interactions in the activation
of peptidoglycan synthases and hydrolases and, second,

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 Reviews

LD-crosslink
Mainly DD-crosslinked wild-type peptidoglycan

Inhibition of outer membrane assembly

Peptidoglycan
β-lactam antibiotic defects

Mutations: aPBP GTase

↑ Ldt expression DD-CPase
↓ Growth rate LDTs

aPBP GTase
β-lactam insensitivity DD-CPase
LDTs

Bypass of PBPs and production of exclusively LD-crosslinks; Peptidoglycan repair; mainly DD-crosslinked peptidoglycan
β-lactam insensitivity and enhanced LD-crosslinks

Fig. 4 | Peptidoglycan remodelling in response to stress. Typical mature peptidoglycan in growing cells features glycan
chains predominantly linked by DD- (or 4–3) crosslinks (with 4–3 referring to the amino acid positions linked in each peptide
stem). A minority of crosslinks are lD (or 3–3), catalysed by lD-transpeptidases (LDTs), which use tetrapeptides as substrates
(see Fig. 1). This minority of lD-crosslinks arises as the cell repairs defects in its peptidoglycan, suffered during envelope
stress. Repair is facilitated by the combined action of class A penicillin-binding protein (aPBP) glycosyltransferase (GTase)
activity , creating a nascent chain; DD-carboxypeptidase (CPase) activity , trimming the terminal D-Ala and thereby creating
a tetrapeptide; and LDT activity , utilizing this tetrapeptide substrate to incorporate the new material into the existing
sacculus. For example, Escherichia coli coli PBP1B cooperates with DD-CPase PBP6a and LdtD to repair peptidoglycan
defects caused during arrested lipopolysaccharide export. The LDT reaction is insensitive to most β-lactam antibiotics, and
with additional mutations boosting LDT expression and reducing growth rate, the lD-mode of peptidoglycan synthesis can
be adopted in order to bypass PBP DD-transpeptidase activity , rendering the cells insensitive to most β-lactams. Areas in
the sacculus with defects are labelled in red; areas with enhanced lD-crosslinked are labelled in blue.

on newly discovered functions of integral membrane
proteins involved in peptidoglycan synthesis and lipid II
transport — that is, the SEDS proteins and MurJ, respec-
tively. The coming years will clarify some of the remain-
ing problems, allowing us to move towards a molecular
picture of sacculus growth. One goal will be to under-
stand how the interplay between the different peptidogly-
can enzymes and multiple interacting cell morphogenesis
proteins and regulators results in the safe expansion of
the stress-bearing sacculus during cell elongation and
cell division. We also need to investigate peptidoglycan
synthesis under different growth and stress conditions
to gain an understanding of how bacteria achieve the
necessary robustness in peptidoglycan growth that
enables them to propagate in different environments.
Finally, we need to know whether non-model bacteria
use additional factors and/or mechanisms to remodel
their sacculus when installing special features such as
cell appendages, when undergoing developmental pro-
cesses or when propagating in particular environmental
niches. Hence, the topic of peptidoglycan growth (and
regulation) will keep us busy for years to come.
Published online 18 May 2020

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Acknowledgements
This work was supported by Wellcome Trust Senior Investigator
Awards (to W.V. (101824/Z/13/Z) and J.E. (209500)).
Author contributions
The authors contributed to all aspects of the article.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Microbiology thanks D.-J. Scheffers,
M. Winkler and the other, anonymous, reviewer(s) for their
contribution to the peer review of this work.
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