MINIREVIEW
Maintaining network security: how macromolecular structures
cross the peptidoglycan layer
Edie M. Scheurwater & Lori L. Burrows
Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, Health Sciences Centre, McMaster
University, Hamilton, ON, Canada
Correspondence: Lori L. Burrows,
Department of Biochemistry and Biomedical
Sciences, Michael G. DeGroote Institute for
Infectious Disease Research, Rm. 4H18, Health
Sciences Centre, McMaster University, 1200
Main St. W., Hamilton, ON, Canada L8N 3Z5.
Tel.: 11 905 525 9140, ext. 22029; fax: 11
905 522 9033; e-mail: [email protected]
Received 23 November 2010; revised 19
January 2011; accepted 20 January 2011.
Final version published online 14 March 2011.
DOI:10.1111/j.1574-6968.2011.02228.x
Abstract
Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and
resisting cellular lysis from high internal turgor pressures. Its integrity is carefully
maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and Nacetylmuramyl-L-alanine amidases. However, its small pore size (2 nm) and
covalently closed structure make it a formidable barrier to the assembly of large
macromolecular cell-envelope-spanning complexes involved in motility and
secretion. Here, we review the strategies used by Gram-negative bacteria to
assemble such macromolecular complexes across the peptidoglycan layer, while
preserving its essential structural role. In addition, we discuss evidence that
suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.
MICROBIOLOGY LETTERS
Editor: Michael Mourez
Keywords
secretion system; motility; peptidoglycan;
twitching; swimming; toxin.
Introduction
The peptidoglycan (murein) layer is an integral component
of the bacterial cell envelope and vital for survival of
most species. Peptidoglycan is an elastic mesh-like net (Koch
& Woeste, 1992; also called the ‘sacculus’) that completely encircles and grows with the cell, providing resistance
to high internal turgor pressures and helping to maintain
a stable cell shape. In order for the peptidoglycan layer
to safely develop with the cell that it encases, a controlled remodeling process involving a number of enzymes
is required to permit its expansion and daughter cell
separation.
Peptidoglycan consists of glycan strands of a repeating
N-acetylglucosaminyl-N-acetylmuraminyl (GlcNAc-MurNAc)
disaccharide that are cross-linked through peptides attached
to the lactyl moiety of MurNAc. Expansion of this heteropolymer involves the incorporation of individual repeat
units (GlcNAc-MurNAc-pentapeptide, Fig. 1, inset) into
the existing sacculus through transglycosylation and transpeptidation reactions, catalyzed primarily by the highFEMS Microbiol Lett 318 (2011) 1–9
molecular-weight penicillin-binding proteins (PBPs) (Vollmer & Bertsche, 2008; Vollmer et al., 2008a). This process
requires the concomitant activities of enzymes that degrade
peptidoglycan to provide space and acceptor sites for
nascent material. These enzymes, whose activities must be
temporally and spatially controlled to prevent autolysis,
include the low-molecular-weight PBPs, lytic transglycosylases (LTs), and N-acetylmuramyl-L-alanine amidases (amidases; reviewed by Vollmer et al., 2008b).
During their life cycle, bacteria express macromolecular
surface structures that are incorporated into their cell
envelopes and peptidoglycan layer (Fig. 1). Examples include structures involved in motility and adhesion (flagella
and pili), secretion of DNA, enzymes, and effectors (type
I–VII secretion systems), conjugation and DNA uptake, and
export of various molecules (tripartite multidrug efflux
pumps). Interestingly, in many cases there are architectural
and sequence similarities between these cell-wall-traversing
systems, specifically between type I secretion (T1S) systems
and multidrug efflux pumps (Koronakis et al., 2004); type II
secretion (T2S) systems, type IV pili (T4P), and the
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E.M. Scheurwater & L.L. Burrows
Outer membrane
Peptidoglycan
Inner membrane
GlcNAc-MurNAcpentapeptide
T1S
T2S
Dedicated Peptidoglycan- ?
degrading enzyme
?
Flagella
T3S
T4S
T6S
T5S
?
?
Yes
?
Yes
Yes
Yes
Yes
LT
Muramidase
LT
Peptidase
LT
LT
Yes
Yes
Yes
Yes
PFO1471
LysM
OmpA/Pal
?
Activity
Peptidoglycan-binding protein?
Motif
T4P
?
OmpA/Pal
Fig. 1. Assembly of motility and secretion systems across the peptidoglycan layer. Multiprotein complexes involved with motility (flagella and T4P) or
secretion (secretion systems T1S–T6S shown) traverse the cell envelope including the peptidoglycan layer. Dedicated peptidoglycan-degrading enzyme
including specialized LTs, muramidases, or peptidases create gaps within the peptidoglycan layer large enough to accommodate these structures. Some
systems also interact with peptidoglycan via specific components containing peptidoglycan-binding motifs, using peptidoglycan as a scaffold or anchor
for assembly and function. Inset: repeat unit of peptidoglycan.
extrusion of filamentous phage (Russel et al., 1997; Russel,
1998; Peabody et al., 2003; Crowther et al., 2005; Ayers et al.,
2010), type III secretion (T3S) systems and flagella (Blocker
et al., 2003; Pallen et al., 2005); type IV secretion (T4S)
systems and conjugation machinery (Alvarez-Martinez &
Christie, 2009; Fronzes et al., 2009; Gillespie et al., 2010);
and type VI secretion (T6S) systems with both T4S systems
and bacteriophage injection machinery (Cascales, 2008;
Leiman et al., 2009; Pell et al., 2009). All of these multiprotein complexes include components in each of the
compartments of the cell envelope that together promote
function at the cell surface.
Because of its architecture, the peptidoglycan layer represents a structural impediment to the assembly of such cellenvelope-spanning multiprotein complexes (Dijkstra &
Keck, 1996a). The size of the pores present within the
sacculus is influenced by the degree of peptide cross-linking,
which changes throughout the life span of a cell (reviewed
by Holtje, 1998); however, the average pore size of peptidoglycan in both Escherichia coli and Bacillus subtilis has been
determined to be approximately 20 Å (Demchick & Koch,
1996). As well, it has been experimentally demonstrated that
proteins of 50 kDa or less can pass through isolated
peptidoglycan sacculi by diffusion (Demchick & Koch,
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1996; Yao et al., 1999; Pink et al., 2000). Proteins or protein
complexes that exceed this size limitation must therefore
circumvent this barrier. Peptidoglycan-degrading enzymes,
particularly dedicated LTs, have been implicated in creating
localized openings within the sacculus for the insertion of
complexes (reviewed in Dijkstra & Keck, 1996a; Koraimann,
2003). However, some systems lack associated peptidoglycan
lytic enzymes, and the ways in which their assembly is
coordinated with peptidoglycan turnover are not obvious.
Further, it is becoming apparent that the efficient function
of some cell-envelope-spanning multiprotein complexes
may require specific components to bind peptidoglycan.
This review will address the mechanisms by which motility
and secretion complexes assemble through and/or associate
with the peptidoglycan layer, with a focus on Gram-negative
bacteria, and discuss the effects of these interactions on
efficient assembly and function.
Preventing security breaches -- controlled
degradation of peptidoglycan
It has been previously noted that general perturbations to
peptidoglycan metabolism can negatively impact bacterial
motility (Stephens et al., 1984). While studying nonmotile
FEMS Microbiol Lett 318 (2011) 1–9
3
Transit of macromolecular structures through peptidoglycan
autolysin-deficient mutants of B. subtilis, Fein (1979) proposed more than 30 years ago that localized peptidoglycan
degradation could facilitate flagellar assembly through the
cell wall. Localized degradation would create space within
the peptidoglycan layer to allow the passage of components
such as the flagellar rod (7.5–11 nm diameter; Hirano
et al., 2001) that would otherwise be too large to pass
through the naturally existing pores (2 nm) within the
peptidoglycan sacculus (Demchick & Koch, 1996). Similarly,
gaps created through the peptidoglycan layer would assist in
the passage of pili, filaments, membrane fusion proteins,
and other structural components of motility and secretion
systems. However, this degradation must be regulated, both
to control its extent and to prevent gaps from being formed
when and where they are not required, thus preventing
accidental lysis.
It is predominantly the activity of LTs that has been
implicated in the process of transenvelope macromolecular
complex assembly (Dijkstra & Keck, 1996a; Koraimann,
2003; Scheurwater et al., 2008). LTs cleave the glycan moiety
between MurNAc and GlcNAc creating 1,6-anhydromuropeptides, unique structures that have been proposed to act
as an acceptor for new material, although their exact role in
peptidoglycan biosynthesis remains unclear (Holtje, 1998).
Interestingly, bacteriophages often use LTs to penetrate the
bacterial cell wall, relying on host enzymes to seal their entry
lesions (Moak & Molineux, 2000) using the energy stored in
the anhydro bond. However, upon completion of their lytic
cycle, they exit the cell using lysozymes (Moak & Molineux,
2000), which hydrolyze the same peptidoglycan bond as LTs
do, but without the creation of anhydromuropeptides. ORFs
encoding enzymes with LT active site-like domains (Blackburn & Clarke, 2001) have been identified within chromosomal or plasmid-borne operons associated with T3S and
T4S systems (Koraimann, 2003). Koraimann (2003) termed
these putative LTs ‘specialized LTs’ to indicate that they have
a unique biological function not associated with basic
peptidoglycan metabolism. The peptidoglycan-lytic activity
of putative specialized LTs has often been demonstrated with
zymograms on peptidoglycan-containing gels. However,
proteins that bind but do not hydrolyze peptidoglycan can
still produce zones of clearing on a zymogram by sequestering peptidoglycan away from the stain; for this reason,
zymograms intended to demonstrate lytic activity should
be interpreted with caution (Dijkstra & Keck, 1996b; Kohler
et al., 2007). Work by Zahrl et al. (2005) and Kohler et al.
(2007) demonstrated cleavage specificity against the MurNAc-GlcNAc linkage for a number of specialized LTs involved in T3S (IpgF, Shigella flexneri; IagB, Salmonella
enterica) and T4S (VirB1, Agrobacterium tumefaciens, Brucella suis; TrbN, Pseudomonas sp.; P19, E. coli plasmid R1;
HP0523, Helicobacter pylori; AtlA, Neisseria gonorrhoeae).
AtlA, one of two N. gonorrhoeae LTs involved in T4S (Kohler
FEMS Microbiol Lett 318 (2011) 1–9
et al., 2005, 2007), was also shown to produce 1,6-anhydromuropeptides, the definitive sign of an LT-catalyzed reaction. Degradation by AtlA does not appear to contribute to
the overall pools of peptidoglycan monomer that N. gonorrhoeae releases to the extracellular environment, suggesting
that its activity is reserved for the creation of localized gaps
to permit T4S system assembly (Kohler et al., 2007).
Although specialized LTs degrade peptidoglycan, their
activities are typically nonessential; loss of the putative LT
in most cases decreases, but does not abrogate, secretion of
effectors and thus virulence. The observed decreases are
often due to a reduction in surface components including
flagellin or needle filaments, pilin (Viollier & Shapiro, 2003;
Hoppner et al., 2004; Yu et al., 2010), and in some cases,
structural components from the inner or outer membranes
(Baron et al., 1997; Viollier & Shapiro, 2003). As most
bacteria encode a number of different LTs, it is likely that
assembly of T3S and T4S complexes can continue, albeit less
efficiently, by taking advantage of temporary breaks in the
sacculus that are created during normal peptidoglycan
metabolism.
While most studies have examined the involvement of
specialized LTs in macromolecular complex assembly, other
peptidoglycan-degrading activities may also be involved in
this process. In fact, three different enzymatic mechanisms
of peptidoglycan cleavage have been associated with flagellar
assembly. FlgJ from S. enterica serovar Typhimurium and its
homologues are required for flagellar rod formation, the
earliest flagellar structure whose assembly would necessitate
a localized opening within the peptidoglycan layer (Nambu
et al., 1999). The C-terminal domain of FlgJ contains a
muramidase domain with similarity to Gram-positive autolysins that hydrolyze the glycosidic bond between MurNAc
and GlcNAc (Nambu et al., 1999; Hirano et al., 2001).
Interestingly, in some bacterial species the functional homologue of FlgJ has a C-terminal peptidase domain active
against the stem peptide, while other flagellar systems lack
a peptidoglycan-active domain all together (Nambu et al.,
2006). In the latter case, it is proposed that the requirement
for localized peptidoglycan degradation is fulfilled by homologues of PleA from Caulobacter crescentus (Nambu et al.,
2006), an LT involved in both flagellar and T4P assembly
(Viollier & Shapiro, 2003).
When operons encoding cell-envelope-spanning macromolecular structures do not encode a discernible peptidoglycan-degrading enzyme, it is possible that one or more
associated peptidoglycan remodeling enzymes are encoded
elsewhere in the genome. Alternatively, some systems may
co-opt the activity of peptidoglycan-degrading enzymes
normally involved in general peptidoglycan metabolism.
ponA, encoding PBP1a, is divergently transcribed from the
pilMNOPQ structural operon for the T4P system of Pseudomonas aeruginosa. This genetic organization was noted as a
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possible link between peptidoglycan biosynthesis and the
assembly of the macromolecular pilus complex (Martin
et al., 1995; Dijkstra & Keck, 1996a). However, our data
show that ponA mutants have wild-type levels of T4Pmediated twitching motility, suggesting that pilus assembly
is unaffected when PBP1a is missing (E.M. Scheurwater and
L.L. Burrows, unpublished data). Interestingly, treatment of
N. gonorrhoeae or Neisseria meningitidis with subminimal
inhibitory concentration levels of penicillin, which inactivates PBPs, caused decreased piliation and adherence to host
cells. Stephens et al. (1984) suggested that penicillin treatment affected assembly or anchorage of pili within the cell
wall. Similarly, the presence of plasmid-borne class A or D
b-lactamases in P. aeruginosa was reported to negatively
affect twitching motility (Gallant et al., 2005). As these
classes of b-lactamases are homologous to low-molecularweight PBPs, it was suggested that they may sequester
peptidoglycan substrates from PBPs, altering peptidoglycan
remodeling and thus T4P assembly and twitching motility
(Gallant et al., 2005).
Irrespective of the type of peptidoglycan-degrading enzyme involved, localized gaps within the peptidoglycan
sacculus are likely created in a controlled manner by the
spatial and/or temporal regulation of the activities of
peptidoglycan-active enzymes. This requirement for the
restriction of lytic activity is evident in the observation that
complementation of phenotypes associated with loss of a
specialized LT can only be achieved with a low copy-number
plasmid, as seen with EtgA, the specialized LT for T3S in
enterohemorrhagic E. coli (EHEC) (Yu et al., 2010). Expression from a higher copy-number plasmid in either the wild
type or mutant backgrounds caused autolysis, reminiscent
of the effects of overexpressing major peptidoglycan-degrading enzymes, and reduced the expression of a number of
T3S components (Yu et al., 2010).
Interactions of components of macromolecular complexes with peptidoglycan-degrading enzymes could assist
in the spatial control of their activity. For example, VirB1 is
the LT associated with the T4S system from A. tumefaciens
and B. suis (Baron et al., 1997; Hoppner et al., 2004). VirB1
interacts with the VirB4 ATPase situated in the inner
membrane (Ward et al., 2002; Draper et al., 2006). Its
processed and secreted VirB1 C-terminus, which lacks LT
activity, may associate with a component of the periplasmspanning channel, VirB9, in addition to being loosely
associated with the cell exterior (Baron et al., 1997). These
associations with the T4S apparatus would serve to restrict
the LT activity of VirB1. As well, it is possible that the
specialized LTs are substrates for their associated secretion
system, as some lack a discernable Sec secretion signal. They
could be secreted by the assembling secretion system into
the periplasm at the place and time that their activity is
required to create a localized gap in the peptidoglycan. In
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E.M. Scheurwater & L.L. Burrows
Pseudomonas syringae, the LTs HrpH and HopP1 are both
T3S substrates that can be translocated into the host (Oh
et al., 2007). In addition to localized peptidoglycan degradation in the bacterium, they may degrade peptidoglycan
fragments that were cotranslocated into the host cell, in
order to prevent recognition by Nod and other immune
receptors and aiding in the infection process (Oh et al.,
2007). FlgJ from S. enterica serovar Typhimurium is secreted
into the periplasm by the type III flagellar export system and
generates breaks in the peptidoglycan sacculus required to
complete the formation of the flagellar rod so that further
assembly of the flagellum can proceed (Nambu et al., 1999).
Although it is the C-terminal domain of FlgJ that is involved
in peptidoglycan hydrolysis, it is the essential N-terminal
domain that acts to cap the flagellar rod. The N-terminal
portion of FlgJ may be important for spatial control of the
lytic activity of FlgJ due to its direct interactions with the
rod, as the C-terminal domain alone is more active in vitro
compared with the full-length protein (Nambu et al., 1999;
Hirano et al., 2001). Also, work with a PleA homologue,
RSP0072 from Rhodobacter sphaeroides, demonstrated that
it interacts with a monofunctional form of FlgJ, which has
only a rod-capping function, despite not being exported by
the type III flagellar export system (de la Mora et al., 2007).
This observation strengthens the idea that the activity of
these enzymes can be regulated by interactions with flagellar
components.
Spatial control can also be achieved through localization
of peptidoglycan-degrading enzymes to specific cellular
sites, for example mid-cell for those associated with division. Although their distribution can vary depending on the
organisms, a number of macromolecular structures associated with motility and secretion are localized to specific
cellular sites, primarily the poles (Weiss, 1971; Scott et al.,
2001; Chiang et al., 2005; Buddelmeijer et al., 2006; Senf
et al., 2008; Morgan et al., 2010). It is plausible that
peptidoglycan-degrading enzymes dedicated to facilitating
the assembly of these structures would show a similar
localization pattern. Such is the case with C. crescentus.
Asymmetric cell division of C. crescentus yields a stalked cell
with a polar holdfast organelle and a swarmer cell with a
single polar flagellum and T4P. Swarmer cells can revert to
the stalked cell form, losing their motility organelles (Viollier & Shapiro, 2003). The LT required for both flagellum
and pilus assembly in C. crescentus, PleA, is colocalized to
the distal pole where pili and flagella are made. Interestingly,
the expression of PleA is concurrent with the appearance of
pili and flagella, indicating that this enzyme is also temporally regulated with cell development (Viollier & Shapiro,
2003). Although not yet experimentally demonstrated, polar
localization of motility and secretion complexes may imply
an assembly process that is associated and/or regulated with
the synthesis of new poles during cell division.
FEMS Microbiol Lett 318 (2011) 1–9
5
Transit of macromolecular structures through peptidoglycan
In general, the expression of bacterial virulence factors is
tightly regulated so that they are produced only when
required, and it is becoming apparent that their associated
peptidoglycan-degrading enzymes are under similar regulation. This scenario would facilitate the controlled production of localized gaps necessary for the assembly of cellenvelope-spanning virulence factors. For example, the activity of specialized LTs appears to be regulated with expression
of T3S structural components. GrlA, a regulator of the LEE
genes in EHEC, appears to negatively regulate production of
the LT EtgA, thus preventing etgA expression before initiation of T3S assembly (Yu et al., 2010; Garcı́a-Gómez et al.,
2011). Pseudomonas syringae encodes three putative LTs
under the control of a Hrp promoter whose expression is
activated by the alternative s factor, HrpL. HrpL is also
important in activation of T3S structural and effector genes
(Oh et al., 2007). Similarly, in the hierarchial expression of
flagellar genes in E. coli and Salmonella sp., flgJ is a class II
gene that is expressed after the initial structural proteins are
synthesized (Kutsukake et al., 1990; Apel & Surette, 2007).
Finally, in Brucella abortus, the LT VirB1 is under the control
of the BvgR/S two component system that regulates expression of the other components of the virB T4S operon
(Martinez-Nunez et al., 2010).
Security enhancements: interactions
with peptidoglycan
Even though peptidoglycan represents a structural barrier
that must be surmounted during assembly of transenvelope
macromolecular structures, it can also facilitate the function
of these systems by stabilizing them within the cell envelope
or by acting as an assembly scaffold. For this to happen,
specific components of the motility and secretion systems
would need to interact with the peptidoglycan layer. These
interactions could contribute to complex assembly and
function in a number of ways: they could sequester substrates away from biosynthetic enzymes and thereby assist in
maintaining a localized gap created by a peptidoglycandegrading enzyme; they could direct assembly and incorporation through the peptidoglycan sacculus at a specific
spatial or temporal point such as at the poles or division
septum during formation; or they could make use of
peptidoglycan as a structural extension of the complex.
Components of motility and secretion systems that contain
known motifs for peptidoglycan binding have been identified, such as the well-studied OmpA-like (Grizot & Buchanan, 2004; Parsons et al., 2006) or LysM motifs (Bateman &
Bycroft, 2000; Buist et al., 2008). These motifs do not
catalyze cleavage of peptidoglycan, but instead are involved
in processes including the association of the outer membrane with the sacculus (Parsons et al., 2006) or promoting
peptidoglycan degradation by mediating substrate binding
FEMS Microbiol Lett 318 (2011) 1–9
(Buist et al., 2008). In proteins associated with flagellar, T4P,
T2S, or T6S systems that contain a peptidoglycan-binding
domain, mutation of key residues for peptidoglycan binding
within these motifs, or deletion of the entire motif, results in
the loss of normal levels of motility or secretion (Muramoto
& Macnab, 1998; Van Way et al., 2000; Aschtgen et al., 2010;
Li & Howard, 2010; Li et al., 2011; Wehbi et al., 2011). The
identification of additional peptidoglycan-binding motifs
that have not yet been characterized is likely. Examples
include PrgH and PrgK, which make up the base of the
T3SS in S. enterica serovar Typhimurium, as well as the
outer membrane lipoprotein InvH. These proteins were
bound to the peptidoglycan layer (Pucciarelli & Garcia-del
Portillo, 2003) even though they lack known peptidoglycanbinding motifs or sorting signals for covalent attachment to
the sacculus. Therefore, depending on unique functional or
structural requirements, a number of different mechanisms
may be used by transenvelope complexes to interact with,
but not degrade peptidoglycan.
The role of peptidoglycan in the resistance to turgor
pressures is well established, but it can also provide support
or counteract the physical forces exerted by macromolecular
structures during the creation of motion. Flagellar rotation,
which has been measured at 100 Hz, (Ohnishi et al., 1994)
requires interactions between the MotAB stator of the
flagellar rotor and the peptidoglycan sacculus to create the
torque necessary to facilitate movement (Doyle et al., 2004;
Kojima et al., 2009). The C-terminal region of MotB
resembles the binding domain of peptidoglycan-associated
proteins such as OmpA or Pal (De Mot & Vanderleyden,
1994). These two proteins form noncovalent associations
with the peptide components of the peptidoglycan sacculus,
linking it to the outer membrane (Parsons et al., 2006), and
Pal also is part of the Tol–Pal system that forms an envelope
spanning complex (reviewed recently by Godlewska et al.,
2009). Interestingly, a chimera of MotB containing a variant
of the peptidoglycan-binding motif from Pal instead of its
native motif was able to facilitate flagellar motility (Hizukuri
et al., 2009), demonstrating that the peptidoglycan interactions, rather than the specific peptidoglycan-binding motif,
were critical for function. Crystal structures of MotB and its
homologue MotY revealed that the peptidoglycan-binding
site is wider than its counterparts in OmpA or Pal (Kojima
et al., 2008; Roujeinikova, 2008). The larger binding site was
suggested to mediate low affinity binding to peptidoglycan,
explaining the transient nature of MotB–peptidoglycan
interactions.
Peptidoglycan can also act as an anchor to counter forces
such as those experienced during pilus-mediated twitching
motility. The forces generated by retraction of a single T4P
can reach 140 pN (Maier et al., 2002), representing one of
the strongest molecular motors identified to date. Unlike
that observed with flagellar motility, the peptidoglycan–
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pilus assembly complex association is unlikely to be transient in nature. To prevent detachment of pili during generation of retraction forces, the basal complex of the pilus
would need to be affixed to the peptidoglycan layer. For
similar reasons, the structural support provided by the
peptidoglycan layer could presumably assist in puncturing
of target cells by the T3S, T4S, and T6S system apparati,
processes that would exert inwardly directed forces on the
bacterial cell envelope.
FimV, a protein containing an LysM peptidoglycan-binding motif, has been implicated in interactions of the T4P
system with the peptidoglycan layer in P. aeruginosa (Semmler et al., 2000). The LysM motif is a ubiquitous domain that
is involved in binding to peptidoglycan and chitin, presumably through direct interactions with the GlcNAc moiety
shared by these two polysaccharides (Buist et al., 2008).
FimV is required for twitching motility, as well as multimerization of the 4 1 MDa outer membrane secretin, PilQ.
Mutants expressing a form of FimV lacking the LysM
domain retain only 30% of wild-type twitching and have
reduced levels of surface piliation and multimeric PilQ
(Wehbi et al., 2011). As interactions of FimV with the
PilMNOP inner membrane assembly complex were inferred
from protein stability experiments, FimV may be involved in
anchoring of the T4P apparatus within the peptidoglycan
layer (Wehbi et al., 2011).
In addition to anchoring macromolecular complexes
within the cell envelope, peptidoglycan could contribute to
complex assembly by acting as a scaffold. SciZ, an inner
membrane component of the Sci-1 T6S system from enteroaggregative E. coli (EAEC), contains a peptidoglycan-binding motif of the OmpA/Pal family and is thought to stabilize
the T6S apparatus (Aschtgen et al., 2010). Most T6SS
identified to date include a protein with a peptidoglycanbinding motif. This protein is typically a SciZ homologue or
is an IcmH-like protein containing an OmpA/Pal-like
peptidoglycan-binding motif (Boyer et al., 2009; Aschtgen
et al., 2010). Alternatively, the latter can contain a
pfam05036 type peptidoglycan-binding motif that is found
in proteins associated with cell-division and sporulation
(Aschtgen et al., 2010). T6SS IcmH-like proteins share
sequence similarity with an inner membrane component of
the T4S system, exemplified by Legionella pneumophila
IcmH, which lacks a peptidoglycan-binding motif (Zusman
et al., 2004). SciZ homologues are found in systems such as
EAEC, where the IcmH-like protein lacks a peptidoglycanbinding motif (Aschtgen et al., 2010). SciZ interacts directly
with the IcmH-like protein, SciP (Aschtgen et al., 2010),
linking the peptidoglycan layer with core inner membrane
components of the T6SS.
The ExeA component of the T2S system of Aeromonas
hydrophila contains a peptidoglycan-binding motif (pfam01471)
similar to that found in SleB, an LT from Bacillus cereus, though
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E.M. Scheurwater & L.L. Burrows
ExeA itself has no lytic activity. The peptidoglycan-binding
activity of ExeA is necessary for the correct localization and
multimerization of ExeD, the T2S outer membrane secretin
(Ast et al., 2002; Howard et al., 2006). Interestingly, ExeA,
which forms an inner membrane complex with ExeB, was
recently shown to form multimers when bound to peptidoglycan (Li & Howard, 2010). This finding suggests that
ExeAB may form a ring-like structure associated with the
peptidoglycan layer through ExeA that acts as a scaffold for
the pseudopilus and other components of the T2S system
(Li & Howard, 2010).
Conclusions
Bacteria have adapted various strategies to permit assembly
of transenvelope complexes through the peptidoglycan layer,
including use of the peptidoglycan layer as a structural
extension of the complex. Despite the paucity of in-depth
studies of this aspect of cell envelope assembly, some
common themes are emerging. It is apparent that a dedicated peptidoglycan-degrading enzyme, which may or may
not be encoded with other components of a particular
complex, is not an absolute requirement for assembly, as
the systems can potentially take advantage of gaps in the
peptidoglycan layer that are created during normal metabolism by peptidoglycan-degrading enzymes. Where dedicated
peptidoglycan-degrading enzymes participate in transenvelope complex assembly, their activities are likely to be under
spatial and temporal control. The participation of dedicated
peptidoglycan-degrading enzymes or peptidoglycan-binding proteins contributes to the efficiency of assembly, as
their loss often impacts the multimerization of outer
membrane secretins and/or the surface expression of the
systems, and on the level of complex activity. The observation that multiprotein complex–peptidoglycan interactions
modulate function is significant, as it implies that peptidoglycan may play roles aside from its vital barrier function.
Delineating the nature of such accessory roles will aid in our
further understanding of the impact of peptidoglycan
metabolism and architecture on bacterial virulence and
physiology.
Acknowledgements
Work in the Burrows laboratory on the intersection of
peptidoglycan metabolism and macromolecular complex
assembly is supported by funding from the Natural Sciences
and Engineering Research Council and the Advanced Food
and Materials Network of Centres of Excellence. E.M.S.
received partial salary support from a Canadian Institutes
of Health Research (CIHR) New Emerging Team grant on
Alternatives to Antibiotics. L.L.B. held a CIHR New Investigator award.
FEMS Microbiol Lett 318 (2011) 1–9
7
Transit of macromolecular structures through peptidoglycan
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