Maintaining network security: how macromolecular ... - Burrows Lab

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 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Outer membrane Peptidoglycan Inner membrane GlcNAc-MurNAcpentapeptide

T1S

T2S

Dedicated Peptidoglycan- ? degrading enzyme

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Flagella

T3S

T4S

T6S

T5S

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LT

Muramidase LT Peptidase

LT

LT

Yes

Yes

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PFO1471

LysM

OmpA/Pal

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Activity

Peptidoglycan-binding protein? Motif

T4P

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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 A˚ (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, 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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

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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 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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

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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 initia´ tion of T3S assembly (Yu et al., 2010; Garc´ıa-Gomez 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– 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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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

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