Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Review

Physiological functions of D-alanine carboxypeptidases in Escherichia coli Anindya S. Ghosh1, Chiranjit Chowdhury1 and David E. Nelson2 1

Department of Biotechnology, Indian Institute of Technology, Kharagpur, District-West Midnapore, West Bengal, PIN-721302, India 2 Department of Biology, Indiana University, Bloomington, Jordan Hall, JHA303A, 1001, E. 3rd Street, Bloomington, IN 47405-3700, USA

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of b-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology. Peptidoglycan and the enzymatic activities of penicillin-binding proteins In Gram-negative bacteria, the peptidoglycan (PG) layer imparts high tensile strength to the bacterial cell wall, which is probably necessary to maintain cell shape in the face of intracellular pressures of several atmospheres [1– 5]. Assembly of PG (murein synthesis) requires the polymerization of glycan chains composed of N-acetylglucosamine (NAG) and N-acetylmuramic acids (NAM) and subsequent crosslinking of these chains via muramyl pentapeptide moieties [6,7]. The former reactions are catalyzed by transglycosylases, and the latter are catalyzed by transpeptidases [5,6,8]. PG remodeling, and possibly some aspects of synthesis, are mediated by carboxypeptidases (enzymes that remove terminal D-alanine from muramyl pentapeptide) and endopeptidases (which hydrolyze the crosslinks between glycan chains) [6,7]. Recently, various PG hydrolases (e.g. amidases [9]) and LD-carboxypeptidases [10] have also been implicated in PG remodeling. Penicillin-binding proteins (PBPs) are a family of enzymes of common evolutionary origin that share their namesake by virtue of their ability to bind to b-lactam antibiotics [2,6,11,12]. b-lactams are substrate analogs of PG constituents, the normal targets of PBPs in vivo [8]. Usually, PBPs are acyl-serine transferases, the actions of which are mediated by an active-site serine moiety [2,13]. Corresponding author: Ghosh, A.S. ([email protected]).

PBP-mediated acylation consists of several concerted steps and the interaction of PBPs with substrate seems to be mediated by specific functional motifs in these enzymes, including a Ser-Xaa-Xaa-Lys tetrad, a Ser-Xaa-Asn triad and a Lys-Thr-Gly triad [7,8,13]. PBPs are classified broadly as either high molecular mass (HMM) or low molecular mass (LMM) [5,8,12,13] based on their relative mobilities during SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel) and their amino acid sequences. In Escherichia coli, the LMM PBPs are present in higher protein copy numbers per cell than the HMM PBPs [14]. The majority of E. coli LMM PBPs are DD-carboxypeptidases (DD-CPases) and/or endopeptidases. Although many phylogenetically diverse eubacteria encode an array of DD-CPases, these enzymes seem to be non-essential for growth in laboratory culture, at least in E. coli [15–17]. DD-CPase LMM PBPs have been studied most extensively in laboratory-adapted strains of E. coli, and the results of these studies are the primary focus of this review. E. coli K-12 encodes at least 12 functional PBPs [5,6,15]. The HMM PBPs (PBP1a, PBP1b, PBP1c, PBP2 and PBP3) are responsible for polymerization of NAG–NAM PG chains (transglycosylation) and inter-strand crosslinking of adjacent PG molecules via peptide side-chains (transpeptidation). PBP1a or PBP1b, PBP2 and PBP3 are essential for bacterial cell elongation, maintenance of cellular morphology and septation [5,6,15,18]. The roles of LMM PBPs [PBP4, PBP6, DacD (also known as PBP6b) and PBP7] are less clear. PBP4 has been reported to be a bifunctional (endopeptidase and DD-CPase) enzyme, and PBP5, PBP6 and DacD are mono-functional DD-CPases [5,6,19,20]. Despite its role during cell septation and having an additive effect on morphology, PBP7 seems to be a DD-endopeptidase [21] with no known DD-CPase activity; hence, it is excluded from further discussion here. Surprisingly, E. coli mutants that lack any or all of the LMM PBPs grow with near normal kinetics in common laboratory media [15]. During the past decade, several investigators have characterized the biochemistry of E. coli DD-CPases and a clearer picture of the substrates and kinetics of these enzymes has emerged. Comparatively, less effort has been directed towards elucidating the physiological functions of DD-CPases in E. coli; how and why these enzymes affect PG function remains murky. Here, we review the physiological functions of E. coli DD-CPases, infer the functions of

0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.04.006 Available online 5 June 2008

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similar enzymes in other bacteria and explore directions for future research in this field. E. coli PBP4 E. coli PBP4, encoded by the dacB gene, is a 457-aminoacid protein that consists of three major domains, as revealed in the recently solved crystal structure [22]. The amino acid sequence of PBP4 is very similar to class C b-lactamases and other DD-CPase LMM PBPs, which is consistent with their common evolutionary origin, but the PBP4 sequence includes a novel region of 200 residues [22,23] (Table 1). However, PBP4 lacks a crucial glutamic acid residue that catalyzes de-acylation in the class A blactamase TEM1, which might explain the observed differences in the in vitro de-acylation rates of these enzymes [22]. PBP4 has been attributed both DD-endopeptidase and DD-CPase [24] activities in vitro, although its DD-CPase activity has been disputed [23,24]. PG fragments from PBP4-treated sacculi contain increased proportions of muropeptide monomers compared with wild-type sacculi, confirming that this enzyme cleaves some classes of interstrand crosslinks in intact PG [25]. However, PBP4 digestion has substantially less effect on muropeptide trimer and tetramer composition, indicating that the endopeptidase activity of PBP4 might be limited to a subset of muropeptide crosslinks. Moderate overexpression of PBP4, unlike PBP5, is tolerated [26], possibly due to these inaccessible crosslinks. Enzymological data to date strongly support the hypothesis that PBP4 primarily functions as a DD-endopeptidase in vivo [20,24]. Loss of PBP4 alone fails to induce either serious growth defects in E. coli or detectable morphological alterations [15]. However, deletion of dacB in a PBP5 mutant background, especially in the absence of PBP7, induces severe morphological defects [19,20]. PBP4 has also been implicated in murein recycling and maturation [27] because overexpression of PBP4 induces AmpC [23], which is thought to be a PG hydrolase [28]. Collectively, these data indicate that the primary function of PBP4 endopeptidase

activity is repair and/or regulation of excessive and/or inappropriate PG crosslinks via the PG-hydrolyzing activity of the enzyme. PBP5 – the best-studied E. coli DD-carboxypeptidase In E. coli, PBP5 (encoded by the dacA gene) is one of the most abundant PBPs [29] and is by far the best-studied LMM PBP. PBP5 shares strong sequence similarity with PBP4 and b-lactamases [30]. PBP5 penicillin binding and DD-CPase activity are robust in vitro and in vivo, and the enzymology of this enzyme has been thoroughly characterized [7,31,32] (Table 1). Most importantly, loss of PBP5 in some backgrounds induces morphological defects in E. coli. This phenotype has facilitated extensive mapping of DD-CPase active-site and accessory motifs, and dissection of the functional domains of LMM PBPs, and has shaped our interpretation of the roles of more poorly characterized members of this enzyme family. Domain architecture of PBP5 High-resolution crystal structures of wild-type PBP5 [7] and a deacylation-defective PBP5 mutant [33] have been solved. Both structures indicate that PBP5 consists of two well-defined domains (amino acids 3–262) and (amino acids 263–356), orientated at right angles to one another. The N-terminal domain contains conserved signature sequence motifs (Ser-Xaa-Xaa-Lys, Ser-Xaa-Asn and Lys-Thr-Gly) that mediate DD-CPase activity. By contrast, the C-terminal domain is b-sheet rich and poorly conserved between PBP5 and other so-called DD-CPases. DD-CPase activity of PBP5 is mediated by the N-terminal domain, whereas the carboxyl domain seems to serve an unknown auxiliary function [11,16]. PBP5 membrane association, which is mediated by a 20-amino-acid C-terminal amphipathic anchor, seems to be crucial to the function of this enzyme in vivo [11] (Box 1). DD-CPase activity of PBP5 PBP5 residues that modulate penicillin-binding and DDCPase activity have been extensively characterized using

Table 1. Features of the E. coli D-alanine carboxypeptidases PBP

Molecular Temporal Structural features and mass expression crystal structure PBP4 49.6 KDa Log phase Three domains. Domains I and II are similar to those of PBP5. Domain III is a novel loop that modulates substrate binding at the active site Two domains. Domain I is PBP5 41.3 KDa Early log phase the enzymatic and morphology domain, domain II is a stalk for domain I PBP6 40.8 KDa

Early stationary phase

Unknown

DacD 43.0 KDa

Mid log phase

Unknown

310

Biochemical activity known DD-endopeptidase, weak DD-CPase

Major DD-CPase

Membrane anchoring C-terminal 18 amino acids, electrostatic interaction

C-terminal 20 amino acids, amphipathic ahelices, hydrophobic interaction DD-CPase, might C-terminal 19 have distinct amino acids, substrate specificity amphipathic ahelices, hydrophobic interaction Minor DD-CPase (5% Similar to PBP4 total) in vivo

Physiological role

Refs

Auxiliary role in morphology maintenance, possible role in PG maturation and recycling, and possible cell-separation function during and after division Maintains normal morphology

[20,22– 24,27,73–75]

Might stabilize stationaryphase PGs. Actual function is not known

[19,51,54, 74,75]

Unknown

[51,74]

[7,11,16,19, 32,33,74,75]

Review Box 1. Comparison between membrane anchors of various E. coli DD-CPases Amino acid content The C-terminal 20 residues of PBP5 [72,73] and 19 residues of PBP6 [54] interact strongly with membranes, whereas the 18 C-terminal residues of PBP4 are only weakly associated with the outer face of the cytoplasmic membrane [73]. Membrane interaction Fourier transform infrared and circular dichroism spectral analyses indicate that the membrane anchors of PBP5 and PBP6 form amphiphilic a-helices in the presence of lipids. A hydrophobicity gradient might mediate penetration of these helices into membranes and promote their interaction with acyl chains of membrane lipids in oblique orientations [73,74]. The PBP4 anchor assumes a different conformation that promotes comparatively weak electrostatic interactions between PBP4 and membranes [74]. It is assumed that Fx[K/R]xxD motifs might mediate the interactions between these C-terminal amphipathic helixes and membranes. PBP5 and PBP6 retain this motif [11], whereas PBP4 does not (Table I). Functionality Membrane anchoring is essential for PBP5-mediated morphological maintenance. Anchors of PBP6 and DacD, but not PBP4 or other unrelated membrane proteins, are functional substitutes for the anchor of PBP5 [11]. However, the physiological utility of PBP4, PBP6 and DacD membrane anchors are unknown.

site-directed mutagenesis and in vitro enzymology. Peptide substrates and penicillin interact with Ser44 and Lys213 of the Ser-Xaa-Xaa-Lys and Lys-Thr-Gly motifs. Mutation of either of these residues abolishes PBP5 penicillin-binding and DD-CPase activities [34,35]. During acylation, Lys47 of the Ser-Xaa-Xaa-Lys tetrad acts as proton acceptor for the nucleophilic attack by Ser44, thereby facilitating the formation of the acyl-enzyme intermediate [7,36–38]. During deacylation of this intermediate, Ser110 of the Ser-Xaa-Asn motif and Lys213 of the Lys-Thr-Gly motif form a hydrogen bridge with a water molecule. This helps to orientate the hydrolytic water molecule towards the carbonyl carbon of the acyl-enzyme complex and facilitate dissociation of the complex during DD-carboxypeptidation [38]. In addition, the Lys213 of the Lys-Thr-Gly motif might serve as a general base in polarizing Ser110 during deacylation of the acyl-enzyme intermediate [38] and act as an electrostatic anchor for substrate binding [36]. Ser110 of E. coli PBP5 is orientated similarly to Tyr159 in the DDpeptidase R61 and might have an equivalent role in deacylation [38,39]. Additional residues including Asp175, His216 and Thr217 (near the Lys-Thr-Gly motif) also regulate DD-CPase activity [34]. Important PBP5 motifs are depicted in Figure 1. Clear clustering of many of these crucial residues around a central peptide-binding pocket indicates that additional residues in this region might mediate substrate specificity and DD-CPase kinetics. PBP5 hydrolyzes penicillin more efficiently than PBP4 does PBP4 and PBP5 share many conserved active-site residues (similar Ser-Xaa-Xaa-Lys and Ser-Xaa-Asn motifs) and retain enzymatic domains of overall similar topology. However, PBP5 deacylates penicillin more rapidly in vitro than PBP4 does [7,22]. Unlike PBP4, PBP5 contains a V-looplike region similar to TEM1 b-lactamases. The spatial

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orientation of His151 in PBP5 is similar to that of Glu166 in TEM1, and this residue probably facilitates proper positioning of the water molecule towards Lys47, which is necessary for deacylation of b-lactams (i.e. hydrolysis of the acyl-enzyme complex) [33] (Figure 1). PBP5 and cell shape PBP5 has a prominent role in maintaining the diameter, contour and morphology of E. coli in vivo. Overexpression of PBP5 converts rod-shaped E. coli to an osmotically stable spherical form [40], whereas loss of PBP5, along with at least two other LMM PBPs, promotes branching and topological distortions [15,41]. Expression of wild-type PBP5 restores near-normal morphology to severely aberrant mutant strains that lack as many as seven PBPs. PBP5-mediated morphological rescue is dependent on both DD-CPase activity and uncharacterized functions of the Cterminal domain [19]. Mapping studies indicate 20 amino acids (200–219) surrounding the PBP5 Lys-Thr-Gly motif that constitute the ‘morphology-maintaining domain’ (Figure 1) and, of these residues, Asp218 and Lys219 are most important for morphological rescue in a sextuple PBP mutant background [16]. PBP5-mediated maintenance of cell shape also requires membrane association via a C-terminal amphipathic helix, although this helix is unnecessary for PBP5 DD-CPase activity (Table 1 and Box 1). The DD-CPase activity of PBP5 seems to be maximal during logarithmic growth [19]. The overexpression of PBP5 during early logarithmic phase induces lysis, but is tolerated as E. coli approaches stationary phase [19]. It has been hypothesized that this lysis is a result of excessive DD-CPase-mediated removal of D-alanine from pentapeptide side-chains, residues that are required for HMM-PBPmediated transpeptidation reactions that crosslink PG strands to one another. Why PBP5 deficiency causes morphological distortions is unclear, but it has been proposed that diminished DD-CPase-mediated PG ‘proofreading activity’ promotes excessive transpeptidation and formation of inappropriate crosslinks [19,42]. PBP5 in bacterial elongation and division Some models of the E. coli cell cycle indicate that bacterial elongation and cell division preferentially use distinct pools of tri- and penta-peptide PG precursors [41,43]. Interestingly, PBP5 seems to be a major regulator of pentapeptide levels in E. coli [44,45]. PBP5 is dispensable for septation and elongation, but recent findings indicate that this protein might have an accessory regulatory role in both processes. First, the morphology of E. coli filaments formed during overexpression of the FtsZ inhibitor, SulA, is PBP5 dependent. Wild-type E. coli forms long straight filaments, whereas filaments of PBP5 mutants are twisted around a central axis and form long helical strands [41,42,46]. Second, loss of PBP5 in an amiC background exaggerates the chaining defects associated with single loss of this septal amidase [9,45]. One interpretation of both results is that increased levels of pentapeptide side-chains in PBP5 mutants elicit formation of inappropriate inter-strand peptide crosslinks. Presumably, these inappropriate crosslinks are not readily repaired 311

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Figure 1. Crystal structure of PBP5 of Escherichia coli. (a) PBP5 contains two domains, domain I and domain II. The proportion of a-helices versus b-strands is similar in domain I, whereas domain II is b-strand rich. In domain I, the Lys47 serves as a proton acceptor during nucleophilic attack by Ser44. Various amino acids, namely, Ser44, Lys213, Asp175, His216 and Thr217 [34], are required for both penicillin-binding and DD-CPase activity, whereas Ser110 and Lys213 contribute to the higher deacylation rate of PBP5. (b) Surface organization of the Ser-Leu-Thr-Lys (SLTK; amino acids 44–47), Ser-Gly-Asn (SGN; amino acids 110–112) and Lys-Thr-Gly (KTG; amino acids 213–215) motifs in the N-terminal domain. (c) Interactions among the amino acids present at the active site are indicated by broken lines. (d) Top view of the 20 amino acids containing the ‘morphology maintenance’ region of PBP5 (blue) where the crucial amino acids Asp218 and Lys219 are located [16]. Modified from 1NZO PDB file (http:// www.rcsb.org; DOI:10.2210/pdb1nzo/pdb) [7].

by PG-remodeling enzymes. In the case of filamentation, extra crosslinks could result in patches of inert PG in the lateral wall that would promote ‘crimping’ and rotation around the axis. At the poles, inert PG crosslinks would result in tangled daughter sacculi that might be difficult to resolve. However, both explanations are largely speculative and elucidation of the role of PBP5 in cell elongation and division will require a better understanding of the sacculus architecture formed during conditions of excess and normal pentapeptide levels. Implications of PBP5 in biofilm formation Loss of dacA elicits only subtle changes in morphology of planktonic E. coli cells, but recent findings indicate that PBP5 and the other LMM PBPs might have more prominent roles in dictating formation of biofilms and multicellular communities. The first link between the LMM PBPs and biofilms was suggested by Gallant and coworkers [47]. Overexpression of TEM1 family b-lactamases impairs the Pseudomonas aeruginosa interaction with matrices that normally trigger planktonic to multicellular conversion. TEM1 presumably diverged from, and 312

is highly similar to, DD-CPases, although it is a weak DD-CPase in vitro. This indicated that TEM1 inhibition of biofilm formation could be mediated by substrate competition between this enzyme and the DD-CPases. Consistent with this interpretation, loss of PBP5 alone or in combination with additional LMM PBPs inhibits biofilm formation in E. coli [47]. Computer modeling of TEM1 blactamases indicates that a putative pentapeptide docking site is present in the active-site cleft of this enzyme [47], which is consistent with the substrate-competition interpretation [48,49]. Moving beyond this enzymological competition hypothesis, it is unclear how loss of DD-CPase function would alter bacterial interactions with substrate or with one another. One possibility is that perturbations in sacculus structure alter the micro-architecture of the bacterial outer membrane, such as flagellar organization, adhesins or secretory complex polysaccharides (e.g. alginate) that mediate multicellular interactions during biofilm formation. The study of DD-CPases in biofilm formation is in its infancy but these exciting preliminary studies indicate that it might be a broadly conserved function of these proteins.

Review Involvement of PBP6 and DacD in different cellular processes, and controversies concerning their functions PBP6 and DacD (PBP6b), which are encoded by dacC and dacD, respectively, share substantial amino acid identity with PBP5 [16,50]. PBP6 (but not DacD) also shares a common transcriptional regulator, BolA, with PBP5 [5,40]. Historically, the functions of PBP6 and DacD have been assumed to be similar to PBP5 [31,51]. However, differences in the activities of these proteins are apparent in vitro (Table 1). For example, PBP6 exhibits weaker DDCPase activity towards some substrates compared with PBP5 [31]. DacD can function as a DD-CPase in vitro, but a dacA-dacB-dacC triple mutant only retains 5% residual DD-CPase activity of wild-type cells, questioning whether this protein is an important player in this activity in vivo [51]. One possibility is that each of the DD-CPase LMM PBPs preferentially acts on specific subsets of PG

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pentapeptide substrates, but this has yet to be explored in a systematic manner. Loss of all three E. coli DD-CPases, individually or in combination, fails to substantially affect cell division or elongation in vitro [15,46]. However, both PBP5 and PBP6 overexpression can restore cell division in an ftsI23 mutant strain (PBP3 mutant cells) [43]. A possible explanation is that increased tripeptide side-chains are formed at the expense of pentapeptide chains (tripeptides are assumed to be the substrates for PBP3 during septation) [52,53], and that PBP5 and PBP6 are involved in such conversions. The idea that PBP6 and DacD can carry out some functions of PBP5 with lower efficiency [19,51] has been termed the ‘substitution hypothesis’. However, several observations argue against the interpretation that PBP6, DacD and PBP5 are functional equivalents of one another in vivo, including: (i) overexpression of PBP6 in either the periplasm or in the cytoplasm is not detrimental [19]; (ii)

Figure 2. Schematic diagram of the involvement of PBPs in PG biosynthesis and remodeling. (a) The HMM PBPs (PBP1a, 1b and 1c) polymerize glycan chains using lipid II as a substrate for transglycosylase activity. (b) PBP2 and PBP3 crosslink peptide side-chains of different PG strands (transpeptidase activity). (c) PBP5 removes the terminal D-alanine from both monomeric and dimeric disaccharide pentapeptide side-chains (the involvement of PBP6 in this DD-CPase activity is controversial), whereas PBP4 and PBP7 cleave crosslinked peptide side-chains (DD-endopeptidase activity) to yield tetrapeptides. (d) Monomeric disaccharide tetrapeptides are then translocated via the permease AmpG to the cytoplasm, where, by the actions of LD-CPase (LdcA), b-N-acetylglucosaminidase (NagZ), amidase (AmpD), murein peptide ligase (Mpl) and other enzymes, muramyl tripeptides are formed (PG recycling). (e) Muramyl tripeptides undergo multiple conversions to form muramyl pentapeptide (NAM) by the action of various murein synthetases MurA-MurF, murein peptide ligase (Mpl) and other enzymes. Undecaprenyl phosphate (UDP) carrier lipids are then attached to NAM and NAG (available in the cytosol) to form lipid II (de-novo synthesis of PG), which is then translocated to the periplasm. Color code: blue, processes that occur over multiple steps; pink, enzymes involved; yellow, reaction name; ?, unconfirmed.

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deletion of PBP6 or DacD does not impact E. coli proliferation or alter morphology [15,40]; (iii) membranebound and soluble PBP6 in periplasmic preparations lacks DD-CPase activity [54]; (iv) the number of pentapeptide side-chains does not increase in dacC mutants during log phase [43,54]; (v) expression of PBP6 and DacD increase during stationary and mid-log phase, respectively [40,51], when pentapeptide substrate levels are low [54]; and (vi) neither PBP6 nor DacD can compensate for the morphology defects caused by the loss of PBP5 [11]. Considering all these results, the substitution hypothesis in its complete form seems unlikely, although a small subset of results is consistent with a lesser version of the idea. It is assumed that PBP6 is involved in tripeptide production [43] (Figure 2) but it is unclear why expression, and presumably activity, of this enzyme increases during stationary phase. Pentapeptide-to-tripeptide conversion involves at least two steps. Removal of the terminal Dalanine from pentapeptides (from penta- to tetra-peptide) seems to be primarily mediated by PBP5 [6,7,31]. The principal mechanism for removal of the second D-alanine residue is unknown, although LD-carboxypeptidase (LDCPase) [6,10] can mediate tetrapeptide-to-tripeptide conversion in some situations. Expression of the LD-CPase ldcA is augmented early in stationary phase in the cytoplasm, and LdcA can cleave tetra- but not penta-peptides [55], implying that LdcA is at least one source of tripeptides. However, generation of tripeptides in the cytoplasm and their use as PBP3 substrates in the periplasm seemingly requires translocation machinery that is, as yet, unknown. By contrast, DD-CPases are likely to convert pentapeptides to tetrapeptides and, therefore, pentapeptide side-chain use is probably a good measure of

DD-CPase activity. The number of pentapeptide side-chains does not increase in PBP6 mutants, indicating that PBP6 does not mediate the first reaction, and there is no solid evidence for the involvement of PBP6 in the conversion of tetrapeptide to tripeptide side-chains. Elucidating the sources of tri and tetra-peptides during logarithmic and stationary phases will be an important step towards understanding the functions of PBP5 and PBP6, and might explain the observation that these enzymes are differentially expressed during stages of growth. In summary, although they possess similar primary structures, PBP5, PBP6 and DacD are likely to have distinct in vivo functions, and possibly function during different phases in bacterial growth. DD-CPases from bacteria other than E. coli It is not known how many bacterial species lack DDCPases, and if these species have novel enzymes that compensate for the absence of DD-CPases. The study of LMM PBPs in other bacteria is clearly essential to knowledge in this arena, but only a few model systems are as tractable as E. coli. Aside from those of E. coli, DD-CPases from Neisseria gonorrhoeae and Myxococcus xanthus are the two most extensively studied enzymes in the family in Gram-negative bacteria. N. gonorrhoeae has at least two DD-CPases (PBP3 and PBP4), and PBP3 seems to be involved in murein biogenesis [56,57] (Table 2). PBP3 of N. gonorrhoeae is similar to PBP4 of E. coli (based on primary amino acid sequence) and seems to have weak transpeptidase and endopeptidase activities in addition to robust DD-CPase activity [56]. The M. xanthus PdcA protein has DD-CPase activity and retains signature DD-CPase

Table 2. DD-CPases of various Gram-negative bacteria, other than E. coli Bacterial species

Gene/protein

Amino acid identity with E. coli DD-CPase

Hemophilus influenzae

dacB/PBP4

H. influenzae

dacA/PBP5

Myxococcus xanthus Neisseria gonorrhoeae

pdcA/PdcA Unknown/PBP3

42.6% with PBP4 16.4% with PBP5 50% with PBP5 20.3% with PBP4 Unknown 24% with PBP4

N. gonorrhoeae

Unknown/PBP4

Proteus mirabilis Pseudomonas aeruginosa

PBP4 (H-form) and PBP5 (L-form) Unknown/PBP4

P. aeruginosa

dacA/PBP5

Salmonella enterica serovar typhimurium

dacA/PBP5

18% with PBP5 25.2% with PBP4 46% with PBP5 17% with PBP4 95% with PBP5

S. enterica serovar typhimurium

dacD/DacD

17.6% with PBP4 47% with PBP5

Vibrio cholerae

Unknown/CPase1

V. cholerae

dacA-1/CPase-2

a

ND, not determined.

314

20.5% with PBP5 15.7% with PBP4 16.9% with PBP5 No such

18.5% with PBP4 17% with PBP5 15.7% with PBP4 56% with PBP5 17.6% with PBP4

Complements E. coli dacA mutant? a ND

Possible function/activity

Refs

DD-CPase/DD-endopeptidase

[76]

Yes

Unknown

[16]

Unknown ND

Endopeptidase and DD-CPase Weak transpeptidase. Acts with PBP4

[58] [56,57]

ND

Necessary for PBP3 function

[56,57]

ND

[77]

ND

PG synthesis in the presence of blactam PG synthesis/unknown

ND

PG synthesis/unknown

[78,79]

Yes

Unknown

[16]

No

Unknown

[16]

Yes

Unknown

[16]

No

Unknown

[16]

[78,79]

Review motifs, although these are arranged differently compared with the E. coli DD-CPases [58] (Table 2). PdcA might be a bifunctional enzyme. PdcA has a C-terminal domain that has amidase activity, and an N-terminal domain that is homologous to Gram-positive DD-CPases [59]. Among the DD-CPases of Gram-positive bacteria, those of Bacillus are the best characterized. Bacillus subtilis seem to encode at least four DD-CPases: PBP4a (dacC), PBP5 (dacA), PBP5* (dacB) and DacF [60,61]. PBP4a is expressed during the stationary phase and is a homolog of E. coli PBP4, but has a unique thiolesterase activity [61,62]. Bacillus PBP5 is expressed during log-phase and promotes PG maturation [61,63]. Some DD-CPases of Bacillus, including PBP5* (which has previously been known as PBP5a), are involved in endospore formation. PBP5* is expressed during sporulation and is involved in spore cortex synthesis, whereas DacF is expressed in early stationary phase within the forespore and might modify PG during sporulation [60,61]. In Listeria monocytogenes, PBP5 regulates cell-wall thickness [64]; in Streptococcus pneumoniae, PBP3 promotes localization of cell-division proteins to the septum [65]. Some bacteria with complex morphologies, including Helicobacter pylori, Caulobacter crescentus and various Mycoplasma spp., lack obvious DD-CPase homologs (see Refs [66,67]) (NCBI-BLAST result, www.ncbi.nlm.nih. gov/blast/, data not shown). This indicates that DDCPases are not always primary determinants of bacterial shape. However, we recently assessed the abilities of diverse LMM PBPs to reverse morphological defects in E. coli dacA mutants. Many E. coli LMM PBP homologs are capable of at least partial rescue of morphological defects in a dacA mutant [16] (Table 2). Considering present limitations in the study of many bacterial species, such surrogate expression strategies are likely to continue to be important for characterization of the functions of diverse LMM PBPs. Concluding remarks and future perspectives PG and PBPs are unique components of bacterial cells, with no eukaryotic homologs (except the PG-binding proteins). PBPs are medically and economically important enzymes because they are the primary targets of b-lactam antibiotics. Although polymerization of PG by HMM PBPs is essential to bacterial growth (Figure 2), the functions of LMM PBPs are dispensable in vitro. However, LMM PBPs, including three highly similar DD-CPases, are redundant (especially in E. coli) and highly conserved in a broad variety of bacteria. These observations imply that LMM PBPs have important, but heretofore unrecognized, physiological roles. Many questions about the functions of LMM PBPs, and especially DD-CPase LMM PBPs, remain unresolved (Box 2). One of the most prominent of these is why E. coli retains three highly similar DD-CPase LMM PBPs. Structural and molecular studies of PBP5 have revealed important details about the basic biochemistry of this enzyme, but have not provided a clear rationale for divergence among PBP5, PBP6 and DacD [16]. Determining whether the cell-shape-maintenance activity of DD-CPase is limited to E. coli or if it is applicable to PBP5 homologs of

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Box 2. Unanswered questions  Why are so many apparently enzymatically redundant and nonessential LMM PBP enzymes retained in diverse Gram-negative bacteria?  If PBP5, PBP6 and DacD have similar enzymatic activities in vitro, why are the latter two unable to restore normal morphology in PBP5 mutant strains?  Do LMM DD-CPases preferentially act on distinct substrates?  Do diverse PBP5 homologs function similarly to E. coli PBP5?  Why is expression of PBP5, PBP6 and DacD temporally regulated?  Does PBP6 supply substrate to PBP3 during cell division?  Does LD-CPase interact directly with DD-CPases to generate tripeptides and/or form a higher-order complex with these enzymes?  Do DD-CPases have roles outside maintenance of morphology?  Do DD-CPases have roles in immune evasion or biofilm formation?  What roles do LMM PBPs have in evolution of clinical b-lactam resistance?

other Gram-negative bacteria will be important to resolving this enigma. Results of at least one recent study indicate that PBP5 homologs from diverse Gram-negative bacteria can restore normal morphology in E. coli [16] (Table 2). Confirming the hypothesis that all three DD-CPase LMM PBPs have distinct physiological functions will require multiple approaches. In vitro, comparison of the differences in 3D structures of various LMM DD-CPases and how these proteins interact with PG mimetic substrates could reveal important enzymological distinctions in this enzyme family. Study in vivo of heterologous complementation of PBP5 mutants with diverse LMM DDCPase homologs from various bacteria should clarify whether these enzymes are functionally diverse outside E. coli. Study of the temporal regulation of DD-CPase expression [40] is also warranted, and would strengthen the assumption that diversity in this enzyme family reflects different contributions by these enzymes to septation and lateral-wall elongation. Finally, approaches that illuminate the 3D architecture of PG-strand placement in sub-regions (lateral wall versus poles) of cell walls could clearly advance the understanding of LMM DD-CPase function and diversity. Recent results from a variety of studies have indicated that LMM DD-CPase PBPs also have roles outside their established functions as modulators of bacterial morphology. First, many eubacteria are commensals of higher organisms and these host organisms have immune systems. PG is a potent immunogen [68] and can elicit strong innate immune responses inhibitory to commensal bacteria [69]. Recent data indicate that PBPs might manipulate PG structure to avoid triggering innate immune responses in mammals [70,71]. Although these studies were conducted in Gram-positive bacteria and related to the functions of HMM PBP, the possibility that LMM PBPs of Gram-negative bacteria serve similar functions is an attractive hypothesis. We speculate that an additional function of LMM PBPs might be regulation of the release of immunogenic PG fragments. It would be exciting to test whether E. coli PBP mutants provoke altered innate immune responses in animal models [69]. Supporting this 315

Review hypothesis, many environmental bacteria that are not associated with higher organisms, such as Caulobacter spp., lack LMM PBPs altogether. This is an attractive area for future investigation. Second, LMM PBPs might mediate formation of microbial communities. In nature, most bacteria do not live in planktonic states. Small perturbations in microbial shape at the single-cell level could have dramatic effects on the 3D architecture of microbial communities [18]. Whether PBPs are needed to maintain an overall morphology that permits community formation, or if PBP mutations inhibit cell-surface localization of factors that promote this phenomenon, is unknown. This is an important area of research considering the implications of biofilm formation in human health (e.g. Pseudomonas on catheters and dental plaques) and in various environmental applications (i.e. bio-shielding of jutes and other biodegradable materials). Although it has been >30 years since Spratt’s seminal report [29] describing the existence of multiple LMM PBPs in E. coli, surprisingly little is known about the in vivo functions of these enzymes (Figure 2). LMM PBPs clearly have prominent roles in morphological maintenance, at least in E. coli, but emerging data indicate that LMM PBPs also function outside this area. Understanding these novel in vivo activities might be crucial to explaining why E. coli and other bacteria invest so much energy in making this apparently dispensable family of redundant enzymes. Acknowledgements We thank Kevin D. Young for the critical reading of the manuscript and for his helpful suggestions. Our work is supported by a grant from the Department of Science and Technology, Government of India, and an Institutional Scheme of Innovative Research and Development grant from the Indian Institute of Technology (Kharagpur) to A.S.G.

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