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Microbiology and Molecular Biology Reviews, December 2003, p. 686-723, Vol. 67, No. 4
1092-2172/03/$08.00+0 DOI: 10.1128/MMBR.67.4.686-723.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria
Francis C. Neuhaus1* and James Baddiley2
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208,1
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom2
Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis.
The wall of the gram-positive bacterium constitutes a multifaceted fabric that is essential for survival, shape, and integrity (493). Macromolecular assemblies of cross-linked peptidoglycan (murein), polyanionic teichoic acids (TAs), and surface proteins function within this envelope. TAs are composed of wall teichoic acid (WTA) and lipoteichoic acid (LTA) (24, 30, 33, 36, 158, 160, 291, 489, 499). WTA is covalently linked to the peptidoglycan, whereas LTA is a macroamphiphile with its glycolipid anchored in the membrane and its poly(glycerophosphate) (Gro-P) chain extending into the wall. Protonated D-alanyl ester residues (Fig. 1), one of the principal substituents of TAs in many low-G+C gram-positive bacteria, are covalently linked to these chains and provide counterions for determining the net anionic charge of the TA.
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FIG. 1. Protonated D-alanyl ester substituent linked to the 2' hydroxyl of a Gro-P unit (sn-glycerol 1-phosphate). Ion pairing of the phosphodiester with the protonated amino group occurs on rotation of the phosphodiester linkage.
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Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic steering of the envelope (15, 76, 125, 130, 284, 319, 474). This matrix is a polyelectrolyte gel with ion-exchange properties required for not only maintaining metal cation homeostasis and control but also assisting in the "trafficking" of ions, nutrients, proteins, and antibiotics (14, 35, 127, 199, 230, 291, 319, 483). The wall matrix is also responsible, in part, for the permeability of proteins (123, 125), the linkage of wall proteins (334, 357), and the presentation of peptidoglycan hydrolases (autolysins) and adhesins (173, 445), as well as being one of the determinants of cell surface hydrophobicity (426). Under conditions of chemiosmosis, a proton gradient further defines the ionic properties of this matrix (256, 268). Within this complex continuum of anionic charge, peptidoglycan provides its stress-bearing role against turgor pressure (267). Thus, the envelope is an organelle that provides the necessary functions needed for cellular growth of the gram-positive cell in its biological niche.
Although not all gram-positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionally similar anionic ones (413, 466). For example, lipomannan is found in place of LTA in Micrococcus luteus (379, 404). Its polyanionic character is determined by succinyl groups esterified to the mannosyl residues. In another example, growth of Bacillus subtilis in phosphate-limited medium results in the replacement of WTA with teichuronic acid, a phosphorus-free polysaccharide containing uronic acid residues (145). Each of these examples illustrates the importance of a wall anionic polymer(s) during the growth of the organism.
Both the structures and biosyntheses of WTA and LTA have been well characterized (22, 158, 160, 201, 293a, 401, 489). However, the functions of TAs within the wall matrix have been more difficult to define. The D-alanyl esters of these polymers, resulting from a single D-alanine incorporation system encoded by the dlt (for "D-alanyl-LTA") operon (209, 367, 383), constitute important substituents for modulating the properties of the envelope in many species. For this reason, knowledge of these ester residues is essential for understanding the functions of TAs in bacterial physiology as well as in host-mediated responses.
The goals of this article are (i) to summarize the structures and functions of D-alanyl-TAs in the envelope; (ii) to describe the mechanism of D-alanine incorporation into TAs; and (iii) to review the role of the D-alanyl esters in antibiotic action, pathogenesis, adhesion, biofilm formation, and virulence. New insights are emerging which provide a greater understanding of the role played by these esters both in the growth of the bacterium and in their function in host-mediated responses. The ability to isolate mutants deficient in D-alanyl esters by targeted mutagenesis provides a tool for addressing these goals. In addition, an understanding of the D-alanine incorporation system provides screening reactions for designing novel antibacterial agents targeted to D-alanyl-TA synthesis.
Since the WTA and LTA of Streptococcus pneumoniae contain phosphorylcholine substituents instead of D-alanyl esters, the review does not address the TAs of this organism; the reader is directed to reference 164.
Structures of WTA and LTA
A review of the literature reveals a wide structural diversity of WTAs in gram-positive bacteria (22, 32, 149, 239, 355). Some of this diversity is confined to the presence and nature of the glycosyl substituents, D-alanyl esters, and repeating units (monomers) (11, 33, 201). The monomers are joined via anionic phosphodiester linkages to form linear chains that constitute 30 to 60% of the cell wall. Two examples are 1,3-glycerol-phosphate (-Gro-P-) and 1,5 D-ribitol-phosphate (-Rbo-P-) (11, 33). WTA is attached to peptidoglycan via the linkage unit (Gro-P)2 or 3ManNAc(ß1-4)GlcNAc-P to C-6 of the MurNAc residues (Fig. 2A) (9, 102, 205, 273).
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FIG. 2. WTA. (A) Linkage unit. (B) Poly(Gro-P)(sn-glycerol 3-P) moiety from B. subtilis 168 and poly(Rbo-P) from S. aureus H. (C) Substituents on poly(Rbo-P) and poly(Gro-P) characteristic of these bacteria.
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The major WTA from B. subtilis 168 is D-alanyl-[
-D-glucosylated poly(Gro-P)], with a chain length of 53 residues (range, 45 to 60) (Fig. 2B) (32, 133, 393). The degree of -D-glucosylation is 0.8, but this has been shown to depend on the age of the cells and the Pi concentration in the growth medium (68, 190). This bacterium also contains a minor WTA, poly (3-O-ß-Glu-GalNAc) (441). The genus Bacillus contains WTA with a variety of repeating units (22, 239). For examples, B. subtilis 168, B. subtilis W23, and Bacillus coagulans contain the monomers -Gro-P-, -Rbo-P-, -6-Gal(1-2)GroP-, respectively.
Staphylococci also contain either -Gro-P- or -Rbo-P- as the repeating unit of WTA. For example, Staphylococcus aureus H contains D-alanyl-[,ß-GlcNAc-poly(Rbo-P)] glycosylated on position 4 of the D-ribitol in either an - or ß-linkage (Fig. 2C) (38, 40). S. aureus Copenhagen contains this WTA with 15% - and 85% ß-GlcNAc-poly(Rbo-P) WTA (430). On the other hand, Staphylococcus cohnii contains poly(Gro-P) WTA with glucosyl substituents (149). Although most WTAs conform to the substituted poly(1,5-Rbo-P) or poly(1,3-Gro-P) structures, there are exceptions; these include examples in which the repeating unit is either -Gro-P-glycosyl-P- or -GlcNAc-P- (18, 20, 149). In addition, other species contain arabitol-P (e.g., Agromyces cerinus) (440) or erythritol-P (e.g., Glycomyces tenuis) (403).
Without exception, the alanyl esters of TA are of the D-configuration (25). In the poly(Rbo-P) WTA of S. aureus H, the D-alanyl ester is found at position 2 of the -Rbo-P-monomer (39, 346). In this WTA, a phosphodiester anionic linkage and the vicinal 3'-OH of the ribitol flank the D-alanyl ester. In contrast, two phosphodiester linkages flank the D-alanyl ester of poly(Gro-P) TAs. When the 2'-OH of glycerol is substituted by a glycosyl unit, e.g., in group D streptococci, the D-alanyl esters are substituents on the sugar (496).
Many LTAs, originally named membrane TAs (17, 31, 291), are macroamphiphiles composed of poly(Gro-P) (attached to C-6 of the nonreducing glucosyl of the glycolipid anchor [type I] [160, 170, 476, 498, 499]). The glycolipid is Glc(ß1-6)Glc(ß1-3)(gentiobiosyl)diacyl-Gro in staphylococci, bacilli, and streptococci (Fig. 3A) (135, 160, 428). The chain length of poly(Gro-P) (Fig. 3B) varies from 14 to 33 and from 5 to 50 Gro-P units in LTA isolated from Enterococcus faecalis and Lactobacillus rhamnosus ATCC 7469, respectively (301, 394). The polydispersity of LTA in these organisms confirmed that observed originally in Streptococcus agalactiae (331). In contrast to this LTA type, Lactococcus garvieae and Clostridium innocuum contain type II and type III LTA, respectively. Type II LTA has a -GalGal-Gro-P- repeating unit, while type III LTA has a -Gal-Gro-P- repeating unit (160). Type I LTA occurs in 84 of 86 strains of oral streptococci (the exceptions are Streptococcus mitis and Streptococcus oralis) (221). Streptococcus sp. strain DSM 8747, which is closely related to S. pneumoniae (with phosphorylcholine TAs), contains type I LTA with an average chain length of 10 Gro-P units partially substituted by D-alanyl esters (417). The chain length distribution varies from 7 to 17, and the glycolipid anchor is a rare 3-O-(ß-D-galactofuranosyl)-1,2-diacylglycerol (417). Based on the side chain substituents of LTA, Bacillus strains/species are divided into group A and group B. In group A (six members), -GlcNAc is linked to the -Gro-P- repeating unit, while in group B (five members) -Gal is linked (Fig. 3C) (239). B. subtilis is an example of group A, and Bacillus megaterium is an example of group B. Neither LTA nor a related anionic polymer was found in Bacillus circulans and Bacillus polymyxa (240). Comparative studies of 13 species of lactobacilli revealed that 8 contain both LTA and WTA while 5 have only LTA (41, 254). All possess a type I D-alanyl-poly(Gro-P)[Glc(ß1-6)Gal(1-2)Glc(1-3) diacylglycerol] (353). A significant fraction of the glycolipid anchor is also Glc(ß1-6)Gal(1-2)6-O-acyl-6Glc(1-3) diacylglycerol (167, 353). While this example implies a defined structure, Fischer (159) has emphasized that microheterogeneity of LTAs is the result of several variables: (i) fatty acid composition, (ii) kind and extent of glycosyl substitution, (iii) length of hydrophilic chain; and (iv) degree of D-alanylation.
In the high-G+C (>55 mol%) subdivision, LTA is generally replaced by lipoglycans (160, 466). For example, Bifidobacterium bifidum contains a macroamphiphile with single Gro-P units attached to the glycan backbone by phosphodiester linkages and substituted with L-alanyl esters (156, 238). The lipoglycan of M. luteus is a mannan substituted with succinyl substituents esterified to approximately 25% of the mannose residues. Sutcliffe (463) proposed that the diversity of these cell surface components might be useful in classification of the high-G+C and low-G+C (<50 mol%) gram-positive bacteria.
Pathways of LTA and WTA Biosynthesis
With the exception of D-alanyl esters, WTA and LTA are assembled via different pathways (489). For example, in organisms that have the repeating unit -Gro-P- for both LTA and WTA (such as B. subtilis 168), the units are derived from different sources. WTA contains sn-glycerol 3-phosphate derived from CDP-glycerol (78, 201), whereas LTA contains sn-glycerol 1-phosphate derived from phosphatidylglycerol (147, 176, 315). Because of the different origins of the Gro-P units, the chains are not stereoisomerically identical. Thus, LTA is not a precursor of WTA.
WTA assembly.
The assembly of WTA requires four phases: (i) synthesis of the (Gro-P)3-ManNAc-GlcNAc-PP-polyprenol carrier (linkage unit carrier) (9, 200, 201, 502), (ii) polymerization of the poly(alditol-P) on this lipid intermediate (78, 154, 183, 198, 205, 294, 326, 431), (iii) glycosylation of this moiety (184), and (iv) attachment of the WTA linkage unit to peptidoglycan. Undecaprenol phosphate is the carrier lipid on which this linkage intermediate is assembled prior to attachment (69, 201, 502). This lipid is identical to that involved in peptidoglycan synthesis (490). Chain elongation in WTA assembly occurs by successive addition of monomer from either CDP-glycerol or CDP-ribitol to the terminal alditol-P that is distal to the linkage unit (235, 259).
The genes (tagABDFEGHO) encoding the enzymes for the assembly of WTA in B. subtilis 168 have been isolated and characterized (Fig. 4) (293a, 401). These are organized into two divergently transcribed operons (divergon), tagAB and tagDEF (224, 329). The one-gene operon tagO and the two-gene operon tag GH, involved in WTA translocation, are both independent of the above divergon (293a, 451). tagF encodes the polymerase responsible for formation of the poly(Gro-P) moiety from CDP-glycerol (399, 431), tagD encodes the glycerol 3-phosphate cytidyltransferase (382), and tagE encodes the enzyme for glucosylation of the poly(Gro-P) from UDP-glucose (Fig. 4). The regulatory elements of the divergon, two 
A-controlled promoters, are further modulated by signals coupled to cell division as well as to growth phase, media richness, Pi concentration, and temperature (328). Evidence pointing to differences in the septal and cylindrical wall in this strain may be correlated with the differential control of WTA synthesis determined by the regulatory elements of this divergon (328). Growth of strains with temperature-sensitive tag mutations (exception tagE) at the restrictive temperature caused an immediate cessation of WTA synthesis, while the synthesis of LTA and phospholipid was not affected (398, 402). In contrast to tagF, no conditional lethal phenotypes were observed for tagE. Thus, glucosylation is not essential for growth. Interestingly, the synthesis of the poly(Rbo-P) WTA of B. subtilis W23 is directed by the tar genes, also organized in two divergently transcribed operons but with different regulation from that observed in B. subtilis 168 (292). In W-23 four promoters are needed for poly(Rbo-P) WTA synthesis, whereas in B. subtilis 168 two promoters are needed for poly(Gro-P) WTA synthesis.
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FIG. 4. Assembly of the glucosylated poly(Gro-P) WTA of B. subtilis 168. TagD, TagE, and TagF are enzymes in WTA synthesis, and TagO, TagA, and TagB participate in linkage unit synthesis. Reprinted from reference 399 and amended with permission of the authors and publisher.
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The synthesis of the linkage unit requires the sequential transfer of GlcNAc-1-phosphate from UDP-GlcNAc (TagO) and ManNAc from UDP-ManNAc (TagA) to polyprenyl phosphate to form lipid 2 (Fig. 4) (9, 205, 293b, 451, 502). Completion of the linkage unit is accomplished by the addition of two or three Gro-P units from CDP-glycerol to this lipid (TagB). In early studies of a putative LTA carrier (LTC) (154, 326), it was observed that poly(Rbo-P) polymerase from S. aureus is strongly inhibited by D-alanyl esters (157, 271). While the role of this LTC in WTA synthesis has not been supported (157, 335), the inhibitory effect of D-alanyl ester residues on the polymerase (169, 271) remains a distinct possibility in regulating the synthesis of WTA on the linkage unit (157). Both the LTC and the linkage unit contain at least two or three Gro-P units. A similar type of inhibitory effect has also been observed in Bacillus cereus, where the glycosyltransferase is inhibited by the D-alanyl esters of the LTA acceptor (442). Short-chain, lipophilic LTA readily accepts D-alanyl esters (90, 155), and thus the (Gro-P)n moiety of the linkage unit lipid, analogous to the terminus of LTC, may also accept D-alanyl esters, resulting in the inhibition of the poly(Rbo-P) or poly(Gro-P) polymerases. Under conditions of high D-alanylation, the assembly of WTA on the linkage unit may be inhibited. Alternatively, under conditions of low D-alanylation, the assembly of WTA may be enhanced. This putative regulatory mechanism clearly warrants further study.
LTA synthesis.
Biosynthesis occurs via the transfer of Gro-P units from phosphatidylglycerol with the formation of elongated LTA and diacylglycerol (80, 147, 272, 469). In Enterococccus hirae ATCC 9790, Gro-P moieties are added sequentially to the glycolipid acceptor, phosphatidylkojibiosyldiacylglycerol (176). This observation is consistent with the in vivo pulse-chase experiments that implicated phosphatidylglycerol as the donor of Gro-P units (147, 185). On the other hand, the discovery of a series of oligophosphoglycerophospholipids derived from phosphatidylglycerol in Streptococcus sanguis suggested that these lipids may be intermediates in the assembly of LTA and that the mechanism of assembly may differ from that observed in E. hirae (92). One of these lipids, phosphatidylglycerophosphoglycerol, is formed from two molecules of phosphatidylglycerol. The resulting diacylglycerol is phosphorylated by diacylglycerol (diglyceride) kinase and reutilized for phosphatidylglycerol synthesis (469). The elongation of the poly(Gro-P) moiety would appear to occur by the distal (external) addition of Gro-P units (80). Using sequential cleavage by phosphodiesterase/phosphatase, Taron et al. (469) found that the newest Gro-P units added were the ones first cleaved from the in vitro-synthesized LTA. Distal addition can occur only while the growing poly(Gro-P) moiety is in contact with the cytoplasmic membrane. The polydispersity of the LTA chain length suggests that chain growth may terminate at any point in elongation (300). Comparative studies of LTA glycosylation in B. subtilis further illustrate aspects of structural diversity. Glycosylation of the -Gro-P- repeating unit (three strains, group A) is the result of GlcNAc transferases that utilize ß-GlcNAc-P-polyprenol (240, 443). Another intermediate, -GlcNAc-P-polyprenol, is the precursor of ß-linked glycosyl groups in several Bacillus species. The mechanism of elongation, the organization of this assembly system, and the attachment of the glycolipid anchor are not well understood.
It was hypothesized that a specific glycerol phosphotransferase targets the glycolipid with a single Gro-P unit and that this modified glycolipid would serve as the growing point for the assembly of the poly(Gro-P) chain (161, 272, 417). In Streptococcus sp. strain DSM 8747, the least abundant glycolipid (monohexosyldiacylglycerol) is selected of the four for the membrane anchor (417). Several short-chain D-alanyl-lipophilic LTAs that could participate in this targeting process have been described (71, 287). To test this mechanism, mutants deficient in glycolipid anchor were sought. The ypfP gene encoding the diglucosyldiacylglycerol synthase, responsible for synthesis of this anchor, was isolated and characterized from S. aureus and B. subtilis (249, 250, 260). Inactivation of ypfP resulted in LTA with diacylglycerol as the membrane anchor (260), an LTA similar to that described by Chiu et al. (92). In 1976, Button and Hemmings (79) observed the loss of the glycolipid anchor in a phosphoglucomutase mutant of Bacillus licheniformis. The poly(Gro-P) was linked, instead, to diacylglycerol. In at least two organisms, B. coagulans and B. megaterium, the glycolipid is also normally replaced by diacylglycerol as the LTA anchor (160, 239). While not proven, these observations are consistent with the assembly of the poly(Gro-P) moiety on phosphatidylglycerol with subsequent transfer to the glycolipid anchor.
The synthesis of peptidoglycan, LTA, and WTA occurs within the proton gradient of the membrane-wall matrix of the growing cell (247, 257). Harrington and Baddiley (204) found that the ionophore valinomycin disrupted this gradient and thereby inhibited the synthesis of WTA and peptidoglycan. In contrast, this ionophore had no effect on LTA assembly. Since each of the phases of WTA synthesis was detected on the outer leaflet of the protoplast membrane (50), it was suggested that an enzyme complex rotates or reorients between the inner and outer leaflets. At least one of these phases most probably utilizes a translocation step facilitated by the ATP-binding cassette transporter (TagGH) encoded by the tagGH operon (293). The dependence of WTA synthesis on the proton gradient (34) is consistent with an energy requirement for a transport system necessary for the translocation of intermediates across the membrane. Whether the proton gradient is coupled to the rotation of an enzyme complex or used to drive a transport system in the translocation of a polymer is not known.
Topography of TAs
Archibald et al. (19) proposed that the chains of WTA in B. subtilis are arranged perpendicular to the surface of the wall. A fraction (50%) of this WTA is located in a "fluffy"-layer region beyond the wall (63, 132). It was postulated that this fibrous layer reflects a region of autolysin-catalyzed degradation of outer nonstressed peptidoglycan (189). A similar organization was also observed in many strains of S. aureus (23, 478). This topography is illustrated in Fig. 5.
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FIG. 5. Topography of WTA and LTA in S. aureus. Peptidoglycan (black lines) and WTA (green symbols) adapted with permission of the author and publisher of reference 478. Topology of LTA (red symbols) and WTA derived from references 19, 158, 189, 481, and 500.
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Many bacteriophages show binding specificity for accessible WTA (12). By using pulsed incorporation of WTA in B. subtilis, it was observed that phage bind initially only to the inner surface of the wall (7, 13). Maximal binding to the cell surface occurred after 0.75 to 1 generation. These findings indicated that the assembly of WTA initiates at the wall-membrane interface at many sites and that this is followed by movement of covalently linked WTA-peptidoglycan through the thickness of the cylindrical wall. At longer times after the pulse, phage binding was detected only at the polar region. Using differential staining of WTA and teichuronic acid, Merad et al. (339) established that in the transition from phosphate-limited to phosphate-replete growth, the new WTA is evenly distributed along the inner surface of the cylindrical region of the B. subtilis cell. Therefore, insertion of new WTA occurs at the membrane concurrently with peptidoglycan in an "inside-to-outside" growth mode (119, 151, 269, 327, 339, 396, 397), with a slower appearance in the cell poles (96).
LTA is found at the interface of the cytoplasmic membrane and wall (157, 291, 489). Using immunoelectron microscopy, Aasjord and Grov (1) established that LTA in S. aureus Cowan not only is attached to the membrane but also penetrates the wall. In Lactobacillus fermentum, an organism that does not contain WTA, one portion of the LTA is exposed on the cell surface while a second portion is concentrated at the membrane (481). The surface-oriented LTA is responsible for the serological specificity of this species (41). Based on turnover experiments, the LTA is released from the cell surface during growth (107, 344, 499). This transient LTA is noncovalently associated with wall components (e.g., peptidoglycan and proteins) through ionic interactions (157, 230). In group A streptococci, the glycolipid moiety of the LTA becomes surface oriented as a result of interaction with the M protein. Thus, one of the determinants of cellular hydrophobicity in this bacterium is the anchoring of LTA with the hydrophobic moiety to the medium (107, 344, 373).
TAs and the Glycocalyx
Many bacteria are enveloped with an additional matrix of polymers known as the glycocalyx (104, 150, 179, 288). This matrix is distal to the wall peptidoglycan and in some cases includes an S layer, capsule, or slime layer. These highly hydrated (99%) structures play roles in adherence, access of macromolecules and ions, and virulence. In several bacteria, coalescence of adjacent glycocalyces leads to biofilm formation (150, 179, 444). The glycocalyx is composed of exopolysaccharides (467), WTA and LTA (74, 234, 376), wall-associated proteins (253), and a variety of membrane constituents (179). A major fraction of the LTA-antibodies-protein A-gold complexes in group B streptococci is located within the glycocalyx (capsule) and organized as long, fibrous threads spanning this structure (376). These threads appear to be fibrillar in the glycocalyces of several organisms. Electron microscopy with the cationic dye ruthenium red defined regions of anionic sites on the fibrils from S. aureus ATCC 6538P (321). In Staphylococcus epidermidis, the solid component of the slime layer is approximately 80% (wt/wt) TA and 20% protein (234). Based on immunochemical labeling studies and LTA turnover experiments, Wicken et al. (497, 501) concluded that LTA would be expected to be a component of the glycocalyx and thus to play an important role in its function. It was proposed that the spatial divisions of the wall and glycocalyx are not rigid but "represent regions in a continuum and individual types of cell-wall-associated polymers may be distributed across the continuum both spatially and also temporally if they are in transit" (501).
Content in WTA and LTA and Relationship to Growth Conditions
The D-alanyl ester content in WTA and LTA is highly variable. The molar ratios of D-alanine to P (degree of D-alanylation) in LTA from a variety of species vary from not detectable to 0.88 (157, 158, 165, 428). WTA generally has a lower ratio of D-alanine to P than does LTA. For example, the ratio of D-alanylation (WTA/LTA) in S. aureus is 0.75 (161, 312). Growth of B. subtilis in two media of different richness resulted in ratios of 0.2 and 0.5 (383).
Although these esters are constituents of TAs in many bacteria, some bacteria appear to lack D-alanyl esters: e.g. E. hirae ATCC 9790 (157), Micrococcus varians ATCC 29750 (157), S. cohnii (428), and S. pneumoniae (158). TA from actinomycetes also contains no D-alanyl esters (355). Type II and type III LTAs do not contain D-alanyl esters. Iwasaki et al. (239) found that LTAs of Bacillus lacking the glycolipid anchors are also deficient in D-alanyl esters. Thus, D-alanyl ester substituents are generally found in the low-G+C subdivision of gram-positive bacteria, mostly those containing type I LTAs.
The D-alanyl ester contents of LTA and WTA of S. aureus and B. subtilis are a function of the pH of the growth medium (21, 145, 146, 312). In the former, the ester content varies from 0.75 at pH 6.1 to 0.07 at pH 8.1, and thus at the higher pH only 9% of the ester content was observed (312). MacArthur and Archibald (312) reasoned that this observation might be incompatible with a regulatory function of D-alanyl-LTA. It was proposed that the newly synthesized D-alanyl-LTA at pH 8.1 is highly substituted and that the ester groups are subsequently lost by base-catalyzed hydrolysis. The newly synthesized molecules would fulfill the functions that require the presence of D-alanyl esters, while the alanine-free molecules would be excreted or utilized when esters are not required. Growth of B. subtilis in media of decreasing pH (i.e., 7 to 5) resulted in a progressive increase in the D-alanyl ester content of WTA (146). Ellwood and Tempest (145) concluded that the increased ester content is necessary for the proper functioning of TA in the cell at higher proton concentrations. This conclusion may be important for interpreting the observation that S. mutans deficient in D-alanine esters loses its acid tolerance response (65).
In addition to medium pH, the degree of D-alanylation is a function of the temperature (233, 370). For example, growth of the facultative thermophile B. coagulans at 55°C resulted in a threefold decrease in ester content compared to cells grown at 37°C. In addition, sublethal heating of S. aureus resulted in a loss of 65% of the D-alanyl ester content of TA. "Repaired" cells contained four times more D-alanine than did the freshly heated cells (233).
The degree of D-alanylation is also affected by growth on media containing increasing concentrations of NaCl. For example, D-alanylation of S. aureus LTA decreased from 0.71 to 0.33 (D-alanine/P) as the NaCl concentration was increased from 0.2 to 7.5% (171, 172). Koch et al. (270) suggested that the high concentration of NaCl directly affects one of the enzymes involved in the synthesis of D-alanyl-LTA. Both NaCl and KCl markedly decreased the D-alanylation of membrane-associated LTA when measured in an in vitro system from L. rhamnosus (414).
In contrast to separate biosynthetic pathways for WTA and LTA, the D-alanyl-ester substituents of WTA are derived from those of D-alanyl-LTA (197). This is consistent with a single dlt operon encoding the machinery of the D-alanine incorporation system (383). One of the remarkable features of the D-alanyl esters of TA is their dynamic turnover. For example, a t1/2 of 37 min was observed for D-alanyl-LTA in S. aureus growing at pH 7. It was postulated that the turnover is an enzyme-catalyzed process (197). In toluene-treated cells of this organism, the D-alanyl esters are lost from LTA and replaced by the ATP-dependent incorporation of new ones (270). "Reesterification" of vacant sites in LTA and WTA maintains the D-alanyl ester content of both TAs. The rate of D-alanyl-LTA synthesis is correlated with the rates of ester loss that occurs through transfer to WTA and through "base-catalyzed" hydrolysis. Thus, from both in vitro and in vivo pulse-chase experiments, it was concluded that the D-alanyl esters of LTA are the precursors of those in WTA (197, 270).
Of major importance to this review is the observation that the growth of B. subtilis under conditions of Mg2+ limitation resulted in elevated levels of WTA (143). Organisms grown under these limiting conditions have a higher capacity and affinity for binding Mg2+ (145). Higher growth rates in Mg2+-limited cells also resulted in an increased amount of TA in the wall. These observations strongly support a requirement for TA in cation assimilation and scavenging from the environment.
Chemical Reactivity
Knowledge of the chemical reactivity and stability of the D-alanyl esters is important for understanding the functions, distribution, and transacylation of these esters in the envelope. They are unusually labile at alkaline pH. For example, at pH 8 and 37°C, the t1/2 of D-alanyl-LTA from L. rhamnosus is 3.9 h (90). In contrast, the t1/2 at pH 6 and 37°C is >10,000 h. When making stability measurements of D-alanyl-LTA from S. aureus, it was observed that >98% of the ester content was recovered after 9 h at 25°C in the pH range from 4.0 to 6.8 (168) but that only 3% of the content was recovered when D-alanyl-LTA was incubated at pH 8. These in vitro studies are consistent with the in vivo growth experiments described in the previous section. Esterification of the carboxyl group of alanine increases the acidity of the protonated amino group by 
2 pKa units (101). Thus, in the pH range from 7 to 8, the concentration of the nonprotonated amino function is increased in D-alanyl-LTA compared with that of the zwitterionic amino acid.
To explain the enhanced base-catalyzed hydrolysis of D-alanyl-LTA, we suggest that in the unprotonated form, the D-alanyl ester forms a transient D-alanyl-phosphodiester intermediate stabilized by a hydrogen bonding to an adjacent 2'-hydroxyl of a Gro-P unit (Fig. 6). This structure would be more susceptible to base-catalyzed hydrolysis and thus would account for the lability of the D-alanyl ester at pHs of >7. Under conditions of protonation, the amino group forms an ion pair with the anionic phosphodiester, hindering the formation of this intermediate and resulting in increased stability of the D-alanyl ester in the range from pH 5 to 7. Thus, the features that determine the reactivity and stability of the ester provide a basis for interpreting a number of experimental observations to be described in this review.
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FIG. 6. Formation of the acyl phosphodiester intermediate. Stabilization by hydrogen bonding of the C-2 hydroxyl of glycerol increases the electrophilicity of the carbonyl carbon. Protonation of the D-alanyl ester would result in ion pair formation with the phosphodiester and thus would inhibit the formation of the intermediate.
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The vicinal hydroxyl groups in D-alanyl-glycerol play a role in determining the stability of this ester (90, 168). Compared with the stability of D-alanyl-LTA from L. rhamnosus at pH 6.0, the t1/2 of D-alanyl-glycerol is 8.8 h at 37°C. The instability of D-alanyl-glycerol at this pH can be partially explained by the inductive effect of the hydroxyl groups that increase the electrophilicity of the carbonyl carbon (75). In addition, hydrogen bonding of the -OH to the oxygen atom of the carbonyl (75, 504) enhances its electrophilicity (Fig. 7A). The ease of acyl migration is illustrated by the postulated cyclic ortho diester shown in Fig. 7B. These suggestions are consistent with the chemical reactivities of hydroxyethyl-D,L-alanate and phosphohydroxylethyl-D,L-alanate in 0.1 M hydroxylamine at pH 7.4 and 37°C (439).
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FIG. 7. (A) Enhanced electrophilicity of the carbonyl carbon in D-alanyl-glycerol by hydrogen bonding to the C-1 hydroxyl of glycerol. (B) Migration of the D-alanyl ester via the cyclic ortho ester intermediate. These structures are based on those proposed for acyl mobility and reactivity in aminoacyl-tRNA (75, 192, 504).
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The reactivity of phosphohydroxyethyl-D,L-alanate in 0.1 M hydroxylamine at pH 7.4 is similar to that of the type 1 LTA (439). In the pH range from 5 to 6, the anionic linkages apparently shield the carbonyl carbon from nucleophilic attack, resulting in a greater stability of the ester while maintaining its reactivity. In D-alanyl-poly (Rbo-P) WTA, the D-alanyl ester is flanked by both a phosphodiester anionic linkage and a vicinal OH group. Thus, the stability of this ester is different from that of D-alanyl poly(Gro-P) LTA and D-alanyl-glycerol. In group D streptococci, the D-alanyl esters of LTA are found on the glucosyl units at either position 3 or position 4 (496). Since no cis-vicinal OH groups are present, these esters are appreciably more stable to alkali than are those of type I D-alanyl-LTA.
In 31P nuclear magnetic resonance (NMR) spectra of L. rhamnosus LTA substituted with D-alanine, phosphorus has two additional peaks (0.57 and 0.62 ppm) in addition to the primary resonance at 0.96 ppm. These shifts would appear to be the result of differences in ion pairing between the amino function of the chiral D-alanyl residue and the two adjacent phosphodiester anionic linkages (45). Using MNDO-PM3 predictions, Arnold and Neuhaus modeled two energy-minimized conformations, one in which the protonated amino group forms ion pairs with the up-chain phosphodiester and one in which the protonated group forms ion pairs with the down-chain phosphodiester (F. Arnold and F. Neuhaus, unpublished observation) (Fig. 8). In the up-chain ion pair, the protonated amino group can form symmetrical hydrogen bonds with the nonbridging oxygens, while in the down-chain ion pair, one hydrogen bond would appear to be formed with the nonbridging oxygen and one is formed with the bridging oxygen of the phosphodiester linkage. Thus, the rotational characteristics of the D-alanyl ester, determined by the cis-trans orientation of the carbonyl oxygen and the C-2 proton of the glycerol, allow for the different interactions in the two conformations. While this review emphasizes the role of the D-alanyl ester in decreasing the net anionic charge of TA, another physical feature that has not been quantitated is the hydrophobic effect contributed by the D-alanyl residues to LTA and WTA. As described below, the increasing lipophilicity of the LTA determined by D-alanyl esters may play a significant role in a number of host responses.
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FIG. 8. Predicted conformations of the D-alanyl ester on (Gro-P)2Gro. Two conformations are shown: up-chain ion pairing (A) and down-chain ion pairing (B). In panel A, the N–O distances are both 2.66 Å. In panel B, the corresponding distances are 2.61 and 4.70 Å. In panel A, the carbonyl oxygen and the C-2 proton of glycerol are cis, and in panel B they are trans. Resonance stabilization in the ester linkage determines a rotational barrier (495) between the two conformers. For this figure, the flanking glycerol residues are truncated. The conformations were calculated by the semi-empirical molecular orbital method, MNDO-PM3 (457, 458; Arnold and Neuhaus, unpublished).
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Distribution in LTA
A key feature of the D-alanyl esters of isolated LTA is their even distribution along the poly(alditol-P) chain. Fischer et al. found that the esterification of LTA from S. aureus occurs within a relatively narrow substitution range and that no alanine-free species are present (168, 171). Stepwise hydrolysis of the LTA revealed a uniform (even) distribution of the D-alanyl esters (168). In additional studies it was observed by hydrophobic interaction chromatography that the molar ratio of D-alanine to P increased from 0.54 to 0.81 as the length of the hydrophilic chain decreased from 39 to 16 Gro-P units (159, 162). An inverse relationship was also found with E. faecalis LTA by using anion-exchange chromatography (302). As the chain length increased, the ratio decreased from 0.53 to 0.23. These observations imply that shorter-chain LTA has a higher degree of D-alanylation than does longer-chain LTA. The implications of this observation are not understood. In Lactobacillus lactis Kiel, the distribution of the D-alanyl esters is correlated with the random distribution of the -D-galactopyranosyl residues of the LTA (434).
A random distribution of D-alanyl esters was also deduced for LTA from L. fermentum and Enterococcus faecium by using 31P NMR (45). D-Alanine incorporation in toluene-treated cells of L. rhamnosus also did not show a gradient of D-alanyl ester label along the poly(Gro-P) backbone even though a gradient of Gro-P addition was easily observed during LTA synthesis (91). The random distribution of D-alanyl esters would appear to have significant implications for the transacylation and redistribution of these esters within the wall.
We propose that transacylation of D-alanyl esters could occur during the isolation and purification of LTA and that the apparent even or random distribution of the esters may, in fact, be the result of events occurring after cell death. From the time of LTA isolation to the time of measuring the distribution of D-alanyl esters, a variety of manipulations, e.g., extraction, purification, and concentrating steps, have been performed. While transacylation has not been measured at each of these steps, migration of D-alanyl esters from short-chain LTA to long-chain LTA was easily measured in protein-free LTA micelles (91). Therefore, the distribution of D-alanyl esters in the TA at the time of exponential or balanced cell growth is probably not really known. It is conceivable that during the time of growth a gradient of D-alanyl esters across the wall may exist, determined by a variety of factors, e.g. the proton gradient (see "Functions of teichoic acids" below). At the time of cell death, this gradient collapses and redistribution of the esters occurs. Clearly, additional experiments are required to assess the importance of this proposal.
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SYNTHESIS OF D-ALANYL-LTA
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Overview
The synthesis of D-alanyl-LTA requires four proteins that are encoded by the dlt operon (Fig. 9). Two of these are the 56-kDa D-alanine:D-alanyl carrier protein ligase (AMP forming) (Dcl) and the 8.8-kDa D-alanyl carrier protein (Dcp). In addition to the genes encoding Dcl (dltA) and Dcp (dltC), dltB and dltD of this operon encode a transport protein (DltB) and a membane protein (DltD) that ensures the ligation of D-alanine to Dcp. Thus, incorporation of D-alanine is accomplished in the two-step reaction sequence:
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FIG. 9. Comparison of the dlt operons from L. rhamnosus, B. subtilis 168, and S. agalactiae. The accession numbers are AF192553 (U43894), X73124, and AJ291784, respectively. In addition, the sequences for dlt from L. monocytogenes (AJ012255), S. mutans (AF051356; AF049357), S. aureus (AF101234; D86240), S. pneumoniae R6 (AE008562), L. lactis (AE006358), S. xylosus (AF032440), S. pyogenes (AE004092), S. gordonii DL1 subsp. Challis (AF059609), and L. plantarum (NC_004567) have been determined. For alignment and comparison of the dlt proteins, the http://genolist.pasteur.fr/SubtiList site is invaluable. Each of the red genes is common to all dlt operons. The green genes in S. agalactiae represent a novel two-component regulatory system (405). The genes in black are not required for D-alanylation. • is the rho-independent terminator.
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In this sequence, D-alanyl-Dcp provides the essential link between the ligase (Dcl) and the D-alanylation of LTA. Transfer of the activated D-alanine from this intermediate requires that the acceptor LTA be membrane associated.
Proteins of the dlt Operon
In 1960, Baddiley and Neuhaus (43) detected an enzyme that activates D-alanine by using a pyrophosphoryl cleavage of ATP. Isolation of the gene encoding the activating enzyme (dltA) (Fig. 9) from L. rhamnosus ATCC 7469 provided the key for identifying the role of this enzyme in D-alanine incorporation (209, 367). The enzyme is a member of a large protein family that both activates and transfers amino or fatty acids via a 4'-phosphopantetheine prosthetic group of a carrier protein or coenzyme A CoA (262). It contains 7 of the 10 highly conserved consensus sequences (A2, A3, A4, A5, A7, A8, and A10) of the nonribosomal peptide synthetases (adenylation domain) (209) described by Konz and Marahiel (278). A heat-stable protein, which was formerly designated the D-alanine:membrane acceptor ligase (309, 414), contains this prosthetic group and functions as the D-alanyl carrier protein (Dcp) (117, 211). Thus, Dcl (DltA) not only activates D-alanine but also ligates the activated ester to the 4'-phosphopantetheine prosthetic group of the carrier protein. Therefore, the activating enzyme is now designated D-alanine:Dcp ligase (AMP forming).
In addition to dltA and dltC, the operon (Fig. 9) contains two additional genes, dltB and dltD. The hydropathy profile of DltB shows a pattern of 12 putative membrane-spanning domains (367). A BLAST search with DltB from L. rhamnosus identified regions of DltB with sequence similarity to a variety of transport proteins in the major facilitator superfamily and ATP-binding cassette family. Three of these proteins include proton antiporters that pump compounds (e.g., tetracycline, glycerol 3-phosphate, and gluconate) from the cytosol at the expense of the proton motive force. Amiloride, a pyrazinoylguanidine inhibitor of Na+ channels (263), prevents the synthesis of D-alanyl-LTA in the in vitro incorporation system when Na+ is the only monovalent cation (367). No effect was observed when K+ replaced Na+. While it is not established, we suggest that one of the functions of DltB is the secretion of unfolded D-alanyl-Dcp. The reversibility of the thermal denaturation of Dcp (485) is consistent with this suggestion.
Comparison of DltB with a variety of O-acyltransferases identified two conserved motifs that may also link this transport protein to a superfamily of membrane-bound O-acyltransferases (219). It is of interest that this family also includes AlgI, involved in the O acetylation of alginate in Pseudomonas aeruginosa (174, 175). Based on low homology to transferases requiring polyprenol, it was proposed that undecaprenol-P might be an intermediate membrane acceptor in the D-alanine incorporation system (211). However, compelling evidence for D-alanyl-P-polyprenol is lacking, and it has therefore been concluded that this lipid is not an intermediate in D-alanine incorporation (F. C. Neuhaus, unpublished observations). Whether DltB functions in the actual secretion of D-alanyl-Dcp, whether it functions as an acyltransferase, or whether it is bifunctional is not known.
The membrane protein, DltD, functions in the selection of the correct carrier protein, Dcp, for ligation with D-alanine (reaction 1 [above]) and in the hydrolysis of mischarged D-alanyl-ACPs (118). As shown in Fig. 10, it is proposed that DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine. The hydrophobic N-terminal sequence of DltD is required to anchor this protein to the membrane, most probably the inner leaflet (118). It had been proposed that DltD functions in the final esterification step (383, 389). However, attempts to implicate DltD as a catalyst in reaction 2 (D-alanyl transfer to membrane-associated LTA) have been unsuccessful (F. C. Neuhaus, unpublished observations). Thus, the protein complex utilizing DltD to bind the cytosolic components, Dcl, Dcp, ATP and D-alanine described in Fig. 10 guarantees the specific ligation of Dcp with D-alanine.
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FIG. 10. Model for the incorporation of D-alanyl ester residues into membrane-associated LTA. DltD provides binding sites for Dcp and Dcl on the cytoplasmic leaflet. DltB provides a putative channel for the secretion of D-alanyl-Dcp to the periplasm where D-alanylation occurs.
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Based on this proposal, one of the paradoxes of the D-alanine incorporation system is explained (Fig. 10). It was observed that the Km for D-alanine, as measured in the assay of the isolated D-alanine-activating enzyme, is 70 mM (43). In contrast, the Km for D-alanine in the synthesis of D-alanyl-LTA is 18 µM (309). Thus, it would appear that the binding of Dcl and Dcp to membrane-associated DltD enhances the affinity of Dcl for D-alanine. This change in Km resulting from the binding of Dcl to DltD would further ensure that Dcp is ligated with D-alanine at the cytosolic concentrations of this amino acid.
D-Alanylation of LTA
The transfer of the D-alanyl residue from D-alanyl-Dcp to LTA requires only that the acceptor LTA be membrane associated (211) (reaction 2). None of the acyl carrier proteins (ACPs) involved in fatty acid metabolism replace the requirement for Dcp, even though Dcl ligates D-alanine to ACPs in the absence of DltD (211, 367). A distinct membrane-acceptor:D-alanyl transferase that catalyzes reaction 2 has not been detected.
Incubation of D-alanyl-Dcp with micellar LTA (Na+ form) resulted in the time-dependent hydrolysis of D-alanyl-Dcp (261). In contrast, D-alanyl-ACP is not hydrolyzed. It was proposed that D-alanyl-Dcp forms a complex with the poly(Gro-P) moiety of LTA and that within this complex a "thioesterase-like" enzyme mimic occurs. Based on the specificity for D-alanyl-Dcp in the D-alanylation of LTA and this "thioesterase-like" reaction, we suggest the presence of a specific binding site in Dcp for LTA (261). Thus, while D-alanyl-Dcp is hydrolyzed in the presence of isolated micellar LTA, transfer of the D-alanyl residue occurs only when membrane-associated LTA is used (211).
Structural studies of Dcp by multidimensional heteronuclear NMR provided the basis for concluding that the carrier protein is a homologue (three-helix bundle) of the ACPs involved in fatty acid, polyketide, and nonribosomal peptide syntheses (485) (Fig. 11). These studies also provided a basis for defining two sites on Dcp: (i) the phosphopantetheine prosthetic group linked to Ser39 at the N terminal of helix II, which is recognized by Dcl, and (ii) a putative binding site utilizing invariant Arg64 for binding the poly(Gro-P) moiety of LTA. The first site is modified by AcpS, the 4'-phosphopantetheine transferase of primary metabolism in B. subtilis (117, 349). The second site, which encompasses a 310-helix (helix II') spanning residues Asp63-Trp67 of Dcp, is, in part, made up of Arg64. The conserved Trp67 plays an important role in positioning helix II', which in turn determines the orientation of Arg64 for participation in the binding of Dcp to the poly(Gro-P) moiety of LTA. This invariant cationic surface residue is missing from ACPs involved in fatty acid metabolism (Fig. 12, arrow).
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FIG. 11. Ribbon diagram of the minimized average structure of apo-Dcp (PDP entry 1HQB). Residues shown in white bury the Trp67 side chain (purple) in the hydrophobic core. Other key residues include the conserved Glu33 and Asp38(red) and Ser39 (yellow), as well as a cluster of basic residues (blue) proximal to the phosphopantetheine attachment site (Arg64, Lys65, and Lys72). Reprinted from reference 485 with permission of the publisher.
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FIG. 12. Putative binding site for the poly(Gro-P) moiety on apo-Dcp. Surface representations, colored according to electrostatic potential, are shown for apo-Dcp (A) and AcpP (PDP entry 1ACP, model 1) (B). Arg64 is conserved in all Dcp proteins, whereas Lys65 in L. rhamnosus Dcp is not conserved. Reprinted from reference 485 with permission of the publisher.
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It is proposed that the transacylation of the activated D-alanyl ester residue from D-alanyl-Dcp to membrane-associated LTA occurs by nucleophilic attack of the 2'-glycerol hydroxyl (R-O:) on the electrophilic carbonyl of D-alanyl-Dcp (261) (Fig. 13). While the binding site on D-alanyl-Dcp is not completely defined, it is further suggested that its interaction with membrane-associated LTA positions the nucleophile (R-O:) for transacylation of the D-alanyl ester to LTA. Thus, the proposed mechanism for reaction 2 does not require a putative membrane acceptor:D-alanyl transferase; only D-alanyl-Dcp and membrane-associated LTA are required.
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FIG. 13. Proposed mechanism for the formation of membrane-associated D-alanyl-LTA from D-alanyl-Dcp. B·· indicates an unknown proton acceptor for generating nucleophile. The electrostatic interaction between D-alanyl-Dcp and the phosphodiester anion may be due to Arg64. Reprinted from reference 261 with permission.
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Transacylation of D-Alanyl Esters
Two features of the D-alanyl esters linked to LTA are that (i) they are precursors of the D-alanyl esters of WTA and (ii) the esters of both WTA and LTA are evenly distributed along their backbone chains in isolated polymers. Based on these features, it was proposed that the esterification of LTA with D-alanine occurs in one of two modes: (i) addition at random or (ii) addition at at specified loci in the poly(Gro-P) chain followed by redistribution of the ester residues to other loci. If (ii) occurs, a mechanism for distributing or transacylating D-alanyl esters between and along TAs must exist. Two observations have provided insights into this process.
In 1985, Childs et al. (91) observed the nonenzymatic transacylation of D-alanyl esters from short-chain D-[14C]alanyl-lipophilic LTA to long-chain hydrophilic LTA. This transacylation required neither ATP nor the components of the D-alanine incorporation system. No evidence for an enzyme-catalyzed transacylation reaction was detected. The only prerequisite for this reaction was the assembly of the donor and acceptor LTA species into a micelle. Since this transacylation was described in micelles of pure LTA with high packing density, the topological organization most probably does not reflect that in the cellular membrane. However, as noted in "Distribution in LTA" (above), this process may play a role in redistributing the D-alanyl esters during purification and isolation procedures, resulting in their uniform or even distribution.
A second observation that provides an insight into the transacylation process was detected in further studies of reaction 2. It was discovered that the D-alanyl esters of membrane-associated D-alanyl LTA are transferred to Dcp in the reversal of this reaction (261) (Fig. 14). Reversal is consistent with the high chemical reactivity of these esters. As in the case of the forward reaction, the reverse is also specific for Dcp. Membranes prepared from Lactobacillus casei 102S with inactivated dltD also synthesize D-[14C]alanyl-Dcp from membrane-associated D-[14C]alanyl-LTA. Thus, the observations presented in Fig. 14 indicate not only that reaction 2 is reversible but also that DltD is not a catalyst in this reaction.
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FIG. 14. Effect of Dcp concentration on the formation of D-Alanyl-Dcp from membrane-associated D-alanyl-LTA. The reaction mixture contained 20 µg of membrane-associated D-[14C]alanyl-LTA and the indicated amounts of Dcp or ACP in 15 µl of reaction mixture. In mixtures containing dltD::cat membranes, dltD was insertionally inactivated (118). The amounts of D-[14C]alanyl-Dcp formed were quantitated by nondenaturing polyacrylamide gel electrophoresis by the method of Heaton and Neuhaus (211). Reproduced from reference 261 with permission.
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Assuming that D-alanyl-Dcp is secreted (translocated) from the inner leaflet of the cytoplasmic membrane to the wall matrix by DltB, two reactions may occur: (i) D-alanylation of LTA and (ii) D-alanylation of the resulting Dcp with D-alanyl ester from an adjacent LTA molecule followed by D-alanylation at yet another LTA site. Thus, transacylation (ii) of D-alanyl residues by this mechanism will distribute the D-alanyl esters along and among the LTA molecules of the wall. As illustrated schematically in Fig. 15, it is proposed that this process of inter- and intrachain transacylation is responsible for modulating the net anionic charge and lipophilicity of the hydrophilic LTA chain. In this way, perturbation of the D-alanyl ester content by either acylation or deacylation at one location in the membrane can be translated to an adjacent location (365). The proposal does not account for differences in the degree of D-alanylation of short-chain and long-chain LTA noted above.
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FIG. 15. Transacylation of D-alanyl ester residues along and among the chains of LTA and WTA. Interchain and intrachain transacylation is illustrated as a mechanism for distributing the esters and for the formation of D-alanyl-WTA from D-alanyl-LTA. Whether this process occurs by the mechanism described in reference 91 or that described in reference 261 has not been determined. A+ represents the D-alanyl ester.
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LTA is located primarily at the membrane-wall interface, with poly(Gro-P) chains extending into the wall. This topography is consistent with the observations by Haas et al. (197), who found that the D-alanyl esters of WTA are derived from those of D-alanyl-LTA. Dcp in the membrane-wall matrix can be utilized for D-alanyl-Dcp synthesis from membrane-associated D-alanyl-LTA. By this mechanism, Dcp can "catalyze" the transfer of the D-alanyl ester from LTA to WTA. The thermodynamic feasibility of this process for distributing esters between and along molecules of LTA and WTA is not well understood.
Organization and Regulation of the dlt Operon
The dlt operon has been characterized in 12 species (Fig. 9). Each of these contains four genes, dltABCD, in a novel organization. dltA and dltB, as well as dltC and dltD, overlap by either 1 or 4 bp. Overlapping stop and start codons in the two pairs of genes are characteristic of most dlt operons examined and, thus, may be the basis for the mechanism of translational coupling that coordinates the expression of these functionally related proteins (375). In addition to dltABCD, Glaser et al. (186) identified a fifth gene (dltE) encoding an oxidoreductase in B. subtilis (Fig. 9). However, inactivation of dltE did not inhibit D-alanylation (383). It has not been established whether a fifth gene encoding an acetyl hydrolase is required for D-alanylation in Streptococcus pyogenes. From these studies and the position of the rho-independent terminator, it was concluded that dlt contains a minimum of four genes encoding the machinery required for D-alanylation.
The dlt operon was also identified in the genome of S. pneumoniae R6 (44). Since this species contains phosphorylcholine esters instead of D-alanyl esters in both LTA and WTA (164), the observation was unexpected. The organization of the operon in this organism is identical to that described above. Gene expression studies indicated that mRNAs of each dlt gene are synthesized under normal laboratory growth conditions (228). Whether there is an additional constituent of the envelope that is D-alanylated remains to be established.
Our understanding of the regulatory elements that control the expression of the dlt operon is fragmentary No single regulatory paradigm has been found that can be applied to the expression of all dlt operons. Apparently, the multiplicity of elements reflects the fact that individual species are adapted to different growth requirements, stresses, and habitats. Nevertheless, a comparison of these elements has provided some insights into the regulation of dlt.
The dlt operon in B. subtilis is part of the x regulon (213). x-dependent promoters precede a variety of genes that affect the composition or metabolism of the cell envelope. In addition to the PX promoter, another regulatory sequence that controls its expression is the global regulator SpoOA, targeted to a DNA-binding recognition sequence, the "OA box," located downstream of the x-dependent promoter (383). In addition to control by SpoOA, AbrB functions in the temporal regulation of dlt transcription. This complex regulatory system reflects, in part, the sporulation capability of this organism and the fact that spore LTA contains no detectable D-alanyl ester. In contrast, the L. rhamnosus dlt operon contains a single putative promoter region (-10 and -35 with a 20-bp spacer) similar to those reported for Lactobacillus species (86, 209).
Poyart et al. (405) discovered two regulatory genes, dltR and dltS, upstream of the dlt operon in S. agalactiae (Fig. 9). These encode putative regulatory and sensor proteins of a two-component regulatory system.
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