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N. F. Gamaleya Institute for Epidemiology and Microbiology,1 N. D. Zelinsky Institute of Organic Chemistry, Moscow, Russia,3 Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Borstel, Germany2
Received 31 March 2004/ Accepted 9 August 2004
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ABSTRACT |
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INTRODUCTION |
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Staphylococci are gram-positive bacteria, and their cell walls are composed of murein (32, 38, 41), teichoic acids (2), and wall-associated surface proteins (20, 26, 30). Stress-bearing murein represents a continuous macromolecular sacculus covering the whole cell. Murein consists of glycan strands, which are cross-linked by peptide bridges furnishing the structural integrity of the sacculus. It is a distinctive feature of staphylococci that the observed degree of murein cross-linking, which was determined as a ratio of bridged peptides to the total amount of all peptide ends in general, is extremely high, on the order of 80 to 90% (16, 35).
Glycan strands in staphylococcal murein are composed of N-acetylglucosamine (GlcpNAc) and N-acetylmuramic acid (MurpNAc) residues that furnish ß-(1
4)-linked disaccharide repeating units, with MurpNAc representing the reducing terminus of the chain. The carboxyl group of each MurpNAc residue is amidated by the stem pentapeptide L-Ala-D-iso-Gln-L-Lys-D-Ala-D-Ala, and the 
-amino group of the lysine residue is substituted with a pentaglycine appendage (37). Thus, each peptide attached to a MurpNAc residue is a branched decapeptide with an amino group on the Gly and a carboxyl group on the D-Ala terminus, and arms of the peptide side chains interact with each other to provide a high degree of murein cross-linking.
Although the general principle of murein structural organization is simple, the muropeptide composition of the staphylococcal sacculi appears very complex, as a standard digestion of sacculi with muramidase (the enzyme that cleaves MurpNAc glycosidic bonds) releases more than 20 distinct components plus an unresolved material. The latter makes up about 50 to 60% of the muropeptide-containing oligomers, with up to 20 repeating units (7, 38). Thus, the major part of staphylococcal murein architecture could be envisaged as being constructed of interlinked glycan and oligopeptide chains, both varying in their lengths.
On electron micrographs, the cell wall of staphylococci appears as a thick (about 20- to 40-nm-thick) homogenous slab (4, 18), and it is not possible to resolve the actual orientation of the glycan strands within the cell wall. Two published models proposed that the glycan strands were arranged in shell-like parallel layers around the plasma membrane. While the first model (37, 41) accounted for a high degree of cross-linking, it was discounted because it is incompatible with the known helical conformation of glycan strands. The second model ascribed to murein an unusual "plywood"-type architecture with consecutive turns of each glycan layer by 60° (21, 22). This type of architecture is difficult to reconcile with the centripetal closure of the septum during cell division.
Based on computer simulation studies, we recently developed a new structural principle of the bacterial murein architecture. The scaffold model considers the cell wall fabrics as a solid gel-like matrix deprived of glycan layers as principal elements of the wall architecture, the glycan chains being oriented perpendicularly to the plasma membrane, and permits a graphic representation of gram-negative bacterial cell walls (11). We have now adapted this model to the specific requirements of the gram-positive cell wall of S. aureus. This tertiary model of the staphylococcal murein is compatible with a high degree of cross-linking and, at the same time, facilitates a structural understanding of essential phenomena of staphylococcal morphogenesis such as consecutive division plane alteration and centripetal septum closure.
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MATERIALS AND METHODS |
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Simulation of the conformation and graphical representation of the major repeating unit of the S. aureus cell wall. The computer-simulated conformation of the repeating fragment composed of the disaccharide GlcpNAc-(ß1-4)-MurpNAc substituted with the branched decapeptide L-Ala-D-iso-Gln-L-Lys(Gly5)-D-Ala-D-Ala was optimized in an MM3-1996 force field by using molecular mechanics calculations (PC Model, version 7; Serena Software, Bloomington, Ind.). As the strands represent extended helices with the symmetry order N = +4 (6), they were approximated as relatively long cylinders with peptide substituents projected outward from each disaccharide unit, and with stem peptides perpendicular to each other. The bridges connecting the adjacent peptide arms were approximated as straight lines with the length of the -(Gly)5-D-Ala- cross-linking fragment, and both interacting peptides retained their antenna-like configuration. All graphical approximations presented in this paper were exclusively made to scale, thus preserving the basic atomic parameters.
Primary conformational analysis of muropeptides, the building blocks of murein. To estimate the conformations of oligomuropeptides usually obtained from staphylococcal sacculi by a standard digestion with muramidase, it is sufficient to consider just the conformation of the tetrameric muropeptide. Since the latter could be regarded as two dimers concatenated via their short and long antenna's arms, respectively, two situations were analyzed as follows. First, each pair of disaccharide units involved in bridging was in a "vis-à-vis" position, i.e., in the vicinity of, and at the same distance from, the plasma membrane, with appropriate peptide arms pointing at each other, while both peptide antennas were in a plane perpendicular to the axis of a disaccharide cylinder. Second, the arms of the peptide antennas were oriented alongside the glycan strands to make the positions of bridged disaccharide pairs shifted in a "ladder"-type fashion along the glycan cylinders. In this type of bridged disaccharide location, the antenna's arms appear in the same plane as the axis of the glycan cylinder.
Simulation of the murein tertiary structure. The algorithm used for murein simulation and the appropriate mathematical apparatus have been described in detail previously (11). In brief, the methodological principles are as follows:
(i) For simulation of murein assembly, chain length distributions expressed in terms of strand numbers (NL) with particular chain lengths (L) are necessary. The observed experimental data on the chain length distribution pattern in the cell wall of S. aureus were published in terms of relative amounts, expressed as percentages (5). The recalculation of the literature data on the relative mass content of the glycan strands to obtain strand numbers was achieved with equation 1:
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(ii) To build up a murein matrix, a square piece of the fabric possessing 22 by 22 strand positions was virtually cut off from the sacculus. The square hole thus obtained possessed 484 positions for the glycan strands and was flanked along the perimeter by the existing fabric to allow incoming strands to cross-link with it, thus making the simulated process in agreement with murein assembly in vivo (16, 39). The maximal height of the matrix was considered to be about 45 disaccharides in order to guarantee the true thickness of the S. aureus cell walls observed in experiments omitting the procedures of fixation and staining (12). To mend the punctured fabric, peptidoglycan helices were randomly selected from the statistical pool and sequentially inserted into the square hole (MurpNAc termini pointing to the membrane) to occupy all 484 positions. Consequently, each disaccharide unit occupied a definite spatial location at a given distance from the membrane. We called these distances "levels, " and the total number of levels within the box was 45. All the strands were inserted into the matrix vertically. Each disaccharide-peptide unit within the matrix was located at a definite height and corresponded to one particular stem peptide level. Further, each position in the matrix was occupied by at least one strand and could also be occupied by additional, shorter strands located above it. As a result, the total number of strands inserted into the matrix exceeded 484.
The distribution of strands in the matrix (probability P as a function of strand length i) was calculated by equation 2, as follows:
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(iii) To achieve a maximal degree of cross-linking within the simulated murein, peptide side chains were oriented along the inserted glycan strands to run in parallel with their axes; the long pentaglycine arms were oriented downward, and the short alanine arms were oriented upward.
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RESULTS |
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Experimentally, the muropeptide fraction represents a complex mixture of branched oligomers of different lengths with up to 20 repeating units; monomers and dimers are almost absent (7, 38). It should be noted that oligomers (at least even-numbered components) comprise chains composed of cross-linked dimers. Therefore, to achieve a general appreciation of the oligomeric muropeptide conformation, it is sufficient to carry out conformational analysis for a tetrameric muropeptide, which consists of two interconnected dimers. To guarantee the formation of a tetramer, the basic murein structure needs only to allow four monomeric muropeptides to be close enough to be cross-linked by two dimeric bridges. If cross-linked disaccharide units are located within the basic murein structure in a "vis-à-vis" manner, i.e., in agreement with the published scaffold model (11), the formation of the tetramer requires that two of four cross-linked disaccharides be arranged in one tetrasaccharide glycan block (the sequence to be split by muramidase) located within one strand while two other monomeric muropeptides belong to different strands (Fig. 2a); the minimal number of strands necessary to accomplish this is three. Thus, within the "vis-à-vis" murein structure, the two bridges are perpendicular to each other but appear in distinct planes about 1 nm (i.e., 1 disaccharide unit) apart. To make a tetrameric muropeptide, the short peptide arm of one bridge must reach the long arm of the other bridge while the two bridges are positioned in separate planes and at a rather long distance. A conventional "vis-à-vis" connection of cross-linked disaccharide pairs, therefore, seems unlikely, because it would necessitate a strong conformational deformation of the structure.
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This requires a quarter-turn rotation of a peptide around its stem, but this conformational transition needs little energy and does not introduce any strain into the structure, as indicated by the conformational analysis of the disaccharide-peptide unit. We conclude that within the murein architecture, the major peptide chains of the oligomeric muropeptides can adopt an undistorted zigzag conformation and may run alongside the glycan strands in a plane roughly perpendicular to the plasma membrane, interconnecting them in a zipper-like manner with a high degree of cross-linking.
Graphic representation of the staphylococcal murein architecture. Having discovered a clue to the possible orientation of the oligopeptide bridges within the murein matrix, we applied the refined principle of the scaffold model (10, 11) to construct the S. aureus murein architecture and oriented both glycan strands and extended oligopeptide chains in a plane perpendicular to the surface of the plasma membrane. Here, the short alanine and the long pentaglycine arms went upwards and downwards, respectively, whereas the MurpNAc termini of the glycan strands pointed to the plasma membrane (Fig. 3).
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Three-dimensional model of staphylococcal murein. To obtain an accurate three dimensional view of the proposed murein architecture, we simulated its assembly (i) by using the experimental chain length distribution (5) and (ii) by following the principle of staphylococcal murein assembly known as "restricted monomer addition" (16). To perform the simulation, three major assumptions were made as follows. First, all strands within the murein of S. aureus are oriented perpendicularly to the plasma membrane. Second, because both the biosynthesis of precursors and concomitant murein assembly proceed in association with the plasma membrane (39), the assumption was made that the MurpNAc termini of the glycan chains point to the plasma membrane while the GlcpNAc termini are oriented outward. Third, since the dynamics of murein biogenesis simultaneously comprises the assembly and turnover processes, and the latter makes the outer surface of the wall appear worn out (18), it was assumed that degraded (short) strands tend to accumulate in the outer part of the sacculus, while those that are not yet split penetrate through the wall. It bears mentioning that the experimentally determined, predominant length of the glycan chains in S. aureus is 3 to 10 disaccharide units and that an average chain length is about 6 disaccharides (5). Therefore, we did not consider chains longer than 30 disaccharide units. The program was adjusted to obtain a maximal degree of possible cross-linking, and consequently, peptide side chains were oriented along the glycan strands to run in parallel with them. Because the nascent strands emerge from the plasma membrane and then attach by their short arms (alanine donor) to the long arm (pentaglycine acceptor) of the preexisting murein (39), the long peptide arms were oriented downward.
The simulation of the murein architecture was started with the assembly of the material that had not yet undergone degradation and thus was free of very short chains. For this purpose, chains with 4 to 30 disaccharide units were selected. A certain number of strands were modeled as still attached to the membrane, while the rest were positioned as already detached. Thus, the strands could become closely interwoven to produce a maximally cross-linked material in proximity to the membrane. Evidently, a certain number of long chain ends protruded upwards from the primary matrix without being cross-linked. The remaining short strands were subsequently inserted into the box above the primary matrix so that the distance between chain ends was 1 or 2 disaccharide units. Synchronously with the strand insertion, all possible cross-links were initiated between adjacent peptides. When the arms of antenna peptides were oriented along the glycan chain, each peptide could interact with two neighbors (one from above and the other from below), thus achieving a high degree of cross-linking between the chains. During the assembly process, shorter chains tended to accumulate in the periphery, corresponding to the structure of material digested by lytic enzymes. The simulated murein matrix had 83% cross-linking, while the published degree of cross-linking for the purified cell wall preparation was 80% (16). When the distance between the glycan chain ends was programmed to be 1 disaccharide unit, the degree of murein cross-linking, as a consequence, was raised to 90%.
The overall visualization of the simulated murein architecture is presented in Fig. 4. The thick fragment of the murein matrix (Fig. 4a) is almost impermeable and produces the impression of a tightly interwoven thicket, which very much resembles the ultrastructure observed on electron micrographs (4, 18). Evidently, there is an immense inconvenience in any two-dimensional representation of the cross-linked murein architecture due to two intrinsic features of the staphylococcal cell wall: its considerable thickness and the high degree of cross-linking. Therefore, to demonstrate the details of the architecture, we selected a rather thin and small fragment from the upper part of the simulated murein matrix for enlargement (Fig. 4b). The porous structure, oligopeptide zigzags, and distinct glycan chains of different lengths can be seen clearly. A stereo view of the selected murein fragment is presented in Fig. 4c.
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DISCUSSION |
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The refined scaffold model of peptidoglycan architecture, when applied to the gram-positive wall of S. aureus, has three distinct advantages over previously discussed concepts: (i) it can account for the extremely high degree of cross-linking (80 to 90%), (ii) it is compatible with the phenomenon of consecutive division plane alternation, and (iii) it can explain appositional growth during centripetal septum closure. No other published structural views of the cell wall architecture can simultaneously accommodate all of these experimentally confirmed biological facts about S. aureus (for examples, see the most recently published figures in references 13 and 38).
Four decades ago it was presumed that glycan chains possess a chitin-like conformation and run parallel to the plasma membrane as well as to each other, thus producing a set of glycan planes. Beneath each plane and at right angles to the glycan chains, there was a plane of peptide chains that were cross-linked at considerable distances; the whole structure made a covalently closed sphere joined by glycan chains in one direction and by oligopeptide chains at 90o to the glycan chains (29, 32, 36, 37, 41). This model was discounted by X-ray diffraction data (6, 21), which proved that glycan strands do not possess a straight chitin-like conformation but represent right-handed helices with 4 disaccharides units per turn. Although there are no conditions under which helices can be introduced into the proposed type of architecture, the "chitin" model is surprisingly present even in today's microbiology textbooks.
In an alternative model, peptidoglycan strands were approximated as long cylinders (21, 22) or stretched ropes (23) around which a continuous and narrow spiral, imitating the discrete peptide substituents, was wound. The approximated strands were arranged parallel to the plasma membrane and to each other to simulate a peptidoglycan layer. To achieve a maximal degree of cross-linking, the layers were proposed to pile one over the other with a 60° turn of each consecutive layer relative to its neighbor. In this way, the murein architecture resembled a plywood-layered structure (21). For its assembly, this model apparently demands strong conformational distortions of the oligopeptide chains to allow consecutive turns of the layers and to accomplish a high degree of cross-linking.
The scaffold model, in contrast, readily guarantees 80 to 90% cross-linking in accordance with published experimental data (16, 35) and implies that all the structural elements of murein architecture are unstressed and adopt energetically minimized conformations of helices and zigzags.
Staphylococci divide in three successively different planes. Each new plane changes its direction at an angle of 90o to the preceding division plane (18, 27, 38), but conserved patches of old wall sectors still exist during division, as visualized recently in spherical cells of Escherichia coli (8). By the principle of glycan strand arrangement proposed by the "chitin" model, the consequences of this phenomenon for the cell wall would be those presented in Fig. 5.
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How do the different models of the staphylococcal murein architecture account for the process of cell wall morphogenesis, as it has been revealed both by advanced electron microscopy (1, 4, 18) and by recent biochemical studies (38, 39)? The spherical walls of staphylococci seem to grow mostly via a zonal mechanism of cross-wall assembly. During cell division there is a clearly exposed leading edge, and septum growth proceeds strictly centripetally, like the iris diaphragm of a camera, from the peripheral edges to the center (1). The septum consists of two newly born walls with a "splitting system" sandwiched in between; each newly born wall represents the primary wall of the daughter cell in its pure form. The ongoing secondary process known as wall "thickening" accompanies the primary wall assembly, and when the septum is completed, the maternal wall becomes cleaved and skinned off along the "stripping" layer to allow daughter cells to be separated (18). The process is repeated in the next cell cycle, with the only distinction that the next division plane is turned by 90°.
The proposed scaffold model is in agreement with the cell wall morphogenesis described above because it implies the appositional attachment of the nascent peptidoglycan strands to the centripetally moving leading edge of the closing septum. Enzymatic cutting of the inner maternal wall into two hemispheres produces two bare leading edges, and their contact with the plasma membrane is initiated by invagination of the cell membrane, thus allowing the nascent strands to interact with the exposed edges of the growing walls. This is possible only if the glycan strands are oriented perpendicularly to the plasma membrane, because any horizontal attachment of nascent strands would be in conflict with the phenomenon of division plane alternation.
As mentioned above, staphylococci possess on their surfaces type-specific teichoic acid (2) and a set of proteins (20), all of which are covalently attached to murein. Teichoic acid (19) and surface-associated proteins (26, 30) are first synthesized in vivo in the form of the corresponding biosynthetic precursors, which are closely related to the precursors of murein biosynthesis (39). These precursors could concomitantly participate in cell wall assembly, i.e., teichoic acid and surface-associated proteins could become attached to murein in the course of its assembly. The scaffold model is in accord with such molecular mechanisms of cell wall assembly because both the teichoic acid and surface protein intermediates could be readily inserted into the nascent peptidoglycan chain during its attachment to the edge of the growing septum. The involvement of all precursors in a zonal process of murein assembly provides teichoic acid and surface proteins with a unique opportunity to reach the bacterial surface directly.
Although the "chitin" model may theoretically account for the centripetal mechanism of staphylococcal cell wall growth, other shortcomings mentioned above have rendered this model obsolete. The "plywood" model seems to contradict the centripetal leading-edge growth of the closing septum. The growing septum is a round plate with a hole in the center, and the movement of the edge of a constricting hole (zone of growth) has to be coherent with that of the constricting membrane, both being strictly centripetal (1, 18). This implies that the orientation of all glycan strands in all layers (if those exist in bacterial murein) of the septum has to correlate unvaryingly with the centripetal edge movement; otherwise the septum will never be closed. In contrast, the plywood model states that glycan strands diverge their orientation in consecutive layers and therefore, by definition, concurrently run both centripetally and centrifugally. In conclusion, only the advanced model described here adequately reflects the biological and physicochemical requirements for the assembly of highly cross-linked murein within the cell wall of S. aureus.
Finally, it is pertinent to consider the relationship between the scaffold-like model originally proposed by us for gram-negative cell walls (11) and that described here for the highly cross-linked cell wall of S. aureus. It is evident that the previous model does not account for a high degree of murein cross-linking, a characteristic feature of gram-positive walls, and therefore cannot serve as a general model. The question arises whether the advanced scaffold model is universally applicable to all types of bacterial walls or whether it applies only in the special situation of gram-positive walls.
With the Gram staining procedure, all bacteria described so far can be divided into three large classes, i.e., positive, negative and indeterminate, while according to the chemical classification (33) of murein structures described thus far, there are about 20 types of murein. Actually, the number of murein types is larger, because particular chemotypes were not included in the list and an additional type of architecture comprising the subsequently discovered form of cross-linking via Dap-Dap bridges was not considered. Despite this complicated situation, it can be stated that the walls of all gram-negative bacteria belong to type A1 and possess the simplest murein structures (33). Here, glycan strands are loosely cross-linked by ordinary bridges, and oligomeric peptide fragments are practically absent. The tertiary structures are therefore similar and can be adequately presented in their entirety by the previously published scaffold model (11). Indeed, even if the advanced scaffold model with a shifted "ladder-type" location of the cross-linked disaccharide units is applied to gram-negative walls, the tensile forces that act to expand the wall material will inevitably revert the construction to the "vis-à-vis" tertiary structure with ordinary peptide bridges oriented parallel to the plasma membrane.
As to the cell walls of gram-positive bacteria, they exhibit a wide diversity from simple to very complex structures with variably high degrees of cross-linking. Staphylococcal cell walls have a rather extraordinary type of architecture, belonging to the most highly cross-linked type; only the advanced scaffold model adequately describes this specific feature. The walls of other gram-positive bacteria exhibit a much lower degree of cross-linking, and the muropeptide fraction of these walls does not contain long oligomeric chains. For example, the walls of bacilli exhibit about 50 to 55% cross-linking, and the major component of the oligopeptide fraction is trimeric and tetrameric muropeptides (17). Furthermore, the arm lengths of the peptide antennas in the walls of other gram-positive bacteria are not as long as those in staphylococci. However, it is likely that the same major principle, i.e., that both glycan and oligopeptide chains tend to run in a plane perpendicular to the plasma membrane, holds for the architecture of all gram-positive walls. As these walls contain sufficient amounts of unbridged disaccharide units along with dimeric and trimeric bridges, their architecture might be of a mixed type, exhibiting the tertiary structural elements of both the basic and advanced scaffold models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Supplemental material for this article may be found at http://jb.asm.org/.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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