The murein [peptidoglycan] sacculus is the essential exoskeletonof all eubacteria [except Mycoplasma species and a few other species] that is needed to withstand the internal cytoplasmicturgor [osmotic] pressure [60, 78] . Murein consists of oligo[GlcNAc-MurNAc]glycan strands that are cross-linked by short peptides and thusform a net-like polymeric structure that surrounds the cytoplasmicmembrane [78] . The sacculus of Escherichia coli is one giantmacromolecule with a molecular mass of more than 3 x 10⁹ Da,which is in the same range as the molecular mass of the chromosomeof this bacterium [2.32 x 10⁹ Da] . Moreover, the sacculus isembedded in the cell envelope by virtue of its location in theperiplasm of gram-negative bacteria . It carries approximately10⁵ molecules of covalently bound lipoprotein [Lpp, Braun'slipoprotein] that links the outer membrane to the murein [6].It has been assumed that the murein glycans and peptides arearranged parallel to the membrane, forming a thin layer in gram-negativespecies and a thick multilayer structure in gram-positive species.This concept was challenged recently by Dmitriev et al . [20, 21], who proposed the scaffold model, in which the murein glycanstrands extend perpendicular to the cytoplasmic membrane . In this communication we first review relevant data on gram-negative murein structure and biosynthesis that were obtained over the past few decades in many laboratories, most of which were obtained from studies of E . coli . Then we discuss these findings with respect to different structural models of the murein sacculus.

 
Size of the murein sacculus. The murein was isolated from gram-negative bacteria by boilingthe cells in a sodium dodecyl sulfate [SDS] solution, followedby purification by enzymatic removal of glycogen and proteins[26, 56, 78] . As visualized by electron microscopy, the purified murein sacculi are bag-shaped structures with the dimensions and form of the bacteria from which they were isolated [Table 1] . Like the rod-shaped cells, the sacculi of E . coli consistof a cylindrical part that is closed by two polar hemisphericalregions . Compared to the length [about 2 to 4 µm] and the diameter [about 0.5 to 1 µm] of the sacculi, the mureinis very thin, which results in the observed appearance of anempty and sometimes crumpled envelope laying flat on a gridused for electron microscopy [18, 22, 78].

 
Thickness of murein. Three methods have been used to measure the thickness of themurein of E . coli: electron microscopy, neutron scattering,and atomic force microscopy . The results obtained by electronmicroscopy were different when different techniques were used[4] . After successive fixation of cells with glutaraldehyde,osmium, and uranyl acetate, followed by dehydration with ethanoland embedding in araldite, De Petris observed a multilayer architecturefor the cell envelope [19] . One layer [the g₂ layer] disappearedcompletely upon treatment with the murein hydrolase lysozymeand was therefore identified as the murein layer . The g₂ layerappeared to be 1.5 to 3 nm thick, whereas isolated murein sacculithat were obtained by boiling cells in an SDS solution and werepurified by treatment with amylase and protease were 1 to 1.2nm thick . Another study revealed a similar thickness [2 to 3nm] for the murein layer in the envelope of E . coli that disappearedafter lysozyme-EDTA treatment [61] . However, these findings were questioned by a study of Hobot et al . [31], in which differentsample preparation techniques were used . For example, glutaraldehyde-fixedcells did not contain a typical murein layer after low-temperaturedehydration, and the authors did not identify a central linethat appeared after staining with uranyl acetate as the mureinlayer . Because of the high water content of isolated murein, Hobot et al . proposed the concept of a periplasmic gel, in which the murein is distributed throughout the periplasm and is more cross-linked near the outer membrane and less cross-linked nearthe cytoplasmic membrane . Phosphotungstic acid [PTA] stainingwas used, which is specific for carbohydrate compounds in amutant lacking periplasmic membrane-derived oligosaccharides.PTA stained the whole space between the inner and outer membranes,which was about 15 nm thick . However, isolated murein sacculithat were obtained from exponentially growing cells and werestained with PTA were thinner [8.8 ± 1.8 nm], and ifisolated sacculi were purified further by treatment with proteaseto release the bound lipoprotein, the thickness after stainingwith PTA decreased to 6.6 ± 1.4 nm [53] . The murein ofcells in the stationary phase was thicker, 10.7 ± 1.1 nm if the cells were grown in rich medium and 9.9 ± 1.3nm if the cells were grown in minimal medium . Employing PTAstaining during autolysis of E . coli led to similar results.However, the fact that a thickness for regularly structuredmurein of about 15 nm [the thickness of the PTA-stained layer]contradicts a number of other experimental data, including themeasured amount of murein per cell, was discussed by Leduc etal . [54] . It was pointed out in two publications that thicknessmeasurements resulting from electron microscopy images shouldbe viewed with caution . First, the thickness of stainable materialin the periplasm is not equivalent to the thickness of murein[79], and second, the measurement indicates the thickness ofthe contrasting metal and not of the murein itself [19] . Recently, cryotransmission electron microscopy was used to visualize the envelope structure of gram-negative bacteria [58] . This techniqueinvolves neither chemical fixation nor staining procedures andis therefore believed to produce fewer artifacts . Murein was visualized by cryotransmission electron microscopy in the periplasm of E . coli K-12 as a thin line below the outer membrane with a thickness of 6.35 ± 0.53 nm [58].

The thickness of isolated murein sacculi was also directly measured by small-angle neutron scattering [52] . The advantages of thismethod were that the sacculi were fully hydrated, they containedno bound lipoprotein, and no staining procedure was used . Thepurified sacculi of exponentially growing E . coli W7 were notuniform in thickness . It was found that 75 to 80% of the surface was 2.5 nm thick and the remaining 20 to 25% was about 7 nm thick . An important finding was that the maximum thickness ofthe sacculi did not exceed 7 nm . Atomic force microscopy allowed determination of the thickness of nonhydrated murein and hydrated murein [81] . As determined by this method, nonhydrated murein of E . coli was 3 nm thick, whereas hydrated murein was 6 nm thick.

Elasticity of the sacculus. The murein net is quite elastic and can reversibly expand andshrink, and this property is mainly due to the flexibility ofthe peptide part [2], whereas the glycan strands are ratherrigid [48] . This flexibility was demonstrated in filamentouscells of strain TOE13, which contain a temperature-sensitiveftsA allele . If the osmotic pressure of filaments of this strainwas suddenly removed by destruction of the cytoplasmic membrane,the length of the filaments decreased by 17%, and it was calculatedthat the surface area of the murein in vivo was 45% greaterthan that in the relaxed state [46] . The elasticity of isolatedmurein sacculi was also estimated by measuring the surface areaby low-angle laser light scattering under different conditions[47] . It was concluded that the surface area of the sacculicould reversibly increase threefold without rupture . Furthermore,atomic force microscopy revealed that sacculi are two- to threefoldmore deformable in the direction of the long axis [elastic modulus,1.5 x 10⁷ to 3 x 10⁷ N/m²; average, 2.5 x 10⁷ N/m²] than inthe direction perpendicular to the long axis [elastic modulus,3.5 x 10⁷ to 6 x 10⁷ N/m²; average, 4.5 x 10⁷ N/m²] [81] . Theelastic modulus is lower for material with greater elasticity. It was suggested that the observed anisotropy in elasticitywas the consequence of the alignment of the murein glycan strandsmainly perpendicular to the long axis of the cell.

Porosity of the murein meshwork. Demchick and Koch determined the size of fluorescently labeleddextrans that could penetrate isolated murein sacculi of E.coli ATCC 11775 . They estimated that the pores in the mureinhad a mean radius of 2.06 nm, which would theoretically allowthe penetration of a globular protein with a molecular massof 22 to 24 kDa [17] . Because in the living cell the mureinis under tension, it was roughly estimated that in vivo thestretched murein may allow free diffusion of globular proteinswith a maximum molecular mass of 50 kDa . In another study itwas demonstrated that EDTA treatment of E . coli cells in combinationwith a hyperosmotic shock released a subset of cytoplasmic proteinsthat were almost identical to the proteins that are able topass through a 100-kDa-cutoff filter [74] . It was speculatedthat this limitation was caused by the molecular sieving propertyof the murein sacculus that was impermeable for proteins withmolecular masses of more than 100 kDa under the osmotic shockconditions.

Amount of murein per cell. The amount of murein per cell was estimated by determining theamount of m-diaminopimelic acid [m-Dap], an amino acid thatis present exclusively in murein . In early work two methodswere used, chemical determination and determination via incorporationof radioactive m-Dap, and these methods gave essentially thesame results [2.7 x 10⁶ m-Dap molecules per cell for strainsHfrH and W945/3282] [7] . Using both methods, workers in a later study determined that the amount was 3.5 x 10⁶ molecules ofm-Dap per cell for strain MC4100 lysA [79] . The surface areaof the same cells was measured by using electron micrographsand was found to be 8.3 or 8.9 µm², indicating that theaverage surface area per disaccharide unit was 2.5 nm² [79]. In a third study with E . coli B/r H266 grown in a variety of different media, an average surface area of 1.6 to 1.8 nm² per disaccharide unit was determined, and this value was found to be nearly constant at different growth rates ranging from 0.40to 2.93 doublings/h [82] . In the m-Dap auxotrophic strain MC6RP3,the amount of murein as measured by incorporation of radioactivem-Dap into SDS-insoluble material could be reduced by 50% withoutany growth defect if the external concentration of m-Dap wasdecreased from 40 to 1.4 µg/ml [68].

Chemical composition of murein. Murein is composed of glycan strands of alternating ß-1,4-linkedGlcNAc-ß-1,4-MurNAc disaccharides that are cross-linkedby short peptides [72] . In E . coli and other gram-negative bacteria,the MurNAc residue at the end of the strand has a 1,6-anhydromodification [34, 69] . The stem peptides that are linked tothe lactyl group of MurNAc consist of two to five amino acids,and the sequence of the pentapeptide is as follows: L-Ala-D-Glu-[]-m-Dap-D-Ala-D-Ala. Most cross-links are formed between the D-Ala at position 4of one stem peptide and m-Dap at position 3 of a second stempeptide of a neighboring glycan strand [DD cross-links], butthere is also a small percentage of cross-links between twom-Dap residues [LD cross-links] . Besides such dimeric cross-links,there is a smaller fraction of trimeric and tetrameric cross-linkedstructures [26].

Length of the murein glycan strands. One GlcNAc-MurNAc disaccharide in the murein is the same lengthas a GlcNAc-GlcNAc disaccharide in crystalline -chitin, namely,1.03 nm [7, 12, 13, 48] . The average degree of oligomerizationof the murein glycan strands of E . coli can be determined intwo different ways: first, by analyzing the length distributionof isolated glycans and second, by quantification of the 1,6-anhydro-MurNAc-containingmuropeptides that are a hallmark of one of the ends of the glycans.Murein glycan strands can be released from isolated murein bytreatment with an amidase from human serum . The glycans canbe purified by cation-exchange chromatography, and the lengthdistribution can be analyzed by C₁₈ reversed-phase chromatography[30] . However, the standard method allows only separation ofglycans that are not longer than 30 disaccharide units . Theaverage length of the glycans containing from 1 to 30 disaccharideunits was 8.9 disaccharide units in strain W7 . Glycans thatare longer than 30 disaccharide units eluted together in onepeak . These long glycans represented about 25 to 30% of thetotal material, and their average length was 45.1 disaccharideunits . The average length of all glycans was estimated to be21 disaccharide units by this method [30] . If the chromatographywas prolonged, a regular pattern of peaks corresponding to glycanswith up to 80 disaccharide units could be resolved [Y . Chenand J.-V . Höltje, unpublished].

The glycan strands in the E . coli murein do not contain a reducing end but do contain a 1,6-anhydro-MurNAc moiety . Therefore, digestion of murein with a muramidase, such as lysozyme or Cellosyl, yields a fraction of muropeptides with 1,6-anhydro-MurNAc representing muropeptides from the end of the glycan strands . Muropeptidescan be separated by reversed-phase high-performance liquid chromatography[26], and the proportion of muropeptides containing 1,6-anhydromuropeptides was shown to depend on the strain and on the growth conditionsand ranged from 3 to 6% of the total muropeptides . For example,strain KN126 from an exponential-phase culture in Luria-Bertanimedium contained 3.88% 1,6-anhydromuropeptides, indicating thatthe average length of the murein glycan strands was 25.8 disaccharideunits . In murein of cells from a stationary-phase culture theaverage length was 17.8 disaccharide units . In cells grown inPB medium the average length was greater, 33.3 disaccharideunits at 30°C and 37.9 disaccharide units at 42°C . Asmentioned above, strain MC6RP3 produced only 50% of the mureinif it was grown in medium containing a low level of m-Dap [68].When cells were grown with 1.4 µg of m-Dap per ml, theaverage length of the murein glycan strands was 24.4 disaccharideunits [corresponding to 4.1% of the 1,6-anhydromuropeptides],which was only slightly shorter than the average length of 28.6disaccharide units [3.5% of the 1,6-anhydromuropeptides] forcells grown with 40 µg of m-Dap per ml, which containedtwice as much murein.

Murein as an ordered structure? The murein glycan strands are similar in chemical structure[and also function] to polysaccharides like chitin and cellulose.Chitin is a crystalline polymer consisting of chains of ß-1,4-linkedGlcNAc residues . By using different physical chemical methods,like X-ray diffraction, infrared spectroscopy, nuclear magneticresonance, and immersion refractometry, it became clear thatmurein differs fundamentally from chitin in that it is not crystalline[1, 8, 9, 35, 48, 49, 51, 57, 62] . The reason for the noncrystallinestate of murein might be the high flexibility and elasticitythat is intrinsic to the peptide part [see above] . On the otherhand, the lack of a crystalline structure does not exclude thepossibility that murein might still be, on average, highly ordered[73].

Orientation of the glycan strands in the sacculus. Sacculi were treated either with an endopeptidase [MepA] thatcleaves both DD- and LD-peptide cross-links [76] or mechanicallyby sonication [75] and examined by electron microscopy . Thepartially digested or fragmented sacculi contained oblong gapsthat were oriented predominantly in the direction perpendicularto the long axis . In contrast, digestion with muramidases [Slt70or lysozyme] did not result in gaps with a preferential orientation.Assuming a layered murein architecture, Verwer et al . concludedthat the material resistant to endopeptidase digestion [namely,the glycan strands] is oriented predominantly perpendicularto the long axis of the cell [76] . However, the results forthe appearance of muramidase-treated sacculi are conflicting.It was found recently by de Pedro et al . that the digestionof murein sacculi with the muramidase Cellosyl produced orientedoblong gaps [18] similar to those seen after endopeptidase treatment[76] . Therefore, it might not be possible to determine the orientationof the murein glycan strands by analyzing electron microscopicimages of partially digested murein [42].

Cross-linkage of murein. A cross-linking [transpeptidation] reaction occurs between thecarboxyl group of a donor peptide and a free amino group ofan acceptor peptide such that the energy for the formation ofthe new peptide bond is generated by the release of the D-alanineat position 5 of the donor peptide . About 50% of the peptidesin the murein of E . coli are part of cross-linked [dimeric,trimeric, or tetrameric] muropeptides . For example, the mureinof exponentially grown strain KN126 contains 51.51% free [non-cross-linkedor monomeric] peptides, 43.27% peptides in dimeric structures,5.02% peptides in trimeric structures, and 0.19% peptides intetrameric [cross-linked] structures [26] . Glauner et al . definedthe degree of cross-linkage as follows: degree of cross-linkage= 100 x [1/2 dimers + 2/3 trimers + 3/4 tetramers]/all muropeptides.By this definition, the degree of cross-linkage is equal tothe molar percentage of peptides that functioned as donors incross-linking reactions and is not the same parameter as [andis much smaller than] the molar percentage of cross-linked peptides,a number that is given frequently in publications . Thus, KN126has a degree of cross-linkage of 25.12%, and 48.48% of all peptideside chains are part of cross-linked structures . The degreesof cross-linkage are similar for different strains and mutantsof E . coli, and analysis of the mureins of nine other gram-negativespecies revealed that in all cases more than 40% of the peptideswere part of cross-links [69] . Only in E . coli cells grown inthe presence of D-amino acids was the cross-linkage dramaticallyreduced to a value of 34% of cross-linked peptides [11] . Pulse experiments showed that newly incorporated material had a lower degree of cross-linkage [16% after a 20-s pulse], but this value increased to the normal value, 25.1%, after a 150-s pulse [25]. The degree of cross-linkage decreased to 23.9% during a 90-min chase . The murein glycan strands have two ends, and it was shownthat not only the 1,6-anhydro-MurNAc end of the glycans [26] but also the GlcNAc ends [71] are predominantly part of cross-linkedstructures.

Growth of the sacculus and murein turnover. Growth of the sacculus occurs by incorporation of new materialby two reactions, transglycosylation and transpeptidation . Thetranspeptidation reaction results in a new cross-link in whichthe donor peptide is characterized by a free -amino group atm-Dap, whereas on the acceptor peptide the -amino group at m-Dapis linked to D-Ala at position 4 of the donor peptide [see above].By determining the radioactive label distribution among donorand acceptor sites of the cross-bridges in pulse-chase experiments,several conclusions could be drawn . First, new [labeled] materialis cross-linked to the existing [old] material [10, 14, 16,25], and this is proposed to occur during elongation of thecell . During cell division, there is also a cross-linking reactionbetween new donor and new acceptor peptides [16, 25] . It appears that free oligomeric murein intermediates are not formed priorto incorporation into the sacculus . Rather, the lipid-linkedprecursors are directly linked to the sacculus without passingthrough an oligomeric stage [28].

Furthermore, during growth of E . coli a dramatic release of murein material from the sacculus [murein turnover] takes place, which is subject to an effective recycling process [27, 29].It was estimated that in one generation 40 to 45% of the mureinof the sacculus is released by the actions of lytic transglycosylases,endopeptidases, and amidases [27].

Growth pattern of the murein glycan strands. By using the m-Dap auxotrophic strain W7 the average lengthof newly synthesized glycans was determined by quantificationof the 1,6-anhydromuropetides in pulse experiments to be 50to 60 disaccharide units [25] . Pulse-chase experiments showed that the average length of the new glycans decreased within5 min to 35 to 40 disaccharide units . Even a longer chase for60 min resulted only in a minor reduction to 31.2 disaccharideunits, which is the characteristic value for uniformly labeledmurein of this strain . Thus, it seems that glycans are firstsynthesized as rather long strands and are then rapidly processedto a length of about 30 disaccharide units . During murein turnover[see above] degradation of glycans seems to occur such thatsome strands are removed completely by digestion, while othersare inert, rather than by continuous shortening of all glycansof the sacculus.

 
Because the three-dimensional architecture of murein cannotbe determined with high resolution by the techniques availableat this time, the structure was modeled on the basis of therelevant experimental data for the physical properties and thechemical composition . As in other scientific fields, the modelswere changed or refined as new techniques were developed andmore data became available . Below, we describe and discuss thedifferent structural models for murein from gram-negative bacteria.For historical reasons, we divide this section into three parts:[i] the first models of the glycan strands and the peptidesand early models of a layered murein, [ii] the more recent [new]model of a layered murein, and [iii] the scaffold model . Therelevant experimental data for the structure of murein and itsconstituents are summarized in Table 1.

First models of the structures of the glycan strands and peptides and early models of a layered murein. The first models included mainly predictions for the conformationof the basic murein constituents, the glycan strands, and thepeptide side chains, as well as predictions for the three-dimensionalarchitecture of the sacculus . The glycans are similar in termsof their primary structure to the strands in chitin in thatthey are ß-1,4-linked GlcNAc oligomers . In the mureinevery second sugar residue carries a lactyl group [the MurNAcresidues] with the peptide side chains at position 3 . In a chitinchain, the glycans are twisted such that successive GlcNAc residuesare rotated 180° relative to each other, and in the most common form, -chitin, adjacent chains run antiparallel [59].A chitin-like tertiary structure was assumed for the mureinglycan strands in the early models [7, 23, 37, 63, 77] [Fig. 1A] . The glycans were modeled as straight rods that run parallel,almost touching each other, with the peptides protruding in the same direction above or below the glycan plane . Some peptides form cross-links to peptides of neighboring strands . With this arrangement only a horizontally layered murein model can be envisioned, in which the glycan strands run parallel to the cytoplasmic membranes . The horizontal models are in accordancewith the conclusion that the murein glycan strands must liepredominantly in the plane of the surface because they are toolong for a vertical arrangement [70] . Furthermore, it was speculated based only on considerations of possible growth mechanisms and without any experimental proof that the glycan strands mightbe arranged perpendicular to the long axis of the cell [66]. Later, indirect evidence for such an arrangement was obtained[81].

 
Conformational calculations of the non-cross-linked pentapeptides revealed that there are several energy-minimized states . However,the favorable conformation is likely to be the one in whichthe pentapeptide is not straight but bends back to the glycanstrand [2] . In two additional studies the authors concludedthat the cross-linked peptide might adopt either a compact ora more extended configuration [63, 77] and that this flexibility of the peptides might explain the observed elasticity of murein.

More recent [new] model of a layered murein. The interpretation of new data obtained by X-ray diffractionstudies, together with stereochemical considerations and quantumchemical studies, led to the conclusion that the glycan strandsin the murein cannot adopt a chitin-like structure with twosugar residues per turn [9, 48, 80] . Instead, the presence ofthe rather bulky lactyl group at MurNAc allows less rotation.About four disaccharide units [eight sugar residues] are requiredfor one turn, and consequently, the peptides protrude from theglycans in a helical pattern [3, 50, 55] [Fig . 1B] . If the glycans are arranged parallel to the membrane, then every second peptide lies in the same plane, and a monolayer of murein can be formed by cross-linking such peptides of neighboring strands [Fig. 2, right side] . One quarter of the peptides would point up, and another quarter would point down, and these peptides could not take part in cross-links . Thus, theoretically, a perfect monolayer with this structure would contain 50% of the peptidesas part of cross-links, and 50% of peptides would not be cross-linked. This is similar to the experimentally observed fraction [40to 50%] of peptides that are part of cross-links [24, 26].

 
In the layered arrangement, one subunit can cover approximately5.2 nm² in the maximally stretched conformation [21] . Consideringthe number of subunits [3.5 x 10⁶ subunits], a maximum totalarea of 18 µm² can be covered, which is about twice themeasured surface area of the cells [79] . In vivo, murein thatis not maximally stretched could cover less surface; therefore,it is likely that the number of subunits allows only one totwo complete layers . The horizontally layered model is in excellentagreement with the thickness of isolated murein [not more than7 nm] determined by small-angle neutron scattering and atomicforce microscopy . The data obtained by small-angle neutron scatteringled to the interpretation that 75 to 80% of the surface of thesacculus is single layered and is 2.5 nm thick, whereas therest is triple layered and has a maximum thickness of 7 nm [52].

A modification of this model was introduced by Koch, who pointed out that in the stress-bearing murein the glycans would notbe straight but would follow a zigzag line [38, 42, 45] andtermed the smallest pore that is formed by two glycan strandsand two peptide cross-links a tessera . A tessera would havethe form of a hexagon [Fig . 3], and it would be more deformablein the directions of the peptides . It was shown experimentallythat sacculi are two- to threefold more deformable in the directionof the long axis [81] . The theoretical elastic constant of aperfect single-layer murein network with the dimensions of thecell consisting of hexagonal tesseras was calculated to be 10⁷ N/m² [5], which is in good agreement with the experimental value,2.5 x 10⁷ N/m² [in the direction of the long axis of the cell]. Thus, Boulbitch et al . [5] concluded that potential defects in the [imperfect] cylindrical network of the real murein [see below] might play only a minor role in determining the elastic properties.

 
Given the experimental data, the layered murein net cannot beperfect for two reasons: [i] compared to the dimensions of thecell, the glycan strands are rather short [average length, 25to 35 nm], and [ii] the percentage of cross-linked peptidesis not the theoretical value [50%] but is slightly less [40to 50%] . Imperfections are holes or slits that are larger thanthe hole of a single tessera or, in other words, consist offused hexagonal tesseras . The distribution of such holes ina layered murein with glycans of the observed length was modeledby Pink et al . [67] . Representing fused tesseras, the largerholes were found to have the form of slits that run predominantlyperpendicular to the long axis of the glycans . If these slitswere distributed over the surface of the sacculus and did notaccumulate at distinct sites, such an imperfect murein would be a stable network [67], and it was concluded that such a mureinwould have a permeability that is consistent with the observeddata [17] . A structurally stable murein network can be modeledwith short glycans consisting of seven disaccharide units and50% cross-linked peptides [Fig . 4] . The structure of the mureinshown in Fig . 4 is far from the structure of the real murein.The latter molecule would have a reduced number of connectedtesseras because the slightly lower level of cross-linkage ismore than compensated for by the three- to fivefold longer glycanstrands . It is not known what number of larger pores and whatmaximum pore size in the layered murein can be tolerated withoutdestroying the integrity of the cell wall . Experimental dataindicate that isolated [relaxed] murein of E . coli has poreswith a mean radius of 2.06 nm [17] . In vivo, the stretched mureinallows penetration of proteins with molecular masses of up to100 kDa [17, 74], indicating that larger pores with a radiusof about 3 nm may exist [according to the formula given in reference 17, a globular protein with a molecular mass of 100 kDa has an estimated radius of 3.1 nm] . The possible existence of larger holes indicates that the murein net is not perfect and is consistent with the data on the glycan length distribution and on the degree of cross-linkage.

 
Because the glycan strands with average lengths are longer thanthe peptide cross-bridges, the arrangement of the glycan strandsis a major structural determinant in a layered murein . One couldenvision the following possibilities for arrangement of theglycan strands in one layer: [i] the glycans run parallel andmainly in the direction of the long axis of the cell, [ii] theglycans run parallel and mainly perpendicular to the long axis,[iii] the glycans run parallel and along helices around thecell surface, [iv] there are patches or areas on the surfacewith regularly arranged parallel glycans, together with areasof random glycan orientation, and [v] the glycans are arrangedin a random orientation without any order . Koch favored an irregularor random structure for the arrangement of the horizontallylayered glycan strands to form a "carded, non-woven fabric "structure [39, 43], and he discussed other models [42] . At thistime, no technique allows direct determination of the orientationof the glycans in the sacculus . Limited fragmentation of sacculiby sonication and different murein hydrolases gave contradictingresults regarding the direction of the slits generated on thesacculi [18, 42, 75, 76] . The anisotropy in elasticity of thesacculi [81] mentioned above would be in accordance with a layeredmurein in which the flexible peptides are arranged predominantlyin the direction of the long axis of the cell and the glycansare predominantly perpendicular to the long axis . If the mureinis the main stress-bearing layer, it was estimated on the basisof the elasticity measurements that the length of the bacteriumwould increase by 12% and the diameter would increase by 8%for every 1 atm of turgor pressure [81].

The murein sacculus is not a static structure . It is enlargedand divided into two sacculi during the cell cycle . Weidel andPelzer pointed out that not only the incorporation of new subunitsbut also the hydrolysis of covalent bonds is required to increasethe surface area of the bag-shaped sacculus [78] . Two major aspects need to be understood with respect to enlargement ofmurein: [i] how is the site of insertion of new material selected,and [ii] what is the mechanism of insertion of precursors intothe sacculus during growth . The selection of the insertion sitesfor new material might be controlled merely by surface tension,as proposed by the surface stress theory of Koch [40, 41, 44].Briefly, it has been proposed that elongation of the rod-shapedstress-bearing sacculus is possible with maintenance of a constantdiameter by random insertion of precursors into the cylindricalpart . This model requires inertness of the polar regions, aproperty that has been proven experimentally [18] . On the otherhand, murein synthesis might be controlled directly or indirectlyby the recently discovered MreB/Mbl proteins that form spiralsat the inner site of the cytoplasmic membrane and that are requiredfor the rod shape in Bacillus subtilis and in other rod-shapedbacteria [15, 36] . There are different models for the mechanismof insertion of new material into the murein of a layered structure.Burman and Park have proposed that local hydrolysis within themurein net precedes the insertion of two newly synthesized andcross-linked glycan strands [10, 65] . On the other hand, the3-for-1 model follows the make-before-break strategy that demandsthat synthetic reactions precede hydrolysis of bonds in thestress-bearing sacculus [38] . Accordingly, three new glycansare synthesized, cross-linked to each other, and linked to bothsides of an existing glycan strand in the sacculus . Upon removalof the so-called docking strand, the new triplet of glycansis inserted into the sacculus [32, 33] . Further experimentalwork is required to determine both the mechanism for selectionof the insertion sites and the mode of insertion of the newmaterial.

Vertical scaffold model. Recently, a novel scaffold model for murein structure was proposed[20, 21], in which the glycan strands extend perpendicular from the cytoplasmic membrane and are cross-linked by peptides that are parallel to the surface of the membrane [Fig . 2, left side].The 1,6-anhydro-MurNAc ends of the glycans were assumed to be located near the cytoplasmic membrane in a region of high cross-linkage, whereas the GlcNAc ends were located close to the outer membrane in a poorly cross-linked region . According to the authors who proposed this model, the murein almost completely fills the periplasmic space . However, several experimental findings donot fit the scaffold model, as discussed below.

E . coli contains about 3.5 x 10⁶ molecules of m-Dap per cell,which are present in murein consisting of glycan strands thatare, on average, 25 to 35 disaccharides long . If an averageof 30 disaccharides is used, this yields a total number of 3.5x 10⁶/30 or 1.17 x 10⁵ glycan strands . According to the authorswho proposed the scaffold model, one unit [one glycan strandoriented perpendicular to the membrane] covers an area of about27 nm² [21] . We realize that this is the maximum possible surfacearea of a unit that was calculated from the length of a maximallystretched peptide cross-link [4.1 nm], the length of one disaccharide[1 nm], and the thickness of one disaccharide [1.1 nm] . With1.17 x 10⁵ glycan strands a maximum total surface of 3.15 µm² could be covered in the maximally stretched scaffold arrangement,which is less than 40% of the surface area of the cell . Thediscrepancy becomes even greater if one considers that strainMC6RP3 could grow with 50% less murein at a low m-Dap concentration.The average glycan strand length was 24.4 disaccharide unitsunder these conditions [68] . It follows that a maximum areaof 1.9 µm² can be covered with scaffold-like murein, whichis only 23% of the cell surface area [assuming that the amountof m-Dap per cell and the average size of the cells are notmuch different from the values for other strains] . We concludedthat E . coli does not contain enough murein for the proposedscaffold murein structure with glycans having the measured lengthdistribution.

According to small-angle neutron scattering and atomic force microscopy experiments, isolated sacculi are rather thin . About75 to 80% of the surface is 2.5 nm thick, and the remaining20 to 25% is at most 7 nm thick [52] . These measurements were obtained with purified murein sacculi that were fully hydratedand not subjected to staining procedures, which could influencethickness measurements determined by electron microscopy . Takingthese facts in account, the scaffold-like murein would consistmostly of glycan strands consisting of 2.5 disaccharide unitsand would contain glycans that have a maximum length of only7 disaccharide units . However, the measured average length ofthe murein glycan strands is 25 to 35 disaccharide units . Giventhe determined average length of the murein glycans, the thicknessof the proposed scaffold-like murein would be 25 to 35 nm, whichis about 10-fold greater than the measured thickness of murein.Furthermore, a high proportion [25 to 30%] of all glycan materialconsists of glycan strands that are longer than 30 disaccharideunits . Thus, many of the murein glycan strands are longer thanthe distance from the cytoplasmic membrane to the outer membrane,which is about 13 to 25 nm [58, 64] . In the attempts to modelboth the planar murein and the scaffold-like murein, the longmurein glycan strands were not included [21] . We concluded thata scaffold-like arrangement with glycan strands arising perpendicularly from the membrane is not in accordance with the observed length distribution of the murein glycan strands and the measured thickness of the sacculus.

The authors who proposed the scaffold model stressed the point that a layered murein with a low percentage cross-linked peptides [33%] and short glycan strands [on average, 12 disaccharideunits] would have large holes [21] . However, both assumptions are far from reality . The glycan strands have an average length of about 30 disaccharide units . High-performance liquid chromatography analysis of muropeptides revealed that about 40 to 50% of the peptides in the murein of E . coli and other gram-negative bacteria are part of cross-links [26, 69] . E . coli grown in the presenceof D-amino acids showed strongly reduced cross-linking; only34% of the peptides were part of cross-links . However, thesecells contained longer glycan strands [average length, 39 disaccharideunits], as determined from the proportion of anhydro-MurNAc-containingmuropeptides [2.6%] [11] . It is possible that the cells can[at least partially] compensate for a low degree of cross-linkageby increasing the length of the glycan strands to stabilizethe murein net.

The murein sacculus is elastic both in vivo and in vitro . As mentioned above, atomic force measurements revealed that the elasticity is greater in the direction of the long axis of thecell and less perpendicular to this direction [81] . This finding does not seem to fit into the scaffold model, in which the elastic peptide bridges point in both directions.

Both ends of the murein glycan strands are subject to greater cross-linkage than internal fragments [26, 71] . This may reflecteither the mechanism of insertion of new glycans or the factthat non-cross-linked glycan ends that do not contribute to the stability of the net are trimmed down by enzymatic degradation to cross-linked structures . This finding is inconsistent with the scaffold model, in which only the 1,6-anhydro-MurNAc endsare located near the cytoplasmic membrane in a zone of highcross-linkage and the GlcNAc ends are localized in a less-cross-linkedzone near the outer membrane [loose ends] [21].

The authors who proposed the scaffold model present a modelin which in one cell cycle "two new walls are synthesized beneaththe old one which is destroyed by lytic enzymes in due course"[20] . However, biochemical studies performed in different laboratorieshave clearly shown that during growth of the sacculus cross-linksare formed between new material and the old murein of the existing sacculus [10, 14, 16, 25], excluding the proposed mechanismof synthesis of two new walls beneath the old wall.

 
In summary, we believe that many experimental results, includingthe amount of murein per cell, the thickness of the sacculus,the analytical data on the length distribution of the glycanstrands, the growth pattern of the glycan strands, the degreeof cross-linkage, and the fact that cross-links are formed betweenthe existing murein and the newly synthesized murein, are inaccordance with a model in which very few layers of glycan strandsare cross-linked by peptides and are arranged parallel to thecytoplasmic membrane [horizontally] . Given the experimentaldata, a scaffold-like murein structure with glycans that extendperpendicularly from the cytoplasmic membrane is highly unlikely.However, our interpretation does not exclude the possibilitythat occasionally or even in particular areas on the sacculusglycan strands may bend out of the horizontal layer to adopt another orientation, but the major, stress-bearing part of the murein is likely to be formed by a planar layer[s] . We hopethat in the future high-resolution techniques will become availablethat allow direct visualization of the orientation of the mureinglycan strands in vivo.

 
* Corresponding author . Mailing address: Universität Tübingen, Fakultät für Biologie, Lehrbereich Mikrobielle Genetik, Auf der Morgenstelle 28, 72076 Tübingen, Germany . Phone: 49-7071-2974635 . Fax: 49-7071-295065 . E-mail: waldemar.vollmer@uni-tuebingen.de .

This publication is dedicated to Uli Schwarz on the occasionof his 70th birthday.

Present address: Galerie Jochen Höltje, Tübingen,72072 Tübingen, Germany.

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