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Revisiting Magnesium Chelation by Teichoic Acid with Phosphorus Solid-State NMR and Theoretical Calculations Jason R. Wickham, Jeffrey L. Halye, Stepan Kashtanov, Jana Khandogin, and Charles V. Rice* Department of Chemistry and Biochemistry, UniVersity of Oklahoma, 620 Parrington OVal, Room 208, Norman, Oklahoma 73019 ReceiVed: October 21, 2008
Teichoic acids are essential components of the Gram-positive bacterial cell wall. One of their many functions is metal binding, a vital process for bacterial growth. With the combination of phosphorus-31 solid-state NMR spectroscopy and theoretical calculations using density functional theory (DFT), we have determined that the binding mode between teichoic acids and magnesium involves bidentate coordination by the phosphate groups of teichoic acid. Measurement of chemical shift anisotropy tensors gave a reduced anisotropy (δ) of 49.25 ppm and an asymmetry (η) of 0.7. DFT calculations with diglycerol phosphate and triglycerol diphosphate model compounds were completed with Mg2+ in anhydrous as well as partially hydrated bidentate and fully hydrated monodentate, bidentate, and bridging binding modes. 31P CSA tensors were calculated from the energy-minimized model compounds using the combined DFT and GIAO methods, resulting in dramatically different tensor values for each binding mode. The anhydrous bidentate chelation mode was found to be a good approximation of the experimental data, an observation that alters the current monodentate paradigm for metal chelation by teichoic acids. Introduction The cell wall of Gram-positive bacteria is mainly composed of peptidoglycan (peptide cross-linked disaccharide polymers) and teichoic acids (long phosphodiester polymers), which are essential to bacterial health. Teichoic acids are suggested to participate in several different aspects of bacterial survival, one being the coordination of metal cations. Of the different functional groups present in teichoic acids (phosphate, alanine, N-acetylglucosamine, hydroxyl, Figure 1), the phosphates are the primary chelation site and have been the subject of numerous investigations into the metal binding chemistry. At physiological pH values, alanine branches are cationic (from NH3+) and the phosphate groups are anionic. This results in the formation of an ion pair that inhibits metal binding, which was demonstrated by a 60% increase in Mg2+ binding when the alanine groups were removed.1 The need for teichoic acids to assist with Mg2+ uptake was demonstrated by studies of B. subtilis bacteria grown in low magnesium concentrations.2,3 These bacteria demonstrated significant increases in the amount of teichoic acids produced in the cell wall in order to maximize metal binding. Bacterial strains with teichoic acids removed have shown a decreased affinity for Mg2+, K+, and Na+ and an inability to bind Ca2+.4 Additionally, while teichoic acids and peptidoglycan are responsible for the binding of Mg2+, K+, and Na+, teichoic acids are solely responsible for the binding of Ca2+. Despite its central role in metal chelation, very little structural data are available to characterize the metal binding chemistry. This lack of information is largely due to samples of this type being unsuitable for diffraction techniques. Nevertheless, two different models have been proposed for the Mg2+:phosphate interaction. Both models rely on monodentate chelation with either a single phosphate (Figure 2A) or bridged between two neighboring phosphates (Figure 2B).5 These models are based * To whom correspondence should be addressed.
on data collected with X-ray photoelectron spectroscopy (XPS) and have remained unchallenged for 35 years. However, by using a combination of 31P solid-state NMR spectroscopic data and theoretical calculations, we show that magnesium ion chelation involves the phosphate groups in a bidentate binding mode, defying the current paradigm. Teichoic acid extends past the cell wall where it can bind metals from the extracellular fluid. The resulting chelation of cations reduces electrostatic repulsion between neighboring phosphate groups. Stabilization in this manner has been observed in the phosphate groups of nucleic acids and lipid membranes. Teichoic acid is a major component (30-60% by mass) of the cell walls6-8 and is found as both a lipid-anchored teichoic acid (lipoteichoic acid, LTA) and a wall-anchored teichoic acid (WTA).6-8 The disaccharide head group of WTA is covalently attached to the peptidoglycan cell wall,8 while LTA is attached via a lipid chain anchored in the phospholipid membrane. Both LTA and WTA contain disaccharide D-glucose head groups with a poly(phosphodiester) chain (Figure 1A-C) extending out past the cell wall.8,10 Depending on the bacterial strain, the backbone of WTA is either the poly(glycerol phosphate), Figure 1B, or poly(ribitol phosphate), Figure 1C. The basic chemical structure of teichoic acids has been determined from proton and carbon liquid-state NMR.11-17 The poly(phosphodiester) chain has 20-50 repeat units,8,12,14,16,18 decorated with D-alanine, Nacetylglucosamine, and hydroxyl groups.18-20 At pH < 7, the D-alanine groups are cationic, the phosphodiesters are anionic, and both the N-acetylglucosamine and hydroxyls are neutral.18-20 This results in incomplete charge neutralization,21 which allows teichoic acid to form ionic bonds with surrounding fluids, dissolved ions, and substrates as well as possible intramolecular contact ion pairs between the D-alanine and phosphate groups. Many antibiotics target the peptidoglycan matrix of the cell wall, but this approach has become less effective; thus, other components of the cell wall have recently become new targets
10.1021/jp809313j CCC: $40.75 2009 American Chemical Society Published on Web 01/27/2009
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Figure 1. Chemical structure of teichoic acids found in the cell wall of Gram-positive bacteria is quite diverse. The polyphosphate backbone is very long, with 20-50 repeat units dependent upon the species of bacteria. The different constituents of teichoic acid facilitate chemical interactions with various ionic species, water, and the cell wall peptidoglycan. LTA is identical for S. aureus and B. subtilis while the WTA of S. aureus has a poly(ribitol phosphate) backbone. The WTA from B. subtilis has a poly(glycerol phosphate) backbone with glucose in place of N-acetylglucosamine. Despite this diversity, the NMR data do not show a difference between the phosphate NMR spectra.
Figure 2. Illustration of the current paradigm for Mg2+ binding with teichoic acid. (A) Monodentate binding of Mg2+ to one phosphate while the other phosphate participates in a contact ion pair with the alanine. (B) Monodentate bridging of Mg2+ between two phosphates in the absence of the contact ion pair. Structures inspired by Baddiley et al.9
for antibiotics. Teichoic acids are one of those new antibiotic targets due to their active role in bacterial defense and regulation of nutrients. They also play a large role in the uptake of nutrients through their coordination of metal ions, which ultimately leads to the intercalation of those ions through the cell wall. The development of antibiotics capable of hindering or preventing the transport of nutrients into bacteria would benefit from an understanding of the coordination structure and intercalation mechanism of metal ions to teichoic acids. Solid-state NMR is ideally suited for gaining detailed molecular level structural information from these amorphous materials. Experimental Section Sample Preparation. Staphylococcus aureus lipoteichoic acid (LTA) was purchased from InvivoGen as a lyophilized
powder. This material was isolated via butanol extraction, and purified with hydrophobic interaction chromatography (HIC), and residual metals were removed by dialysis with EDTA. Bacillus subtilis wall teichoic acid (WTA + peptidoglycan) was obtained from cells isolated from media using the following protocol.22-24 Cells were disrupted using a French press and washed with doubly distilled water. The cell wall was then centrifuged at 15 000g for an additional 20 min. Treatment with 3% Triton X-100 at 37 °C (30 min) followed by 2% SDS (37 °C, 30 min) was used to remove the cytoplasmic membrane. Nucleic acids were removed with trypsin (200 µg/mL) and RNase/DNase (100 µg/mL) at 37 °C for 18 h. Spectroscopic analysis (260 and 280 nm) verified removal of nucleic acids. Both LTA and WTA were dissolved in a 60 mM MgCl2 (SigmaAldrich) solution of double distilled water, mixed for 24 h, and lyophilized. Because both teichoic acid and peptidoglycan have metal binding sites,1-5,9 the high concentration of MgCl2 was chosen to occupy all possible binding sites, guaranteeing a uniform signal. Solutions of 20 and 40 mM MgCl2 did not perturb the phosphate 31P CPMAS spectrum (not shown). NMR Experiments. Solid-state NMR experiments were performed using a three-channel NMR spectrometer (1H ) 300 MHz, UnityInova, Varian, Inc.) and a 5 mm, three-channel, magic-angle-spinning NMR probe (Varian APEX design). CPMAS experiments data were collected and analyzed for anisotropic and asymmetric data. CP-MAS experiments of TA and TA:Mg2+ samples were collected with 100 000 scans, 1 s repetition rate, 3000 Hz spinning rate, and contact time of 1000 µs with 1H decoupling at an RF power level of 50 kHz. Drive and bearing gas were provided by dry compressed air. 31P chemical shift data were referenced to an external standard of 85% phosphoric acid (0 ppm). The temperature was 25 °C. Data acquisition and spectral analyses were accomplished using
SSNMR and DFT Study of Teichoic Acids
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Figure 3. 31P CPMAS NMR spectra of (A) LTA, (B) LTA with 60 mM MgCl2, (C) WTA, and (D) WTA with 60 mM MgCl2. Spectra A and C are identical, demonstrating that the peptidoglycan in the WTA sample does not disturb the phosphate chemical environment. Likewise, metal chelation by LTA and WTA give identical spectra (B and D). Thus, the model compounds do not have to account for peptidoglycan interactions.
VnmrJ 1.1D (Varian, Inc.) and the Herzfeld-Berger Analysis Software Package,25 respectively. DFT Calculations. Computational study was conducted using Gaussian 0326,27 and ADF programs.28,29 Previous theoretical calculations suggest that the density functional B3LYP30,31 combined with 6-21G+* basis can accurately reproduce the geometry and thermodynamics of Mg2+ complexes.32,33 Thus, we performed full geometry optimization using B3LYP and 6-21++G(2d,2p) basis set. The NMR calculations were conducted with the GIAO method at the same level of theory. The hydrated bridging and partially hydrated bidentate structures proved difficult to optimize under Gaussian 03 and were optimized to a minimum energy by ADF. All structures were examined with a frequency check via Gaussian 03 prior to NMR parameter calculations and found to have no negative frequencies, indicating an actual minimum and not a saddle point in the energy minimization. According to the Haeberlen-Mehring convention,34,35 the isotropic chemical shift (δiso), reduced chemical shift anisotropy (δ), and asymmetry parameter (η) were calculated with eqs 1-3. The δiso, δ, and η parameters were then entered into the SpecTrum Analysis of Rotating Solids (STARS) program (Varian, Inc.) to generate simulated spectra for each model. All δiso values (theoretical and experimental) are referenced against the δiso value of H3PO4.
Results and Discussion Phosphorus-31 CPMAS NMR spectra were collected for lipoteichoic acid and cell wall (wall teichoic acid plus pepti-
doglycan) shown in Figure 3A,C. These produced identical 31P CPMAS spectra with a δiso (0 ppm) and a series of spinning side bands that map out δ (41.92 ppm) and η (0.67) values characteristic of teichoic acids. Subsequent experiments with LTA and WTA coordinated with Mg2+ gave different 31P CPMAS NMR spectra (Figure 3B,D). Changes in the spectra demonstrate a change in the chemical environment of the teichoic acid (LTA and WTA) phosphate groups after Mg2+ binding. In addition to an increase in line width (700 Hz vs 1400 Hz), metal chelation alters the isotropic chemical shift value (0 vs -8.15 ppm). The upfield shift results from increased shielding of the phosphorus atoms from strong interactions of the magnesium ion. It is also known that coordination of cations causes upfield shifts in 13C NMR spectra as well.36,37 Finally, Mg2+ binding causes significant changes to the spinning sideband intensities which, in turn, are reflected in the magnitudes of the principal chemical shift anisotropy (CSA) tensor components, δxx, δyy, and δzz. The ability to obtain numerical values of the tensors provides a quantitative assessment of structural changes upon binding. For instance, δzz lies along the PdO bond in the neutral phosphate, and the magnitude of the δzz vector can be used indirectly to describe changes in the PdO bond length.38,39 However, with metal chelation, the δzz tensor component hybridizes, incorporating the two phosphorus:oxygen bonds not involved in the phosphodiester backbone. As this obscures the previously bond-specific data, a more in-depth tensor analysis must be completed before any changes in specific bond lengths can be made with any exactitude. Therefore, to circumvent this quandary and clarify the binding structure of Mg2+ to teichoic acid, quantum mechanical models were created and used to simulate the NMR spectra for monodentate and bidentate binding structures. As our data describe the phosphate:metal interaction, diglycerol phosphate was used as a model compound to simulate the teichoic acid/Mg2+ binding. Figure 4 shows the energyminimized structures of model compounds with Mg2+ chelation in bidentate and monodentate modes. For monodentate binding, the 1.94 Å Mg-O bond distance is similar to values found with ATP:Mg2+ and theoretical calculations of Mg2+.40 For the model compound with the bidentate Mg2+ coordination, the 2.0 Å Mg-O bond distance agrees with other model compounds.41-44 From the energy-minimized structure, the δxx, δyy, and δzz components of the CSA tensor can be used to calculate the reduced anisotropy (δ) and asymmetry parameter (η) (Table 1). The isotropic chemical shift (δiso) can also be calculated, but as this figure relates more to what is generally bound to the 31P
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Figure 4. Energy-minimized structures of teichoic acid models with (a) monodentate [MgCl]+, (b) bidentate Mg2+, (c) bridging Mg2+, (d) hydrated monodentate [MgCl]+, (e) hydrated bidentate Mg2+, (f) cis-hydrated bridging Mg2+, and (g) trans-hydrated bridging Mg2+ models. Note: all hydrated models incorporate four waters to fill the 6-coordinate Mg2+ hydration sphere.
TABLE 1: Magnetic Shielding Tensors (σii) and the Corresponding Reduced CSA Anisotropy (δ) and Asymmetry Parameters (η) Calculated Using the GIAO B3LYP/6-311++G(2d,2p) NMR Method index
model structure
σxx/ppm
σyy/ppm
σzz/ppm
δ/ppm
η
(a) (b) (c) (d) (d) (e) (f) (f) (g) (g) (h) (i) (j)
LTA (experimental LTA with MgCl2 (experimental) monodentate bidentate bridging (P1) bridging (P2) hydrated monodentate hydrated bidentate hydrated cis-bridging (P1)-monodentate hydrated cis-bridging (P2)-monodentate hydrated trans-bridging (P1)-monodentate hydrated trans-bridging (P2)-monodentate partial hydrated bidentate · 1H2O partial hydrated bidentate · 2H2O partial hydrated bidentate · 3H2O
34 904 19.807 172.3300 237.6320 177.8540 228.2900 173.1372 180.4097 165.1311 168.3700 132.3879 169.9863 182.4287 183.1880 184.5971
7.015 -3.281 231.6010 267.3500 230.7640 273.7520 218.6017 214.7936 202.3434 192.0625 191.7056 216.3346 217.3205 213.1856 216.6556
-41.919 -40.989 458.7690 332.6050 461.2520 323.3760 306.5426 264.2741 316.8967 331.4085 400.9320 322.7152 253.8804 243.3682 232.2194
41.919 49.253 256.80 80.11 256.94 72.35 110.67 66.67 133.16 151.19 238.89 129.55 54.01 45.18 31.59
0.67 0.70 0.35 0.56 0.31 0.94 0.62 0.77 0.42 0.24 0.37 0.54 0.97 1.00 1.52
nucleus and not to the specific binding mode, this information was left out. The theoretical reduced anisotropy (δ) values for the anhydrous and hydrated monodentate chelation models (δ ) 256.80 and 110.67 ppm) are significantly different and do not reflect the phosphate structure of our experimental sample (δ ) 49.253 ppm), but the δ values for the bidentate models (δ ) 80.11 and 66.67 ppm) are much closer to the experimental value. To illustrate the values in Table 1, NMR spectra were simulated using the theoretical δ, δiso, and η values (Figure 5).
Note that although partially hydrated bidentate tensor data are included in Table 1, they clearly do not fit the experimental profile and, thus, were not included in the simulated spectra. The bidentate spectrum resembles the experimental data while the monodentate spectrum is significantly different in chemical shift span and the peak intensities. The Baddiley model also shows chelation by two phosphate groups. A model compound was created with Mg2+ chelation in a bridging fashion. Here, according to the energy-minimized structures, the phosphates
SSNMR and DFT Study of Teichoic Acids
Figure 5. Simulated NMR spectra generated from the energyminimized structures of the (a) monodentate [MgCl]+, (b) bidentate Mg2+, (c) bridging Mg2+, (d) hydrated monodentate [MgCl]+, (e) hydrated bidentate Mg2+, (f) cis-hydrated bridging Mg2+, and (g) transhydrated bridging Mg2+ models. From the frequency and intensity of the isotopic peak (iso) and spinning sidebands, the bidentate model is the best description of Mg2+ chelation by teichoic acid. Thus, if monodentate binding is present, the concentration is very small compared to that of the bidentate mode of binding.
of the anhydrous compound bind the metal in a bidentate manner while the hydrated phosphates show monodentate chelation. The CSA tensors show dissimilarities due to differences in the bidentate linkages (Figure 4d). Using the STARS simulation program, we created a simulated NMR spectra of the bridging models with a 1:1 ratio of P1:P2. The spectra clearly do not resemble the experimental data and show that if the monodentate chelation is present, it is at an extremely low concentration and not detected in our NMR data. Thus, the Baddiley model is incorrect for Mg2+ chelation in lyophilized samples. It has been demonstrated that simulated NMR spectra of energy-minimized structures can be a very effective method of determining chemical interactions.30 Fry et al. used SS-NMR spectroscopy and ab initio Gaussian 03 calculations to characterize the binding structure of the mononucleotide dAMP on alumina.30 By means of 31P static and CPMAS NMR spectra, CSA tensors were extracted via the Herzfeld-Berger method45 and compared to simulated data. Combined with REDOR NMR studies, they found a bidentate binding mode between dAMP and octahedral surface alumina groups.30 The Hodgkinson-Elmsley tensor reliability analysis shows that for a reduced anisotropy of 49.25 ppm (5975.35 Hz on a 300 MHz NMR spectrometer), the optimal spinning speed would be roughly 2300 Hz, as it has been shown that optimal reliability in anisotropy calculations occur when νr/δ ) 0.39.46 As it stands, our spinning speed of 3000 Hz results in a νr/δ ratio of 0.50, which does not reduce the reliability of the reduced anisotropy significantly. In addition, although calculations of the asymmetry parameter (η) increase significantly as the spinning speed drops to zero, the same results provided by Hodgkinson-Elmsley show that the static spectra show only minor improvements in situations where η > 0.2. The analysis also shows that the reliability of a particular parameter is inversely proportional to the standard deviation of that parameter such that r(δ) ) δ/s. Therefore, although the results shown here are not the maximum
J. Phys. Chem. B, Vol. 113, No. 7, 2009 2181 of reliability, a slight decrease in the reduced anisotropy (δ) reliability would also result in an increase in the standard deviation by the same factor. Gram-positive bacteria rely on peptidoglycan and teichoic acids to bind metals from extracellular fluids.8 After binding, metals move to the lipid bilayer where they enter the cytoplasm and are used for numerous biochemical processes. The teichoic acids are nearly 100% responsible for calcium uptake, whereas they contribute only 50% to K+, Na+, or Mg2+ binding. However, decades of research have yet to reveal the basic chemical interactions that enable metal binding within the cell wall. The work reported here is a significant step toward unraveling this mystery, and the results are necessary to understand bacterial virulence, formulate antibiotic targets, and advance basic knowledge of bacterial biochemistry. The main obstacle in understanding metal chelation is the lack of structural data needed to create an atomic scale mechanism. Although the macroscopic binding trends have been characterized, structural biology was impossible due to the lack of viable samples for X-ray crystallography and the inability to use solution-state NMR. In the early 1970s, X-ray photoelectron spectroscopy was used with samples of teichoic acids bound to Mg2+ ions in order to decipher the binding mode of the Mg2+ atom. This tool measures the energy of electrons ejected from the Mg2+ atom. Because the electrons are also in chemical bonds, the electronic energy of the Mg2+ interactions to teichoic acid is altered according to the binding mode. The results were interpreted as monodentate binding by a single oxygen of the phosphate group (Figure 2A).9 In 1975, Lambert et al. revisited the binding of Mg2+ with teichoic acid of Lactobacillus buchneri and found that the metal:phosphate binding ratio was 0.4:1.5 This was interpreted as Mg2+ binding via two phosphate groups (Figure 2B). While studying Mg2+ binding to teichoic and teichuronic acids, the initial metal:phosphate ratio was about 1:2.47 However, Doyle et al. showed that the formation of contact ion pairs and cation binding were dependent on pH.48 At physiological pH, contact ion pairs with D-Ala reduce the number of phosphate groups available for metal chelation. This was verified by chemically removing the alanyl groups, which increased the Mg2+:phosphate ratio from 0.35 to 1.0.5 Nevertheless, these studies cannot differentiate between monodentate and bidentate binding. Our data and calculations clearly demonstrate that chelation with either one or two phosphate groups occurs in a bidentate manner. The 31P CPMAS NMR spectrum is a direct indication of the phosphate binding mode, whereas the XPS is an indirect reflection of chelation by the phosphate oxygen atoms. Baddiley et al. did not describe how they determined the monodentate binding model from the data. XPS bombards the cation with high-energy X-rays to eject electrons, whose kinetic energy is measured. When bound to LTA, the Mg 2s electron binding energy was similar to that found in a sample of magnesium phosphate (MgHPO4 · 3H2O), which shows monodentate chelation. The differences could arise because of the ultrahigh vacuum (10-9 Torr) required in XPS may remove water from the solvation sphere of the ions and alter the chemical environment. Our lyophilization requires a more modest vacuum (10-4 Torr) and removes only excess solvent. Rather than using an inorganic mineral, comparisons should be made with alkyl phosphates, whose structure more closely resembles the phosphodiester backbone of teichoic acids. Bidentate chelation by the phosphate groups has precedence in experimental and theoretical studies of nucleic acids and associated model compounds. The low resolution of DNA crystallographic data
2182 J. Phys. Chem. B, Vol. 113, No. 7, 2009 prevents accurate determination of metal chelation chemistry. Binding can occur with the bases and/or phosphate groups. Calculations by Marynick and Schaefer examined metal: phosphate bonds with model complexes of dimethyl phosphate.42 The cations Li+, Na+, K+, Be2+, Mg2+, and Ca2+ yielded energyminimized structures with bidentate coordination.42,43 Realizing that the ion pairing could result from electrostatic and covalent interactions, Liebmann et al. reported that Mg2+ complexation involved bidentate binding with C2V symmetry of dihydrogen phosphate.41 Schneider et al. also found dimethyl phosphate preferentially binds Na+ in a bidentate manner.44 This arrangement was also seen when two Mg2+ cations were bound to a larger model compound, CH3OP(O2)O(CH2)3OP(O2)OCH3. Conclusions Teichoic acids were mixed with MgCl2, which resulted in six possible coordination schemes: anhydrous and hydrated monodentate, bidentate, and bridging. In order to determine the correct binding motif, we compared our experimental data with simulated NMR spectra from energy-minimized structures. We found very good agreement between the spectra and simulated spectra for both the anhydrous and hydrated bidentate models, but not for the monodentate model. Thus, our data strongly support a bidentate binding of the Mg2+ cation and are inconsistent with the previously accepted monodentate model.5 Acknowledgment. This work is supported by the Oklahoma Center for the Advancement of Science and Technology (OCAST) and the University of Oklahoma. Jason Wickham was supported by a “Graduate Assistance in Areas of National Need” (GAANN) Fellowship from the Department of Education Grant No. P200A030196 awarded to the Department of Chemistry and Biochemistry, University of Oklahoma. We thank Dr. Phillip Klebba and his group for the use of their facilities and the availability of their staff to assist with sample preparation. We also thank Scott Boesch (University of Oklahoma) and Albert Strasheim (University of Stellenbosch) for their technical assistance with the installation of our cluster. Supporting Information Available: Figure 6 showing the partially hydrated bidentate structures with (h) 1H2O, (i) 2H2O, and (j) 3H2O and Figure 7 showing the simulated partially hydrated bidentate spectra with (h) 1H2O, (i) 2H2O, and (j) 3H2O. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Heptinstall, S.; Archibald, A. R.; Baddiley, J. Teichoic acids and membrane function in bacteria. Nature (London) 1970, 225, 519–21. (2) Ellwood, D. C. Wall content and composition of Bacillus subtilis varniger grown in a chemostat. Biochem. J. 1970, 118, 367–73. (3) Ellwood, D. C.; Tempest, D. W. Effects of environment on bacterial wall content and composition. AdV. Microb. Physiol. 1972, 7, 83–117. (4) Beveridge, T. J.; Murray, R. G. E. Sites of metal deposition in the cell wall of Bacillus subtilis. J. Bacteriol. 1980, 141, 876–87. (5) Lambert, P. A.; Hancock, I. C.; Baddiley, J. Influence of alanyl ester residues on the binding of magnesium ions to teichoic acids. Biochem. J. 1975, 151, 671–6. (6) Ginsburg, I. Role of lipoteichoic acid in infection and inflammation. Lancet Infect. Dis. 2002, 2, 171–9. (7) Graham, L. L.; Beveridge, T. J. Structural differentiation of the Bacillus-subtilis-168 cell-wall. J. Bacteriol. 1994, 176, 1413–21. (8) Neuhaus, F. C.; Baddiley, J. A continuum of anionic charge: structures and functions of D-alanyl-teichoic acid in Gram-positive bacteria. Microbiol. Mol. Biol. ReV. 2003, 67, 686–723. (9) Baddiley, J.; Hancock, I. C.; Sherwood, P. M. A. X-Ray photoelectron studies of magnesium ions bound to the cell walls of Gram-positive bacteria. Nature (London) 1973, 243, 43–5.
Wickham et al. (10) Peschel, A. How do bacteria resist human antimicrobial peptides. Trends Microbiol. 2002, 10, 179–86. (11) Roethlisberger, P.; Iida-Tanaka, N.; Hollemeyer, K.; Heinzle, E.; Ishizuka, I.; Fischer, W. Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae. Eur. J. Biochem. 2000, 267, 5520–30. (12) Stadelmaier, A.; Morath, S.; Hartung, T.; Schmidt, R. R. Synthesis of the first fully active lipoteichoic acid. Angew. Chem., Int. Ed. 2003, 42, 916–20. (13) Potekhina, N. V.; Shashkov, A. S.; Evtushenko, L. I.; Senchenkova, S. y. N.; Naumova, I. B. A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280. Carbohyd. Res. 2003, 338, 2745–9. (14) Morath, S.; Geyer, A.; Spreitzer, I.; Hermann, C.; Hartung, T. Structural decomposition and heterogeneity of commercial lipoteichoic acid preparations. Infect. Immun. 2002, 70, 938–44. 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