Methods for Acquisition and Assignment of Multidimensional High

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Methods for Acquisition and Assignment of Multidimensional High-Resolution Magic Angle Spinning NMR of Whole Cell Bacteria Wei Li,* Robin E. B. Lee, Richard E. Lee, and Jinghu Li

Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee 38163

High-resolution magic-angle spinning (HR-MAS) NMR was developed in late 1990s, and it has evolved quickly for the study of a variety of biological matrixes. Recently, it has been used as an effective means to study the cell wall structures of intact bacteria. 1H-13C heteronuclear single quantum coherence (HSQC) HR-MAS NMR can provide rapid analysis of the cell wall structure in live bacterial cells, thus allowing observation of drug effects, gene mutation, species differentiation, and environmental effects. However, this rapid analysis is dependent on having an established framework of HR-MAS NMR experiments and a detailed assignment of the whole-cell NMR spectra. This study examines parameters and describes strategies for the effective application of 2D and 3D HR-MAS NMR techniques to assign and study bacterial cell wall structures using Mycobacterium smegmatis as a model organism. Important parameters for successful whole-cell HR-MAS NMR studies, including pulse sequences, rotor synchronization, acquisition times, labeling strategies, temperature, number of cells, and cell viability, are described. A four-prong approach is presented for assignment of the complex whole-cell spectra, including the use of 3D HCCH-TOCSY and HCCH-COSY HR-MAS NMR.

High-resolution magic-angle spinning (HR-MAS) NMR, initially developed in late 1990s, has been used to characterize various biological samples1-8 and for the study of gene mutation and drug * To whom correspondence should be addressed. Phone: (901) 448-7532. Fax: (901) 448-6828. E-mail: [email protected]. (1) Shockcor, J. P.; Holmes, E. Curr. Top. Med. Chem. (Hilversum, Netherlands) 2002, 2, 35-51. (2) Kurhanewicz, J.; Swanson, M. G.; Nelson, S. J.; Vigneron, D. B. J. Magn. Reson. Imag. 2002, 16, 451-463. (3) Griffin, J. L.; Bollard, M.; Nicholson, J. K.; Bhakoo, K. NMR Biomed. 2002, 15, 375-384. (4) Taylor, J.; Wu, C.; Cory, D.; Gonzalez, R.; Bielecki, A.; Cheng, L. Magn. Reson. Med. 2003, 50, 627-632. (5) Cheng, L. L.; Chang, I. W.; Smith, B. L.; Gonzalez, R. G. J. Magn. Reson. 1998, 135, 194-202. (6) Cheng, L. L.; Chang, I. W.; Louis, D. N.; Gonzalez, R. G. Cancer Res. 1998, 58, 1825-1832. (7) Waters, N. J.; Garrod, S.; Farrant, R. D.; Haselden, J. N.; Connor, S. C.; Connelly, J.; Lindon, J. C.; Holmes, E.; Nicholson, J. K. Anal. Biochem. 2000, 282, 16-23. (8) Keshari, K. R.; Zektzer, A. S.; Swanson, M. G.; Majumdar, S.; Lotz, J. C.; Kurhanewicz, J. Magn. Reson. Med. 2005, 53, 519-527. 10.1021/ac050906t CCC: $30.25 Published on Web 08/11/2005

© 2005 American Chemical Society

treatment in mammalian cells.1,9,10 By spinning at the magic angle (54.7°), the contribution from magnetic susceptibility and chemical shift anisotropy to the NMR spectral broadening is significantly reduced.11,12 Therefore, high-resolution NMR spectra can be obtained noninvasively for various biological matrixes. Until recently, only a limited number of 1D and 2D HR-MAS NMR studies had been reported for whole-cell bacteria.13-15 We previously reported using 2D and 3D HR-MAS NMR techniques to study mycobacterial cell wall structures, the effects of antibacterial drug treatment, and verification of gene mutations, demonstrating that multidimensional HR-MAS NMR techniques have the potential to be powerful tools in systems biology studies using live cells.16 However, direct application of commonly used multidimensional techniques optimized for solution NMR often do not work well due to the unique geometry of the NMR sample location, the high spin rate, and the inherent heterogeneous properties of live cells. Establishing the conditions under which these experiments can perform efficiently and provide valuable information is crucial. In this study, we selected Mycobacterium smegmatis as a model organism to develop a series of techniques for the effective application of HR-MAS NMR to study whole bacterial cells. M. smegmatis has traditionally been used as a model organism to study the mycobacterial cell wall physiology due to its rapid growth rate and low pathogenesis.17,18 Mycobacteria have a unique cell wall rich in carbohydrates and lipids. The major components of the mycobacterial cell wall have been well-studied and their (9) Lehtimaeki, K. K.; Valonen, P. K.; Griffin, J. L.; Vaeisaenen, T. H.; Groehn, O. H. J.; Kettunen, M. I.; Vepsaelaeinen, J.; Ylae-Herttuala, S.; Nicholson, J.; Kauppinen, R. A. J. Biol. Chem. 2003, 278, 45915-45923. (10) Coen, M.; Lenz, E. M.; Nicholson, J. K.; Wilson, I. D.; Pognan, F.; Lindon, J. C. Chem. Res. Toxicol. 2003, 16, 295-303. (11) Cheng, L. L.; Ma, M. J.; Becerra, L.; Ptak, T.; Tracey, I.; Lackner, A.; Gonzalez, R. G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6408-6413. (12) Weybright, P.; Millis, K.; Campbell, N.; Cory, D. G.; Singer, S. Magn. Reson. Med. 1998, 39, 337-345. (13) Storseth, T. R.; Hansen, K.; Skjermo, J.; Krane, J. Carbohydr. Res. 2004, 339, 421-424. (14) Szymanski, C. M.; Michael, F. S.; Jarrell, H. C.; Li, J.; Gilbert, M.; Larocque, S.; Vinogradov, E.; Brisson, J.-R. J. Biol. Chem. 2003, 278, 24509-24520. (15) Hanoulle, X.; Wieruszeski, J. M.; Rousselot-Pailley, P.; Landrieu, I.; Baulard, A. R.; Lippens, G. Biochem. Biophys. Res. Commun. 2005, 331, 452-458. (16) Lee, R. E. B.; Li, W.; Chatterjee, D.; Lee, R. E. Glycobiology 2005, 15, 139151. (17) Lee, R. E.; Brennan, P. J.; Besra, G. S. Curr. Top. Microbiol. Immunol. 1996, 215, 1-27. (18) Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29-63.

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structures elucidated by degradative analysis.19,20 The wealth of information known about the mycobacterial cell wall and its diverse structure made it an ideal choice for HR-MAS NMR method development in the study of live bacterial cells. Additionally, establishment of a platform of HR-MAS NMR analytical techniques and assignment of the whole-cell spectra for mycobacteria may prove to be a valuable tool for the study of mycobacterial pathogens. These methods and baseline information can be used to study mycobacterial response to anti-invectives, gene mutation, and environmental factors, as well as investigation of the relationship between cell wall structure and virulence in different mycobacterial and other related species. Herein, we report the optimization of NMR parameters for bacterial whole-cell NMR experiments, factors affecting acquisition of quality whole-cell spectra, a four-prong approach for assignment of the complex whole-cell spectrum, and strategies for the manipulation of whole-cell NMR experiments to probe cell wall structure. EXPERIMENTAL DETAILS Mycobacterial Strains and Growth Conditions. Mycobacterium smegmatis (mc2155) was grown at 37 °C in Middlebrook 7H9 with 0.05% Tween 80 (Difco), 0.2% glycerol, and 10% albumindextrose supplement (ADC). All 13C-labeled compounds were obtained from Cambridge Isotope Laboratories, Inc. For universal 13C labeling, the main carbon sources (0.2% glycerol and 0.2% glucose) were replaced with 0.2% [13C]-glycerol and 0.2% [13C]glucose. For selective labeling of lipids, sodium acetate-1,2-[13C] was added to 7H9-ADC growth media at a final concentration of 3 mg/mL. For selective labeling of the carbohydrate anomeric carbons, 0.2% glucose was replaced with 0.2% glucose-1-[13C]. Cultures were grown to an OD600 of 0.6 and diluted in 13C-labeled media to an OD600 of 0.05. Cells were then harvested (3700g, 10 min, 4 °C) when the OD600 reached ∼0.6 to 0.8. Viability of mycobacterial cells was tested before and after NMR analysis (2 h at 2.5 kHz) by plating serial dilutions of cells on Middlebrook 7H11 solid media supplemented with 10% OADC. Viability was determined by comparison of the resulting colony forming units (CFU). Standards of Cell Wall Components. Arabinogalactan (AG, Mycobacterium bovis BCG) was a generous gift from Dr. McNeil in the department of Microbiology, Immunology and Pathology at Colorado State University. Purified lipoarabinomannan (ManLAM, Mycobacterium tuberculosis, H37Rv) was obtained from Colorado State University under the NIH, NIAID Contract N01 AI-75320 entitled Tuberculosis Research Materials and Vaccine Testing. Preparation of Cells for NMR Analysis. After harvesting, the cell pellets were washed three times in a volume of D2O (Cambridge Isotope Laboratories, Inc., MA) representing 20% of the culture volume. The pellets were resuspended in a final volume of 2 µL of D2O per mg of cells (wet weight). A 50-µL aliquot of cell suspension (∼1.3 × 1011 cells) was loaded into a 4-mm gHX glass MAS rotor (Varian NMR Inc, Palo Alto, CA) and sealed. (19) Brennan, P. J. Tuberculosis (Edinb) 2003, 83, 91-97. (20) Escuyer, V. E.; Lety, M. A.; Torrelles, J. B.; Khoo, K. H.; Tang, J. B.; Rithner, C. D.; Frehel, C.; McNeil, M. R.; Brennan, P. J.; Chatterjee, D. J. Biol. Chem. 2001, 276, 48854-48862.

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NMR Spectroscopy. HR-MAS NMR. All HR-MAS NMR experiments were performed on a Varian Unity Inova 500-MHz spectrometer equipped with a 4-mm gHX Nanoprobe (Varian NMR Inc, Palo Alto, CA). Rotor spinning speed was computercontrolled with a general accuracy of (2 Hz. Temperature was controlled with a general accuracy of (0.1 °C. Individual experimental parameters (90° pulse width, recycle delay, and solvent saturation parameters) were optimized for each experiment. 1D proton spectra were acquired at 295 K using a rotorsynchronized Carr-Purcell-Meibom-Gill (CPMG) pulse sequence with water suppression.21 The total spin-spin relaxation delay was 40 ms. Rotor speed was set at 2.5 kHz except during experiments for the optimization of rotor speed, in which case the rotor speed was adjusted from 1 to 2.5 kHz. Rotor synchronization optimization was performed first with a 13C, 15N-ubiquitin (ASLA Biotech Ltd., Latvia) protein standard, and subsequent optimization with whole M. smegmatis cells. During these experiments, transmitter power levels were adjusted to achieve a 50kHz RF strength for proton (90° pulse is 4.95 µs) and 25-kHz RF strength for carbon (90 ° pulse is 9.9 µs). The gradients in the pulses were adjusted to be multiples of one rotor spin period (0.5 ms at spin 2000 Hz). 2D 1H-13C HR-MAS heteronuclear single quantum coherence (HSQC) spectra were acquired using standard pulse sequences. Rotor speed was set to 2.5 kHz, and data were recorded at 295 K except during variable temperature experiments, in which case the HSQC spectra were acquired at 280, 290, 300, and 310 K. Typically, 256 increments were acquired for 13C-labeled cells, with 2-8 scans for each increment. Longer experiments at 512 increments and 24-48 scans were also conducted during method development. Spectra for unlabeled cells were acquired using 256 increments and 144 scans. 3D HCCH-TOCSY (total correlation spectroscopy) spectra were acquired using an adiabatic mixing sequence.22 The spinning speed was set at 2 kHz. 1001(t3) × 70 (t2) × 48 (t1) complex data points were acquired with 24 scans per increment and a total TOCSY mixing time of 57ms. 3D HCCH-COSY spectra were acquired at 2 kHz spinning, and 600 (t3) × 64 (t2) × 64 (t1) complex data points were acquired with 8 scans/increment. Linear prediction was used in both indirect dimensions with zero-filling before Fourier transformation. All data were processed with Varian VNMR 6.1C software with shifted sinebell weighting functions. Solution Phase NMR. 2D 1H-13C HSQC NMR of AG and LAM soluble standards was performed using a Varian Unity Inova 500MHz spectrometer equipped with a triple resonance probe (trpfg) (Varian NMR Inc, Palo Alto, CA). RESULTS AND DISCUSSION NMR Parameters and Rotor Synchronization. Proper rotor synchronization with radio frequency (RF) and gradient parameters has been reported to be critical for CPMG and TOCSY under MAS conditions.23,24 To study rotor synchronization effects on HSQC and optimize the signal intensity in whole-cell HR-MAS (21) Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958, 29, 688-691. (22) Kay, L. E.; Xu, G. Y.; Singer, A. U.; Muhandiram, D. R.; Formankay, J. D. J. Magn. Reson. 1993, B101, 333-337. (23) Wieruszeski, J. M.; Montagne, G.; Chessari, G.; Rousselot-Pailley, P.; Lippens, G. J. Magn. Reson. 2001, 152, 95-102. (24) Zektzer, A. S.; Swanson, M. G.; Jarso, S.; Nelson, S. J.; Vigneron, D. B.; Kurhanewicz, J. Magn. Reson. Med. 2005, 53, 41-48.

Figure 1. First increment signal intensity of 2D and 3D HR-MAS NMR experiments as a function of rotor spinning rate. All the gradients are set to integers of 0.5 ms corresponding to 2 kHz. Table 1. Relative Intensity of the First Increment as a Function of Spin Rate for HR-MAS NMR Experiments with M. smegmatis Hz

HSQC (1.17 ppm)

gHSQC (1.17 ppm)

HCCH-COSY (4.02 ppm)

HCCH-TOCSY (4.02 ppm)

2500 2250 2000a 1750 1500 1000

1.04 1.03 1.00 0.96 0.96 0.84

0.69 0.92 1.00 0.10 0.23 0.67

0.99 0.39 1.00 0.51 0.06 0.03

0.22 0.17 1.00 0.79 0.05 0.05

a The intensities were normalized relative to the intensity at 2000 Hz in each of the experiments.

NMR experiments, the first increment of HR-MAS NMR sequences was measured as a function of rotor spin rate for HSQC, gHSQC, HCCH-TOCSY, and HCCH-COSY experiments. The results are shown in Figure 1. While HSQC was almost insensitive to the spinning rate, its gradient version, gHSQC showed much greater rotor synchronization dependence (Table 1). The slight increase seen in signal intensity of HSQC spectra with increasing spin rate was due to the better averaging of anisotropic interactions at higher spin rates. By contrast, the intensity of gHSQC signals suffered substantial reduction at spin rates far away from rotor synchronization, with 90% loss of signal intensity at 1.75 kHz. The signal intensity was strongest at a spin rate of 2 kHz, at which both the RF and the gradients were well-synchronized with rotor spinning. Clearly, HSQC proved to be much more robust and should be the choice for whole-cell HR-MAS NMR studies. The first increment of both 3D HCCH-COSY and HCCHTOCSY experiments also showed very strong rotor spin rate dependence. The strongest signal intensities were observed at a spin rate of 2 kHz (rotor synchronization), whereas at lower spin rates of 1 and 1.5 kHz, the signals were almost completely lost (Table 1). Therefore, parameters for 3D HR-MAS NMR experi-

ments should be carefully adjusted to address rotor synchronization to achieve the best signal intensity. It was interesting to compare the signal intensity in the spectra from whole cell bacteria and a solution of doubly labeled 13C,15Nubiquitin (data not shown). While they both had almost no signal at 1 kHz and 1.5 kHz, at a higher spin rate of 2.5 kHz, the first increment of 3D HCCH-TOCSY of ubiquitin solution was almost completely gone, but in the whole-cell sample, substantial signal intensity was still detected (relative signal strength of 22%). This could be explained by the microscopic heterogeneous environments molecules experience in whole cells, whereas those in solution experience a homogeneous environment. These heterogeneous environments may create local variations of magnetic field or gradient that the molecules experience; therefore, the rotor synchronization effect in whole cells may not be as sensitive as that in homogeneous solution. Further experiments may be designed to take advantage of this to probe cell wall structure. Since a glass MAS rotor was used for these experiments, spin rates above 2.5 kHz could not be performed without compromising the integrity of the MAS rotor. It is expected that greater rotor speeds (for example, 4 kHz, which has a rotor spinning cycle of 0.25 ms), would provide greater signal intensities, since the synchronized spinning speed will average the macroscopic inhomogeneities more efficiently. However, cell integrity may need to be considered at the strong relative centrifugal force (RCF) experienced at higher spin rates.4 It is possible that cells subjected to the high gravitational forces during HR-MAS may suffer mechanical stress, reduced integrity, and structural distortion or rearrangement of cell wall structures and the intracellular environment. To ensure the RCF values incurred in this study did not cause cellular lysis of M. smegmatis, cells were tested before and after an acquisition time of 2 h at 2.5 kHz, 22 °C, by plating serial dilutions of cells on solid media. The number of viable cells was found to decrease by 96 scans to produce a 5790 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

spectrum with valuable data, as compared to 8 scans for spectra displayed in 3a, c, and d. Although this study addressed the detection of carbohydrates and lipids in bacterial cell walls, the techniques outlined in this report may also have implications for detecting other macromolecules for whole-cell NMR spectroscopy. Dotsch and co-workers have done extensive work on detecting conformation and dynamics of some overexpressed proteins in whole cells using liquidstate NMR probes.26-29 They explored different labeling strategies with 15N or 13C and their effects on the protein NMR spectral (26) Serber, Z.; Corsini, L.; Durst, F.; Dotsch, V. Methods Enzymol. 2005, 394, 17-41. (27) Serber, Z.; Straub, W.; Corsini, L.; Nomura, A. M.; Shimba, N.; Craik, C. S.; Ortiz de Montellano, P.; Dotsch, V. J. Am. Chem. Soc. 2004, 126, 71197125. (28) Serber, Z.; Ledwidge, R.; Miller, S. M.; Dotsch, V. J. Am. Chem. Soc. 2001, 123, 8895-8901.

Figure 4. HR-MAS HSQC of M. smegmatis whole cells at (a) 7, (b) 17, (c) 27, and (d) 37 °C. Panel set I displays the anomeric signals (δH 4.78-5.37, δC 97.8-108.3), and circles denote the t-R-manp (δH 4.94, δC 102.0), 2-6-R-manp (δH 5.06, δC 98.1), and 6-R-manp (δH 4.80, δC 99.3) residues. Panel set II displays the C2-C6 carbohydrate signals (δH 3.20-4.50, δC 59.0-88.0), and circles denote the C4 signals of t-R-manp (δH 3.98, δC 70.0) and 2,6-R-manp (δH 3.93, δC 78.4) residues.

quality. 15N or 13C selective labeling with specific amino acids showed great advantage to suppress the excessive background signals. The results from this HR-MAS NMR study also show the potential usefulness of selective labeling in discriminating different types of macromolecules. The experimental methods outlined in this report may be applicable to the study of other macromolecules, such as proteins in suitable biological systems. The flexibility of molecules in the cell wall is also of interest. There is some evidence indicating that properly designed experiments may be used to assess the flexibility of cell wall ultrastructure. Interestingly, when lyophilized cells or lyophilized cell wall preparations were analyzed by HR-MAS, they first had to be suspended in a volume greater than the volume used for analysis. For example, 10 mg of lyophilized cells was suspended in 1 mL of D2O and allowed to rest for 10 min. The sample was then centrifuged and resuspended in 100 µL D2O. If the lyophilized cells were simply resuspended into the final sample volume and were not given time to fully hydrate, certain signals in the spectra were very weak or absent completely, while other signals remained strong. However, the cells first suspended in a 10× volume of D2O resulted in signals equivalent to those of freshly grown cells. This indicates that analyses of molecules in the wall are subject to T2 effects and that molecules in an extremely rigid environment may not be detected in the spectra using the parameters reported herein. Additionally, the signal for the C-5 of β-araf of AG which has the mycolic acids attached to it is only visible in the whole-cell spectra of cells grown on solid media, not in the spectra of cells grown in liquid media. This may be due to a difference in the flexibility and ultrastructure of the cell wall when grown in different environments. Further experiments may be designed to take advantage of the differential mobility of cell wall molecules. Acquisition of spectra over a range of temperatures may also be used to probe the ultrastructure of the cell wall. It was (29) Serber, Z.; Keatinge-Clay, A. T.; Ledwidge, R.; Kelly, A. E.; Miller, S. M.; Dotsch, V. J. Am. Chem. Soc. 2001, 123, 2446-2447.

hypothesized that some cell wall components may have restricted motion at lower temperatures such that the T2 times are so short that signals for these molecules cannot be observed. These signals may appear and disappear as the temperature increases or decreases. Additionally, the chemical shifts from cell wall components were expected to display effects of temperature dependence on the basis of their differential solvent accessibility and macromolecular interactions. In the intact cells, the outer layer components are more accessible to the solvent and were expected to be more sensitive to the temperature changes. The inner layer components, which are more or less shielded by the outer layer, may be less sensitive to the temperature changes. To explore the utility of temperature variation for analysis of cell wall structure, HSQC was performed at four temperatures, 7, 17, 27, and 37 °C using whole M. smegmatis cells. (Figure 4). As expected, the signal intensity of all signals was lower at 7 °C and increased as the temperature increased; however changes in relative intensities between different molecules were observed. All four spectra were referenced in chemical shift and number of contours to the anomeric signal for β-araf (δH 5.04, δC 100.57). It was then observed that the signals corresponding to the mannose residues (t-R-manp, 6-R-manp, 2,6-R-manp) in the mannose core of LAM were not present at 7 °C, but appeared in increasing intensity as the temperature was raised. This was of interest because we had previously observed during whole-cell HR-MAS analysis of M. smegmatis and M. bovis BCG a disparity between the intensity of the mannose residues in these mycobacterial strains. LAM exists in mycobacteria as parietal or cellular LAM. It is thought that cellular LAM is inserted into the plasma membrane via its lipid anchor, whereas the parietal LAM is localized near the surface. The evidence suggested that perhaps not only did M. smegmatis have significantly less LAM associated with its cell wall than M. bovis BCG, but also the flexibility of the mannose residues in the LAM may be restricted. This would be expected if M. smegmatis had predominantly cellular LAMs inserted into the plasma membrane and the mannose core Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

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traversed through the rigid peptidoglycan. At 7 °C, the signals corresponding to the 2,6-R-manp, t-R-manp, and 6-R-manp residues were not observed in the spectrum. At 17 °C, only the t-R-manp residue was observed; however, at 27 and 37 °C, all three anomeric signals could be observed, and they increased in intensity from 27 to 37 °C. These results indicated an increase in flexibility of the molecule. Other signals in the cell wall were affected, such as the C-5 and C-6 of the galactan residues (5-β-galf: C-5 δH, 3.86; δC, 75.6; C-6 δH, 3.69; δC, 61.0. 6-β-galf: C-5 δH, 3.88; δC, 69.3; C-6 δH, 3.54, 3.79; δC, 69.1), which normally are uniformly strong irrespective of growth conditions, desiccation, or species diversity. However, in some cases, a decrease in the relative signal intensity was seen as the temperature increased (unassigned signal δH 4.39, δC 77.4). From these data, it was determined that careful consideration into the acquisition temperature was required, depending on the molecule of interest, and that further analysis is warranted on the T2 effects to establish whether this can be exploited for probing of the cell wall ultrastructure. CONCLUSION This study reports methods for the optimization and use of HR-MAS NMR for analysis of cell wall structures using intact bacterial cells. Further development in whole-cell HR-MAS NMR techniques may provide a more precise 3D structural model of bacterial cell walls by studying macromolecular conformations and elucidating their interactions in vivo. The key to the successful application of any whole cell NMR studies lies in improving the sensitivity and specificity. With the advances in NMR instrumenta-

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tion, particularly the continuous development of the cryogenic probes, and with the rapid development of techniques in molecular biology, HR-MAS whole-cell NMR may become a unique tool to study conformation, dynamics, and interactions among biologically important macromolecules in their native states. Studies to measure nuclear Overhauser effect (NOE) of macromolecules using various 3D NMR techniques in whole cells are currently underway in our laboratory. Although this study used mycobacteria as a model organism for method optimization, the strategies outlined in this report are applicable to other bacteria and fungi species. Complementary to genomic, proteomic, and classical biochemical techniques, whole-cell HR-MAS NMR may prove to be a powerful analytical tool in drug development, gene function assignment, and the study of virulence mechanisms. ACKNOWLEDGMENT We gratefully acknowledge helpful discussions with Dr. Richard Kriwacki and Dr. Weixing Zhang of St. Jude Children Research Hospital and Dr. Michael McNeil at Colorado State University. This work was supported in part by NIH grant AI054798 (R.E.L. and W.L.) and the College of Pharmacy at the University of Tennessee Health Science Center.

Received for review May 23, 2005. Accepted July 19, 2005. AC050906T