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Originally published In Press as doi:10.1074/jbc.M302216200 on April 28, 2003

J. Biol. Chem., Vol. 278, Issue 29, 26497-26504, July 18, 2003
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Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan*

Tetsuya Yagi {ddagger}, Sebabrata Mahapatra, Katarína Mikusová §, Dean C. Crick and Patrick J. Brennan 

From the Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-1682

Received for publication, March 4, 2003 , and in revised form, April 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-({alpha}1–3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cell envelope of Mycobacterium tuberculosis is composed of a conventional plasma membrane and a cell wall proper unique to some genera within the Actinomycetales order, consisting of a core of arabinogalactan (AG),1 peptidoglycan (PG), and mycolic acids interspersed with a variety of free lipids, lipoglycans, and proteins (1); there is also evidence for polysaccharides on the outer face of the cell wall (2). The mycolic acids are attached to the nonreducing ends of the arabinogalactan, whereas the reducing ends are covalently attached to the cross-linked peptidoglycan via phosphoryl-N-acetylglucosaminosyl-rhamnosyl linkage units (P-GlcNAc-Rha). This massive structure, the mycolate-arabinogalactan-peptidoglycan-complex (MAPc), is the basis of many of the physiological and pathogenic features of M. tuberculosis and the site of susceptibility and resistance to many of the anti-tuberculosis drugs (3).

Biosynthesis of this complex commences with attachment of the residues of the linkage unit, GlcNAc-1-P and Rha, donated by UDP-GlcNAc and dTDP-Rha, respectively, to a polyprenyl phosphate (Pol-P) carrier lipid (4). Formation of the linkage unit is followed by the sequential addition of galactofuranosyl (Galf) units donated by UDP-Galf, to provide simple Pol-P-P-linked AG intermediates (4). The bulk, if not all, of galactan biosynthesis is catalyzed by a membrane-associated bifunctional galactosyltransferase capable of adding the alternating 5- and 6-linked Galf units (5, 6).

The demonstration that the direct donor of the arabinofuranosyl (Araf) units of the cell wall core is decaprenyl-P-Araf (7) and that 5-P-ribosyl-PP (PRPP) is a precursor of decaprenyl-P-Araf (8) now provides us with the means to characterize the subsequent polymerization steps in AG biosynthesis and the final ligation of the AG lipid-linked intermediates to PG to generate the fully formed cell wall core.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of UDP-Galp Mutase, dTDP-Rha, P[14C]RPP, and UDP-MurNAc-L-Ala-D-Glu-meso-DAP-D-Ala-D-Ala (UDP-MurNAc-pentapeptide)—Escherichia coli BL21 (DE3) (Stratagene, Cedar Creek, TX) was transformed with plasmid pORF6 containing Rv3809c as described (5). The recombinant UDP-Galp mutase was prepared and assayed as described (5); the concentration of protein in the final preparation was 2.0 mg per 100 µl. dTDP-Rha and P[14C]RPP were prepared from dTDP-Glc (9) and D-[U-14C]glucose (8), respectively, and were generous gifts from Dr. M. R. McNeil (Colorado State University).

For the synthesis of UDP-MurNAc-pentapeptide, UDP-MurNAc was first prepared by a two-step coupled enzymatic conversion of UDP-GlcNAc to UDP-MurNAc (10) and identified through negative ion fast atom bombardment mass spectrometry as follows. The recombinant E. coli MurC, MurD, MurE, and MurF were overexpressed and purified from E. coli ER2566 as described (11) but using the Impact CN system (New England Biolabs) following the manufacturer's instructions. The purified enzymes were dialyzed extensively against 50 mM Tris-HCl (pH 8.0) containing 10 mM MgCl2 and 10% glycerol (v/v), and aliquots were stored at –80 °C. Reaction mixtures (30 ml) containing UDP-MurNAc (250 µM), L-Ala, D-Glu, DAP, D-Ala-D-Ala (1 mM each), TAPS (50 mM, pH 8), MgCl2 (5 mM), ATP (2.5 mM), and MurC, MurD, MurE, and MurF (75 µg/ml each) were incubated at 30 °C overnight and deproteinated by ultrafiltration, and the filtrate was loaded on a 10-ml Q-Sepharose (Amersham Biosciences) column equilibrated with 20 mM ammonium acetate. The bound material was eluted with a 20–1000 mM gradient of ammonium acetate. Fractions were monitored at A262 for the presence of UDP-containing compounds. Fractions containing UDP-MurNAc-pentapeptide were identified by TLC on silica gel plates in 2-butyric acid/1 M NH4OH (5:3), utilizing UV absorption and ninhydrin for detection. These fractions were pooled and lyophilized to remove buffer, and the final product, UDP-MurNAc-pentapeptide, was analyzed by mass spectrometry as described (11, 12). In most syntheses, the rate of conversion of UDP-MurNAc to UDP-MurNAc-pentapeptide was about 80%.

Preparation of Chalaropsis Muramidase—Chalaropsis sp. ATCC 16003 (American Type Culture Collection, Manassas, VA) was grown at 25 °C in medium consisting of glucose at 40 g/liter and peptone at 10 g/liter for 5 days (13, 14). The secreted muramidase was adsorbed from crude culture filtrates with Amberlite CG-50-H+ (Sigma) buffered at pH 5.0; protein was eluted from the matrix with 0.5 M ammonium acetate and the muramidase was precipitated with ammonium sulfate at 70% saturation (13, 14). The precipitate was redissolved in 10 mM ammonium acetate (pH 6.5). After dialysis to remove residual ammonium sulfate, the sample was passed over a Sephadex G-75 column (Amersham Biosciences), and fractions containing muramidase activity (measured by the reduction in A610 of Staphylococcus aureus whole cell suspension (13, 14)) were pooled. Purity was checked by SDS-PAGE, and the enzyme preparation showed a single band in SDS-PAGE gels stained with Coomassie Brilliant Blue R250. Yield from 10 liters of culture was about 200 mg of enzyme. One unit of enzyme was defined as the amount of enzyme that decreased the A610 of a S. aureus cell suspension at a rate of 0.008 OD/min.

Preparation of an Enzymatically Active Cell Envelope Fraction from M. smegmatis—M. smegmatis mc2155 cells were grown in nutrient broth to midlog phase (4), harvested, and stored at –70 °C until required. Approximately 8 g of bacteria (wet weight) were washed with a buffer containing 50 mM MOPS (pH 8.0), 5 mM 2-mercaptoethanol, and 10 mM MgCl2 (buffer A), resuspended in 24 ml of buffer A at 4 °C, and subjected to probe sonication as described (4). The sonicate was centrifuged at 27,000 x g for 15 min at 4 °C, and the pellet, containing the cell envelope, was resuspended in buffer A to a final volume of 16 ml. Percoll was added to achieve a 60% suspension, and the mixture was centrifuged at 27,000 x g for 60 min at 4 °C. The particulate, upper band was collected, washed twice with buffer A, resuspended in buffer A to a protein concentration of 15–20 mg/ml, and used as the enzyme source in all experiments.

Reaction Mixtures for [14C]Gal Labeling and Fractionation of Reaction Products—The basic reaction mixtures for assessing [14C]Gal incorporation into lipid-linked AG precursors were prepared as follows. UDP-[U-14C]Galp (1 µCi; 3.5 nmol; 289 mCi/mmol; PerkinElmer Life Sciences) was dried under a stream of N2, dissolved in 38 µl of buffer A, and incubated with 2 µl of the UDP-Galp mutase preparation (0.13 mg of protein) at 37 °C for 15 min. Other reagents and buffer A were added to yield a final volume of 320 µl containing a 10.8 µM mixture of UDP-[U-14C]Galp and UDP-[U-14C]Galf, 60 µM UDP-GlcNAc, 20 µM dTDP-Rha, 100 µM ATP, and the envelope enzyme fraction (2 mg of protein). The reaction mixtures were incubated at 37 °C for the indicated period of time. In some cases, 60 µM PRPP and/or 200 µM UDP-MurNAc-pentapeptide were also included in the reaction mixtures. In the case of the ligation assays, these [14C]Gal labeling reaction mixtures containing both PRPP and UDP-MurNAc-pentapeptide were incubated at 28 °C for appropriate periods.

After incubation, reaction mixtures were extracted with CHCl3/CH3OH (2:1), the resultant pellet was washed thoroughly with 0.9% NaCl and extracted with CHCl3/CH3OH/H2O (10:10:3), followed by "E-soak" (water/ethanol/diethyl ether/pyridine/ammonium hydroxide; 15:15:5:1:0.017) (15) as described (16). The CHCl3/CH3OH (2:1) extract was partitioned with water (17). The backwashed lower (organic) phase was dried under a stream of N2, and the residue was dissolved in 200 µl of CHCl3/CH3OH/H2O/NH4OH (65:25:3.6:0.5) prior to liquid scintillation counting and analysis by TLC. In order to obtain a completely insoluble residue, rich in MAPc, the E-soak insoluble pellet was extracted three times with boiling 60% methanol containing 0.1% ammonium hydroxide.

To examine product-precursor relationships between lipid-linked intermediates and the insoluble residue, [14C]Gal-labeled CHCl3/CH3OH/ H2O (10:10:3)-soluble lipid-linked polymers were synthesized using the basic reaction conditions for [14C]Gal labeling described above. These enzymatically synthesized [14C]Gal-labeled compounds (~300,000 dpm/assay) were dried under N2, and resuspended in 100 µl of buffer A by bath sonication. Fresh enzyme (2 mg of protein), cold UDP-Galp preincubated with UDP-Galp mutase, UDP-GlcNAc, dTDP-Rha, ATP, PRPP, UDP-MurNAc-pentapeptide, and buffer A were added to achieve the same concentrations and volume used in the ligation assays. The resulting mixture was bath sonicated and incubated for periods of time up to 16 h. Reaction mixtures were extracted as described above.

Labeling of the Arabinan Component of AG with P[14C]RPP—The basic reaction mixture contained 3.3 µM P[14C]RPP (~600,000 dpm), 60 µM UDP-GlcNAc, 20 µM dTDP-Rha, 60 µM UDP-Galp preincubated with UDP-Galp mutase (as described above), 100 µM ATP, enzyme (2 mg of protein), and buffer A in a total volume of 320 µl. Reaction mixtures were incubated at 37 °C for 2 h, and fractionation of the reaction products was conducted as described above for [14C]Gal labeling.

Analysis of the Insoluble Residue—The insoluble residue, enriched in MAPc, was subjected to base treatment with 2 ml of 0.5% KOH in methanol for 4 days at 37 °C with gentle stirring. After washing three times with methanol, the methyl esters of the mycolic acids were removed with two diethyl ether extractions. The residual pellet was dried under N2 and digested with 100 µg/ml of Proteinase K (Roche Applied Science) in 250 µl of 10 mM sodium acetate (pH 7.5) at 37 °C overnight. Radioactivity released into the supernatant from the insoluble pellet by Proteinase K treatment was quantitated by liquid scintillation counting and subjected to sugar analysis as described below. After washing with 10 mM sodium acetate buffer, the residual pellet was treated with 2.5 units of purified Chalaropsis muramidase in 250 µl of 10 mM sodium acetate (pH 5.0), 500 units/ml of lysozyme in 10 mM Tris-HCl buffer (pH 7.5), or Proteinase K at 37 °C overnight. Aliquots of radiolabeled materials solubilized by these treatments were subjected to liquid scintillation counting and sugar analysis.

Analysis—In order to facilitate size exclusion chromatography of the polyprenyl-P-linked polymers, the CHCl3/CH3OH/H2O (10:10:3)-soluble, E-soak-soluble, and Chalaropsis muramidase-solubilized materials were hydrolyzed in mild acid as follows to selectively cleave the prenyl phosphate. Samples were suspended in 50 µl of 1-propanol by bath sonication, followed by 100 µl of 0.02 N HCl, and the resulting mixture was incubated for 30 min at 60 °C (18, 19). After neutralization with 10 µl of 0.2 N NaOH, the released water-soluble products were applied to a Biogel P-100 column (1 x 118 cm), equilibrated, and eluted with 100 mM ammonium acetate (pH 7.0). SDS-PAGE analysis of enzymatically radiolabeled products was done using Novex® 10–20% Tricine gels (Invitrogen) under conditions recommended by the manufacturer. After electrophoresis, samples were blotted to nitrocellulose membranes, which were dried at room temperature, and subjected to autoradiography. CHCl3/CH3OH (2:1)-soluble materials were analyzed on silica gel TLC plates developed in CHCl3/CH3OH/NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23), which were then subjected to autoradiography. For [14C]sugar analysis, samples were subjected to acid hydrolysis in 2 M CF3COOH for1hat120 °C. Hydrolysates were analyzed on silica gel TLC plates (silica gel G60, aluminum-backed; EM Science, Gibbstown, NJ), developed in pyridine/ethyl acetate/glacial acetic acid/water (5:5:1:3), and autoradiography. Radioactive spots were identified by comparative chromatography with standard sugars. Protein concentrations were estimated using the BCA protein assay reagent (Pierce).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis of Polyprenyl-P-linked Intermediates—We previously established a cell-free assay system using membranes from M. smegmatis for the synthesis of the simple Pol-P-P-GlcNAc, Pol-P-P-GlcNAc-Rha, and Pol-P-P-GlcNAc-Rha-(Galf)1–4 intermediates in AG biosynthesis (4). The evidence for the nature of these products was based on solubility in organic solvents, susceptibility to mild acid hydrolysis, the presence of the appropriate radiolabeled sugar, and pulse-chase experiments (5). These experiments led to the identification of one of the galactosyltransferases involved in galactan synthesis, a bifunctional enzyme capable of adding the majority of the alternating 5- and 6-linked Galf units (5, 6). In the present study, we modified this cell-free system in order to demonstrate the sequential synthesis of the simpler glycolipid intermediates, followed by polyprenyl-P-P-linked galactan and arabinan intermediates, and in vitro ligation of these lipid-linked arabinogalactan intermediates to PG. The cell wall-membrane fraction of M. smegmatis, the source of endogenous glycosyltransferases and polyprenyl-P, was supplemented with UDP-GlcNAc, dTDP-Rha, the precursors of LU, and UDP-[14C]Galp and UDP-Galp mutase as the source of the Galf units of galactan. Reaction products were extracted with the organic solvents, CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), and "E-soak" (15), and finally with boiling 60% methanol containing 0.1% ammonium hydroxide to remove residual soluble material, providing the insoluble MAPc-containing cell wall core. The incorporation of [14C]Galf from UDP-[14C]Galp into these four fractions is shown in Table I. Analysis of the CHCl3/CH3OH (2:1)-, CHCl3/CH3OH/H2O (10:10:3)-, and E-soak-soluble materials for 14C-labeled sugars showed that [14C]Gal was the sole radioactive sugar component (data not shown). TLC analysis of CHCl3/CH3OH (2:1)-soluble materials revealed a hierarchical array of glycolipids previously identified (5) as polyprenyl-P-P-GlcNAc-Rha-Galf, polyprenyl-P-P-GlcNAc-Rha-(Galf)2, and polyprenyl-P-P-GlcNAc-Rha-(Galf)3,4 (a mixture of tri-Galf- and tetra-Galf-containing glycolipid intermediates) (Fig. 1). No further simple glycolipid intermediates were observed in the more polar CHCl3/CH3OH/H2O (10:10:3) extract; the bulk of its radioactivity remained at the origin of the TLC plate (results not shown), supporting the evidence that this fraction contained the polyprenyl-P-P-GlcNAc-Rha-AG intermediates (5) (see below). The addition of exogenous PRPP and UDP-MurNAc-pentapeptide, precursors of arabinan and peptidoglycan synthesis, respectively, stimulated incorporation of [14C]Gal into the MAPc-containing residue in an additive manner with a concomitant reduction of radioactivity in the CHCl3/CH3OH/H2O (10:10:3) and E-soak extracts (Table I), suggesting that the polyprenyl-P-P-GlcNAc-Rha-(Gal)1–4 and the polyprenyl-P-P-GlcNAc-Rha-AG intermediates in these extracts were precursors of the mature PG-bound AG. However, the increase in insoluble material is not fully matched by a concomitant decrease in the other fractions. There is a substantial loss of radioactivity from the CHCl3/CH3OH/H2O (10:10:3) and E-soak extracts in incubations containing the additional precursors UDP-MurNAc-pentapeptide and PRPP. This is presumably due to a shortage of endogenous lipid carrier, required for de novo PG and decaprenyl-P-Araf synthesis when the UDP-MurNAc-pentapeptide and PRPP precursors are added.


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  		TABLE I
Influence of UDP-MurNAc-pentapeptide and PRPP on the incorporation of [14C]Gal into organic solvent-soluble and insoluble fractions

 


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  		FIG. 1.
TLC fractions from incubations containing either UDP-[14C]Gal or P[14C]RPP. Reaction mixtures contained a cell envelope preparation from M. smegmatis mc2155 (2 mg of protein) with 60 µM UDP-GlcNAc, 20 µM dTDP-Rha, 100 µM ATP, and 60 µM PRPP in 320 µl of buffer A and either 1 µCi (3.5 nmol) of UDP-[U-14C]Galp preincubated with the UDP-Galp mutase preparation or 3.3 µM P[14C]RPP (replacing the PRPP). Reaction mixtures were subjected to serial extractions with organic solvents as described under "Experimental Procedures." Aliquots of the CHCl3/CH3OH (2:1)-soluble materials labeled with UDP-[14C]Galp (lane 1) or P[14C]RPP (lane 2) were applied to TLC plates developed in CHCl3/CH3OH/NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23) and subjected to autoradiography.

 

Arabinan Polymerization Steps in AG Biosynthesis—To define the steps leading to the synthesis of the arabinan component of AG, cell-free reactions containing P[14C]RPP as the ultimate precursor of Araf were prepared in bulk and extracted with CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), and E-soak. Parallel reactions containing UDP-[14C]Galp were run, and similar extracts were prepared. Complete acid hydrolysis and TLC analysis for radioactive sugar showed that all of the [14C]Gal remained as such, and the majority of the P[14C]RPP radiolabel was converted into [14C]Ara-containing material; a minority appeared as [14C]ribose, apparently from intermediates of an unidentified riban (8). TLC of the CHCl3/CH3OH (2:1)-soluble products showed a preponderance of polyprenyl (C50)-P-Araf in this fraction (7) (Fig. 1). Mild acid hydrolysis of the [14C]Ara-labeled CHCl3/CH3OH/H2O (10:10:3)-soluble and E-soak-soluble lipid polymers to remove the presumed polyprenyl-P and subsequent gel filtration showed considerable overlap but not complete coincidence in the profiles of these two sets of [14C]Ara-containing polymers (Fig. 2). Profiles were similar to those of the [14C]Gal-labeled polymers, labeled, released, and extracted under similar conditions (Fig. 2). In both cases, the E-soak-extractable material appeared to be slightly but reproducibly larger than that extractable with CHCl3/CH3OH/H2O (10:10:3), pointing to the presence of a population of polyprenyl-P-linked AG intermediates, partially resolvable by the two extractants. These lipid-linked intermediates were also analyzed by Tricine SDS-PAGE (Fig. 3). Overnight exposure of autoradiograms revealed a population of [14C]Gal-labeled CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble lipid-linked polymers, whereas no images were seen in lanes containing [14C]Ara-labeled lipid-linked polymers, presumably due to lower labeling efficiency when using P[14C]RPP as the precursor. However, after 14 days of exposure, the [14C]Ara-containing CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble lipid-linked polymers were also visible on the autoradiograms and showed a degree of heterogeneity similar to that of the [14C]Gal-labeled material. The E-soak soluble [14C]Ara- and [14C]Galf-containing lipid polymers again appeared to be larger than the CHCl3/CH3OH/H2O (10:10:3)-soluble material, supporting the trend seen in the size exclusion analysis. Analysis for radioactive sugar content in these extracts confirmed the sole presence of [14C]Ara and [14C]Gal in the respectively labeled polymers.



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  		FIG. 2.
Gel filtration analysis of acid hydrolyzed, radiolabeled lipid-linked polymers. The elution profiles of [14C]Gal-labeled CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble materials are shown in A and B, respectively. Elution profiles of the [14C]Ara-containing CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble materials are shown in C and D, respectively. Incubations were conducted as described in Fig. 1.

 


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  		FIG. 3.
Tricine SDS-PAGE analysis of radiolabeled, lipid-linked polymers. Autoradiograms were exposed overnight (A) or for 14 days (B). Lane 1, [14C]Gal-containing CHCl3/CH3OH/H2O (10:10:3)-soluble materials; lane 2, [14C]Ara-containing CHCl3/CH3OH/H2O (10:10:3)-soluble materials; lane 3, [14C]Gal-containing E-soak-soluble materials; lane 4, [14C]Ara-containing E-soak-soluble materials. [14C]Gal- or [14C]Ara-containing lipid-linked polymers were prepared as described in the legend to Fig. 1. Identical volumes of each [14C]Gal- or [14C]Ara-containing material were dried, 10 µl of Tricine SDS buffer was added, and the mixture was boiled for 3 min and applied to Novex® 10–20% Tricine gels. After electrophoresis, those materials were electroblotted to a nitrocellulose membrane and exposed to x-ray film at –70 °C for the indicated periods. The migration positions of protein molecular weight markers are indicated at the left.

 

Evidence for in Vitro Ligation of AG to PG—The basic cell-free systems capable of catalyzing the synthesis of lipid-linked polymer intermediates of AG do not allow appreciable transfer of intermediates from the Pol-P carrier to PG. However, the addition of UDP-MurNAc-pentapeptide as a precursor of PG synthesis and longer incubation times resulted in linear incorporation of radioactivity into the insoluble residue over 16 h. Interestingly, incorporation of radioactivity into the E-soak-soluble fraction reached a plateau after 1 h (Fig. 4). Size exclusion, Tricine SDS-PAGE, and sugar analysis of the CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble products from 16-h incubations revealed that lipid-linked [14C]Gal-labeled polymers similar to those generated in the shorter incubation periods described above had been synthesized. Thus, incubation times up to 16 h were used for further characterization of this first time demonstration of in vitro ligation of cell wall polysaccharide to PG.



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  		FIG. 4.
Time-dependent incorporation of radioactivity into organic solvent soluble and insoluble fractions. Incorporation of [14C]Gal from UDP-[14C]Gal into CHCl3/CH3OH- (2:1), CHCl3/CH3OH/H2O- (10:10:3) and E-soak-soluble materials (A–C). D, incorporation of [14C]Gal into the insoluble residue. Reaction mixtures contained 1 µCi (3.5 nmol) of UDP-[U-14C]Galp preincubated with the UDP-Galp mutase preparation (0.13 mg of protein) at 37 °C for 20 min and 60 µM UDP-GlcNAc, 20 µM dTDP-Rha, 100 µM ATP, 60 µM PRPP, and 200 µM UDP-MurNAc-pentapeptide in 320 µl of buffer A and were incubated at 28 °C for the indicated periods of time. The reaction products were extracted serially with CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), E-soak, and boiling 60% methanol containing 0.1% ammonium hydroxide, to yield the insoluble residue as described under "Experimental Procedures."

 

Previously, we had demonstrated that Pol-P-P-[14C]GlcNAc and Pol-P-P-[14C]GlcNAc-Rha are precursors of more glycosylated versions of lipid-linked polymer intermediates that are soluble in CHCl3/CH3OH/H2O (10:10:3) and E-soak (5). Experiments in which [14C]Gal-prelabeled CHCl3/CH3OH/H2O (10:10:3)-soluble lipid-linked polymers were incubated with fresh enzyme and cold precursors for AG and PG synthesis were conducted. A comparison of radioactivity distributed among the three fractions showed that a linear decrease in the amount of [14C]Gal-labeled CHCl3/CH3OH/H2O (10:10:3)-soluble compounds (i.e. the lipid-linked polymers) was accompanied by an increase in the radioactivity found in both the E-soak- and the MAPc-containing residue (Fig. 5). The amount of radioactivity lost from the CHCl3/CH3OH/H2O (10:10:3) fraction was approximately equivalent to that appearing in the other fractions, indicating that the [14C]Gal-labeled CHCl3/CH3OH/H2O (10:10:3)-soluble precursors were converted into larger, more heavily glycosylated and insoluble products. Tricine SDS-PAGE analysis showed a size shift from CHCl3/CH3OH/H2O (10:10:3)-soluble precursor to E-soak-soluble products similar to that seen in Fig. 3. A small amount of radioactivity (0.4%) was also found in the CHCl3/CH3OH (2:1) fraction, suggesting that some degradation of the CHCl3/CH3OH/H2O (10:10:3)-soluble lipid-linked polymers had occurred.



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  		FIG. 5.
Incorporation of radiolabel from enzymatically synthesized CHCl3/CH3OH/H2O (10:10:3) soluble material into insoluble residue. A time-dependent decrease in [14C]Gal-containing CHCl3/CH3OH/H2O (10:10:3) soluble material is shown in A with a concomitant increase in [14C]Gal-containing material in the E-soak soluble fraction (B) and the insoluble fraction (C). [14C]Gal-containing CHCl3/CH3OH/H2O (10:10:3)-soluble lipid-linked polymers were prepared under ligation assay conditions as described under "Experimental Procedures." Aliquots containing 300,000 dpm of [14C]Gal were dried under N2; 100 µl of buffer A was added to each tube, and the lipid-linked material was emulsified by bath sonication. Fresh cell envelope fraction (2 mg of protein) with cold AG and PG precursors (namely UDP-Galp preincubated with the UDP-Galp mutase preparation, UDP-GlcNAc, dTDP-Rha, ATP, PRPP, UDP-MurNAc-pentapeptide, and buffer A) were added to the same concentrations as for the ligation assay in a total volume of 320 µl and incubated for the indicated periods of time. Reaction mixtures were extracted sequentially with CHCl3/CH3OH (2:1), CHCl3/CH3OH/H2O (10:10:3), E-soak, and boiling 60% methanol with 0.1% ammonium hydroxide.

 

Sugar analysis of the insoluble MAPc-containing residue revealed the presence of both [14C]Gal and [14C]Glc, indicating that some randomization of the radiolabel had occurred over the long incubation period and that both sugars had been incorporated into that fraction (Fig. 6). After base treatment of the final residue to remove mycolic acids, the pellet was treated with Proteinase K, which resulted in 60–65% solubilization of radiolabel without apparent loss of the volume of the pellet. Sugar analysis of the solubilized radioactive compounds revealed [14C]Gal and [14C]Glc in similar amounts. A second treatment with Proteinase K did not solubilize more of the remaining radioactivity. However, when the remaining insoluble residue was subjected to treatment with purified Chalaropsis muramidase, an enzyme known to hydrolyze the {beta}-1,4 linkage of PG and previously utilized for hydrolysis of mycobacterial peptidoglycan (20), ~90% of the remaining radioactivity was solubilized with significant loss of pellet volume. The radiolabeled sugars solubilized by muramidase treatment were identified as predominantly [14C]Gal and a smaller amount of [14C]Glc. Treatment with lysozyme released about 45% of the radioactivity in the insoluble pellet, with a visible reduction in pellet volume. Gel filtration analysis of the solubilized materials with purified Chalaropsis muramidase using a BioGel P-100 column (Fig. 7) showed that the radioactive material was larger than the polymers released from lipid-linked polymer intermediates of AG extracted by the various solvents (Fig. 2). Taken together, these results strongly suggest that the newly synthesized [14C]Gal-labeled AG was ligated to PG; in addition, the increased synthesis in the presence of UDP-MurNAc-pentapeptide may indicate that active PG synthesis is required for ligation.



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  		FIG. 6.
Analysis of radioactive compounds incorporated into the insoluble pellet. A flow chart tracking radioactivity incorporated from UDP-[14C]Galf through the analytical steps is shown on the left. Autoradiograms showing the results of sugar analysis of each of the indicated fractions are shown on the right (relative proportions of the sugars were estimated and are indicated in parentheses). The MAPc-containing residue was first hydrolyzed with 0.5% KOH in methanol, followed by extraction with diethyl ether as described under "Experimental Procedures" in order to remove covalently attached mycolic acids. The residual pellet was then dried under N2, digested with 100 µg/ml Proteinase K in 250 µl of 10 mM sodium acetate (pH 7.5) at 37 °C overnight. After washing with 10 mM sodium acetate buffer (pH 5.0), the residual pellet was treated with 2.5 units of purified Chalaropsis muramidase in 250 µl of 10 mM sodium acetate (pH 5.0) at 37 °C overnight. Sugar analysis of the insoluble residue, materials solubilized by Proteinase K and Chalaropsis muramidase was performed as described under "Experimental Procedures."

 


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  		FIG. 7.
Gel filtration profile of radiolabeled material solubilized by Chalaropsis muramidase. Aliquots (6000 dpm) of radiolabeled material solubilized by Chalaropsis muramidase after removing mycolic acids and treatment with Proteinase K were made up to 600 µl with 100 mM ammonium acetate (pH 7.0), applied to a Biogel P-100 column and eluted with 100 mM ammonium acetate (pH 7.0). Fractions of 1 ml were collected and counted. Mild acid hydrolysis of the radiolabeled material prior to gel filtration did not change the elution profile.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Synthesis of AG begins with the linkage unit, in a manner analogous to that of the cell wall teichoic acids of Gram-positive bacteria (21). GlcNAc-1-P is transferred to Pol-P from UDP-GlcNAc catalyzed by an as yet unidentified GlcNAc-1-P phosphotransferase; the closest homolog of wecA (formerly rfe) is Rv1302 (Fig. 8) (4, 5). This event is sequentially followed by the addition of Rha donated by dTDP-Rha, which is catalyzed by the rhamnosyl transferase Wbbl (Rv3265c) (22),2 and the addition of Galf residues donated by UDP-Galf. The only galactofuranosyl transferase recognized to date, Rv3808c, is reported to be a bifunctional enzyme capable of adding alternating 5- and 6-linked Galf residues (5, 6) and is probably responsible for the synthesis of bulk galactan; whether it is responsible for the synthesis of all of the galactan, especially the initial units, is not clear. The products of the emb operon were originally implicated in the transfer of the D-Araf units from the decaprenyl-P-Araf donor to the growing polymer (23); however, mutants in which embA and embB were inactivated by homologous recombination showed a selective deletion of the terminal {beta}-D-Araf-(1->2)-{alpha}-D-Araf extensions from the 3-position of the terminal, branching 3,5-linked {alpha}-D-Araf residues of AG (24), suggesting a role beyond simple arabinofuranosyl group transfer. Employing differential labeling with UDP-[14C]Gal and P[14C]RPP, as the sources of galactan and arabinan, respectively, we have now defined new aspects of the gross polymerization steps leading to the synthesis of AG and MAPc. Specifically, arabinan is added to galactan at the polyprenyl-linked stage, prior to ligation to PG, and the polyprenyl-linked AG polymers are intermediates in the synthesis of MAPc (Fig. 8). However, the evidence that these CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble intermediates are polyprenyl-P-linked is based on lipid solubility and susceptibility to mild acid hydrolysis (5), and is not unequivocal. Thus, the possibility of other intermediary steps, including transfer of intermediates to different carriers in the later stages of AG assembly, is possible.



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  		FIG. 8.
Proposed pathway for the biosynthesis of mycobacterial AG and ligation to PG. Synthesis of AG begins with the addition of a GlcNAc 1-phosphate to prenyl phosphate, a reaction catalyzed by an as yet unidentified GlcNAc 1-phosphate transferase, which is followed by the sequential addition of individual sugar residues. Rv3265c (WbbL) is a rhamnosyl transferase that adds a Rha residue from TDP-Rha. Rv3808c was identified as a bifunctional galactosyltransferase responsible for the synthesis of the majority of the galactan (5, 6). The arabinosyltransferases may be encoded by the ethambutol resistance genes, embA to -C (31). Ligation of AG to PG is described in this paper and is catalyzed by an unidentified enzyme.

 

Most significantly, we demonstrated ligation of the polyprenyl-linked [14C]Gal-labeled AG to PG in a cell-free system. The ligation of AG to PG involves the transphosphorylation of the terminal GlcNAc-1-P of the LU of the AG polymer from its polyprenyl-P carrier to the 6-position of the N-glycolylmuramic acid residues of PG (25). The reaction closely resembles the ligation of teichoic acid and other anionic polymers to PG in a wide range of Gram-positive bacteria and is a prime candidate for antibiotic intervention in the case of Staphylococcus and Streptococcus infections (21). In Bacillus subtilis 168, the attachment of cell wall teichoic acid or teichuronic acid to PG has been demonstrated in vivo and required the simultaneous synthesis of both polymers (26). This ligation has also been achieved in toluenized cells of B. subtilis W23 under conditions that reduced cell wall autolytic activity and was independent of de novo PG synthesis (27). Using cell wall-membrane preparations, Ward et al. (28–30) demonstrated the ligation of teichuronic acid to PG by formation of a phosphodiester bond between the reducing GalNAc terminus of the teichuronic acid and the 6-hydroxyl groups of muramic acid residues in the glycan chain of PG; a linkage unit was not involved, in contrast to that found for teichoic acids. Concomitant synthesis of both polymers was apparently necessary (30).

In general, the criterion for ligation of Gram-positive cell wall anionic polymers to peptidoglycan was insolubility in 4% boiling SDS (27–30). However, this treatment precludes the subsequent use of muramidase, susceptibility to which is clearly diagnostic for ligation of a target polymer to PG. Therefore, in the present work, extraction with refluxing 60% methanol containing 0.1% ammonium hydroxide was introduced; this treatment appeared to be more stringent than SDS extraction in that 60% more radioactivity was solubilized. It should be noted that Proteinase K treatment liberated compounds containing both [14C]Glc and [14C]Gal from the final solvent-insoluble pellet. Obviously, the long incubation times required for ligation resulted in epimerization of UDP-[14C]Gal to UDP-[14C]Glc and dispersal of the [14C]Glc. This observation suggests the association of glucosylated and galactosylated proteins with the cell wall core. Alternatively, the source may be residual glycans, which have been identified on the surface of mycobacteria (28), or glycogen storage granules (29), although the mechanism by which Proteinase K mediates the release of this type of material is not clear. Following Proteinase K digestion, the remaining radioactivity associated with the insoluble residue was successfully solubilized by Chalaropsis muramidase treatment. Chalaropsis muramidase is a specific enzyme that hydrolyzes the {beta}-1,4 linkage of PG (14, 20) much like lysozyme, which also liberated radioactivity from the insoluble pellet although at a much slower rate. These results provide strong evidence that the muramidase-solubilized radioactivity was ligated to PG, a conclusion supported by the observation that the material released by the Chalaropsis muramidase is larger than the lipid-linked polymers as judged by gel filtration analysis.

The ligation reaction is clearly complex, requiring the admixture of enzymes from different cellular compartments present in wall-membrane preparations. Although there has been no detailed study of the organization of cell wall synthetic enzymes at the molecular level in Gram-positive bacteria or mycobacteria, diverse investigations of Gram-positive bacteria (21, 26, 33–36) lead to the conclusion that the cytoplasmic membrane contains ordered assemblies of the enzymes of polymer and PG synthesis together with shared anchor lipids. Attachment of PG and anionic polymer almost certainly occurs at the outer surface of the membrane. However, in the case of mycobacteria, membranes alone will not accomplish the task. Despite the availability of the genome of B. subtilis, the gene(s) involved in teichoic acid ligation remain unidentified, although tagA and tagB were regarded as candidates at one time (37).

It should be noted that mycolic acids were removed at the first step in analysis of the final residue from the M. smegmatis incubations in order to facilitate effective digestion with Chalaropsis muramidase. Thus, it remains to be seen whether they are attached to AG before or after ligation to PG. However, recent observations through whole cell labeling with different precursors suggest that mycolylation of arabinan termini follows ligation of AG to PG (38).

The previous identification of a bifunctional membranous galactofuranosyl transferase, the fact that the embA-C operon may encode the capability of both Araf addition and polymerization, the present development of cell-free AG polymerization, and subsequent ligation assays now set the stage for the complete enzymatic definition of AG-PG formation and its genetic basis.


   FOOTNOTES
 
* This work was supported by NIAID, National Institutes of Health, Grants AI-18357 and AI-46393 (to P. J. B.) and AI-49151 (to D. C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported in part by the Japan Health Sciences Foundation. Present address: Dept. of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan. Back

§ Present address: Dept. of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, Bratislava 842 15, Slovakia. Back

To whom correspondence should be addressed. Tel.: 970-491-6700; Fax: 970-491-1815; E-mail: Patrick.Brennan{at}ColoState.edu.

1 The abbreviations used are: AG, arabinogalactan; Galf, galactofuranose; Araf, arabinofuranose; Galp, galactopyranose; GL, glycolipid; LU, linker unit; MOPS, 4-morpholinepropanesulfonic acid; PG, peptidoglycan; PRPP, 5-phosphoribose-pyrophosphate; Rha, rhamnose; UDP-MurNAc-pentapeptide, uridine diphosphoryl-N-acetylmuramate-L-Ala-D-Glu-meso-DAP-D-Ala-D-Ala; Tricine, N-[2-hydroxy-1,1-bis(hydoxymethyl)ethyl]glycine; MAPc, mycolate-arabinogalactan-peptidoglycan-complex; Pol-P, polyprenyl phosphate; MurNAc, N-acetylmuramic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; DAP, 2,6-diaminopimelic acid. Back

2 The cell wall arabinogalactan linker formation enzyme, dTDP-Rha: GlcNAc 1-diphosphoryl polyprenol rhamnosyltransferase, is essential for mycobacterial viability (J. A. Mills, K. Motichka, M. Jucker, H. P. Wu, B. C. Uhlic, R. J. Stern, M. S. Scherman, V. D. Vissa, W. Yan, M. Kundu, and M. R. McNeil, unpublished data). Back


   ACKNOWLEDGMENTS
 
We thank Dr. Michael R. McNeil, Michael S. Scherman, and their colleagues for preparation of materials and helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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