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Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan*

Tetsuya Yagi‡, Sebabrata Mahapatra, Katarína Mikušová§, Dean C. Crick, and Patrick J.

Brennan ¶

Department of Microbiology, Immunology and Pathology, Colorado State University,

Fort Collins, Colorado 80523-1682

‡ Present address: Department of Bacterial Pathogenesis and Infection Control, National

Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo, 208-0011, Japan

§ Present address: Dept. of Biochemistry, Faculty of Natural Sciences, Comenius University,

Mlynska dolina CH-1, 842 15 Bratislava, Slovakia.

*This work was supported by the National Institute of Allergy and Infectious Diseases,

NIH, Grants AI-18357 and AI-46393 to P. J. B. T. Y. was partially supported by the Japan

Health Sciences Foundation. 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.

¶ To whom correspondence should be addressed. Telephone: (970) 491-6700;

Fax: (970) 491-1815; E-mail: Patrick.Brennan@ColoState.edu

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 28, 2003 as Manuscript M302216200 by guest on Septem

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Running title: Ligation of Mycobacterial Arabinogalactan to Peptidoglycan

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SUMMARY

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan

chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(αα 1-3)-Rha linkage

unit (LU) and esterified to a variety of mycolic acids at the non-reducing 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 anti-bacterial drugs.

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INTRODUCTION

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), 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 non-reducing 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.

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EXPERIMENTAL PROCEDURES

Preparation of UDP-Galp Mutase, dTDP-Rha, P[14C]RPP and UDP-MurNAc-L-

Ala-D-Glu-mesoDAP-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 FAB-MS as follows. The recombinant E. coli MurC, MurD,

MurE, and MurF were over-expressed and purified from E. coli ER2566 as described (11) but

using the Impact CN system (New England Labs, Beverly, MA) 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 until

use. 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, deproteinated by

ultrafiltration, and the filtrate loaded on a 10 ml Q-Sepharose 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-peptide were identified by TLC on silica gel plates in

2-butyric acid–1M NH4OH (5:3), utilizing UV absorption and ninhydrin for detection. These

fractions were pooled and lyophilized to remove buffer, and the final product,

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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, Rockville, MD) was grown at 25°C in medium consisting of glucose

at 40 g/l and peptone at 10 g/l for 5 days (13, 14). The secreted muramidase was adsorbed

from crude culture filtrates with Amberlite CG-50-H+ (Sigma-Aldrich, St. Louis, MO)

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 percent 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

Pharmacia Biotech AB, Sweden) and fractions containing muramidase activity [measured by

the reduction in OD610 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 l of culture was

about 200 mg of enzyme. One unit of enzyme was defined as the amount of enzyme that

decreased the OD610 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 mid-log 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

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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 nmoles;

289 mCi/mmol; NEN Life Science Products, Inc., Boston, MA) 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, 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.

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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 (approximately

300,000 dpm per 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 (ca. 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 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 (Boehringer Mannheim, Indianapolis, IN) 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

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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.

Analytical Procedures—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-solublized 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 µ l 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

radiolabelled products was done using Novex 10-20% Tricine gels (Invitrogen, Carlsbad, CA)

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 for 1 h at 120 °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, Rockford,

IL).

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RESULTS

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 lipid

solubility, susceptibility to mild acid hydrolysis, the presence of the appropriate radiolabelled

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 1. 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 hierarchal 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

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of tri-Galf and tetra-Galf-containing glycolipid intermediates) (Fig. 1). No further simple

glycolipid intermediates were observed in the more polar CHCl3/CH3OH/H20 (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 and E-soak extracts

(Table 1), 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/H20 and E-soak extracts in incubations containing the additional precursors

UDP-MurNAc 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 and PRPP

precursors are added.

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 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

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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 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.

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.

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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.

Previously we had demonstrated that a mixture of Pol-P-P-[14C]GlcNAc and

Pol-P-P-[14C]GlcNAc-Rha are precursors of more glycosylated versions of lipid-linked

polymer intermediates which 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.

Sugar analysis of the insoluble MAPc-containing residue revealed the presence of

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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 β-1,4 linkage of PG and previously utilized for hydrolysis of

mycobacterial peptidoglycan (20), approximately 90% of the remaining radioactivity was

solubilized with significant loss of pellet volume. The radiolabelled 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 solublized

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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DISCUSSION

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 likely

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 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

β-D-Araf-(1→2)-α-D-Araf extensions from the 3-position of the terminal, branching

3,5-linked α-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/H20 and E-soak solunble 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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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 teichioc 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

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[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 solublized by Chalaropsis muramidase treatment. Chalaropsis

muramidase is a specific enzyme which hydrolyzes the β-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 cyloplasmic 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

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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 sets the stage for the complete enzymatic definition of AG-PG formation

and its genetic basis.

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Acknowledgements—We thank Dr. Michael R. McNeil, Michael S. Scherman, and their

colleagues for preparation of materials and helpful discussions.

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2. Crick, D. C., Mahapatra, S., and Brennan, P. J. (2001) Glycobiology 11, 107R-118R

3. Crick, D. C., and Brennan, P. J. (2000) Curr. Opin. Anti-Inf. Invest. Drugs 2, 154-163

4. Mikušová, K., Mikuš, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol.

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7. Wolucka, B. A., McNeil, M. R., de Hoffmann, E., Chojnacki, T., and Brennan, P. J. (1994)

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8. Scherman, M. S., Kalbe-Bournonville, L., Bush, D., Xin, Y., Deng, L., and McNeil, M.

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9. Lee, R., Monsey, D., Weston, A., Duncan, K., Rithner, C., and McNeil, M. (1996) Anal.

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13. Hash, J. H. (1963) Arch. Biochem. Biophys. 102, 379-388

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15. Angus, W. W., and Lester, R. L. (1972) Arch. Biochem. Biophys. 151, 483-495

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17. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 479-509

18. Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002

19. Turco, S. J., Wilkerson, M. A., and Clawson, D. R. (1984) J. Biol. Chem. 259, 3883-3889

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20. Wietzerbin, J., Das, B. C., Petit, J. F., Lederer, E., Leyh-Bouille, M., and Ghuysen, J. M.

(1974) Biochemistry 13, 3471-3476

21. Archibald, A. R., Hancock, I. C., and Harwood, C. R. (1993) in Bacillus subtilis and

Other Gram-Positive Bacteria; Biochemistry, Physiology and Molecular Genetics

(Sanenshein, A. L., Hoch, J. A., and Losick, R., eds.), pp. 381-410, ASM, Washington,

D.C.

22. McNeil, M. R. (1999) in Genetics of Bacterial Polysaccharides (Goldberg, J. B., ed)

pp. 207-223, CRC Press, Boca Raton, FL

23. Belanger, A.E., Besra, G. S., Ford, M. E., Mikušová, K., Belisle, J. T., Brennan, P. J., and

Inamine, J. M. (1996) Proc. Natl. Acad. Sci. USA 93, 11919-11924

24. 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., and Chatterjee, D. (2001) J. Biol. Chem. 276,

48854-48862

25. Baulard, A. R., Besra, G. S., and Brennan, P. J. (1999) in Mycobacteria: Molecular

Biology and Virulence (Ratledge, C., and Dale, J., eds.) pp. 240-259, Blackwell Science,

Oxford, U. K.

26. Mauck, J., and Glaser, L. (1972) J. Biol. Chem. 247, 1180-1187

27. Hancock, I. C. (1981) Eur. J. Biochem. 119, 85-90

28. Ward, J. B. (1974) Biochem. J. 141, 227-241

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29. Ward, J. B. (1981) Microbiol. Rev. 45, 211-243

30. Ward, J. B., and Curtis, C. A. M. (1982) Eur. J. Biochem. 122, 125-132

31. Schwebach, J. R., Glatman-Freedman, A., Gunther-Cummins, L., Dai, Z., Robbins, J. B.,

Schneerson, R., and Casadevall, A. (2002) Infect. Immun. 70, 2566-2575

32. Ratledge, C. (1982) in The Biology of the Mycobacteria, Vol. 1 (Ratledge, C., and

Stanford, J., eds), pp. 105-271, Academic Press, London

33. Anderson, R. G., Baddiley, J., and Hussey, H. (1972) Biochem. J. 127, 11-25

34. Fiedler, F., and Glaser, L. (1974) J. Biol. Chem. 249, 2690-2695

35. Leaver, J., Hancock, I. C., and Baddiley, J. (1981) J. Bacteriol. 146, 847-852

36. Watkinson, R. J., Hussey, H., and Baddiley, J. (1971) Nature New Biol. 229, 57-59

37. Pooley, H. M., and Karamata, D. (1994) in Bacterial Cell Walls (Ghuysen, J.-M., and

Hakenbeck, R., eds), pp. 187-198, Elsevier Science, B.V., Amsterdam

38. Hancock, I. C., Carman, S., Besra, G. S., Brennan, P. J., and Waite, E. (2002)

Microbiology 148, 3059-3067

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FOOTNOTES

1The abbreviations used are: Araf, arabinofuranose; AG, arabinogalactan; Galf,

galactofuranose; Galp, galactopyranose; GL, glycolipid; LU, linker unit; MOPS,

4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PG,

peptidoglycan; PRPP, 5-phosophoribose-pyrophosphate; Rha, rhamnose; dTDP,

deoxythymidine diphosphate; UDP-MurNAc-pentapeptide, uridine

diphosphoryl-N-acetylmuramate-L-Ala-D-Glu-mesoDAP-D-Ala-D-Ala; Tricine,

N-[2-hydroxy-1,1-bis(hydoxymethyl)ethyl]glycine.

2Unpublished data of Mills, J. A., Motichka, K., Jucker, M., Wu, H. P., Uhlic, B. C.,

Stern, R. J., Scherman, M. S., Vissa, V. D., Yan, W., Kundu, M., and McNeil, M. R. The cell

wall arabinogalactan linker formation enzyme, dTDP-Rha:- D-GlcNAc-diphosphoryl

polyprenol, -3-L-rhamnosyl transferase, is essential for mycobacterial viability.

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Table 1. Influence of UDP-MurNAc-PP and PRPP on the incorporation of [14C]Gal into organic solvent soluble and insoluble fractions.

*Values shown in parentheses are percent increase in radioactivity incorporated into the

insoluble residue relative to that seen in the basic reaction. The basic reaction mixture

consisted of 60 µM UDP-GlcNAc, 20 µM dTDP-Rha, 100 µM ATP, 1 µCi of UDP-[14C]Galp

preincubated with UDP-Galp mutase for 20 min at 37 °C, and enzyme (2 mg of protein) in 320

µl of buffer A. UDP-MurNAc-PP and PRPP were added at a final concentration of 200 µM

and 60 µM, respectively. After a 2 h incubation at 37 °C, reaction mixtures were subjected to

serial extractions with organic solvents to yield an insoluble residue as described in

"Experimental Procedures".

Reaction Mixture CHCl3 /CH3OH (2:1)

CHCl3 /CH3OH/H2O (10:10:3)

E-soak Insoluble Residue

(dpm/fraction)

Basic + UDP-MurNAc-PP + PRPP + UDP-MurNAc-PP and PRPP

16,000

14,000

17,000

13,000

400,000

280,000

240,000

190,000

270,000

110,000

190,000

140,000

2,200

3,000 (36%)*

3,700 (68%)

4,600 (109%)

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FIGURE LEGENDS

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 in

"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-1M ammonium acetate-H2O (180:140:9:9:23) and subjected to

autoradiography.

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 material are shown in panels A and B, respectively. Elution profiles of the

[14C]Ara containing CHCl3/CH3OH/H2O (10:10:3)- and E-soak-soluble material are shown in

panels C and D, respectively. Incubations were conducted as described in Fig. 1.

Fig. 3. Tricine SDS-PAGE analysis of radiolabeled, lipid-linked polymers.

Autoradiograms were exposed overnight (panel A) or for 14 days (panel 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

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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 were added, 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 side of the panel.

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 (panels

A-C respectively). Panel D shows 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-PP

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 in "Experimental Procedures."

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

panel A with a concomitant increase in [14C]Gal-containing material in the E-soak soluble

fraction (panel B) and the insoluble fraction (panel C). [14C]Gal-containing

CHCl3/CH3OH/H2O (10:10:3)-soluble lipid-linked polymers were prepared under ligation

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assay conditions as described in “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 of 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.

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 of the figure. Autoradiograms showing the results of sugar

analysis of each of the indicated fractions are shown on the right of the figure (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 in “Experimental Procedures” in order to remove covalently

attached mycolic acids. The residual pellet was then dried under N2, digested with 100 µg/ml

of 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 done as described in "Experimental Procedures."

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Fig. 7. Gel filtration profile of radiolabeled material solublized by Chalaropsis

muramidase. Aliquots (6000 dpm) of radiolabeled material solublized 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.

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 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-C (31). Ligation of AG to PG is described in this paper and is catalyzed by an

unidentified enzyme.

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1 2

Origin

Polyprenyl-P-Araf (DPA)

Solvent front

Polyprenyl-P-P-GlcNAc-Rha-GalfPolyprenyl-P-P-GlcNAc-Rha-Galf2Polyprenyl-P-P-GlcNAc-Rha-Galf3

1 2

Origin

Polyprenyl-P-Araf (DPA)

Solvent front

Polyprenyl-P-P-GlcNAc-Rha-GalfPolyprenyl-P-P-GlcNAc-Rha-Galf2Polyprenyl-P-P-GlcNAc-Rha-Galf3

Figure 1

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20 40 60 80

Rad

ioac

tivity

(dp

m)

0

50

100

150

200

250

Fraction

20 40 60 80

0

50

100

150

200

250

300 20 40 60 80

Radioactivity (dpm

)0

50

100

150

200

250

300

350

20 40 60 80

0

50

100

150

200

250

300

A

B

[14

C]Gal

CHCl3/CH3OH/H2O

(10:10:3)

[14C]Ara

CHCl3/CH3OH/H2O

(10:10:3)

[14

C]GalE-soak

[14

C]AraE-soak

C

D

Void Void

VoidVoid

Figure 2

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1 2 3 4 1 2 3 4

A B49.0

36.4

24.7

13.1

9.3

MWmarkers(kDa)

Figure 3

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X Data

0 4 8 12 16

0

20000

40000

60000

X Data

0 4 8 12 16

0

100000

200000

300000

400000

Time (h)

0 4 8 12 16

Rad

ioac

tivity

Inco

rpor

ated

(dp

m)

0

20000

40000

60000

80000

0 4 8 12 16

Radioactivity Incorporated (dpm

)

0

20000

40000

60000

CHCl3/CH3OH

(2:1)CHCl3/CH3OH/H2O

(10:10:3)

E-soak Insoluble residue

A

C

B

D

Figure 4

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180000

200000

220000

240000

50000

60000

70000

80000

CHCl3/CH3OH/H2O

(10:10:3)

E-soak

Ra

dio

act

ivity

De

tect

ed

(d

pm

)

0 4 8 12 16

0

1000

2000

3000 Insoluble residue

Time (h)

A

B

C

Figure 5

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Insoluble material(56,000 dpm)

Methanolic KOH treatment (residue, 42,000 dpm)

Residue(17,000 dpm)

Chalaropsismuramidasetreatment

Solublized material(25,000 dpm)

Glu (25%)

Gal (75%)

Glu (25%)

Gal (75%)

Glu (25%)

Gal (75%)

Glu (25%)

Gal (75%)

Glu (50%)

Gal (50%)

Glu (50%)

Gal (50%)

Proteinase Ktreatment

Solublized material(16,000 dpm)

Figure 6

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Fraction

20 40 60 80

Rad

ioac

tivity

(dp

m)

0

100

200

300

400

Void

Figure 7

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Polyprenyl-P

Polyprenyl-P-P-GlcNAc

Polyprenyl-P-P-GlcNAc-Rha

Polyprenyl-P-P-GlcNAc-Rha-Galf(x)

Polyprenyl-P-P-GlcNAc-Rha-Galf(x)-Araf(y)

Peptidoglycan-P-GlcNAc-Rha-Galf(x)-Araf(y)

GlcNAc 1-phosphoryltransferase

Rhamnosyl transferase(Rv3265c)

Galactofuranosyl transferase(Rv3808c)

ArabinofuranosylTransferases

(EmbA-C)

Ligase

UDP-GlcNAc

UMP

dTDP-Rha

dTDP

UDP-Galf

UDP

Polyprenyl-P-Araf

Polyprenyl-P

Peptidoglycan

Polyprenyl-P

Figure 8

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BrennanTetsuya Yagi, Sebabrata Mahapatra, Katarína Mikušová, Dean C. Crick and Patrick J.Polymerization of mycobacterial Arabinogalactan and ligation to peptidoglycan

published online April 28, 2003J. Biol. Chem. 

  10.1074/jbc.M302216200Access the most updated version of this article at doi:

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