Adjuvant Activity of Mycobacterial Fractions

The addition of adjuvant (Bacillus Calmette–Guérin-cell wall skeleton; BCG-CWS) to the culture medium substantially increased the immune response of mouse spleen cells to immunization with heterologous erythrocytes and hapten–protein conjugate in vitro. Cell walls of mycobacteria, nocardia and corynebacteria and their cell wall constituents were used as adjuvants. In the present case, it was found that cell walls of mycobacteria, nocardia and corynebacteria markedly facilitated primary humoral response of mouse spleen cells to heterologous erythrocytes in vitro. The adjuvant effect of BCG-CWS was present only in the formation of 19 S antibody in both primary and secondary responses, but not in that of 7 S antibody in vitro. Primary antihapten response of mouse spleen cells against dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) also succeeded when BCG-CWS was added to the culture medium, and it was found that BCG-CWS increased the helper activity of carrier specific helper T cells in vitro where a double chamber system, separated by a cell-impermeable nucleopore membrane, was used. This result suggested that BCG-CWS acts on T cells, resulting in the release of soluble factor(s) from T cells capable of exerting an adjuvant effect. Furthermore, mucopeptide moiety of BCG-CWS retained some adjuvant activity, but other cell wall constituents, such as mycolic acid, arabinose mycolate, and arabinogalactan, did not show any adjuvant effect in vitro. These results strongly imply that mucopeptide moiety of BCG-CWS plays an important role in the development of adjuvanticity.     

Branched arabino-oligosaccharides isolated from sugar beet arabinan

Sugar beet arabinan consists of an α-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with α-(1,2)- and/or α-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an α-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted α-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted α-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.     

Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.     

Mode of action of Chrysosporium lucknowense C1 α-l-arabinohydrolases

The mode of action of four Chrysosporium lucknowense C1 α-L-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers.     

Purification and properties of two type-B alpha-L-arabinofuranosidases produced by Penicillium chrysogenum

Two distinct extracellular alpha-L-arabinofuranosidases (AFases; EC 3.2.1.55) were purified from the culture filtrate of Penicillium chrysogenum 31B. The molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa (AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 degrees C and were stable up to 50 degrees C. Enzyme activities of AFQ1 and AFS1 were highest at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1. Both enzymes displayed broad substrate specificities; they released arabinose from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and arabino-oligosaccharides. AFS1 also showed low activity towards p-nitrophenyl-beta-D-xylopyranoside. An exo-arabinanase, which catalyzes the release of arabinobiose from linear arabinan at the nonreducing terminus, acted synergistically with both enzymes to produce L-arabinose from branched arabinan.     

Substrate specificity of the alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The alpha-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1-->5)-alpha-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [alpha-(1-->5)-linked] of the arabinan backbone rather than the arabinosyl side chain [alpha-(1-->3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1-->5)-Araf, T-Araf, (1-->3, 5)-Araf and (1-->3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1-->5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [alpha-(1-->5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the alpha-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this alpha-L-arabinofuranosidase can only hydrolyse alpha-L-arabinofuranosyl residues of arabinan.     

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.     

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.     

ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis

The function of a putative glycosyltransferase (At2g35100) was investigated in Arabidopsis (Arabidopsis thaliana). The protein is predicted to be a type 2 membrane protein with a signal anchor. Two independent mutant lines with T-DNA insertion in the ARABINAN DEFICIENT 1 (ARAD1) gene were analyzed. The gene was shown to be expressed in all tissues but particularly in vascular tissues of leaves and stems. Analysis of cell wall polysaccharides isolated from leaves and stems showed that arabinose content was reduced to about 75% and 46%, respectively, of wild-type levels. Immunohistochemical analysis indicated a specific decrease in arabinan with no change in other pectic domains or in glycoproteins. The cellular structure of the stem was also not altered. Isolated rhamnogalacturonan I from mutant tissues contained only about 30% of the wild-type amount of arabinose, confirming the specific deficiency in arabinan. Linkage analysis showed that the small amount of arabinan present in mutant tissue was structurally similar to that of the wild type. Transformation of mutant plants with the ARAD1 gene driven by the 35S promoter led to full complementation of the phenotype, but none of the transformants had more arabinan than the wild-type level. The data suggest that ARAD1 is an arabinan alpha-1,5-arabinosyltransferase. To our knowledge, the identification of other L-arabinosyltransferases has not been published.     

Purification, functional characterization, cloning, and identification of mutants of a seed-specific arabinan hydrolase in Arabidopsis

This work describes the purification and characterization of an enzyme that exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) as a putative beta-D-xylosidase that belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3 hydrolysed synthetic substrates such as p-nitrophenyl-alpha-L-arabinofuranoside and p-nitrophenyl-beta-D-xyloside with similar catalytic efficiency. XYL3 released L-arabinose from (1-->5)-alpha-L-arabinofuranobiose, arabinoxylan, sugar beet arabinan, and debranched arabinan. The enzyme hydrolysed both arabinosyl-substituted side group residues and terminal arabinofuranosyl residues (1-->5)-alpha-linked to the arabinan backbone. This indicates that XYL3 is able to degrade all terminal arabinosyl residues and suggests that it participates in the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by semi-quantitative RT-PCR, in-situ hybridization and a promoter-GUS fusion demonstrated that AtBX3 was specifically expressed in the seed endosperm at the globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera found that arabinan, the XYL3 substrate, was also present in this seed tissue. T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the alpha-L-arabinofuranosidase and beta-D-xylosidase activities corresponding to XYL3. Mutants showed reduced seed size and are delayed in seedling germination compared with the wild type.     

Structural investigations of an arabinan from grape juice

An arabinan has been isolated from grape juice and purified by chromatography on polyamide and repeated ethanol precipitations. The structural identity of the arabinan was established by enzymatic degradation of the polysaccharide with a purified -L-arabinofuranosidase and methylation analysis. The results obtained suggest that the arabinan consists of an (1 → 5)-linked backbone of L-arabinofuranosyl residues to which sidechains of L-arabinose are attached in the 3-position.     

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

AIMS: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. MATERIALS AND METHODS: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. RESULTS: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. CONCLUSIONS: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.     

Postoperative adjuvant immunotherapy of gastric cancer with BCG-cell wall skeleton

The clinical effectiveness of immunotherapy with the cell wall skeleton of Bacillus Calmette-Guérin was assessed in a study involving 140 consecutive patients with gastric cancer, who were gastrectomized at a single institution from January 1976 through December 1978. These patients were randomized by an envelope method after operation and divided into three treatment groups: 'control', 'chemotherapy', and 'chemotherapy plus immunotherapy with BCG-CWS.' Only two patients who died during surgery were excluded, and a survey of survival periods was made on the remaining 138 patients in January 1982. As a result, statistically significant differences in the survival rate curve were observed between the control and chemotherapy plus immunotherapy groups (P less than 0.01), and between the chemotherapy and chemotherapy plus immunotherapy groups (P less than 0.05). These results emphasize effectiveness of BCG-CWS as an adjuvant immunotherapeutic agent in gastrectomized cancer patients.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii alpha-L-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant alpha-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn(83)-Thr-Thr and Asn(202)-Ser-Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn(83), Asn(202), and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn(202) may contribute to thermostability and catalysis.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N-terminal recognition region and a C-terminal synthetic region

The arabinans of the mycobacterial cell wall are key structural and immunological polymers in the context of arabinogalactan (AG) and lipoarabinomannan (LAM) respectively. The three homologous membrane proteins EmbA, EmbB and EmbC are known to be involved in the synthesis of arabinan but their biochemical functions are not understood. Herein we show, that synthesis of LAM, but not AG, ceases after inactivation of embC in Mycobacterium smegmatis by insertional mutagenesis. LAM synthesis is restored upon complementation with the embC wild-type gene. Previously we have shown that the synthesis of the arabinan of AG is affected by embA or embB disruption. Thus the Emb proteins are capable of differential recognition of the galactan or mannan acceptors prior to appropriate arabinosylation. In addition, a combination of genetic and biochemical approaches have allowed us to assign some specific functions to the regions of emb gene products. Complementation of the embCmacr; mutant with a hybrid gene encoding the N-terminus of EmbC and the C-terminus of EmbB resulted in LAM with a lower molecular weight than the wild-type LAM. Structural studies involving enzyme digestion, chromatography and mass spectrometry analyses revealed that the arabinan of the 'LAM' formed in the hybrid was of AG kind rather than LAM type of arabinan.