Cell Wall Arabinan Research Articles

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or β-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to β-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

Structural insight into mycobacterial cell wall biosynthesis by AftB, a potential anti-TB drug target.

Tuberculosis (TB) is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a significant health problem worldwide. The success of Mtb as the leading pathogen can largely be attributed to its atypical multilayered cell wall, which is highly impermeable and confers natural protection against attack from the immune system and many antibiotics. Due to its unique character, the mycobacterial cell wall has become the most widely exploited target of anti-TB drugs. AftB is an arabinofuranosyltransferase that functions in the terminal step of cell wall arabinan biosynthesis, catalyzing the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) onto the cell wall polysaccharides. AftB is essential for Mtb's survival and is deemed a potential new drug target for TB. Here we report the atomic-resolution structures of AftB obtained by cryo-EM in both apo and donor substrate analog bound forms. The apo structure revealed that AftB contains 11 transmembrane helices belonging to the GT-C glycosyltransferase superfamily and a periplasmic segment of a novel fold which might direct the binding of polysaccharides acceptor. AftB forms a large putative substrate-binding cavity, with one side facing the periplasmic domain and the other facing the lipid bilayer. The substrate-bound structure further unmasked the nature of the active site. Using synthetic AftB donor and acceptor substrates, we recapitulated the enzymatic reaction in vitro. And the functional analysis using AftB variants further proved the importance of the active site revealed from our structures. We proposed that AftB catalyzes the glycosylation reaction by utilizing a conserved aspartate residue as the base catalyst. Collectively, our work provides a structural basis for understanding the molecular mechanism of AftB and a blueprint for developing new drugs to combat the increasing threat caused by Mtb.

The Gene and Regulatory Network Involved in Ethambutol Resistance in Mycobacterium tuberculosis.

Ethambutol (EMB) is used in combination with isoniazid and rifampicin for the treatment of tuberculosis caused by Mycobacterium tuberculosis. However, the incidence of EMB resistance is alarming. The EMB targets the cell wall arabinan biosynthesis. It is important to comprehensively understand the molecular basis of EMB to slow down the drug resistance rate of EMB. This study summarized the genes implicated in EMB resistance, regulatory network and the pharmacoproteomic effect of EMB in M. tuberculosis. Many of the genes related to EMB are implicated in membrane components, drug efflux, lipid metabolism, ribosome, and detoxification. The differential response model may help to design a novel anti-tuberculosis antibiotic.

Bacterial cell wall modification by glycolipid probes

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. The structures of mammalian glycans can be perturbed through metabolic incorporation, in which non‐natural sugars are taken up by cells, converted to nucleotide‐sugar intermediates, and incorporated via biosynthetic pathways. Applications of metabolic incorporation in bacteria are complicated by the diversity of pathways bacteria use to acquire, metabolize, and catabolize monosaccharide intermediates. We developed a strategy to alter glycan structure that avoids intracellular processing using lipid‐linked glycans, intermediates directly used in glycan biosynthesis. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified glycolipid donors whose addition restores cell wall arabinan, demonstrating that non‐natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. Our lipid‐based probe could rescue wild type cells treated with an inhibitor of cell wall biosynthesis. We then focused on incorporating derivatives with chemical handles for labeling such that bacterial glycans can be captured and visualized. To this end, we have generated the first probe to label arabinose‐containing structures in mycobacteria and corynebacteria.Support or Funding InformationNIH: RO1‐AI063596, NSERC PGS‐D

Bacterial Cell Wall Modification with a Glycolipid Substrate.

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. Mammalian glycans can be modulated using metabolic incorporation, a process in which non-natural sugars are taken up by cells, converted to nucleotide-sugar intermediates, and incorporated into glycans via biosynthetic pathways. These studies have revealed that glycan intermediates can be shunted through multiple pathways, and this complexity can be heightened in bacteria, as they can catabolize diverse glycans. We sought to develop a strategy that probes structures recalcitrant to metabolic incorporation and that complements approaches focused on nucleotide sugars. We reasoned that lipid-linked glycans, which are intermediates directly used in glycan biosynthesis, would offer an alternative. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified synthetic glycosyl donors whose addition restores cell wall arabinan, demonstrating that non-natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. The addition of an isotopically labeled glycan substrate facilitated cell wall characterization by NMR. Structural analysis revealed that all five known arabinofuranosyl transferases could process the exogenous lipid-linked sugar donor, allowing for the full recovery of the cell envelope. The lipid-based probe could also rescue wild-type cells treated with an inhibitor of cell wall biosynthesis. Our data indicate that surrogates of natural lipid-linked glycans can intervene in the cell's traditional workflow, indicating that biosynthetic incorporation is a powerful strategy for probing glycan structure and function.

The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

Mechanistic Strategies for Catalysis Adopted by Evolutionary Distinct Family 43 Arabinanases

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

Benzothiazinones Mediate Killing of Corynebacterineae by Blocking Decaprenyl Phosphate Recycling Involved in Cell Wall Biosynthesis

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.

Development of a plate-based scintillation proximity assay for the mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis

A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD have been characterized and implicated to be involved in the cell wall arabinan assembly. These arabinosyltransferases are essential for the viability of the organism and are logically valid targets for developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug, targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among these arabinosyltransferases, the terminal β-(1→2) arabinosyltransferase activity has been associated with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmembrane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide, α- d-Ara f-(1→5)-α- d-Ara f-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was identified as the β-(1→2) arabinofuranose adduct. When synthetic tri- and tetra-saccharides were used as substrates, a mixture of products containing both β-(1→2) and α-(1→5) linkages were formed. Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan l. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non-cellulosic polysaccharides of embryos. Arabinose levels decline to approximately 15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.

Partial redundancy in the synthesis of the d-arabinose incorporated in the cell wall arabinan of Corynebacterineae

The major cell wall carbohydrate of Corynebacterineae is arabinogalactan (AG), a branched polysaccharide that is essential for the physiology of these bacteria. Decaprenylphosphoryl-D-arabinose (DPA), the lipid donor of D-arabinofuranosyl residues of AG, is synthesized through a series of unique biosynthetic steps, the last one being the epimerization of decaprenylphosphoryl-beta-D-ribose (DPR) into DPA, which is believed to proceed via a sequential oxidation-reduction mechanism. Two proteins from Mycobacterium tuberculosis (Rv3790 and Rv3791) have been shown to catalyse this epimerization in an in vitro system. The present study addressed the exact function of these proteins through the inactivation of the corresponding orthologues in Corynebacterium glutamicum (NCgl0187 and NCgl0186, respectively) and the analysis of their in vivo effects on AG biosynthesis. We showed that NCgl0187 is essential, whereas NCgl0186 is not. Deletion of NCgl0186 led to a mutant possessing an AG that contained half the arabinose and rhamnose, and less corynomycolates linked to AG but more trehalose mycolates, compared with the parental strain. A candidate gene that may encode a protein functionally similar to NCgl0186 was identified in both C. glutamicum (NCgl1429) and M. tuberculosis (Rv2073c). While the deletion of NCgl1429 had no effect on AG biosynthesis of the mutant, the gene could complement the mycolate defect of the AG of the NCgl0186 mutant, strongly supporting the concept that the two proteins play a similar function in vivo. Consistent with this, the NCgl1429 gene appeared to be essential in the NCgl0186-inactivated mutant. A detailed bioinformatics analysis showed that NCgl1429, NCgl0186, Rv3791 and Rv2073c could constitute, with 52 other proteins belonging to the actinomycetales, a group of closely related short-chain reductases/dehydrogenases (SDRs) with atypical motifs. We propose that the epimerization of DPR to DPA involves three enzymes that catalyse two distinct steps, each being essential for the viability of the bacterial cells.

Dissection of cell wall arabinans in relation to cell functions and plant growth

Dissection of cell wall arabinans in relation to cell functions and plant growth

Transfer of the First Arabinofuranose Residue to Galactan Is Essential for Mycobacterium smegmatis Viability

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic beta-d-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-octyl and beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when alpha-d-Manp-(1-->6)-alpha-D-Manp-(1-->6)-alpha-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.

A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps

Mycobacteria, including Mycobacterium tuberculosis, are characterized by a unique cell wall rich in complex lipids, glycolipids, polyketides, and terpenoids. Many of these metabolites have been shown to play important roles in mycobacterial virulence and their inherent resistance to many antibiotics. Here, we report the development of a new simple method for global analysis of these metabolites using two-dimensional (1)H-(13)C heteronuclear single quantum coherence nuclear magnetic resonance. The major advantages of this method are as follows: the small amount of sample and the minimal sample manipulation required; a relatively short procedural time; and the ability to rapidly attain a qualitative and quantitative lipid profile of a mycobacterial sample in which the majority of the clinically relevant lipids can be observed simultaneously. The effectiveness of this method is demonstrated in four different areas of major concern to the mycobacterial research community: i) adaptive changes in cell wall lipids as a result of drug treatment; ii) analysis of gene function; iii) characterization of new mycobacterial species; and iv) analysis of the production of virulence factors in clinical isolates of M. tuberculosis. This method is complementary to mass spectrometry-based lipidomic technologies and provides an urgently needed tool to gain a better understanding of the role of lipids in mycobacteria pathogenesis.

Characterization of a Distinct Arabinofuranosyltransferase in Mycobacterium smegmatis

The D-arabinans in Mycobacterium are essential, extraordinarily complex entity comprised of d-arabinofuranose residues which are rarely found in nature. Despite the well-recognized importance of the mycobacterial arabinan, delineation of the arabinosylation process has been severely hampered due to lack of positively identified arabinosyltransferases. Identification of genes involved in arabinan biosynthesis entailed the use of ethambutol (EMB), a first-line antituberculosis agent that is known to inhibit cell wall arabinan synthesis. The three genes (embA, embB, and embC) encode novel membrane proteins, implicated as the only known mycobacterial arabinosyltransferases to this date. We have now adapted a multifaceted approach involving development of convenient arabinosyltransferase assay using novel synthetic acceptors to identify arabinosyltransferase/s that will be distinct from the Emb proteins. In our present work, Mycobacterium smegmatis mc(2) 155 (WTMsm) was used as a model to study the biosynthesis of cell wall arabinan. In an in vitro assay, we demonstrate that transfer of only alpha-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [alpha-D-Araf](2)-3,5-alpha-D-Araf-(1-->5)-alpha-d-Araf-(1-->5)-alpha-D-Araf with an octyl aglycon. Higher molecular weight (up to Ara(10)) oligomers were also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of these products revealed that isomeric products were formed and initiation and elongation of arabinan can occur either on the 5-arm or 3-arm of the branched 3,5-alpha-D-Araf. Individual embA, embB, and embC knockout strains retained this alpha-1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol. This particular enzyme function is distinct from the function of the Emb proteins.

Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan, and the anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB. Herein, we describe the identification and characterization of a novel arabinofuranosyltransferase, now termed AftB (Rv3805c), which is essential in Mycobacterium tuberculosis. Deletion of its orthologue NCgl2780 in the closely related species Corynebacterium glutamicum resulted in a viable mutant. Analysis of the cell wall-associated lipids from the deletion mutant revealed a decreased abundance of cell wall-bound mycolic acids, consistent with a partial loss of mycolylation sites. Subsequent glycosyl linkage analysis of arabinogalactan also revealed the complete absence of terminal beta(1 --> 2)-linked arabinofuranosyl residues. The deletion mutant biochemical phenotype was fully complemented by either Mt-AftB or Cg-AftB, but not with muteins of Mt-AftB, where the two adjacent aspartic acid residues, which have been suggested to be involved in glycosyltransferase activity, were replaced by alanine. In addition, the use of C. glutamicum and C. glutamicumDeltaaftB in an in vitro assay utilizing the sugar donor beta-D-arabinofuranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor alpha-D-Araf-(1 --> 5)-alpha-D-Araf-O-C(8) as a substrate confirmed AftB as a terminal beta(1 --> 2) arabinofuranosyltransferase, which was also insensitive to EMB. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and Mt-AftB represents a potential new drug target.

Identification of a Novel Arabinofuranosyltransferase (AftA) Involved in Cell Wall Arabinan Biosynthesis in Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.

Development of a Quantitative Assay for Mycobacterial Endogenous Arabinase and Ensuing Studies of Arabinase Levels and Arabinan Metabolism in Mycobacterium smegmatis

Treatment of either Mycobacterium tuberculosis or M. smegmatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [(14)C]glucose, followed by a "chase" with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.

Cell wall arabinan is essential for guard cell function.

Stomatal guard cells play a key role in the ability of plants to survive on dry land, because their movements regulate the exchange of gases and water vapor between the external environment and the interior of the plant. The walls of these cells are exceptionally strong and must undergo large and reversible deformation during stomatal opening and closing. The molecular basis of the unique strength and flexibility of guard cell walls is unknown. We show that degradation of cell wall arabinan prevents either stomatal opening or closing. This locking of guard cell wall movements can be reversed if homogalacturonan is subsequently removed from the wall. We suggest that arabinans maintain flexibility in the cell wall by preventing homogalacturonan polymers from forming tight associations.