Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Functional analysis of fasciclin-like arabinogalactan in carotenoid synthesis during tomato fruit ripening.

Carotenoids are important pigmented nutrients synthesized by tomato fruits during ripening. To reveal the molecular mechanism underlying carotenoid synthesis during tomato fruit ripening, we analyzed carotenoid metabolites and transcriptomes in six development stages of tomato fruits. A total of thirty different carotenoids were detected and quantified in tomato fruits from 10 to 60 DPA. Based on differential gene expression profiles and WGCNA, we explored several genes that were highly significant and negatively correlated with lycopene, all of which encode fasciclin-like arabinogalactan proteins (FLAs). The FLAs are involved in plant signal transduction, however the functional role of these proteins has not been studied in tomato. Genome-wide analysis revealed that cultivated and wild tomato species contained 18 to 22 FLA family members, clustered into four groups, and mainly evolved by means of segmental duplication. The functional characterization of FLAs showed that silencing of SlFLA1, 5, and 13 were found to contribute to the early coloration of tomato fruits, and the expression of carotenoid synthesis-related genes was up-regulated in fruits that changed phenotypically, especially in SlFLA13-silenced plants. Furthermore, the content of multiple carotenoids (including (E/Z)-phytoene, lycopene, γ-carotene, and α-carotene) was significantly increased in SlFLA13-silenced fruits, suggesting that SlFLA13 has a potential inhibitory function in regulating carotenoid synthesis in tomato fruits. The results of the present study broaden the idea of analyzing the biological functions of tomato FLAs and preliminary evidence for the inhibitory role of SlFLA13 in carotenoid synthesis in fruit, providing the theoretical basis and a candidate for improving tomato fruit quality.

Compartmentalization of galactan biosynthesis in mycobacteria.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

Mycobacterium tuberculosis Raf kinase inhibitor protein (RKIP) Rv2140c is involved in cell wall arabinogalactan biosynthesis via phosphorylation.

Mycobacterium tuberculosis Rv2140c is a function unknown conserved phosphatidylethanolamine-binding protein (PEBP), homologous to Raf kinase inhibitor protein (RKIP) in human beings. To delineate its function, we heterologously expressed Rv2140c in a non-pathogenic M. smegmatis. Quantitative phosphoproteomic analysis between two recombinant strains Ms_Rv2140c and Ms_vec revealed that Rv2140c differentially regulate 425 phosphorylated sites representing 282 proteins. Gene ontology GO, and a cluster of orthologous groups COG analyses showed that regulated phosphoproteins by Rv2140c were mainly associated with metabolism and cellular processes. Rv2140c significantly repressed phosphoproteins involved in signaling, including serine/threonine-protein kinases and two-component system, and the arabinogalactan biosynthesis pathway phosphoproteins were markedly up-regulated, suggesting a role of Rv2140c in modulating cell wall. Consistent with phosphoproteomic data, Rv2140c altered some phenotypic properties of M. smegmatis such as colony morphology, cell wall permeability, survival in acidic conditions, and active lactose transport. In summary, we firstly demonstrated the role of PEBP protein Rv2140c, especially in phosphorylation of mycobacterial arabinogalactan biosynthesis proteins.

Systems identification and characterization of ß-glucuronosyltransferase genes involved in arabinogalactan-protein biosynthesis in plant genomes.

Utilizing plant biomass for bioethanol production requires an understanding of the molecular mechanisms involved in plant cell wall assembly. Arabinogalactan-proteins (AGPs) are glycoproteins that interact with other cell wall polymers to influence plant growth and developmental processes. Glucuronic acid, which is transferred to the AGP glycan by ß-glucuronosyltransferases (GLCATs), is the only acidic sugar in AGPs with the ability to bind calcium. We carried out a comprehensive genome-wide analysis of a putative GLCAT gene family involved in AGP biosynthesis by examining its sequence diversity, genetic architecture, phylogenetic and motif characteristics, selection pressure and gene expression in plants. We report the identification of 161 putative GLCAT genes distributed across 14 plant genomes and a widely conserved GLCAT catalytic domain. We discovered a phylogenetic clade shared between bryophytes and higher land plants of monocot grass and dicot lineages and identified positively selected sites that do not result in functional divergence of GLCATs. RNA-seq and microarray data analyses of the putative GLCAT genes revealed gene expression signatures that likely influence the assembly of plant cell wall polymers which is critical to the overall growth and development of edible and bioenergy crops.

Structural characteristics, antioxidant properties and antiaging activities of galactan produced by Mentha haplocalyx Briq.

An antioxidant-active polysaccharide (WMP) with a molecular weight of 26.91 kDa was isolated from Mentha haplocalyx Briq by water extraction, ethanol precipitation and gel filtration. According to HPLC, methylation, GC-MS, and 1D/2D nuclear magnetic resonance spectroscopy, WMP is a heteropolysaccharide composed of Gal (84.2 %), Glc (9.8 %), Man (2.8 %) and Ara (3.2 %) with (1→6)-α-d-Galp and (1→4, 6)-α-d-Galp residues in the backbone and (1→6)-α-d-Galp and (1→6)-α-d-Glcp residues in the branch. The branch point was located at C-4 of (1→4, 6)-α-d-Galp residue with a branching degree of 19.71 %. WMP showed remarkably high scavenging ability for 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals, ferrous ion chelating activity and ferric reducing powder in vitro. In vivo result showed that WMP oral administration substantially increased the activities of antioxidant enzymes, including SOD, GSH-Px and CAT, and decreased MDA levels in the serum and liver of d-Gal-induced aging mice. Therefore, WMP can be an effective natural antioxidant.

Comparative genomics reveals differences in algal galactan biosynthesis and related pathways in early and late diverging red algae.

Red algae are a major source of marine sulfated galactans. In this study, orthologs and inparalogs from seven red algae were analyzed and compared with the aim to discover differences in algal galactan biosynthesis and related pathways of these algae. Red algal orthologs for putative carbohydrate sulfotransferases were found to be prevalent in Porphyridium purpureum, Florideophytes and Bangiophytes, while red algal orthologs for putative chondroitin sulfate synthases, sulfurylases, and porphyranases /carrageenases were found exclusively in Florideophytes and Bangiophytes. The acquirement of these genes could have happened after the divergence from Cyanidiales red algae. Cyanidiales red algae were found to have more number and types of putative sulfate permeases, suggesting that these genes could have been acquired in adaptation to the environmental stresses and biogeochemistry of respective habitats. The findings of this study shed lights on the evolution of different homeostasis mechanisms by the early and late diverging red algal orders.

Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

Effects of glycerol concentrations on the bioproduction of PGHX by Agrobacterium HX1126.

PGHX is a polymer of ß (1-3)-galactose which posses the gel-forming property. As previously reported in the flask culture experiment, the crude PGHX (24.9 g/L, 48.2% in yield) with the maximum gel strength of 957 g/cm2 can be generated. However, PGHX produced in the stirred bioreactor had no gel-forming property when using the same medium. Hence, the effects of different glycerol concentrations on both the yield and the gel-forming property of PGHX were investigated and the reason for gel-forming property losing was explored. We proposed a new strategy for the production of PGHX with enhanced gel formation in the stirred bioreactor by mediating both the concentration of carbon source and the duration of fermentation. As a result, we managed to obtain the crude PGHX (22 g/L, 42.4% in yield) with the maximum gel strength of 438 g/cm2 at 56 h in the bioreactor. This strategy would help the enhancement of PGHX yield in the industrial production.

Spatio-temporal localization of selected pectic and arabinogalactan protein epitopes and the ultrastructural characteristics of explant cells that accompany the changes in the cell fate during somatic embryogenesis in Arabidopsis thaliana.

During somatic embryogenesis (SE), explant cells undergo changes in the direction of their differentiation, which lead to diverse cell phenotypes. Although the genetic bases of the SE have been extensively studied in Arabidopsis thaliana, little is known about the chemical characteristics of the wall of the explant cells, which undergo changes in the direction of differentiation. Thus, we examined the occurrence of selected pectic and AGP epitopes in explant cells that display different phenotypes during SE. Explants examinations have been supplemented with an analysis of the ultrastructure. The deposition of selected pectic and AGP epitopes in somatic embryos was determined. Compared to an explant at the initial stage, a/embryogenic/totipotent and meristematic/pluripotent cells were characterized by a decrease in the presence of AGP epitopes, b/the presence of AGP epitopes in differentiated cells was similar, and c/an increase of analyzed epitopes was detected in the callus cells. Totipotent cells could be distinguished from pluripotent cells by: 1/the presence of the LM2 epitope in the latest one, 2/the appearance of the JIM16 epitope in totipotent cells, and 3/the more abundant presence of the JIM7 epitope in the totipotent cells. The LM5 epitope characterized the wall of the cells that were localized within the mass of embryogenic domain. The JIM8, JIM13 and JIM16 AGP epitopes appeared to be the most specific for the callus cells. The results indicate a relationship between the developmental state of the explant cells and the chemical composition of the cell walls.

Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deaths per year, along with the emergence of multi- and extensively-drug resistant strains of Mtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent 'Achilles heel' that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.

Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis.

Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

High level expression of ß-mannanase (RmMan5A) in Pichia pastoris for partially hydrolyzed guar gum production.

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel ß-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization<7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6×104Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-d-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable ß-mannanase for PHGG production and some useful information of PHGG are provided in this paper.

Characterisation of a gene encoding a membrane protein that affects exopolysaccharide production and intracellular Mg2+ concentrations in Ensifer meliloti.

Exopolysaccharides play an important role in the physiology of a bacterial cell. Ensifer meliloti is capable of producing at least two types of exopolysaccharides (EPS): succinoglycan and galactoglucan. In E. meliloti, EPS are best known for their role in mediating interaction with its symbiotic hosts. It was previously shown that high concentrations of Mg2+ or K+ were capable of suppressing the mucoid phenotype associated with galactoglucan production in an expR+ derivative of Rm1021. In an attempt to determine how Mg2+ regulates galactoglucan production, SRmD363 was mutagenised and screened for mutants which were visibly mucoid at high concentrations of magnesium. Tn5 mutations in genes exoX, emmB, phoC and SMc00722 were isolated. SMc00722 is annotated as a hypothetical transmembrane protein that is conserved in the α-proteobacteria. Characterisation of SMc00722 in Rm1021 showed that the increased mucoidy was due to succinoglycan. Strains carrying mutations in SMc00722 showed increased biofilm production, and were more sensitive to high Mg2+ concentrations and deoxycholate. In addition, we show that strains carrying a mutation in SMc00722 have elevated intracellular Mg2+ concentrations. Taken together, the data are consistent with the hypothesis that SMc0722 may play a role in maintaining intracellular magnesium concentration, and we suggest that this gene be tentatively annotated as mhrA (magnesium homeostasis related).

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676.

Physico-chemical properties of a galactan exopolysaccharide (EPS) produced by Weissella confusa KR780676 isolated from an idli batter were studied. In our previous paper, we had reported the isolation and characterization of a linear galactan (containing α-(1→6)-linked galactose units) isolated from an acidic fermented food. In this manuscript, we are reporting the physico-chemical properties viz., Colour evaluation (L* 81.79, a* 3.15, b* 15.04), flow properties (compressibility index 38.46%), syneresis, light transmittance (50.7% for 5days), pasting properties (temperature 64.6°C), texture (hardness 9.876N) and particle size (549.81dnm) for the galactan were carried out. This galactan also possessed high oil absorption capacity (5.09mL/g) with good emulsifying activity (69.4%) and emulsion stability up to 15days. Flocculating activity of 65.3-81.7% was observed with 0.2mg/mL EPS in a wide range of pH (2.0-12.0). These functional properties will make the galactan EPS a good candidate for use as thickening and gelling agent and emulsifier to form long-term emulsions in food, pharmaceutical and cosmetic products, as well as for bio-treatment of wastewater and hydrocarbon-polluted environments.

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans.

BACKGROUND: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. RESULTS: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. CONCLUSIONS: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food.

An exopolysaccharide (EPS) producing strain PUFSTM055 isolated from Idli batter (an Indian traditional cereal-legume based fermented food) was identified as Weissella confusa KR780676. The strain was shown to produce 17.2g/L (dry weight) of EPS in 2% sucrose supplemented MRS broth and the EPS was characterized. HPTLC analysis confirmed the presence of galactose monomers, indicating the homopolysaccharide nature of EPS. Fourier-transform infrared spectroscopy and nuclear magnetic resonance analysis revealed that the EPS was found to be a novel linear galactan containing α-(1→6)-linked galactose units. Scanning electron microscopy of the EPS revealed the presence of porous and spongy starch-like granules. Topographical examination of EPS by atomic force microscopy revealed that the EPS formed densely packed mesh-like structure with irregular spherical lumps. The EPS also showed high thermal stability with a degradation temperature of 287.5 °C and melting point at 274.65 °C. EPS was semi-crystalline with crystallinity index of 0.23 and showed 100% water solubility index. These characteristics of the EPS would make it a promising hydrocolloid for food industries as bio-thickeners, stabilizers and also as an encapsulating material for delivery of food bioactive compounds. This is the first study reporting the galactan compose of the EPS from lactic acid bacteria.

Assembly of the Mycobacterial Cell Wall.

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens, claiming over 1.3 million lives worldwide in 2013. The emergence of multidrug-resistant and extensively drug-resistant isolates has prompted the need for new drugs and drug targets. M. tuberculosis possesses an unusual cell wall dominated by lipids and carbohydrates that provides a permeability barrier against hydrophilic drugs and is crucial for its survival and virulence. This large macromolecular structure, termed the mycolyl-arabinogalactan-peptidoglycan complex, and the phosphatidyl-myo-inositol-based lipoglycans are key features of the mycobacterial cell wall. Assembly of these cell wall components is an attractive target for the development of chemotherapeutics against tuberculosis. Herein, we focus on recent biochemical and molecular insights into these complex molecules of M. tuberculosis cell wall.

Arabinogalactan biosynthesis: Implication of AtGALT29A enzyme activity regulated by phosphorylation and co-localized enzymes for nucleotide sugar metabolism in the compartments outside of the Golgi apparatus.

Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize not only to the Golgi cisternae but also to smaller compartments, which may be a part of the unconventional protein secretory pathway in plants. In Poulsen et al., (1) we have demonstrated increased targeting of AtGALT29A to small compartments when Y144 is substituted with another amino acid, and we implicated a role for Y144 in the subcellular targeting of AtGALT29A. In this paper, we are presenting another aspect of Y144 substitution in AtGALT29A; namely, Y144A construct demonstrated a 2.5-fold increase while Y144E construct demonstrated a 2-fold decrease in the galactosyltransferase activity of AtGALT29A. Therefore, the electrostatic status of Y144, which is regulated by an unknown kinase/phosphatase system, may regulate AtGALT29A enzyme activity. Moreover, we have identified additional proteins, apyrase 3 (APY3; At1g14240) and UDP-glucuronate epimerases 1 and 6 (GAE1, At4g30440; GAE6, At3g23820), from Arabidopsis thaliana that co-localize with AtGALT31A in the small compartments when expressed transiently in Nicotiana benthamiana. These proteins may play roles in nucleotide sugar metabolism in the small compartments together with arabinogalactan glycosyltransferases.