Clustered genes required for the synthesis of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

As demonstrated with alr2835 (hepA) and alr2834 (hepC) mutants, heterocysts of Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, must have an envelope polysaccharide layer (the Hep+ phenotype) to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Transpositions presumptively responsible for a Fox- phenotype were localized in open reading frames (ORFs) near hepA and hepC. A mutation in each of nine of these ORFs was complemented by a clone bearing only that single, intact ORF. Heterocysts of the nine mutants were found to lack an envelope polysaccharide layer. Complementation of mutations in alr2832 and alr2840 may have resulted from recombination. However, alr2825, alr2827, alr2831, alr2833, alr2837, alr2839, and alr2841, like hepA and hepC, are required for a Hep+ Fox+ phenotype.     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Identification of genes controlled by the manganese response regulator, ManR, in the cyanobacterium, Anabaena sp. PCC 7120

A computational search was carried out to identify additional binding sites for the manganese response regulator, ManR, in the genome of Anabaena sp. PCC 7120. This approach predicted ManR binding sites: the promoter regions of the genes of all3575-alr3576 and the gene of alr5134 from Anabaena sp. PCC 7120. Electrophoretic mobility shift assays confirmed that the ManR of Anabaena sp. PCC 7120 specifically bound to the promoter regions of all3575-alr3576 and alr5134.     

Identification of ten Anabaena sp. genes that under aerobic conditions are required for growth on dinitrogen but not for growth on fixed nitrogen

Heterocysts are specialized cells required for aerobic fixation of dinitrogen by certain filamentous cyanobacteria. Numerous genes involved in the differentiation and function of heterocysts in Anabaena sp. strain PCC 7120 have been identified by mutagenizing and screening for mutants that require fixed nitrogen for growth in the presence of oxygen. We have verified that 10 Anabaena sp. genes, all1338, all1591, alr1728, all3278, all3520, all3582, all3850, all4019, alr4311, and all4388, identified initially by transposon mutagenesis, are such genes by complementing or reconstructing the original mutation and by determining whether the mutant phenotype might be due to a polar effect of the transposon. Elucidation of the roles of these genes should enhance understanding of heterocyst biology.     

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.     

A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).     

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.     

PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.     

Novel ATP-driven pathway of glycolipid export involving TolC protein

Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.     

Blood group A glycolipid antigen expression in kidney, ureter, kidney artery, and kidney vein from a blood group A1Le(a-b+) human individual. Evidence for a novel blood group A heptaglycosylceramide based on a type 3 carbohydrate chain

Kidney, ureter, kidney artery, and kidney vein tissue were obtained from a single human transplant specimen. The donors erythrocyte blood group phenotype was A1Le(a-b+). Total non-acid glycolipid fractions were isolated and individual glycolipid components were identified by immunostaining thin layer plates with a panel of monoclonal antibodies and by mass spectrometry of the permethylated and permethylated-reduced total glycolipid fractions. The dominating glycolipids in all tissues were mono- to tetraglycosylceramides. In the kidney, ureter, and artery tissue less than 1% of the glycolipids were of blood group type, having more than 4 sugar residues. In contrast, 14% of the vein glycolipids were of blood group type, and the dominating components were type 1 chain blood group H pentaglycosylceramides and A hexaglycosylceramides. Trace amounts of structurally different blood group A glycolipids (type 1 to 4 core saccharide chains) with up to 10 sugar residues were found in the kidney, ureter, and vein tissues, including evidence for a novel blood group A heptaglycosylceramide based on the type 3 chain in the vein. The only detected A glycolipid antigen in the artery tissue was the blood group A difucosyl type 1 chain heptaglycosylceramide (ALeb) structure. Blood group Lewis and related antigens (Lea, Leb, and ALeb) were expressed in the kidney, ureter, and artery, but were completely lacking in the vein, indicating that the Le gene-coded alpha 1-4-fucosyltransferase was not expressed in this tissue. The X and Y antigens (type 2 chain isomers of the Lea and Leb antigens) were detected only in the kidney tissue.     

The structural basis of blood group A-related glycolipids in an A3 red cell phenotype and a potential explanation to a serological phenomenon

Glycolipids from the red cells of a rare blood group A subgroup individual, expressing the blood group A(3) phenotype with the classical mixed-field agglutination phenomenon, A(2(539G>A))/O(1) genotype, and an unusual blood group A glycolipid profile, were submitted to a comprehensive biochemical and structural analysis. To determine the nature of blood group A glycolipids in this A(3) phenotype, structural determination was carried out with complementary techniques including proton nuclear magnetic resonance (1D and 2D), mass spectrometry (MS) (nano-electrospray ionization/quadrupole time-of-flight and tandem mass spectrometry) and thin layer chromatography with immunostaining detection. As expected, total blood group A structures were of low abundance, but contrary to expectations extended-A type 2 and A type 3 glycolipids were more dominant than A hexaglycosylceramides based on type 2 chain (A-6-2 glycolipids), which normally is the major A glycolipid. Several para-Forssman (GalNAcβ3GbO(4)) structures, including extended forms, were identified but surmised not to contribute to the classic mixed-field agglutination of the A(3) phenotype. It is proposed that the low level of A antigen combined with an absence of extended branched glycolipids may be the factor determining the mixed-field agglutination phenomenon in this individual.     

Differentiation-associated changes of glycolipid composition and metabolism in mouse myeloid leukemia cells. Induction of globotriaosylceramide and a galactosyltransferase

This study was to find out whether induction of special glycolipids or glycosyltransferases for glycolipid synthesis which might be involved in the cell functions occurred during the differentiation. Mouse myeloid leukemia cell line (M1-), the differentiated cells (M1+), and a subcloned cell line (Mm1) were used for this purpose. Gangliotriaosylceramide (GA2) was the major glycolipid component in M1- cells. As a result of differentiation of M1- into M1+ cells, globotriaosylceramide (CTH) was newly induced as the main glycolipid, while GA2 decreased to a minor component. GA2 was found to be the main glycolipid in Mm1 cells but no CTH was recognized. All precursor glycolipids and glycosyltransferases required to complete the biosynthetic pathway glucosylceramide (CMH) leads to lactosylceramide (CDH) leads to GA2 leads to gangliotetraosylceramide (GA1) leads to sialosylgangliotetraosylceramide (GM1b) were found in M1- and also in Mm1 cells. A galactosyltransferase activity for CTH synthesis from CDH increased 10 fold during the differentiation. The induction of CTH in M1+ cells could be attributed to the increase of the galactosyltransferase activity. Both CTH as a surface marker and the galactosyltransferase as an enzyme marker are proposed as valuable markers of differentiation in M1- cells. Besides the galactosyltransferase, N-acetylglucosaminyltransferease involved in the formation of lactotriaosylceramide (amino-CTH) increased up to 3 fold during the differentiation. The increase of the enzyme activity seemed to be responsible for the biosynthesis of lactoneotetraosylceramide (paragloboside) which appeared in M1+ cells.     

Glycolipids of recent clinical isolates of Mycobacterium tuberculosis: chemical characterization and immunoreactivity

Five distinct glycolipids were readily detected in isolates of Mycobacterium tuberculosis. Spectroscopic methods and chemical degradation techniques allowed the structural identification of four of these glycolipids. The specific phenolic glycolipid antigen previously characterized from the Canetti strain was found in all the strains examined, with identical structural features (triglycosyl phenol phthiocerol dimycocerosate). The other three glycolipids identified were acylated trehaloses: penta-acyl trehalose (containing phthienoyl substituents), tetra-acyl trehalose 2'-sulphate (with C40-C50 hydroxyphthioceranoyl substituents) and diacyl trehalose 2'-sulphate (with C16 and C18 substituents). The two latter glycolipids as well as the phenolic glycolipid immunoreacted with whole-cell antiserum, indicating their surface location. The occurrence of these glycolipid antigens in recent clinical isolates suggests their possible utilization in the serodiagnosis of tuberculosis and the rapid identification of M. tuberculosis with specific antisera.     

Mycobacterial lipomannan induces granuloma macrophage fusion via a TLR2-dependent, ADAM9- and beta1 integrin-mediated pathway

Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the beta(1) integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.