New insights into the structure of (1→3,1→6)-β-D-glucan side chains in the Candida glabrata cell wall

β-Glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.     

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

The enzyme α-1,6-mannosyltransferase (OCH-1) is required for the synthesis of galactomannans attached to the N-linked oligosaccharides of Neurospora crassa cell wall proteins. The Neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. A carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. Analysis of the integral cell wall protein from wild-type and och-1 mutant cells showed that the mutant cell wall had reduced protein content. The och-1 mutant was found to secrete 18-fold more protein than wild-type cells. Proteomic analysis of the proteins released by the mutant into the growth medium identified seven of the major cell wall proteins. Western blot analysis of ACW-1 and GEL-1 (two glycosylphosphatidylinositol [GPI]-anchored proteins that are covalently integrated into the wild-type cell wall) showed that high levels of these proteins were being released into the medium by the och-1 mutant. High levels of ACW-1 and GEL-1 were also released from the och-1 mutant cell wall by subjecting the wall to boiling in a 1% SDS solution, indicating that these proteins are not being covalently integrated into the mutant cell wall. From these results, we conclude that N-linked mannosylation of cell wall proteins by OCH-1 is required for their efficient covalent incorporation into the cell wall.The fungal cell wall is an important organelle that protects the cell from various environmental stresses. It is a dynamic structure that interacts with the environment and is modified to accommodate growth, cell division, and development. Fungal cell walls have been shown to contain β-1,3-glucan, α-1,3-glucan, β-1,6-glucan, mixed β-1,3/β-1,4-glucans, chitin, and mannan/galactomannan (6, 19). These polysaccharide polymers constitute 80 to 85% of the cell wall mass, while glycoproteins constitute the remaining 15 to 20% (6). The cell wall glycoproteins are required for vital functions, like structural support, signal transduction, biofilm formation, and cell wall biosynthesis. In the case of pathogenic fungi, the cell wall is critical for the invasion of host tissues (8). Because of their accessibility and the crucial functions they perform, cell wall proteins could be important targets for the development of antifungal therapeutics.The glucan and chitin cell wall polymers are synthesized by enzyme complexes (glucan synthases and chitin synthases) that are associated with the plasma membrane. Glucan and chitin are vectorially passed into the cell wall space during synthesis and cross-linked together in the cell wall space. The mannan and galactomannan present in the cell wall are found as glycoconjugates on cell wall proteins. Mannosylation of cell wall proteins occurs in the endoplasmic reticulum (ER) and Golgi apparatus at O-linked and N-linked glycosylation sites. In Saccharomyces cerevisiae, mannosylation of N-linked glycosylation is initiated by the addition of an α-1,6-linked mannose residue by Och1p (33). In the filamentous fungus Neurospora crassa, the structure of the galactomannan associated with N-linked sites has not been definitively determined, but N. crassa has most of the enzymes defined in yeast for the mannosylation of N-linked oligosaccharides (14). An analysis of N-linked oligosaccharides from N. crassa glycoproteins showed that the glycoproteins are modified by the addition of short α-1,6-mannans with short α-1,2-mannose branches that are terminated by galactofuranose residues (31, 32). The N. crassa posttranslational modifications appear to differ from those found in S. cerevisiae by having shorter mannan chains and by the presence of terminal galactofuranose residues.Mannosylation of glycoproteins has been extensively studied in yeast. In S. cerevisiae, OCH1 encodes the α-1,6-mannosyltransferase enzyme that mediates the addition of the initial α-1,6-mannose in the synthesis of long mannans which are attached to the N-linked oligosaccharides (22, 33). Knockout mutants of OCH1 are viable but exhibit a temperature-sensitive growth pattern and are sensitive to cell wall perturbation reagents (34). Mutants for Candida albicans homologs of OCH1 had near-normal growth rates but were much less virulent (3). Mass spectrometry analysis of glycoproteins from the S. cerevisiae och1 and C. albicans och1 mutants showed that the α-1,6-mannose core was absent (3, 33). In Kluyveromyces lactis, the KlOCH1 gene has been shown to be important for cell wall organization and to give a hypersecretion phenotype (37). OCH1 mutants have also been identified in Pichia angusta, Yarrowia lipolytica, Pichia pastoris, and Schizosaccharomyces pombe, and these mutants have cell wall-related phenotypes (2, 9, 17, 38). However, a recent report of OCH1 knockout mutants of Aspergillus fumigatus indicates that these mutants do not have a cell wall-defective phenotype (18).Mannosylation of cell wall proteins has not been extensively studied in filamentous fungi. We report on the characterization of the N. crassa knockout mutant of the α-1,6-mannosyltransferase, och-1. The mutant was generated by the Neurospora genome knockout project (10). The N. crassa och-1 mutant has a severe growth defect and exhibits a tight colonial phenotype. We demonstrate that the och-1 mutant exhibits a defect in cell wall biosynthesis. A carbohydrate analysis of the mutant cell wall showed a drastic reduction in mannose and galactose content with a compensatory increase in the glucose content. The och-1 cell wall also showed a reduced cell wall protein content as assessed by a Coomassie brilliant blue dye binding assay and by proteomic analysis. Protein secretion assays showed that the mutant releases large amounts of cell wall protein into the growth medium. We demonstrate that the och-1 mutant is defective in covalently cross-linking known cell wall proteins into the cell wall matrix. Our data demonstrate that the N-linked galactomannan, which is built upon the mannose residue added by OCH-1, is required for the incorporation of cell wall proteins into the cell wall matrix.     

A Mutational Analysis of Killer Toxin Resistance in Saccharomyces Cerevisiae Identifies New Genes Involved in Cell Wall (1 -> 6)-β-Glucan Synthesis

Recessive mutations leading to killer resistance identify the KRE9, KRE10 and KRE11 genes. Mutations in both the KRE9 and KRE11 genes lead to reduced levels of (1 -> 6)-β-glucan in the yeast cell wall. The KRE11 gene encodes a putative 63-kD cytoplasmic protein, and disruption of the KRE11 locus leads to a 50% reduced level of cell wall (1 -> 6)-glucan. Structural analysis of the (1 -> 6)-β-glucan remaining in a kre11 mutant indicates a polymer smaller in size than wild type, but containing a similar proportion of (1 -> 6)- and (1 -> 3)-linkages. Genetic interactions among cells harboring mutations at the KRE11, KRE6 and KRE1 loci indicate lethality of kre11 kre6 double mutants and that kre11 is epistatic to kre1, with both gene products required to produce the mature glucan polymer at wild-type levels. Analysis of these KRE genes should extend knowledge of the β-glucan biosynthetic pathway, and of cell wall synthesis in yeast.     

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

The enzyme γ-glutamyltranspeptidase 1 (GGT1) is a conserved member of the N-terminal nucleophile hydrolase family that cleaves the γ-glutamyl bond of glutathione and other γ-glutamyl compounds. In animals, GGT1 is expressed on the surface of the cell and has critical roles in maintaining cysteine levels in the body and regulating intracellular redox status. Expression of GGT1 has been implicated as a potentiator of asthma, cardiovascular disease, and cancer. The rational design of effective inhibitors of human GGT1 (hGGT1) has been delayed by the lack of a reliable structural model. The available crystal structures of several bacterial GGTs have been of limited use due to differences in the catalytic behavior of bacterial and mammalian GGTs. We report the high resolution (1.67 Å) crystal structure of glutamate-bound hGGT1, the first of any eukaryotic GGT. Comparisons of the active site architecture of hGGT1 with those of its bacterial orthologs highlight key differences in the residues responsible for substrate binding, including a bimodal switch in the orientation of the catalytic nucleophile (Thr-381) that is unique to the human enzyme. Compared with several bacterial counterparts, the lid loop in the crystal structure of hGGT1 adopts an open conformation that allows greater access to the active site. The hGGT1 structure also revealed tightly bound chlorides near the catalytic residue that may contribute to catalytic activity. These are absent in the bacterial GGTs. These differences between bacterial and mammalian GGTs and the new structural data will accelerate the development of new therapies for GGT1-dependent diseases.     

Conformational Studies of Dextran [α-(1⇒6)-D-Glucan]

Abstract

A theoretical conformational study of dextran, a (l?6)-linked α-D-glucan polysaccharide, has been made to allow an explicit comparison with earlier results on pustulan, the corresponding (1 ?6)-linked β-D-glucan. The nonbonded, torsional and hydrogen bond contributions to potential energy were calculated as a function of rotational angles φ, ψ, and ω The (φ, ψ, ω)-space of the disaccharide and of helices contain many local energy minima with very small energy differences. A comparison of (1?6)-α-D-glucans with (1?6)-β-D-glucans indicates significant differences in conformational behavior. Specifically, our results shed light on the fact that dextran does not gel, whereas pustulan does. The difference in tendency to gel may be related to the fact that dextran has no particularly favored conformations with structural regularity whereas pustulan does.     

Conformation of (1→3)-α-D-Glucan Tribenzoate

The molecular conformation of (1→3)-α-D-glucan tribenzoate (TBG) was studied by X-ray diffraction measurements coupled with a conformational analysis. Although the fiber pattern obtained was of very low crystallinity, the presence of a meridional reflection at the 5th layer line indicated that the TBG molecule took a five-fold helical conformation with a 19.63 A fiber repeat. A conformational analysis on the five-fold helix, which was done by calculating van der Waals’ repulsion energy between non-bonded atoms comprising the TBG chain, suggested that the most preferable energy-based conformation was –5/1, a left-handed five-fold helix.     

TGFβ Activated Kinase-1: New Insights into the Diverse Roles of TAK1 in Development and Immunity

A number of recent publications have examined the role of TAK1 in model systems ranging from fly to mouse. Rather than fit into a clearly defined linear molecular pathway, TAK1 seems to act in a signaling nexus that responds to a variety of upstream signals, including inflammatory molecules and developmental cues. TAK1 then influences a number of downstream processes ranging from innate immune responses to patterning and differentiation via JNK, NFκB, and TCFβ-catenin signaling. These differences in function are not simply a matter of cell type. For example, NFκB signaling in a particular cell may or may not require TAK1 depending on the nature of the activating signal. Interestingly, the multi-task functionality of TAK1 is conserved between vertebrate and invertebrate species. Studies of TAK1 in multiple experimental systems is likely to reveal more roles for this kinase and also elucidate mechanisms by which other signaling molecules fulfill diverse signaling roles. Here we provide an overview of the data concerning TAK1 from its discovery to more recent findings and provide a synthesis of the conclusions that have arisen from the multiple model systems and experimental approaches.     

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-d-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.     

New Insights into the in situ Microscopic Visualization and Quantification of Inorganic Polyphosphate Stores by 4’,6-Diamidino-2-Phenylindole (DAPI)-Staining

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.Key words: DAPI, polyphosphate, fluorescence, fluorimetry     

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria: PimB�� IS AN ESSENTIAL ENZYME OF MYCOBACTERIUM SMEGMATIS*

Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB′ (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB′ exclusively transfers to the 6-position. Moreover, whereas PimB′ can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM₁) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM₁ product of PimB′. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB′ indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB′. Importantly, genetic evidence is provided that pimB′ is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac₁PIM₂, a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB′, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis.PIMs³ are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes.⁴ They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3–6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc²155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimB′, the enzyme responsible for the transfer of the second Manp to the 6-position of the myo-Ins ring of PIM₁, still remains controversial. The Rv0557 protein of Mycobacterium tuberculosis H37Rv (PimB; MSMEG_1113 in M. smegmatis mc²155) was originally characterized as PimB′ (7). However, the lack of an Rv0557 ortholog in the genome of Mycobacterium leprae and the fact that the disruption of this gene in M. tuberculosis Erdman did not significantly affect the biosynthesis of PIMs suggest that compensatory activities exist in the bacterium or that Rv0557 serves another primary function (8, 9). Somewhat supporting the latter hypothesis, the ortholog of Rv0557 in Corynebacterium glutamicum (NCgl0452, renamed mgtA) was implicated in the mannosylation of a novel glycolipid (1,2-di-O-C₁₆/C_18:1-(α-d-mannosyl)-(1→4)-(α-d-glucopyranosyluronic acid)-(1→3)-glycerol), and Rv0557 from M. tuberculosis was reported to functionally complement for this enzyme in a C. glutamicum knock-out mutant (10). However, to our knowledge this mannosylated glycolipid has never been reported in mycobacteria, and it remains unclear whether PimB serves a similar physiological function in Mycobacterium spp.More recently, Lea-Smith et al. (11) have shown that the biosynthesis of Ac₁PIM₂ from Ac₁PIM₁ in C. glutamicum is catalyzed by NCgl2106 (Cg-PimB′). Disruption of the NCgl2106 gene totally abolished Ac₁PIM₂ production in the mutant, arguing against the existence of a compensatory activity associated with the corynebacterial PimB enzyme. Although Ac₁PIM₂ production in Cg-pimB′ and Cg-pimB′/Cg-pimB knock-out mutants was restored upon complementation with the M. tuberculosis Rv2188c gene (11, 12), direct evidence that Rv2188c carried out the same physiological function in mycobacteria has been lacking. Moreover, in light of the recent work by Torrelles et al. (9) showing an involvement of pimB (Rv0557) in the synthesis of LM and LAM in M. tuberculosis Erdman and of the demonstrated relaxed substrate specificity of the M. tuberculosis PimB (Rv0557) and PimB′ (Rv2188c) enzymes expressed in C. glutamicum (12), whether or not pimB and pimB′ could compensate for one another in mycobacteria remained open to speculation.Both PIM₁ and PIM₂ can be acylated with palmitate at position 6 of the Manp residue transferred by PimA by the acyltransferase MSMEG_2934 (orthologous to Rv2611c from M. tb) to form Ac₁PIM₁ and Ac₁PIM₂, respectively (13). Ac₁PIM₂ can further be acylated at position 3 of the myo-Ins ring by an as yet unidentified acyltransferase to yield Ac₂PIM₂. Importantly, Ac₁PIM₂ and Ac₂PIM₂ are among the most abundant forms of PIMs found in mycobacteria and are considered both metabolic end products and intermediates in the biosynthesis of more polar forms of PIMs (PIM₅ and PIM₆), LM, and LAM.In this work, clear evidence is provided that PimB′ (MSMEG_4253 in M. smegmatis mc²155) is the α-ManT responsible for the biosynthesis of PIM₂ from PIM₁ in mycobacteria and that no other ManT can compensate for a deficiency in this enzyme in M. smegmatis. Like PimA (5), PimB′ is essential to the growth of M. smegmatis. Cell-free assays using purified PimA and PimB′ and M. smegmatis membrane preparations provide new insights into the sequential events leading to the synthesis of the early forms of PIMs in mycobacteria.     

Characterization of the CaENG1 Gene Encoding an Endo-1,3-β-Glucanase Involved in Cell Separation in Candida albicans

The Candida albicans CaENG1 gene encoding an endo-1,3-β-glucanase was cloned by screening a genomic library with a DNA probe obtained by polymerase chain reaction using synthetic oligonucleotides designed according to conserved regions found between two Saccharomyces cerevisiae endo-1,3-β-glucanases (Eng1p and Eng2p). The gene contains a 3435-bp open reading frame (ORF), capable of encoding a protein of 1145 amino acids (124,157 Da), that contains no introns. Comparison of the ScEng1p sequence with partial C. albicans genomic sequences revealed the presence of a second protein with sequence similarity (the product of the Ca20C1.22c ORF, which was named CaENG2). Disruption of the CaENG1 gene in C. albicans had no dramatic effects on the growth rate of the strains, but it resulted in the formation of chains of cells, suggesting that the protein is involved in cell separation. Expression of CaENG1 in S. cerevisiae cells afforded a 12-fold increase in the 1,3-β-glucanase activity detected in culture supernatants, showing that the protein has similar enzymatic activity to that of the S. cerevisiae Eng1p. In addition, when the C. albicans protein was expressed under its native promoter in S. cerevisiae eng1 mutant cells, it was able to complement the separation defect of this mutant, indicating that these two proteins are true functional homologues.     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Insights into the Structure and Metabolic Function of Microbes That Shape Pelagic Iron-Rich Aggregates (“Iron Snow”)

Microbial ferrous iron [Fe(II)] oxidation leads to the formation of iron-rich macroscopic aggregates (“iron snow”) at the redoxcline in a stratified lignite mine lake in east-central Germany. We aimed to identify the abundant Fe-oxidizing and Fe-reducing microorganisms likely to be involved in the formation and transformation of iron snow present in the redoxcline in two basins of the lake that differ in their pH values. Nucleic acid- and lipid-stained microbial cells of various morphologies detected by confocal laser scanning microscopy were homogeneously distributed in all iron snow samples. The dominant iron mineral appeared to be schwertmannite, with shorter needles in the northern than in the central basin samples. Total bacterial 16S rRNA gene copies ranged from 5.0 × 10⁸ copies g (dry weight)⁻¹ in the acidic central lake basin (pH 3.3) to 4.0 × 10¹⁰ copies g (dry weight)⁻¹ in the less acidic (pH 5.9) northern basin. Total RNA-based quantitative PCR assigned up to 61% of metabolically active microbial communities to Fe-oxidizing- and Fe-reducing-related bacteria, indicating that iron metabolism was an important metabolic strategy. Molecular identification of abundant groups suggested that iron snow surfaces were formed by chemoautotrophic iron oxidizers, such as Acidimicrobium, Ferrovum, Acidithiobacillus, Thiobacillus, and Chlorobium, in the redoxcline and were rapidly colonized by heterotrophic iron reducers, such as Acidiphilium, Albidiferax-like, and Geobacter-like groups. Metaproteomics yielded 283 different proteins from northern basin iron snow samples, and protein identification provided a glimpse into some of their in situ metabolic processes, such as primary production (CO₂ fixation), respiration, motility, and survival strategies.