Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.     

Reassessment of the status of Lymantria albescens and Lymantria postalba (Lepidoptera: Erebidae: Lymantriinae) as distinct ‘Asian gypsy moth’ species,using both mitochondrial and nuclear sequence data

For regulatory purposes, the name ‘Asian gypsy moth’ refers to a group of closely related Asian Lymantria species and subspecies whose female moths display flight capability, a trait believed to confer enhanced invasiveness relative to the European gypsy moth, Lymantria dispar dispar, whose females are flightless. Lymantria albescens and Lymantria postalba are Asian gypsy moths occurring in the southern Ryukyu Islands and in the northern Ryukyu and adjacent Kyushu and Shikoku Islands of Japan, respectively. Although once considered subspecies of L. dispar, their status as distinct species, relative to the latter, is now well established. While postalba was subsequently considered a subspecies of L. albescens, largely on the basis of differences in forewing ground colour in males, both taxa were later given distinct species status by Pogue & Schaefer (2007) following their revision of the genus Lymantria. Here, we re-examined the validity of this revised status through the sequencing of a large portion of the mitochondrial genome (c. 60%) and multiple nuclear marker genes [elongation factor 1-alpha (Ef-1α), wingless (Wgl), internal transcribed spacer 2 (ITS-2), ribosomal protein S5 (RpS5)] in representative specimens of both taxa and other Lymantria species, including L. monacha, L. xylina, L. mathura and members of the L. dispar + L. umbrosa clade. A comparison of the number of substitutions in these genomic regions among the taxa we considered showed lower or equivalent variation between L. albescens and L. postalba compared with subspecies of L. dispar, for mitochondrial and nuclear sequences, respectively. This finding was reflected in the maximum likelihood trees generated independently for mitochondrial and nuclear data, where L. albescens and L. postalba formed, in both analyses, a short-branch sister clade basal to the L. dispar + L. umbrosa clade. We further sequenced three markers [cytochrome c oxydase 1 (COI), EF-1α, Wgl] in multiple L. albescens–L. postalba specimens collected along a south-to-north transect across the Ryukyu Arc and observed no clear distinction among the sampled specimens as a function of taxonomic designation. We conclude that L. albescens and L. postalba form a single species, with postalba representing a darker-winged morph along an apparent south-to-north wing colour cline. Accordingly, L. postalba is relegated to synonymy under L. albescens ( syn.n. ).     

Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.     

A cascade synthesis, in vitro cholinesterases inhibitory activity and docking studies of novel Tacrine-pyranopyrazole derivatives

In this work, we describe the preparation of some new Tacrine analogues modified with a pyranopyrazole moiety. A one-pot multicomponent reaction of 3-methyl-1H-pyrazol-5(4H)-one, aryl(or hetero)aldehydes, malononitrile and cyclohexanone involving a Friedländer condensation led to the title compounds. The synthesized heterocyclic analogues of this molecule were evaluated in vitro for their AChE and BChE inhibitory activities in search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable AChE inhibitory activities with IC₅₀ values ranging from 0.044 to 5.80?µM, wherein compounds 5e and 5j were found to be most active inhibitors against AChE with IC₅₀ values of 0.058 and 0.044?µM respectively. Molecular modeling simulation on AChE and BChE receptors, showed good correlation between IC₅₀ values and binding interaction template of the most active inhibitors docked into the active site of their relevant enzymes.     

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂ glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.Trypanosoma brucei is a protozoan parasite with two main proliferative stages in its life cycle: the procyclic form that grows in the tsetse fly midgut, and the bloodstream form that causes African sleeping sickness in humans and nagana in cattle. The bloodstream form is covered in a densely packed layer of 5 × 10⁶ glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) dimers. This coat protects the parasites from the alternative pathway of complement-mediated lysis, shields other cell surface proteins from the host immune system, and by the process of antigenic variation allows these parasites to persist for long periods in the host bloodstream (16, 54). The trypanosome genome contains several hundreds of silent VSG genes, most of which are pseudogenes in subtelomeric arrays (40). T. brucei evades host-acquired immunity through differential activation of these genes, which encode immunologically distinct GPI-anchored glycoproteins with one to three N glycosylation sites (27, 43).Protein N glycosylation is the most common covalent protein modification in eukaryotic cells (25). N-glycans contribute to “quality control” in the endoplasmic reticulum (ER) through a series of oligosaccharide-processing and lectin-binding reactions that contribute to protein folding and the targeting of misfolded glycoproteins for degradation (24, 47, 58, 65). As nascent protein chains enter the ER lumen, they are modified covalently in most eukaryotes by the addition of the Glc₃Man₉GlcNAc₂ core glycan via the action of oligosaccharyltransferase (OST). After deglucosylation by α-glucosidases I (GI) and II (GII), misfolded glycoproteins can be reglucosylated in the ER by the UDP-Glc:glycoprotein glucosyltransferase (UGGT), recreating the same monoglucosylated trimming intermediate generated by GII (9, 64, 66). UGGT behaves as a sensor of glycoprotein conformation and is a key constituent of ER quality control (50, 61). Calnexin and calreticulin are ER-resident lectin-like quality control chaperones that recognize the monoglucosylated glycans on glycoproteins and help them to fold properly through their close association with the oxidoreductase ERp57 (49). On reaching the proper tertiary structure, the glycoproteins are still substrates of GII but no longer of UGGT. Properly folded molecules, thus liberated from the lectins, are then free to continue their transit to the Golgi apparatus (64). When exposure to the folding machinery in the ER is not sufficient to promote a native conformation, proteins are eventually degraded by ER-associated degradation (49, 64).Most eukaryotes under conditions of stress, such as heat shock, undergo an unfolded protein response (UPR) that is triggered by sensing unfolded proteins in the ER. The UPR typically leads to increased expression of ER quality control components, such as calnexin and calreticulin and the ER chaperone Gpr78/BiP, as well inhibition of protein synthesis and cell cycle arrest (53, 57, 60).In contrast to the situation in most other eukaryotes, none of the trypanosomatid dolichol-linked oligosaccharides are capped with glucose residues, as these parasites do not synthesize the sugar donor dolichol-phosphate-glucose for these reactions (41, 59). The mature dolichol-phosphate-oligosaccharide species used for transfer to protein vary according to trypanosomatid species (17, 51, 52, 56). Therefore, in these organisms, monoglucosylated glycans are exclusively formed through UGGT-dependent glucosylation (12). Furthermore, trypanosomatids lack calnexin, which binds and participates in the refolding of glucosylated proteins, and it has been suggested that differences in the N-glycan precursor have profound effects on N-glycan-dependent quality control of glycoprotein folding and ER-associated degradation (4). These protozoa do not present a conventional OST complex and express only the catalytic stt3 protein subunit that, at least for the Trypanosoma cruzi and Leishmania major enzymes, shows little specificity toward the structure of the dolichol-phosphate-oligosaccharide donor (4, 11, 26, 31, 32, 45). In the case of T. brucei, while the insect-dwelling procyclic form makes and transfers Man₉GlcNAc₂-phosphate-dolichol (1), previous work from our group showed that the bloodstream form of the parasite transfers both Man₉GlcNAc₂ and Man₅GlcNAc₂ to VSG in a site-specific manner (29). Regarding ER folding and quality control, although in vitro assays have shown that T. cruzi and higher eukaryotic UGGTs exclusively glucosylate high-mannose glycans in misfolded glycoproteins (66), in T. brucei the UGGT and GII enzymes use Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂, respectively, as their substrates in the processing of VSG variant 221 (VSG221) (29). However, it could not be concluded from that study whether this apparent preference for atypical biantennary Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂ structures reflected the substrate specificity of the enzymes or the location of the glycosylation site in the VSG polypeptide chain (30).In this work, we further analyze the specificity and function of the UGGT/GII quality control system of T. brucei by analyzing the non-VSG N-glycans of our α-GII null mutant and creating and characterizing a T. brucei UGGT null mutant.     

Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)

Vol. 57 No. 11, 2006,     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.     

A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.