Characterization of the Enzymes Encoded by the Anthrose Biosynthetic Operon of Bacillus anthracis

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β-l-rhamnose α-1,3-l-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, rod-shaped soil bacterium that forms spores when deprived of essential nutrients (15). Spore formation begins with an asymmetric septation that divides the developing cell into a forespore compartment and a larger mother cell compartment, each of which contains a copy of the genome. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (10). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (17). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (21).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost layer of the spore, the exosporium, which serves as a semipermeable barrier to potentially harmful macromolecules (8, 25) and as the vital first point of contact with the immune system of an infected host (11, 18, 30). The exosporium of B. anthracis and of closely related species, such as Bacillus cereus and Bacillus thuringiensis, is comprised of a paracrystalline basal layer and an external hairlike nap (1). The basal layer contains approximately 20 different proteins (20, 23), while the filaments of the nap are formed by trimers of a single collagenlike glycoprotein called BclA (2, 26). The central region of BclA contains a large number of GXX repeats, and the region varies in length in naturally occurring strains of B. anthracis, resulting in hairlike naps of differing lengths (22, 27). Most of the GXX repeats are GPT, and many of the threonine residues are glycosylated. Two major oligosaccharide side chains are present, a pentasaccharide and a trisaccharide, and both are linked to the protein through reducing terminal N-acetylgalactosamine (GalNAc) residues (3). Several studies have demonstrated that the oligosaccharides are antigenic and are exposed on the surface of Bacillus anthracis spores (14, 29). This makes them prime targets for both detection devices and immunoprophylaxis.We previously reported our use of hydrazinolysis to release BclA oligosaccharides from exosporium preparations (3). The primary product was a tetrasaccharide that formed as a result of the undesirable loss of the reducing terminal GalNAc residue of the pentasaccharide, a process called “peeling.” We determined that the oligosaccharide consisted of a linear chain of three rhamnose residues with a novel deoxyamino sugar at its nonreducing terminus. This unusual sugar, 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, was given the trivial name anthrose.Rhamnose is the major sugar present in both the trisaccharide and the pentasaccharide, and a four-gene rhamnose biosynthetic operon was previously identified (22). Previously, we proposed a pathway for anthrose biosynthesis (Fig. ​(Fig.1)1) and identified a four-gene operon (Fig. ​(Fig.2)2) that is essential for its biosynthesis (5). An in-frame deletion of the first gene of the operon reduced the amount of anthrose by approximately 50%, whereas the deletion of any one of the other three genes totally abolished anthrose synthesis. Here, we describe the characterization of the altered oligosaccharide side chains of the four deletion mutants. We also cloned several genes that we predicted are involved in anthrose biosynthesis and demonstrated that the gene products possessed the expected biochemical activities.Open in a separate windowFIG. 1.Proposed biosynthetic pathway of anthrose. The pathway utilizes dTDP-4-keto-6-deoxy-α-d-glucose, an intermediate in rhamnose biosynthesis, and methylcrotonyl-CoA, derived from leucine catabolism. (Modified from reference 5.)Open in a separate windowFIG. 2.Anthrose operon and flanking genes. The four genes of the anthrose operon are antA (BAS3322), antB (BAS3321), antC (BAS3320), and antD (BAS3319). The operon is flanked by genes that encode a putative collagenase (BAS3323) and a putative methyltransferase (BAS3318). (Modified from reference 5.)     

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore''s DPA once rapid DPA release had begun (ΔT_release) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ΔT_release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination ≥15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T_lag, between mixing spores with nutrient germinants and the beginning of ΔT_release. (iv) A number of factors decrease spores'' T_lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25, 29, 30). The germinants that most likely trigger spore germination in the environment are low-molecular-weight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore''s inner membrane that recognize their cognate germinants in a stereospecific manner (17, 24, 25, 29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6, 29, 30). Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B. subtilis spore germination by binding of l-alanine or l-valine to the GerA GR. However, many GRs cooperate such that binding of germinants by ≥2 different GRs is needed to trigger germination (2, 29): for example, the triggering of B. subtilis spore germination by the binding of components of a mixture of l-asparagine, d-glucose, d-fructose, and K⁺ ions (AGFK) to the GerB and GerK GRs. The binding of nutrient germinants to GRs triggers subsequent events in germination, although how this is accomplished is not known.The first readily measured biochemical event after addition of nutrient germinants to Bacillus spores is the rapid release of the spore''s large depot (∼10% of spore dry weight) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus its chelated divalent cations, predominantly Ca²⁺ (Ca-DPA), from the spore core (25, 29). Ca-DPA release then results in the activation of two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, which hydrolyze the spore''s peptidoglycan cortex layer (16, 22, 27, 29). CwlJ is activated by Ca-DPA as it is released from the spore while SleB is activated only after most DPA is released (17, 20, 22, 26, 27). Cortex hydrolysis ultimately allows the spore core to expand and take up more water, raising the core water content from the 35 to 45% of wet weight in the dormant spore to the 80% of wet weight characteristic of growing cells. Full hydration of the spore core then allows enzyme action, metabolism, and macromolecular synthesis to resume in the now fully germinated spore.Germination of spores in populations is very heterogeneous, with some spores germinating rapidly and some extremely slowly (4, 5, 9, 11, 13-15, 19, 26, 31, 32). Where it has been studied, the reason for this heterogeneity has been suggested to be due to a variable lag period (T_lag) between the time of mixing spores with a germinant and the time at which rapid DPA release begins, since once rapid DPA release begins, the time required for release of almost all DPA as well as for subsequent cortex hydrolysis is generally rather short compared to T_lag values in individual spores (5, 11, 13-15, 19, 26, 31, 32). The times required for DPA release and cortex hydrolysis are also similar in wild-type spores with both very short and long T_lag values (5, 15, 19, 27). The reasons for the variability in T_lag times between individual spores in populations are not known, although there are reports that both activation of spores for germination by a sublethal heat treatment (heat activation) as well as increasing concentrations of nutrient germinants can shorten T_lag values (12, 14, 15, 18, 32). However, there has been no detailed study of the causes of the variability in T_lag values between very large numbers of individual spores in populations.In order to study the heterogeneity in spore germination thoroughly, methods are needed to follow the germination of hundreds of individual spores over several hours. Initial studies of the germination of individual spores examined a single spore in a phase-contrast microscope and followed the germination of this spore by changes in the core''s refractive index due to DPA release and core swelling (14, 15, 32, 34). However, this method is labor-intensive for gathering data with hundreds of individual spores. More recently, confocal microscopy and then surface adsorption and optical tweezers have been used to capture single spores, and germination events have been followed by methods such as Raman spectroscopy to directly measure DPA release, as well as phase-contrast microscopy and elastic light scattering (3, 5, 9, 10, 19, 26). While the latter recent advances have allowed accumulation of much information about germination, collection of this type of data for large numbers of individual spores is still labor-intensive, although use of dual optical traps (35) and perhaps multiple traps in the future may alleviate this problem. However, phase-contrast microscopy plus appropriate computer software has recently allowed the monitoring of many hundreds of individual spores for several hours, with automated assessment of various changes in the cells during the period of observation (19). In the present work, we have used both phase-contrast and differential interference contrast (DIC) microscopy to monitor the germination of many hundreds of individual spores of three Bacillus species adhered on either an agarose pad or a glass coverslip for 1 to 2 h. This work, as well as examination of times needed for release of most DPA once rapid DPA release has begun during germination of individual spores under a variety of conditions, has allowed detailed examination of the effects of heat activation, nutrient germinant concentration, GR numbers per spore, and individual CLEs on spore germination heterogeneity and on values of T_lag for individual spores.     

Synthesis, characterization, and cytotoxicity of some novel glycosyl thiazol-2-imines as antitumoral agents

A series of novel glycosyl thiazol-2-imines (3a-g) have been synthesized regioselectively in good yields from the hydrolysis of thiazol-2(3H)-imine-linked glycoconjugates. The glycosyl thiazol-2-imines were evaluated for their antitumor activity against Hela (cervical carcinoma), HCT-8 (colon carcinoma) and Bel-7402 (liver carcinoma). Among the compounds screened, 1-benzoyl-4-(4-nitrophenyl)-3-β-d-glucopyranosyl-thiazol-2(3H)-imine (3c) was found to be the most active compound against HCT-8.     

F-box Protein Arabidillo-1 Promotes Lateral Root Development by Depressing the Functioning of GA<Subscript>3</Subscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, Arabidillo-1 and Arabidillo-2 have great sequence homology to Dictyostelium and metazoan β–catenin/Armadillo, which are important to animal and Dictyostelium development. Arabidillo-1 and Arabidillo-2 promote lateral root formation redundantly in Arabidopsis. Here, we showed that gibberellins (GA₃) has a greater inhibitory effect on lateral root growth from the null mutant arabidillo-1 than from the wild type, suggesting that the mechanism for Arabidillo-1-regulated modulation of lateral root proliferation is associated with GA₃-metabolic or signaling pathways. Our yeast two-hybrid analysis demonstrated that Arabidillo-1 interacts with ASK2 and ASK11, and that ASK2 can bind with the F-box domain of Arabidillo-1. Therefore, Arabidillo-1 is involved in the ubiquitin/26S proteasome-mediated proteolytic pathway. Based on these results, we conclude that Arabidillo-1 can degrade some positive regulator of the GA₃ signaling pathway through selective protein degradation of ubiquitin/26S. Moreover, that process is believed to be the mechanism for Arabidillo-1 promotion of lateral root development in Arabidopsis.     

IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Recruitment of active glycogen synthase kinase-3 into neuronal lipid rafts

Glycogen synthase kinase (GSK)-3beta has emerged as a key molecule that regulates neuronal apoptosis. To examine the molecular mechanism(s) through which GSK-3beta regulates this process, we studied the subcellular localization of GSK-3beta following exposure of the cells to well-characterized apoptotic stimuli. Here, we report that the induction of apoptosis by withdrawal of serum and potassium triggers dephosphorylation of GSK-3beta at serine 9 and subsequent translocation of these molecules into neuronal lipid raft microdomains. Inhibition of GSK-3beta by small molecule inhibitors blocks specific phosphorylation of lipid raft associated protein Tau. Consistent with the notion that the lipid raft domains may serve as a platform for the cellular signaling complexes, disruption of lipid rafts protected neurons from apoptosis induced by withdrawal of serum and potassium as well as by HIV-1 Tat. Our observations reveal novel interaction of GSK-3beta and raft domains, and suggest that such interaction could contribute to neuronal apoptosis.     

Repletion of atypical protein kinase C following RNA interference-mediated depletion restores insulin-stimulated glucose transport

The role of atypical protein kinase C (aPKC) in insulin-stimulated glucose transport in myocytes and adipocytes is controversial. Whereas studies involving the use of adenovirally mediated expression of kinase-inactive aPKC in L6 myocytes and 3T3/L1 and human adipocytes, and data from knock-out of aPKC in adipocytes derived from mouse embryonic stem cells and subsequently derived adipocytes, suggest that aPKCs are required for insulin-stimulated glucose transport, recent findings in studies of aPKC knockdown by small interfering RNA (RNAi) in 3T3/L1 adipocytes are conflicting. Moreover, there are no reports of aPKC knockdown in myocytes, wherein insulin effects on glucose transport are particularly relevant for understanding whole body glucose disposal. Presently, we exploited the fact that L6 myotubes and 3T3/L1 adipocytes have substantially different (30% nonhomology) major aPKCs, viz. PKC-zeta in L6 myotubes and PKC-lambda in 3T3/L1 adipocytes, that nevertheless can function interchangeably for glucose transport. Accordingly, in L6 myotubes, RNAi-targeting PKC-zeta, but not PKC-lambda, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; more importantly, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-lambda. Conversely, in 3T3/L1 adipocytes, RNAi constructs targeting PKC-lambda, but not PKC-zeta, markedly depleted aPKC and concomitantly inhibited insulin-stimulated glucose transport; here again, these depleting/inhibitory effects were rescued by adenovirally mediated expression of PKC-zeta. These findings in knockdown and, more convincingly, rescue studies, strongly support the hypothesis that aPKCs are required for insulin-stimulated glucose transport in myocytes and adipocytes.     

Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.