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Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular-weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other α-hydroxyacids, as acceptor substrate. In sharp contrast to the structures reported for the GlcNAc—1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon site hybridization study as required for growth, sporulation, or germination, suggesting that the biosynthesis of BSH could represent a target for development of novel antimicrobials with broad spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.
In his recent review on the management of oxidative stress in Bacillus, Zuber (1) concludes that the metabolites that function in redox buffering and thiol homeostasis, and their influence on the oxidative stress response, are not well understood. Earlier work from this laboratory (2) demonstrated that CoASH provided the major low-molecular weight thiol redox buffer in Bacillus anthracis, replacing GSH as had previously been demonstrated for Staphylococcus aureus (3). The likelihood that CoASH plays an important functional role in redox buffering and thiol homeostasis is strengthened by the demonstration that both B. anthracis (4) and S. aureus (3, 5-7) have NAD(P)H-dependent coenzyme A-disulfide reductases (CoADRs).1 We have also shown that the type III pantothenate kinase, which is unusual in its insensitivity to feedback inhibition by CoASH (2), is essential for growth of B. anthracis (8). CoADR, the chimeric CoADR-RHD protein (9), and the type III pantothenate kinase represent three well-characterized adaptations to the CoASH-based redox buffer system for this Gram-positive pathogen.
A new unknown thiol, originally referred to as U12, was also identified in the B. anthracis extract (2); a mass of 398 Da was reported for U12-SH. By combining analytical chemical approaches with mass spectrometry and NMR, the structure of U12 has been determined to be N-cysteinyl-α-D-glucosaminyl L-malate, and U12 has been renamed bacillithiol [BSH (10)]. BSH is present at intracellular concentrations of 0.1-0.35 mM 2 in a number of Bacillus species, including B. anthracis, Bacillus subtilis, and Bacillus megaterium, as well as in S. aureus. While none of the four B. subtilis strains represented in the NCBI database have orthologs of CoADR or any of its isoforms (9), B. anthracis, B. megaterium (11), and S. aureus (3) maintain both CoASH and BSH redox buffer systems, in the absence of GSH. Pöther et al. (12), in a recent study with S. aureus demonstrating that the majority of reversible protein thiol oxidations observed during treatment of cells with diamide are based on S-thiolations with Cys rather than protein disulfide formation, have concluded that Cys also functions as an important thiol redox buffer in S. aureus.
In an initial study of the biosynthesis and functions of BSH (13), we have demonstrated that the B. subtilis YpjH protein (BsuBshA), the enzyme orthologous to B. anthracis ORF BA1558, catalyzes the synthesis of N-acetyl-α-D-glucosaminyl L-malate (GlcNAc-malate) from UDP-GlcNAc and L-malate; the product structure has been confirmed by mass spectrometry (10, 13). We have also demonstrated that the BA1557 gene product (BaBshB) catalyzes the deacetylation of GlcNAc-malate (13), providing the free 2-amino group of the GlcN moiety required for the Cys ligation step that is proposed to complete the synthesis. Deletion of the B. subtilis yllA locus, predicted to be involved in the biosynthetic pathway and initially suggested to encode this putative ligase, does eliminate BSH production; the recombinant protein, however, does not catalyze ATP-dependent BSH formation from Cys and GlcN-malate, as assayed in vitro.
In the absence of functional information, Ruane et al. (14) have reported the structure of the ORF BA1558 (identified in this work as BaBshA) apoenzyme, refined at a resolution of 3.1 Å. Although this enzyme, like the MshA glycosyltransferase that produces GlcNAc-Ins-P in the first step of mycothiol (MSH) biosynthesis (15, 16), is a member of the GT-B and GT4 fold (17) and Carbohydrate-Active enZymes (18) families, respectively, structural homology searches using the MshA apoenzyme structure were unsuccessful in identification of other GT-B family members, including ORF BA1558. This indicates a major conformational difference between the two apoenzymes, despite the similarity in reactions catalyzed. Fadouloglou et al. (19) reported the crystal structure of the Zn2+-binding BcZBP protein from Bacillus cereus, as refined at a resolution of 1.8 Å. BcZBP is the enzyme orthologous to BaBshB, the GlcNAc-malate deacetylase, with 97% sequence identity. A recent kinetic analysis (20) with GlcNAc and several GlcNAc oligomers indicated that, although the biological function and natural substrate for BcZBP were unknown, the enzyme had optimal activity with (GlcNAc)2 (kcat/Km = 3.3 × 107 M-1s-1 at 37°C). The enzyme is a member of the PIG-L superfamily represented by GlcNAc-phosphatidylinositol deacetylase (21), the MshB deacetylase (22) of the MSH biosynthetic pathway, and the mycothiol S-conjugate amidase (23). The active-site Zn2+ of BcZBP is buried at the bottom of a ca. 12 Å deep cavity on the hexamer surface, and Arg140 is prominently positioned at the entry of the cavity. Both R140A and R140E mutants exhibit kcat/Km values 0.2-0.3% that of wild-type enzyme (20). His12, Asp15, and His113 provide the protein ligands to the Zn2+, and Asp14 has been proposed as an acid-base catalyst in the hydrolase reaction. A model of a BcZBP-GlcNAc complex reveals that this substrate is considerably smaller than the volume of the cavity; a hydrophilic patch consisting of the side chains of Asp108, Asn150, and Tyr194 marks an unoccupied region proximal to the Zn2+. The Y194F mutant specificity constant, which is <0.1% that of wild-type (20), reflects a dominant effect on Km. In particular, the anomeric C1-OH of GlcNAc also appears to be accommodated by a relatively large space within the cavity. For the BcZBP apoenzyme structure, three very mobile loops have recently been implicated in determining active-site accessibility and regulating substrate specificity (24).
Given the goal of better defining the thiol redox buffer in B. anthracis and characterizing potential new targets for the development of antimicrobial agents that are selective against B. anthracis, S. aureus, and other Gram-positive pathogens that rely on the BSH-based redox buffer system, we report here detailed kinetic analyses of the BaBshA glycosyltransferase and BaBshB deacetylase. The kinetic studies are interpreted in view of the ORF BA1558 and BcZBP crystal structures, and we also provide the structure of the BaBshA-UDP-malate complex, refined at a resolution of 3.3 Å, which demonstrates the absence of any major conformational change on ligand binding. In addition we show that deletion of the BAS1445 locus encoding BshA in B. anthracis Sterne generates a BSH-deficient strain appropriate for testing in a murine model of inhalational anthrax.
The B. anthracis Sterne 34F2 strain (pXO1+ pXO2-) used in these studies was obtained from Dr. Philip Hanna, University of Michigan Medical School. Cultures were grown and maintained in BHI broth (Difco) and on solid media containing 15 g/L of agar. For preparation of parental and mutant endospores, cultures were grown at 37°C on BHI plates containing antibiotics as appropriate. A single colony was inoculated into 3 mL of BHI broth containing antibiotics as required, and this culture was taken as the inoculum (5%, v/v) into 75 mL of fresh modified G (sporulation) medium (25) without antibiotic, after 8-12 h of growth. After 4 days' growth at the appropriate temperature endospores were collected by centrifugation, and residual vegetative cells were killed by a 30-min incubation at 65°C. Pellets were washed 3-4 times in deionized water, and endospores were stored (in deionized water) at ambient temperature. The purity of the spore preparations was confirmed by phase-contrast microscopy, and spore titers were determined by serial dilution. Escherichia coli strains DB3.1 (26) and GM272 were used for the cloning and propagation of pBKJ236 and pBKJ223 (27), respectively. DB3.1(pBKJ236) was cultured and maintained in LB broth or on solid media supplemented with 250-400 μg/mL erythromycin. GM272(pBKJ223) was cultured and maintained in LB broth or on solid media supplemented with 15 μg/mL tetracycline.
In-frame deletion mutants of the genes encoding BaBshA (bshA, loci BA1558 and BAS1445 in Ames and Sterne strains, respectively), BaBshB (bshB, loci BA1557 and BAS1444, respectively), BaCoADR [cdr, loci BA1263 (4) and BAS1170, respectively] and BaCoADR-RHD [cdr2, loci BA0774 (9) and BAS0736, respectively] were constructed following the markerless gene replacement protocol (27), using the pBKJ236 and pBKJ223 plasmids. The procedure followed in this work is described in detail for the BASΔ1445 mutant. A modified chromosomal segment containing the BAS1445 deletion was constructed by first amplifying the flanking upstream and downstream regions (>500 bp each) using primer sets BAS1445up (FWD/REV) and BAS1445dwn (FWD/REV), respectively (Table S1). The two amplified products were then ligated using overlap PCR with BAS1445up FWD/BAS1445dwn REV, and the resulting deletion fragment was TA cloned into pCR2.1 (Invitrogen) before subcloning into pBKJ236 digested with NotI. This plasmid, pBKJ236::BAS1445′ was electroporated into methylation-deficient (dam- dcm-) E. coli GM272. E. coli GM272(pBKJ236::BAS1445′) (LB plus 250 μg/mL erythromycin), E. coli helper strain 459 (LB plus 100 μg/mL ampicillin), and B. anthracis Sterne were grown overnight, and aliquots (75 μL) from washed samples of each culture were mixed and spotted onto BHI agar before incubating for 48 h at room temperature. The accumulated growth was resuspended in fresh LB broth, and 150 μL of the suspension was dispensed in a "straight line" on one quadrant of a BHI agar plate supplemented with 5 μg/mL erythromycin and 80 U/mL polymyxin B. After the line was allowed to dry, cells were streaked to isolation and incubated at room temperature for 48 h. Isolated EryR colonies were cultured overnight, again at room temperature, before diluting 1:1000 in fresh BHI broth plus erythromycin and shifting to 37°C, a nonpermissive temperature for plasmid replication. Following this overnight incubation, integrants were isolated using the "straight line" procedure described above, but with incubation at 37°C on BHI agar plus erythromycin. A single EryR colony was grown out, to A600 ca. 0.2, in 25 mL of LB plus glucose (28) at 37°C, with shaking at 100 rpm; cells were harvested by centrifugation at 4°C, resuspended and washed in ice-cold electrotransformation buffer (29), and transferred (400 μL) to a chilled 0.4-cm electrode-gap cuvette. After 10 min incubation on ice, with 5 μL of unmethylated pBKJ223 [isolated from E. coli GM272(pBKJ223)], cells were electroporated at 2.4 kV, 200 Ω and 25 μF. After pulsing, cells were placed back on ice, and 700 μL of ice-cold LB plus glucose was added; the mixture was transferred to a microcentrifuge tube, and cells were allowed to recover for 3 h at 30°C before plating on BHI agar plus 10 μg/mL tetracycline and incubating overnight at 37°C. Several single TetR colonies selected after repeatedly mixing and streaking to isolation were patched onto BHI agar plus erythromycin and BHI agar plus tetracycline and incubated overnight at 37°C. Clones with the desired TetR EryS (loss of pBKJ236) phenotype were validated by colony PCR, repurified by streaking, and patched onto BHI agar with and without tetracycline. Clones cured of TetR (loss of pBKJ223) were again validated by colony PCR to confirm the BAS1445 deletion. ΔbshB, Δcdr, and Δcdr2 mutants were constructed following the same protocol; the ΔcdrΔcdr2 double mutant utilized the Δcdr strain in the initial conjugation step.
An active culture grown to A600 of 0.5 (mid-log) was used to inoculate (1% v/v) 5 mL aliquots of fresh BHI broth containing fosfomycin at incremental concentrations. The wild-type Sterne strain showed robust growth (static conditions in 17 × 100 mm polystyrene tubes) at all fosfomycin concentrations (0-1,000 μg/mL). The ΔbshA mutant grew only in the control (no fosfomycin) culture and at the lowest fosfomycin concentration tested (200 μg/mL), but not at concentrations ≥400 g/mL. The ΔbshB mutant was sensitive to fosfomycin at 800 μg/mL, but not at ≤400 μg/mL.
The codon-optimized BA1558 and BA1557 genes, encoding BaBshA and BaBshB, respectively, were synthesized by GenScript (Piscataway, NJ) and subcloned into pET28a(+) (Novagen). Both genes were expressed in E. coli BL21(DE3) cells using auto-induction media at 37°C (30); BshA was expressed with an N-terminal His-tag (see "Results"), while BshB had a C-terminal His-tag (-LEH6). All steps of both purifications were carried out at 4°C. Harvested cells were disrupted using an Avestin EmulsiFlex-C5 homogenizer, and nucleic acids were removed by addition of 2% streptomycin sulfate and centrifugation (27,000g for 20 min). For BshA, the clarified extract was loaded onto a 25-mL Ni Sepharose High Performance column (GE Healthcare, Piscataway, NJ) in 50 mM sodium phosphate, pH 8.0, containing 0.3 M NaCl and 20 mM imidazole; the protein was eluted with a stepwise increase of imidazole, to 250 mM. Peak fractions were pooled and loaded onto a Q-Sepharose HP column after dialysis against 20 mM HEPES, pH 7.5, containing 50 mM NaCl. The protein was eluted with a linear 375-mL gradient of 0.05 → 1 M NaCl, and the BaBshA pool was dialyzed against the Q-Sepharose loading buffer before concentrating and storing in aliquots at -80°C. For BaBshB, the immobilized metal-ion affinity chromatography column was first converted to the Zn2+ form (31); the clarified extract was loaded onto the 25-mL Zn Sepharose column in 50 mM sodium phosphate, pH 8.0, containing 0.3 M NaCl and 20 mM imidazole. The protein was eluted with a stepwise increase of imidazole, to 250 mM. Peak fractions were pooled and loaded onto a Q-Sepharose HP column after dialysis against 25 mM potassium phosphate, pH 7.0. The protein was eluted with a linear 375-mL gradient of 0 → 1 M NaCl, and the purified BaBshB protein was dialysed against 25 mM Tris-HCl, pH 8.0, containing 50 mM NaCl, concentrated by ultrafiltration, and brought to a final concentration of 20% (v/v) glycerol before being frozen in aliquots at -80°C.
The cysteinyl-tRNA synthetase gene (cysS, BA0089) was amplified from genomic B. anthracis DNA using primers designed with NcoI and XhoI linkers that allowed cloning into pET28a. The recombinant C-terminal His-tagged CysS protein was expressed in E. coli BL21(DE3); cultures grown to mid-log phase in LB plus kanamycin at 37°C were induced with lactose for 3 h. Harvested cells were resuspended in 1.5 volumes of degassed 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and were lysed with a French press. The extract was clarified by centrifugation and applied to a Ni2+-charged metal-ion affinity column (GE Healthcare) equilibrated with the lysis buffer; the column was washed with this buffer containing 20 mM imidazole before the recombinant CysS protein was eluted with 100 mM imidazole. Pooled fractions were diluted with 5 volumes of lysis buffer and loaded onto a HiTrap Q XL column (GE Healthcare); the column was washed with the lysis buffer before eluting the protein with a linear 50-mL gradient of 0 → 1 M NaCl. The cysteine adenylation activity of B. anthracis CysS was assayed using the ATP-32PPi exchange reaction in the presence of 50 μM Cys (32).
GlcNAc-malate required for kinetic analysis of BaBshB was produced on a preparative scale by incubating 0.5 mg BaBshA at 37°C with 50 μmol each of UDP-GlcNAc and L-malate (substrates purchased from Sigma) in 5 mL of 25 mM HEPES, pH 7.5, containing 100 mM NaCl, 10 mM MgCl2, and 1 mM 2-mercaptoethanol. The reaction was sampled at indicated times; samples were processed as described by Newton et al. (15) for identification of the MshA-catalyzed reaction products (using unlabeled substrates) and were analyzed by HPLC with A260 detection for UDP-GlcNAc and UDP. The conversion of UDP-GlcNAc → UDP was complete in 2 h. The GlcNAc-malate product (50 μmol, ca. 15 mg) was processed for preparative HPLC purification by 1) adding 1 volume of acetonitrile to the reaction mixture and incubating at 60°C for 15 min, 2) clarifying by centrifugation, and 3) adjusting the supernatant pH→3 with trifluoroacetic acid after reducing the volume to 2 mL in a SpeedVac. A Vydac 218TP1022 (1 × 25 cm) reversed-phase preparative column was used with a linear gradient from 0→20% methanol in 0.1% trifluoroacetic acid, over 40 min (flow rate of 5 mL/min), with online detection by mass spectrometry. Fractions containing GlcNAc-malate (ESP-, m/z 336) were combined, and the separation was repeated to obtain the purified product, which was adjusted to pH 6 with NaOH and lyophilized.
Synthetic GlcNAc-malate was maintained at -70°C in H2O at a concentration of 80 mM and contained ca. 1 equiv sodium trifluoroacetate from the HPLC solvent. The sodium trifluoroacetate content was determined in the quantitative pH titration described above. In addition, the disodium GlcNAc-malate content determined by the BaBshB—Accu-Tag method (see below) accounts for ca. 50% of the total mass of the lyophilized material. 1H- and gCOSY NMR spectra were recorded on a Jeol ECA500 at 500 MHz and were referenced to residual solvent (HDO) at 4.8 ppm. The 1H NMR of GlcNAc-malate in D2O (trifluoroacetate is not observable) confirmed the absence of other organic impurities. 13C spectra were collected on a Varian X500 (125 MHz) equipped with an XSens cold probe. All spectra were collected at 23°C. High resolution mass spectrometry analysis was provided by the UCSD Chemistry and Biochemistry Mass Spectrometry Facility using a Thermo Scientific LTQ Orbitrap XL mass spectrometer operating in positive ion mode ESP ionization.
The kinetic properties of BaBshA were first analyzed from initial rates of UDP-GlcNAc → UDP conversion, using the A260-based HPLC assay described above (15) for monitoring GlcNAc-malate synthesis. Initial rates were determined over a range of UDP-GlcNAc concentrations (0→25 mM) at a fixed concentration of 2 mM L-malate (ca. 15 × Km, see "Results"); for each individual assay at 37°C [in 25 mM HEPES, pH 7.5, containing 100 mM NaCl, 10 mM MgCl2, and 1 mM 2-mercaptoethanol], the reaction was sampled four times in order to establish the observed rate of UDP formation. Similarly, initial rates were measured over a series of L-malate concentrations (0→3.2 mM) at a fixed concentration of 3 mM UDP-GlcNAc (ca. 5 × Km). Results representing the mean of triplicate determinations were fit to the Michaelis-Menten equation [equation 1 (33)] where Vmax is the apparent maximal velocity, A is the concentration of the varied substrate, and KA is the Michaelis constant (apparent) for substrate A.
Data described with this single variable equation were analyzed using KaleidaGraph.
In order to analyze the steady-state kinetic mechanism with several fixed concentrations of UDP-GlcNAc and L-malate, respectively, the continuous spectrophotometric assay for glycosyltransferases (34) was adapted, using a 96-well format with a POLARstar OPTIMA (BMG LABTECH) microplate reader. For each assay at 37°C, 30 ng of BaBshA was incubated in 25 mM HEPES, pH 7.5, containing 50 mM KCl, 10 mM MgCl2, 7.5 U pyruvate kinase and 15 U lactate dehydrogenase (both enzymes purchased from Sigma), 0.8 mM phosphoenolpyruvate, and 0.15 mM NADH in a final volume of 200 μL. The concentration of L-malate was varied over the range 0.08-1.2 mM at fixed concentrations of 0.1-1.5 mM UDP-GlcNAc. Reactions were initiated by addition of enzyme and were performed in duplicate. These results (for varying [L-malate]) were fit to the reciprocal form of the velocity equation for a rapid equilibrium random system [equation 2 (33)], using GraFit version 5.
where Don = UDP-GlcNAc and Acc = L-malate. α is a factor representing the effect each substrate has on the binding of the second substrate. Controls showed that the omission of 2-mercaptoethanol and substitution of 50 mM KCl for 100 mM NaCl in the coupled assay had no effect on initial velocity. In addition, any competitive UDP-GlcNAc hydrolysis reaction (measured in the absence of L-malate) was negligible.
The kinetic parameters for BaBshB were determined from initial rates of GlcNAc-malate → GlcN-malate conversion, using an HPLC assay for the AccQ-Tag derivative of the GlcN-malate product, as monitored by fluorescence (13, 31, 35). Initial rates were determined over a series of GlcNAc-malate concentrations (0→2 mM); for each assay at 37°C [in 50 mM HEPES, pH 7.5, containing 50 mM NaCl and 1 equiv of sodium trifluoroacetate per mol GlcNAc-malate], the reaction was sampled at intervals in order to establish the rate of GlcN-malate formation. Results representing the mean of triplicate determinations were fit to equation 1 as described above. Bacillithiol S-conjugate amidase activity was measured using the methylbimane derivative of BSH (BSmB) purified as described (10). The reaction was sampled at intervals; aliquots were processed for analysis, using an adaptation of the fluorescence-based HPLC assay for CySmB (+ GlcN-malate) product formation described previously (23). The initial rate of CySmB formation was determined by sampling the reaction four times in the presence of 320 μM BSmB.
After purification BaBshA was buffer-exchanged into 20 mM HEPES, pH 7.5, containing 150 mM NaCl plus 5 mM dithiothreitol, and concentrated to 16 mg/mL for crystallization. Crystals were obtained in 0.2 M Mg(HCO2-)2 containing 15% PEG3350 with 1 mM each of UDP and L-malate. Crystals were cryoprotected with mother liquor supplemented with 25% glycerol and flash-frozen in liquid nitrogen. BaBshA crystals, which appeared after 4-5 days, diffracted to 3.3 Å. Data sets were collected at SER-CAT beamline 22-BM (Advanced Photon Source, Argonne National Laboratory). Data were indexed and scaled using HKL2000 (36). Molecular replacement was performed using PHASER (37), and model refinement was carried out using the PHENIX software suite (38). The model was adjusted manually using COOT (39) as well as 2Fo-Fc and Fo-Fc electron density maps. The UDP and L-malate model and definition files were generated with PHENIX.
All manipulations of B. anthracis Sterne strains were carried out under Biosafety Level 2 conditions at Wake Forest University School of Medicine and at University of California, San Diego. The detailed methods for culture growth, sample preparation, and analysis, including the determination of thiol redox status, are given in Nicely et al. (2) and Newton et al. (10).
Sequence analyses were performed with the NCBI and PDB databases using BLASTP, and the resources of the Carbohydrate-Active enZymes database (18) were also utilized in this work. Structure superpositions and sequence alignments were performed using DALILITE (40), PyMOL (41) , CLUSTALW (42), and ESPript (43), respectively. Figures of protein molecules and residues were prepared using PyMOL.
Given the structural similarity between bacillithiol and mycothiol (Figure 1) and the well-defined pathway for MSH biosynthesis in Mycobacterium tuberculosis and other Actinobacteria (44, 45), three distinct enzymatic steps have been considered in the pathway for BSH biosynthesis (Figure 2). The first reaction produces GlcNAc-malate from UDP-GlcNAc and L-malate, as indicated:
We have demonstrated (13) that the B. subtilis YpjH gene product catalyzes the synthesis of GlcNAc-malate as given in reaction (1). In B. anthracis the BA1558 locus is the ortholog of B. subtilis ypjH; as shown in the preparative scale assay (Figure S1), ORF BA1558 catalyzes the production of UDP and GlcNAc-malate when incubated with UDP-GlcNAc and L-malate. Isolation of the GlcNAc-malate product from this preparative scale reaction allowed its confirmation by 1H and 13C NMR (Figure S2) and high resolution mass spectrometry. The proton NMR spectra (Figure S2A) indicated that the GlcNAc-malate was pure (see "Experimental Procedures") and contained the L-malate and D-glucosamine resonances found in bacillithiol (10). The glucosamine N-acetyl group is validated by the acetyl protons (singlet, 2.04 ppm) and the carbon resonances at 174.3 and 21.9 ppm (Figure S2B). The carbon 2 proton (3.9 ppm) of glucosamine (nitrogen bonded carbon) is identified in the gCOSY spectra (Figure S2A) by correlation to the anomeric proton. The anomeric proton (4.89 ppm) coupling constant (3.8 Hz) indicated this was the α-anomer of the malyl glycoside as predicted by the structure of bacillithiol. The high-resolution mass spectra of this compound gave a mass of 360.0902 for the sodium ion of GlcNAc-malate (360.0901, δ 0.3 ppm) corresponding to a formula of C12H19NO10Na, consistent with the product of BaBshA. Confirmation of the novel D-glucosaminyl L-(α)-malate moiety validates the structure of bacillithiol from Deinococcus radiodurans (10) and shows that it is identical to bacillithiol produced in B. anthracis. ORF BA1558 is the BshA glycosyltransferase in B. anthracis and is therefore designated BaBshA (Figure 2).
When the structure of the M. tuberculosis GlcNAc-Ins deacetylase MshB [PDB entry 1Q74 (22)] was used to query the Protein Data Bank with DALILITE, the highest score (Z = 21.6) was observed with the Zn2+-binding BcZBP protein [PDB entry 2IXD; rmsd = 2.3 Å, 24% identity (19)] from B. cereus. This deacetylase is 97% identical in sequence to the B. anthracis ortholog, ORF BA1557. We have recently demonstrated (13) that recombinant ORF BA1557 catalyzes the efficient deacetylation of GlcNAc-malate → GlcN-malate, as given in reaction (2):
The preparative scale reaction, using GlcNAc-malate prepared by enzymatic synthesis with BaBshA and purified by HPLC (see "Experimental Procedures"), allowed isolation of the GlcN-malate product, and deacetylation of the substrate was confirmed by ESP mass spectrometry. ORF BA1557 is the BshB GlcNAc-malate deacetylase in B. anthracis and is therefore designated BaBshB. We have now identified the enzymatic activities, in vitro, for the BaBshA glycosyltransferase and the BaBshB deacetylase. Thus, from UDP-GlcNAc and L-malate precursors, these enzymes combine to produce GlcN-malate, UDP, and acetate; GlcN-malate has also been identified by HPLC analysis of cell-free extracts from B. subtilis (13), confirming this aspect of the BSH biosynthetic pathway as presented in Figure 2 for B. anthracis. While we have not attempted to identify GlcNAc-malate in vivo, these combined results provide very strong evidence that the biosynthetic scheme presented in Figure 2 for BshA and BshB represents the intracellular process in B. anthracis.
These functional assignments, as confirmed experimentally, leave the enzyme(s) responsible for the proposed ATP-dependent Cys:GlcN-malate ligase reaction (Figure 2) unidentified. The mshC gene product in M. tuberculosis is a second cysteinyl-tRNA synthetase isoform that differs from the functional CysS enzyme primarily through the absence of a 76-residue C-terminal extension that is responsible for recognition and specific binding of the tRNACys anticodon (46, 47); the Cys:GlcN-Ins ligase catalyzes the ATP-dependent formation of the amide linkage in the ultimate MSH product (+ AMP + PPi). BLASTP analysis of the B. anthracis genome, however, fails to identify any second CysS isoform, and the ORF BA0089 (annotated as cysteinyl-tRNA synthetase) protein as expressed is active in the standard tRNA synthetase assay. Although deletion of the B. subtilis yllA locus eliminates BSH production with a concomitant increase in intracellular GlcN-malate (the BshB product), the recombinant B. subtilis YllA and S. aureus CysS proteins both fail to catalyze ATP-dependent production of BSH from Cys and GlcN-malate in an in vitro assay (13).
Passalacqua et al. (48) have recently demonstrated that the seven genes corresponding to BA1554-BA1560 (Ames strain) are transcribed as a single mRNA unit in B. anthracis Sterne (BAS1441-BAS1447); this experimental result is consistent with results obtained earlier with the operon prediction algorithm developed by Bergman et al. (49). Given the involvement of both BSH and CoASH in the B. anthracis thiol redox buffer (2, 10), we note that the BA1562-BA1564 loci (panBCD) encoding three key enzymes in the synthesis of the CoASH precursor pantothenate (50) are separated by a single ORF from the bsh operon and have been demonstrated to be cotranscribed together with BA1565 in a second tetracistronic operon (48). In the event that candidate bshC loci might be clustered within the bsh operon, we analyzed the seven protein products for structural homologs of known function, using the PDB and NCBI databases. Table 1 summarizes details of the structural and functional annotations for each of the ORFs in the bsh and pan operons. Although there may be a functional linkage between BSH production and methylglyoxal synthase [ORF BA1556 (51)] activity (see "Discussion"), we find no evidence for candidate bshC loci in either bsh or pan operons in B. anthracis.
In contrast to the soil bacterium B. subtilis, which lacks the CoASH thiol redox buffer (9), the redundancy provided by the CoASH and BSH redox buffers in B. anthracis and S. aureus may provide these two pathogens with some advantage in virulence and/or survival, given the hostile environment posed by host defense mechanisms (52, 53). The results described above predict that individual deletions of the bshA and bshB genes in B. anthracis Sterne (loci BAS1445 and BAS1444, respectively) will disrupt the BSH biosynthetic pathway at the points of GlcNAc-malate and GlcN-malate precursor production, respectively. The two mutants were prepared using the markerless deletion method developed by Janes and Stibitz (27) and were analyzed for low-molecular-weight thiol content in parallel with wild-type B. anthracis Sterne and deletion mutants lacking the cdr and cdr2 genes that encode BaCoADR (4) and the chimeric CoADR-RHD protein (9). The major conclusions from the analytical data presented in Table 2 are as follows: 1) deletion of the bshA locus eliminates BSH production, 2) deletion of the bshB locus does not eliminate BSH production but reduces it by 30% relative to wild-type, 3) BSH redox status (BSH/BSSB) strongly favors the reduced thiol in vegetative B. anthracis cells, and this is not affected by deletions of the cdr and/or cdr2 loci, 4) the value for CoASH is higher (1.9±0.4 μmol/g dry residual weight) than that reported previously [0.87±0.11 μmol/g (2)]; this is attributed to the different wild-type strains used in the two analyses, 5) Cys is a major low-molecular weight thiol, consistent with the recent report of Pöther et al. (12), for S. aureus, and 6) thiosulfate (S=SO32-), a principal source of sulfur for aerobic bacteria (54) and donor substrate for the sulfurtransferase rhodanese (55), is a major inorganic sulfur compound in B. anthracis.
In B. subtilis the primary fosfomycin resistance determinant FosB functions as a thiol-dependent S-transferase that produces a thioether conjugate from the thiol and the antibiotic; until very recently, however, the enzyme had only been demonstrated to work quite poorly in vitro (56), with Cys as thiol substrate. Using a zone of inhibition assay we have recently demonstrated (13) that a B. subtilis mutant deficient in BSH is as sensitive to fosfomycin as a fosB mutant, leading to the conclusion that FosB is a BSH S-transferase. We tested the sensitivity of wild-type and ΔbshA mutant strains of B. anthracis to fosfomycin in liquid culture. While the wild-type strain grew well at 1,000 μg/mL fosfomycin, the bshA mutant did not grow at fosfomycin concentrations ≥400 g/mL. B. anthracis ORF BA4109 is annotated as the FosB protein, and we propose that this enzyme requires BSH as a cofactor for conjugation in the detoxification of fosfomycin (Scheme 1).
Our results with the ΔbshB mutant clearly indicate that other protein(s) capable of recognizing and deacetylating GlcNAc-malate are expressed in B. anthracis. Fadouloglou et al., in their earlier structural analysis of BcZBP (19), had identified ORF Bc3461 as a BcZBP paralog; more recently, this analysis was extended (24) with the identification of the BA630 (BaBshB), BA425 (ORF BA3524), and BA758 (ORF BA3888) orthologs in the B. anthracis genome (Figure 3). ORF Bc3461 has now been expressed and characterized (20); when assayed in parallel with BcZBP, the enzyme shows a preference for (GlcNAc)2 and (GlcNAc)3 as deacetylase substrates. kcat/Km(GlcNAc)2 = 5.6 × 106 M-1s-1, or ca. 20% that of BcZBP. While its biological role is unclear, ORF Bc3461 has potent deacetylase activity. Figure 3 gives a structure-based sequence alignment for BcZBP, BaBshB, ORF BA3888, ORF BA3524, and ORF Bc3461. While BA3888 and BA3524 have limited sequence similarity to BcZBP (26-28% identity), the latter protein is 95% identical in sequence to Bc3461; all three conserve the three Zn2+ protein ligands identified in BcZBP as well as the proposed active-site base and charge-relay dyad (His110/Asp112). These conserved features identify these as Zn2+-dependent hydrolases3; Figure 3 also identifies a series of conserved insertions and deletions that distinguish the true BshB deacetylases BcZBP and BaBshB from the three paralogs. These include a 10-residue insert I1 (relative to BcZBP) after Ser45, two small inserts (1-2 residues) following BcZBP Lys82 and Asp108, and two deletions D1 and D2 of 4-6 residues following Ile129 and Phe205, respectively. The I1 insert falls within the hypermobile BcZBP active-site loop L46, and the D1 deletion falls within the L135 loop identified in the molecular dynamics study (24). Fadouloglou et al. have implicated these loops (plus loop L185) in active-site accessibility and substrate specificity. Further consideration of proposed ORF BA3524 and BA3888 catalytic functions is presented in a following section.
Using a coupled assay, Vetting et al. (16) reported that the dimeric CgMshA catalyzes the formation of GlcNAc-Ins-P via a sequential (ternary complex) kinetic mechanism in which the donor substrate UDP-GlcNAc almost certainly binds first. The steady-state kinetic parameters determined at pH 7.8, 25°C, yield Km(UDP-GlcNAc) = 0.21 mM, Km(Ins-1-P) = 0.24 mM, and kcat = 12.5 s-1. Structural analyses of the apoenzyme and the binary UDP complex demonstrate that nucleotide binding leads to a major rotation of the C-terminal domain relative to the N-terminal domain. kcat/Km(UDP-GlcNAc) is 6 × 104 M-1s-1, and a superposition of the CgMshA apoenzyme and UDP complex structures gives an rmsd of ca. 11 Å, underscoring the magnitude of the conformational change in the UDP complex.
Using an HPLC assay for the UDP product, we demonstrated that the BaBshA reaction was saturable with respect to both donor and acceptor substrates (Figure 4). At pH 7.5 and 37°C, in the presence of 100 mM NaCl, the apparent kinetic parameters (at 2 mM L-malate) for UDP-GlcNAc are Km = 0.37 mM, kcat = 28 s-1, and kcat/Km = 7.6 × 104 M-1s-1. D-Malate, other α-hydroxyacids such as glycolate and D-lactate, and Ins-1-P gave no more than 0.5% of the activity with L-malate when assayed at 0.1 mM (Table S2). The coupled spectrophotometric assay was then employed in an analysis of the steady-state kinetic mechanism for BaBshA. As given in Figure S3, the family of reciprocal plots obtained with varying [L-malate] at different fixed [UDP-GlcNAc] intersects below the x-axis. This suggests that 1) the kinetic mechanism conforms to a rapid equilibrium random bireactant system (33), as contrasted with an ordered system, and 2) the binding of either substrate to free enzyme increases Kapp for the second substrate. From the primary plot and secondary slope and intercept replots, we have determined the kinetic parameters: Km(UDP-GlcNAc) = 0.22 mM, Km(L-malate) = 58 μM, kcat = 15 s-1, and α = 2.6. kcat/Km(UDP-GlcNAc) = 6.8 × 104 M-1s-1, similar to that reported for CgMshA at 25°C.
Figure 5 gives a structure-based sequence alignment for CgMshA and the three known functional BshA enzymes. An initial description of the BaBshA donor substrate binding environment, based on an overlay of the apoenzyme structure with those of the UDP—2-deoxy-2-fluoroglucose complex of the GT4 glucosyltransferase WaaG and the PimA—GDP-mannose complex, identified BaBshA His120, Lys211, Glu282, and Glu290 within the active site (14). Figure 5 demonstrates that His120 is conserved in the functional BshA enzymes; the equivalent of this His (His118) is essential for PimA activity (57). Lys211, interacting with the distal phosphate oxygens of UDP, and the two Glu residues, which recognize the sugar 4-OH and the ribose-2′-OH and 3′-OH, respectively, in the PimA complex (57), are conserved in all four sequences. Guerin et al. demonstrated that other residues within the PimA Glu274-Ile278 signature motif, notably Ser275, Phe276, and Ile278, provide main chain hydrogen bonds to the bound mannose sugar moiety, and this motif (BaBshA Glu282-Leu286, with Leu replacing Ile) is conserved in the BshA and MshA enzymes. Vetting et al. (16) have identified four CgMshA residues (Lys78, Tyr110, Thr134, and Arg154) that coordinate the phosphate of bound Ins-1-P in the ternary complex. As represented in Figure 5, CgMshA Tyr110, Thr134, and Arg154 are conserved in all three known functional BshAs, with the single exception of a conservative Arg→Lys substitution in SaBshA; CgMshA Lys78 is replaced by Val in the sequence alignment. When the BaBshA apoenzyme is used to query the PDB database with DALILITE, the closed UDP (and UDP + Ins-1-P complex) structures for CgMshA give the highest Z-scores [ca. 37; rmsd = 2.4-2.5 Å (respective A-chains)]. Without adjustments, the superposition represented in Figure 6 gives a clear view of the donor substrate binding environment in BaBshA, as described above. In addition, the overlay demonstrates that CgMshA-UDP His133 is the structural equivalent of BaBshA His120 and identifies BaBshA Ile204 (main chain), Asn206, Asp263, and Glu282 as likely recognition elements for the UDP pyrophosphate and for the ribose-2′-OH and uracil oxygens, respectively. There is no evidence for any conformational change on binding of UDP to BaBshA, based on the model deduced from this overlay.
The kinetic parameters for BaBshB were determined using an HPLC assay for the AccQ-Tag derivative of the GlcN-malate product, as monitored by fluorescence (13, 31); Figure 7 gives the resulting initial velocity plot and the fit to the Michaelis-Menten equation. At pH 7.5 and 37°C, in the presence of 50 mM NaCl, the kinetic parameters are Km(GlcNAc-malate) = 0.16 mM, kcat = 42 s-1, and kcat/Km(GlcNAc-malate) = 2.6 × 10 M-1s-1. We also analyzed the deacetylase activity of BaBshB with GlcNAc, at a fixed concentration of 0.15 mM. The observed rate of GlcN production is ca. 10-5 that measured with GlcNAc-malate at the same concentration; the L-malate moiety of GlcNAc-malate is therefore a major determinant of substrate recognition and/or transition state stabilization with BaBshB. When BaBshB was assayed for amidase activity with BSmB (→ CySmB + GlcN-malate), the observed rate at 0.15 mM substrate is only ca. 2 × 10-5 that for GlcNAc-malate deacetylation.
In order to test the model for the enzyme-UDP complex described above, and in an attempt to identify the binding determinants for L-malate, we undertook a crystallographic study with the N-terminal His-tagged BaBshA protein. This His-tag (MGSH6SSGLVPRGSHMASMTGGQQMGRGS-) differs from that for the ORF BA1558 construct (MGSSH6-) analyzed by Ruane et al. (14), which crystallized in space group P21; there were 12 monomers organized into three tetramers within that asymmetric unit. BaBshA crystals belonging to space group P41 were obtained in 0.2 M Mg(HCO2-)2 containing 15% PEG3350 with 1 mM each of UDP and L-malate. The structure determined by molecular replacement using the ORF BA1558 apoenzyme coordinates has eight monomers arranged as a dimer of tetramers within the asymmetric unit (Figure 8); gel filtration analysis of the apoenzyme, in the presence of dithiothreitol, suggests that the biological unit for BaBshA is the dimer. Refinement at 3.3 Å resolution led to a final model with reasonable statistics (Table 3). The final refined structure is missing the residues of the N-terminal His-tag and Met1, as well as residues 11-13, 42-47, and 60-63; the short internal segments correspond closely to three of the four exposed regions in the final ORF BA1558 model (14). Within the BaBshA asymmetric unit, the final model includes two UDP, two malate, and one Mg2+ per tetramer; Vetting et al. (16) reported that Ins-1-P soaks of CgMshA-UDP crystals resulted in a complex with two UDP, two Mg2+, and only one Ins-1-P per dimeric asymmetric unit. The dimeric (substrate-bound) unit of the BaBshA-UDP-malate complex gives an overall Cα rmsd of 1.1 Å for 371 atoms when compared with the corresponding dimeric unit of the ORF BA1558 apoenzyme (Figure 9). In addition, the two subunits per tetramer that are complexed with UDP and malate are covalently linked via an intersubunit Cys241-Cys241′ disulfide (Figure 8); this disulfide is not observed with the complementary unbound subunits within that tetramer, which appear to reflect crystal packing. Focusing on one of the subunits containing bound UDP and malate, Figure 10 gives a final composite omit Fo-Fc map in the vicinity of the substrates, together with the refined model. Considering the 3.3 Å resolution of the BaBshA complex, we have also worked with the possibility of modeling glycerol in two conformations as an alternative to malate. Two conformations of glycerol could be modeled into the density; however, after refinement, additional positive density appears. Upon placing malate in the electron density, no additional Fo-Fc density appears.
Bacillithiol is only the second malyl glycoside reported in the literature to date (10, 58), and the interactions and specificity determinants for L-malate recognition are of significant interest, particularly given the recent report that L-malate is a second preferred carbon source in B. subtilis (59), strongly contributing to repression of the uptake of alternate substrates. An overlay of the CgMshA—UDP-Ins-1-P and BaBshA-UDP-malate complexes (not shown) demonstrates that the positioning and geometry of the Ins-1-P 3-OH and malate 2-OH, relative to the distal phosphates of the respective UDPs, are similar. Neither malate carboxylate is close to the position of the Ins-1-P phosphate, however. Although this interpretation is limited by the 3.3 Å resolution of the BaBshA complex, it is consistent with a chemical mechanism [Scheme 2 (16, 60)] involving nucleophilic attack by the malate 2-OH on the anomeric carbon of UDP-GlcNAc.
The superposition of the two complexes also demonstrates that two residues in the CgMshA phosphate coordination site, Tyr110 and Thr134 (see Figure 5), are structurally conserved in BaBshA. BshA Val64 does occupy the same approximate position as MshA Lys78, but the respective Arg138 and Arg154 residues are not equivalent. Figure 11 gives a stereo representation of the BaBshA-UDP-malate active site, focusing on important polar interactions with both malate and UDP. In particular, this analysis identifies Thr122-OH, Asn206-Nδ2 and -Oδ1, and a distal phosphate oxygen (O1B) of UDP as ligands to the malate C1-carboxylate. The substrate C2-OH interacts with Gly15-N and with both O1B and O2B oxygens of the uridine-pyrophosphate, and Ser16-O stabilizes the C4-carboxylate. All four BaBshA residues are conserved in the other known functional BshA enzymes (Figure 5). It has previously been proposed (61) that an active-site His functions in glycogen phosphorylase and related glycosyltransferases to stabilize an oxocarbenium transition state (e.g., via interaction with His377-O in glycogen phosphorylase) such as that considered for CgMshA. This is thought to be especially important for those GT enzymes operating with negatively-charged (e.g., malate, Ins-1-P) acceptor substrates (61), and the conservation of His120 in the BaBshA catalytic center appears to be an important factor in this regard.
With regard to UDP binding, the structure of the BaBshA ternary complex confirms several aspects of the model deduced from the overlay described in Figure 6. The very favorable comparison of apoenzyme and ternary complex structures (Figure 9) demonstrates that there is no major conformational change on substrate binding to BaBshA.
In this work we have defined the functions of ORF BA1558, previously annotated as a glycosyltransferase, group 1 family protein, and ORF BA1557, previously annotated as a hypothetical protein, as the N-acetyl-α-D-glucosaminyl L-malate synthase (BaBshA) and deacetylase (BaBshB), respectively. The GlcNAc-malate product of BaBshA has been confirmed by 1H and 13C NMR and by high resolution mass spectrometry; deacetylation by BaBshB gives GlcN-malate, as confirmed by ESP mass spectrometry. The kinetic parameters have been determined for both enzymes. BaBshA is specific for L-malate and does not recognize either D-malate or other α-hydroxyacids as acceptor substrates. BaBshA appears to follow a rapid equilibrium random bireactant mechanism in which the binding of either substrate increases Kapp for the second substrate (33). Vetting et al. (16) reported that CgMshA followed a sequential mechanism in which UDP-GlcNAc was proposed to bind first, i.e., as in a rapid equilibrium ordered bireactant scheme. However, Segel has shown that the family of reciprocal plots (1/ν versus 1/[Acc], at several fixed [Don]) for such an ordered sequential mechanism intersects on the y-axis, in the case where substrate A (e.g., UDP-GlcNAc) binds first (33). An examination of the data (16) indicates, in contrast, that reciprocal plots of 1/ν versus 1/[Ins-1-P] for CgMshA intersect in the second quadrant, very close to the x-axis. We suggest that BaBshA and CgMshA both follow rapid equilibrium random bireactant mechanisms; they differ primarily in the fact that α ~1 for CgMshA. While binding of either substrate to BaBshA has the effect of increasing Kapp for the second substrate (α = 2.6), this interpretation does not apply to CgMshA.
The respective kcat/Km(UDP-GlcNAc) values of 6.8 × 104 M-1s-1 (37°C) and 6 × 104 M-1s-1 (25°C) correspond to free energies of activation [ΔGT‡ (62)] of 11.3 kcal/mol and 10.9 kcal/mol. This quantitative similarity is significant, in that Vetting et al. (16) have reported a large conformational change between the CgMshA apoenzyme and UDP complex. Apoenzyme crystals were prepared with an N-terminal His-tagged protein and were of space group P31, while crystals of the complex were prepared with a C-terminal His-tagged protein and were of space group I422. While a crystallographic argument has been presented against the possibility that the apoenzyme conformation does not represent the functional enzyme, this remains an open question. In particular, searches using the CgMshA apoenzyme with the SSM server yielded no significant structural homologs among the GT-B fold family. There is no major conformational change on UDP binding to BaBshA; the possibility of a conformationally distinct BaBshA—UDP-GlcNAc complex also appears unlikely, as the superposition of the BaBshA apoenzyme with the monomeric PimA—GDP-mannose structure reveals similar conformational states (Z = 33.6, rmsd = 3.0 Å). In view of the Km(UDP-GlcNAc) value of 0.22 mM for BaBshA, the intracellular concentration of ca. 2.4 mM reported for B. megaterium (63) predicts that ca. 8% of the enzyme is present in the apo form in the in vivo steady state. With respect to the BaBshA complex, crystal packing stabilizes a closed tetrameric protein form (two UDP and two malate per tetramer; Figure 8) in which bound UDP is solvent inaccessible. Similarly, Vetting et al. showed that crystals of the dimeric CgMshA-UDP complex, when soaked with high concentrations of UDPGlcNAc, do not exchange bound UDP (16).
Using a transposon site hybridization assay protocol, Day et al. (64) concluded (without the benefit of the functional assignments presented in this work) that the bshA and panC genes, as well as BA1559 (pcnB, Table 1), were individually required for optimal growth, sporulation, or germination in B. anthracis strain ΔAmes. As we have demonstrated that deletion of the bshA locus in B. anthracis Sterne eliminates BSH production, this suggests that BSH is important for one or more of these aspects of the life cycle. In this context, we have recently demonstrated that the B. subtilis bshA mutant sporulates with an efficiency ca. 1% that of the wild-type strain (13). In their transcriptional profiling of the B. anthracis life cycle, in which five distinct temporal waves of gene expression were identified from germination through sporulation, Bergman et al. (65) showed that the bshA and bshB genes encoding the first two enzymes in the BSH biosynthetic pathway are upregulated together in waves II and III; the panBCD genes are upregulated in wave V of the germination→sporulation cycle. panB and panC are also upregulated between 1 and 2 h postinfection within host macrophages (52).
We have described the gene products for all seven loci in the bsh operon, which is upregulated during that part of the life cycle corresponding to early outgrowth → rapid growth (65). Aside from the GlcNAc-malate synthase and deacetylase enzymes, we find no evidence for coding sequences that provide for the enzymatic conversion of GlcN-malate → BSH. In connection with the demonstration that BSH is important for fosfomycin resistance in B. anthracis, the coexpression of the mgsA gene encoding methylglyoxal synthase is of interest. Methylglyoxal is cytotoxic in millimolar concentrations and is also mutagenic (51). Still, many bacteria produce methylglyoxal synthase, with a functional implication as a bypass of glycolysis active under conditions of Pi starvation. In most microorganisms, methylglyoxal is detoxified via GSH-dependent conversion to D-lactate; however, we have demonstrated that GSH is absent in B. anthracis (2). Both methylglyoxal (51) and fosfomycin (66) are potent C3-electrophiles known to react with GSH, and our evidence strongly suggests that BSH is the cofactor for FosB in B. anthracis. We have recently demonstrated, using a zone of inhibition assay, that a B. subtilis bshA mutant has a significantly increased sensitivity to methylglyoxal (13). We conclude that BSH may also be the cofactor for an S-transferase reaction converting methylglyoxal to lactoylbacillithiol [CH3CHOHCO-SCH2CH(NH3+)CO-NGlc-malate]. A possible fate of these BSH conjugates is discussed below.
As indicated previously, BshB is both a structural and functional homolog of M. tuberculosis MshB; when assayed in parallel with synthetic GlcNAc-Ins, however, MshB gives kcat of only 0.49 s-1 (31), with kcat/Km(GlcNAc-Ins) = 1.44 × 103 M-1s-1. Both values are ca. 1% of the respective parameters with BaBshB (acting on GlcNAc-malate as substrate). The difference in BshB and MshB catalytic efficiencies is approximately that required by rapidly dividing cells to maintain stable levels of BSH or MSH, respectively, given the 50-fold difference in generation times for B. anthracis and M. tuberculosis (0.5 and 24 h). We have shown that the B. anthracis ΔbshB mutant produces BSH at ca. 70% of the wild-type level, and this has been attributed to an overlapping substrate specificity for two BaBshB paralogs, ORFs BA3524 and BA3888. In the closely related B. cereus 14579, the ORF Bc3461 ortholog of BA3524 has been demonstrated (20) to catalyze deacetylation of both (GlcNAc)2 and (GlcNAc)3, and we propose that this enzyme and BcZBP similarly have overlapping specificities for GlcNAc-malate deacetylation in B. cereus. A BcZBP-deficient mutant would still be expected to produce significant BSH, through the action of ORF Bc3461.
The substrate specificity of the MshB deacetylase overlaps that of the M. tuberculosis mycothiol S-conjugate amidase (Mca), which functions in the MSH-dependent detoxification of thiol-reactive drugs and metabolites by cleaving the Cys-GlcN amide bond of the S-conjugate to give AcCySR plus GlcN-Ins. The optimal amidase substrate for Mca is MSmB, yielding AcCySmB + GlcN-Ins; kcat/Km = 9 × 104 M-1s-1 at 37°C (23), and MshB catalyzes amide cleavage with this substrate, though with kcat/Km = 460 M-1s-1 (31), less than 1% of the value observed with Mca. The M. tuberculosis Rv1170 mutant deficient in MshB continues to produce MSH at ca. 20% of the wild-type level (67), and this has been attributed to the weak deacetylase activity observed with purified Mca in vitro for GlcNAc-Ins [kcat/Km = 0.3 M-1s-1 (23)], ca. 10-4 that of MshB (see above). In addition to providing for GlcNAc-malate deacetylation in the absence of BshB (see above), ORFs Bc3461, BA3524, and BA3888 may function primarily as paralogs of Mca that catalyze bacillithiol S-conjugate amidase reactions related to xenobiotic detoxification. The S-conjugates of fosfomycin and methylglyoxal described above represent two potential substrates (Scheme 3). The charged GlcN-malate product of the amidase reaction can be recycled for BSH synthesis, and a number of AcCySR products from the analogous amidase reactions with mycothiol S-conjugates are known to be lost from the cell (44, 45). Rukmana et al. (68) have recently reported that the Spx regulon is induced in B. subtilis cells challenged by the antibiotics enduracin and bacitracin, suggesting that disulfide stress is occurring under these conditions, but the Spx regulatory system appears to be independent of BSH (13).
Bacterial growth and proliferation, especially during infection of the host, often involves a hostile environment (52, 53) that challenges the defensive mechanisms of the pathogen (e.g., B. anthracis or S. aureus). Among the physiological mechanisms for maintaining thiol-disulfide redox homeostasis are the important functions of thiols such as GSH (69, 70) and MSH (44, 45). B. anthracis (2), S. aureus (3), and B. megaterium (11) are among a number of Gram-positive bacteria that lack both of these thiols. It is clear from several studies, perhaps most closely associated with the Beckwith laboratory (69, 70), that redundancy in these systems is the rule, particularly in E. coli; mutants lacking either TrxR or glutathione reductase grow normally (70, 71).
Figure 12 gives the present working scheme for cytoplasmic disulfide reducing and oxidizing pathways in our model Gram-positive pathogen, B. anthracis; the same or very similar system applies in S. aureus and in the soil bacterium B. megaterium. The three branches include CoASH/CoADR, Trx/TrxR, and bacillithiol (BSH). The recent work of Pöther et al. (12) indicates that cysteine (CySH) represents a fourth branch in S. aureus, accounting for most protein (mixed) disulfide formation during diamide stress. While a specific bacillithiol disulfide reductase has not been identified, the BSH/BSSB redox status is very reduced (redox ratio of 84 versus 2.8 for CySH/CySSR) in vegetative B. anthracis cells (Table 2); this ratio is unchanged in the Δcdr and Δcdr2 mutants, and neither CoADR nor CoADR-RHD is a reductant of oxidized bacillithiol (BSSB). There is, however, no direct evidence as to whether the B. anthracis Trx/TrxR system might reduce BSSB. In both Arabidopsis (72) and Drosophila (73), for example, the Trx/TrxR system either constitutes a functional backup or substitutes for glutathione reductase. In E. coli, the Grx-SSG reductase activity of AhpC* maintains the reduced GSH pool at essentially wild-type levels in ΔgorΔtrxB strains (70).
We recently demonstrated (8) that the B. anthracis cysK-1 locus encoding cysteine synthase A is part of the tricistronic coaX operon that also encodes the type III pantothenate kinase. The B. anthracis pathway from pantothenate → CoASH (Figure 13), as well as the components of the de novo pantothenate biosynthetic pathway (including the panBCD genes of the pan operon; Table 1), have now been established (2, 74). In this work we describe the first two steps in BSH biosynthesis for B. anthracis and the effects of deleting the respective genes on BSH production. While the identification of the proposed Cys:GlcN-malate ligase, the biological functions of the B. subtilis YllA and ORF BA3524 proteins, and the description of the reductase(s) that maintain the reduced status of BSH in vegetative B. anthracis cells remain as priority goals, the materials necessary for genetic analysis of the relative roles of the CoASH/CoADR and BSH systems in both intracellular thiol-disulfide redox homeostasis and in virulence are presently being exploited.
We gratefully acknowledge Dr. Robert Fahey, who first identified bacillithiol and led the structure determination, for his generous support and for many very helpful discussions. We thank Dr. Philip Hanna for hosting one of us (C.P.) in his laboratory and contributing to the construction of mutant strains, Dr. James La Clair for help with NMR work, and Ms. Kelly Lo and Mr. Austin Dosch for technical assistance.
†This work was supported by National Institutes of Health (NIH) Grants GM-35394 (A.C.), AI-78924 (M.R.R.), and AI-72133 (G.L.N.; Robert C. Fahey, Principal Investigator).
‡Coordinates have been deposited with the Protein Data Bank under the file name 3MBO.
1Abbreviations: CoADR, coenzyme A-disulfide reductase; CoADR-RHD, coenzyme A-disulfide reductase isoform with a C-terminal rhodanese homology domain; BSH, bacillithiol; BshA, N-acetyl-α-D-glucosaminyl L-malate synthase; GlcNAc-malate, N-acetyl-α-D-glucosaminyl L-malate; BshB, N-acetyl-α-D-glucosaminyl L-malate deacetylase; GlcN-malate, α-D-glucosaminyl L-malate; MshA, N-acetyl-α-D-glucosaminyl 1-D-myo-inositol-3-phosphate synthase; Ins, myo-inositol; MSH, mycothiol; GT, glycosyltransferase; BcZBP, Bacillus cereus zinc-binding protein; MshB, N-acetyl-α-D-glucosaminyl 1-D-myo-inositol-3-phosphate deacetylase; BHI, Brain Heart Infusion; LB, Luria-Bertani; EryR, erythromycin-resistant; TetR, tetracycline-resistant; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HPLC, high-performance liquid chromatography; ESP, electrospray; gCOSY, gradient correlation spectroscopy; RSmB, methylbimane derivative of thiol, RSH, from reaction with monobromobimane; Mca, mycothiol S-conjugate amidase; TrxR, thioredoxin reductase; Trx, thioredoxin; Grx, glutaredoxin.
2Intracellular concentrations are calculated from BSH contents (μmol/g dry weight), using a value of 2 μL/mg dry weight for the cell volume (Ref 2).
3Purification of the recombinant BcZBP protein employed Ni-NTA chromatography and resulted in Zn2+-containing BcZBP crystals (Ref 19), as analyzed by both X-ray fluorescence and X-ray diffraction. Similar purification of the MshB protein (Ref 31), however, yielded a preparation with no Zn2+ and 0.82 mol Ni2+ per subunit.
Figures S1 and S2, depicting preparative-scale production and NMR characterization of GlcNA-cmalate, Figure S3, providing reciprocal plots of BaBshA activity, and Tables S1 and S2, providing the primer sequences used in the study and describing the acceptor substrate specificity for BaBshA. This material is available free of charge via the Internet at http://pubs.acs.org.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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subset: commoncrawl


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This week, Phineas and Ferb branches out into an unusual medium...unusual in that it's one that isn't as robust as it used to be. Thanks to the rise of electronic media such as video games and the Internet, magazines geared for children haven't been as successful as they used to be. Nickelodeon magazine ended publication in 2009 due to the decline in magazine sales and Disney's own children's magazine, Disney Adventures, ceased in 2007.
But the New York Times reports that Disney is giving a try to replicating the success they have in Europe with magazines based on their familiar characters by launching a Phineas and Ferb magazine this week. A test issue last year "greatly surpassed" Disney's expectations, and expects that "kids want" the magazines featuring familiar Disney characters, and "moms will pay for them." Will Disney's plan succeed? Only time will tell.


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The Undergraduate Research and Creative Activities Award in the College of Arts and Sciences at Western Michigan University:
- Increases the opportunities for undergraduates to collaborate with faculty members in professional activities outside the classroom
- Provides undergraduate students with opportunities for hands-on research or creative activities experience with faculty from the College
- Recognizes and honors academic excellence and potential
- Provides opportunities for undergraduates to present research at academic conferences
The joint student-faculty projects represent the diversity of scholarly activity in the College. Projects have ranged from English editorial assistants to molecular biology research.
Undergraduate students will generally be expected to have:
- Completed at least 30 credit hours
- Declared a major or minor in a department or program within the College
- Good academic standing
The College of Arts and Sciences Undergraduate Research and Creative Activities support may not be held during the same term as a research and creative activities award administered by the Lee Honors College. Award recipients may request renewal for one additional term after providing a progress report for their project.
Award selection
Up to 60 awards of up to $500 may be used by the students as a stipend or to defray the cost of the project. Work associated with the award may be conducted during the fall or spring semesters or either summer sessions. Recipients of the award must be enrolled during the future semester for which they are requesting funds. All awards are subject to final budgetary approval and authorization by the dean of the College.
Note: In addition to the College funding, projects can be sponsored by regional industry, government and hospitals.
Feb. 1 and Sept. 30
This support will be offered for the deadlines listed as long as funds are available.
All sections of both the student application and faculty portion form must be completed to be considered for the award.
Student application cover sheet
Submit to:
College of Arts and Sciences Office of the Dean
2304 Friedmann Hall, Mail Stop 5308
Kalamazoo MI 49008-5308 USA
Note: If you submit your application electronically, emails from the WMU Webmail Plus account of a faculty member and department chair or director may serve in place of signatures.
Please do not hesitate to contact the College of Arts and Sciences dean's office with any questions or concerns.


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Welcome to the ineedhits Search Engine Marketing blog, where we share the latest search engine and online marketing news, releases, industry trends and great DIY tips and advice.
The Super Bowl is a huge event for both sporting fans and marketers alike. Every year the world's largest brands spend millions of dollars for a TV spot during the game which is one of the most watched events in the U.S.
To help smaller businesses claim their share of super bowl sales, Yahoo! have released some super bowl sales tips for online advertisers.
I've provided a summary of their advice below:
While the game may still be two weeks away, it's good to get everything sorted early because some consumers will be looking to purchase these items already!
Keep up to date with the latest from our blogs.
Google Rolls Out New Local Ad Features
The Google AdWords team has rolled out some new features for Local Business Ads which aim to make it easier ...


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Cloud computing is the most innovative technology that serves enterprises towards augmented capacity of their businesses. Business agility and scalability are the key components that have become essential for the growth of businesses and can be achieved by using cloud technology. It is being considered as the way to attain on demand swiftness and consistent scalability in operations. The researches so far have proclaimed that cloud based solutions are expected to cater disruptive advantages. It has been predicted that there are only few enterprise decision makers that are prepared to adapt into the cloud delivery model.
This latest trend is being used in several sectors due to the increasing demands of people based on future oriented mobility. This has even served in the field of sports sector and continually strive to cater better user experience. Cloud system has the potential to respond quickly during any sports event or when the tournament is about to get started. It has helped users in predicting the ways that players need to follow to increase their possibility of winning against their opponents. There have been Bay area that has ensured to serve riders by operating wireless network that transmit mobility solutions and services to travelers underground. Use of cloud mobility solutions catered developers to briskly develop and deliver projects at a faster rate.
Given this context, it has been highly adaptable among e-commerce websites. These are using cloud based mobility solutions so that they may provide customized offers, economical price and promotions in real time. It becomes feasible for them to create new services that will allow consumers to take products online. Businesses are growing at a rapid pace by taking a leap towards the cloud. Following are the recent trends by which cloud computing is driving innovation:
Interlinking system of records with future generation systems: Enterprises are consolidating the systems that depend on internal functioning to the systems as mails etc. to connect with customers, business partners and employees.The main objective behind using this methodology was to gain traction of large amounts of data compiled by an organization. This scalable approach is helping in mobilizing this trend.
Use of cloud technology to speed innovation: The transformation to new business strategy is developing foundation in such a manner so that it becomes feasible for them to turn their business models. New and enhanced apps or business processes are created when enterprises take services and data together to bind them in a single process. The data are safeguarded by making use of an open cloud environment as it is considered to be the reliable process.
Augmenting services in a hybrid cloud environment: By automating cloud services, enterprises can actually monitor the way their services are being used to ensure privacy and security. They can work with multiple number of communities, create applications and develop tech based business models. This is the most effective approach for those enterprises that used to think that they are losing control over their revenue accommodated in centralized data centers.
Cloud computing technology is not only for tech savvies. IT professionals also give an equal response to the ways that cloud offers to create enhanced business models. Cloud has emerged as a powerhouse of innovation and from technology perspective it is being adapted for efficiency and cost savings at an initial phase.
Original: http://www.mobileappsprogramming.com/2014/09/08/how-has-cloud-computing-led-to-major-innovations/
By: Ridhima Gupta
Posted: September 8, 2014, 1:52 pm
For more than a decade, Mobile Programming LLC, a Certified Mobile Application Development Company, has been providing high-end custom mobility solutions to its clients. We strive hard to provide...


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Manage capacity constraints proactively.
Supplier capacity risk is one of several factors supply chain managers need to evaluate in order to ensure the uninterrupted flow of parts and products necessary to service fluctuating customer demand. A lack of visibility to supplier capacity constraints can directly lead to lost revenue and market share, and declines in customer satisfaction and shareholder value if you cannot respond to supply shortages and demand surges.
Resilinc Supplier Capacity Management provides a proactive capacity management system to ensure you have the necessary supplier capacity at a part-level to meet fluctuating demand forecasts. Analytics focus you on high-risk suppliers and parts based on risk thresholds you define, while alerts, mitigation workflows, and complementary event monitoring services facilitate on-going capacity management excellence.


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Photos are for members only!!
Peachmate is a 100% free sex site for adults looking for fun with no strings attached.
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I have a so called thirst for life being always positive, joyful, curious, adventurous and goal-oriented. Also I am open, reliable, responsible, very loving and caring. I love arts, music, growing pot flowers, reading books on self-improvement. I am very active and like outdoors. I do swimming, jogging and aerobics regularly and lead a healthy way of life.


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Elizabeth Mansfield appeared on Coronation Street in August & September 1999 and June 2000 as Joan Wakefield, the mother of Alison Wakefield (later Webster).
After briefly attending Elmhurst Ballet School, she went on to train at the Guildhall School of Music and played at rep theatres up and down the country. In the late 1970s she became involved in the Red Ladder Theatre when the company moved to Leeds. She joined them initially for one production but stayed for five years. In the early 1980s when her daughter was still an infant, she trained in opera for three years at the City of Leeds College of Music.
As well as appearing on television and on stage, she was with the BBC Radio Drama company for eighteen months and has since founded her own company, Ensemble.
Other credits include roles in The Bill, Casualty, Heartbeat, Emmerdale and the films The Slipper and the Rose: The Story of Cinderella (1976) and Lost Along the Way (2008). On stage she has appeared in numerous productions including The Mother, The Threepenny Opera, A Better Day, Hymn to Love; The Happy Haven, Hay Fever, The Merchant of Venice, The Cherry Orchard, Rosencrantz and Guildenstern are Dead, A Midsummer Night's Dream, Guys and Dolls, Cabaret, My Fair Lady and the original production of Godspell.


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Strength and Power
This is an excellent warm-up that practises good ball skills whilst getting players 'switched on' in terms of movement, speed and ball control. Players should get a good feel of the pace of the ball when they take the shot at goal – the 'race' adds pressure.
Arrange the players in pairs and tell them to react to your whistle. You need balls in each part of the warm-up.
- Whistle 1 – the players sprint into the first area where the first one to the ball must keep it and hold the other player off. After 15 seconds the coach whistles again…
- Whistle 2 – the players leave the ball and sprint into the second area, again trying to be first to the ball and hold the other player off. After 15 seconds the coach whistles again.
- Whistle 3 – the players react and sprint to get a first time shot at goal. The players then become servers. The servers now jog back to the starting position. The whistles work on a conveyor-belt effect. On each whistle a new pair is entering an area that the previous pair has just left.
Speed and Agility Ladder
This five minute fitness drill can be used during your training sessions for a quick break to help coaching points sink in, or as an incentive for a drinks break.
Speed ladders are excellent for player speed and fitness but if you haven't got one you can mark out the rungs of the ladder with cones.
- Forward hops – 3 in 1 out
- Hop forward on one leg
- One hop in each square
- Every 3 hops step once out of the ladder onto the other leg
- Continue this sequence until ladder is complete
- Ground contact on balls of feet. Repeat 5 times.
- Rest 60 seconds between repetitions.


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Triad Dragons featuring Julie Zorrilla – "Chasing Shadows"
After rapidly appearing on the scene in May with their remix of Ellie Goulding's "I Know You Care," Denver-based trio Triad Dragons have released their first track on a major label. "Chasing Shadows" featuring former American Idol contestant Julie Zorrilla is out now on Beatport via Manufactured Music, the imprint owned and operated by Manufactured Superstars. Listen to the new single after the jump.
A catchy, sing-a-long dance track perfect for summer festivals and hot club nights, "Chasing Shadows" features Julie's unique and powerful vocal chops and a chiming melody full of piano riffs and deep bass wobbles. Already at #75 on Beatport's Progressive House chart after just 3 days, "Chasing Shadows" was debuted with a live performance at the legendary Red Rocks Amphitheater during Global Dance Festival on July 20.
Triad Dragons is the newly launched EDM group consisting of Ha Hau, John Lee and Kevin Alves. Each with considerable, individual accomplishments in the Colorado dance music scene as promoters and DJs, they have pooled their talents to launch a brand new musical endeavor.


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Wellness & Nutrition Programs
You don't need to be a runner or a walker to commit to improving your overall well-being!
Take a healthy step towards improving your overall wellness in a supportive, small group setting. Our program topics include healthy eating, stress management, exercise for beginners, weight loss and more.
The face-to-face group style programs will hold you accountable as you commit to improving your health. Our groups are led by local experts in the health and wellness industry who will empower you to make positive, sustainable changes and help you reach your personal wellness goals.
Program offerings vary by store location; please explore below to find out what wellness offerings are available near you!
Upcoming Groups
Check back for future groups!


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FreshDirect Helps City Students Experience Farm Life
The online grocer's educational effort gives children hands-on experience in crop cultivation, dairy farming.
The online grocer's educational effort gives children hands-on experience in crop cultivation, dairy farming.
Online grocer FreshDirect and Into the Outside, a nonprofit organization that provides New York City students in low-income areas more access to educational opportunities, marked their continuing partnership with a trip enabling 45 children in Brooklyn's Red Hook neighborhood to enjoy fresh fields and pastures new.
The second-grade students, all from PS 27 Agnes Y. Humphrey School for Leadership in Red Hook, visited Satur Farms and Catapano Dairy Farm on Long Island to learn about crop cultivation and dairy farming. In the second unit of a specially designed Seed-to-Salad curriculum created by Into the Outside and FreshDirect, children discover the journey food takes from the farm to the plate, under the tutelage of local farm owners.
At Satur Farms the students observed the differences in urban vs. rural farming methods, comparing the acreage with a local farming project where they've been harvesting vegetables in Brooklyn. At Catapano Dairy Farm the children took part in milking demonstrations, sampled homemade cheese, and learned about the various breeds of animals at the farm.
The partnership between Into the Outside and FreshDirect has been in place since
March 2008.
Long Island City, N.Y.-based FreshDirect serves most of Manhattan and locations in Queens, Brooklyn, Staten Island and the Bronx, as well as parts of New Jersey and Westchester and Nassau counties.


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Athenaeum building celebrates 170 years
Australian Broadcasting Corporation
Broadcast: 11/11/2009
Tomorrow marks the 170th birthday of one of the nation's historic cultural landmarks. Melbournes Athenaeum building has, in one form or other, provided education and entertainment for the Victorian colony as it became a city; and along the way, documented its growth.
TranscriptKERRY OBRIEN, PRESENTER: Tomorrow marks the 170th birthday of one of the nation's historic cultural landmarks.
Melbourne's Athenaeum building has, in one form or another, provided education and entertainment. For the Victorian colonies it became a city and along the way documented its growth.
The building's original library and theatre still draw devotees and as Lisa Whitehead reports, a loyal band of volunteers.
KEVIN QUIGLEY, ATHENAEUM PRESIDENT: There's nothing like us that has been here from day one, four years after the boat pushed ashore, here we are. It's a thread that runs through the life of Melbourne.
LISA WHITEHEAD, REPORTER: In the heart of Melbourne's CBD, the Athenaeum is a celebrity in disguise, the oldest cultural icon in the city, but barely noticed.
MARJORIE DALVEAN, VOLUNTEER HISTORIAN: People of Melbourne walk past this area and they have no idea what it is.
RAY LAWLER, PLAYWRIGHT: It seemed to me to be a place that absolutely, or breathes Melbourne, I suppose, culture.
LISA WHITEHEAD: Just four years after Melbourne was founded, the colony built a Mechanic's Institution, one of the first in the world, a place where the working class could meet and learn.
KEVIN QUIGLEY: People think of it as Wild West sort of place where these hearty types drank and rushed about, but Melbourne was freely settled. It was a city of people who wanted to better themselves - entrepreneurs. And the Mechanic's Institution was that innovative idea that had grown up in Edinburgh and London about providing an opportunity for education for the working people.
LISA WHITEHEAD: Mark Twain lectured there. Later, other buildings were added and a theatre to host classic plays. And it adopted its more bourgeois friendly title of the Athenaeum. Crucially from the start there was the library, the first to offer affordable lending to the working man. And it still attracts devotees.
Former University lecturer Margaret Bowman, 89, comes in every Wednesday, along with her dog to join an enthusiastic band of volunteers sorting through the archives.
MARGARET BOWMAN, FORMER UNIVERSITY LECTURER: Doing research is something that I find actually I enjoy more than anything. Every old lady needs to have a project and now I've got a project.
MARJORIE DALVEAN: Margaret, Christine has just found out that Alfred Deakin was a member here from 1874 to 1877.
This place is not flashy, we've never been flashy. But book lovers walk in here and they know this is the place for them.
ARCHIVAL FOOTAGE: Old times and old names. The Athenaeum theatre in Melbourne for more than 40 years has been one of the city's best known cinemas.
LISA WHITEHEAD: In the 20th century, the theatre surrendered to the new craze of talking pictures, and one particular fan was famous Australian playwright Ray Lawler. At 13, he dropped out of school to work in a Footscray factory and two years later his first trip to the glamorous Athenaeum cinema hinted at the education he was missing.
RAY LAWLER: It just had a style about it which I responded to, I think. I was looking for something and this seemed to be part of it.
Ray Lawler went on to write "Summer of the Seventeenth Doll" and found literary fame overseas.
About a century after it had started as an educational place for the working man, Ray Lawler had, in effect, become an Athenaeum graduate.
RAL LAWLER: If they had been looking for the sort of person that they were hoping to encourage along the way, I suppose I would have been somebody that might have fitted the mould, you know.
LISA WHITEHEAD: In time, the cinema was returned to its theatrical roots.
FRANK THRING, 1977: It has a great resemblance to the Theatre Royal in Hobart which Larry Olivier has called the best theatre he's ever worked in. And it's almost identical. It is the true Victorian playhouse. The horseshoe shaped thing: stalls, dress circle and gallery. And you're close to the audience and they're close to you. Marvellous feeling.
LISA WHITEHEAD: Today, it's still a theatre. But time has brought compromises. The once vaunted art gallery has now covered its windows and become a comedy club and performance space.
TV and suburban life have eaten away at the library membership. It offers an online service now, and a recent federal government grant will pay for the upkeep of its gracious interior, including the 1930s elevator Ray Lawler used to ride. For him, it's money well spent on history quietly made and discreetly observed.
RAY LAWLER: It's the lack of awareness, I think, that people don't know what they've got here. They've really got the whole history of Melbourne almost.
KEVIN QUIGLEY: It was a similar organisation in Sydney but we are the only one that's got a continual lineage on the same spot. We started here and we're still here and we'll be here for another 100 years.
KERRY O'BRIEN: Lisa Whitehead on a great Melbourne landmark.


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Category Archives: Ghost in the Park news
Are Speech to Text Programs Useful to Writers?
Are Speech to Text Programs Useful to Writers? I belong to a writers group called 20booksto50k. The ideology behind it, is that by the time one has published twenty books, one can be making 50k a year. It's not quite … Continue reading
Writing News
The year of writing dangerously continues! I finished Nick of Time! It's done, it's had a couple of re-writes, and it's off to the editor. Once I get it back and make corrections, it's next up at Kindle Scout. I … Continue reading
Rejection. What a grim and depressing idea. The word itself seems cold and desolate. Whether you are an actor auditioning for a favorite role, or a single guy with a crush on the woman in the next cubical, no one … Continue reading
Posted in getting published, Ghost in the Park news, writing, Writing Advice Tagged absolute write, ferrets, rejection, writing advice 28 Comments
Moving Sucks
Moving What can I say about moving? Moving sucks. Moving out of a seven room apartment into two rooms at your aging parents house sucks ass. Moving when you're nearing fifty, when you have a family emergency that gives you … Continue reading
Posted in Change, Ghost in the Park news, Moving, Uncategorized, Wyrd House Tagged change, Ghost in the Park, moving, wyrd house 6 Comments
When it rains it pours
Ok, I know I just posted, but I'm still amazed, so I want to share more fully with you what happened today. The past few weeks, I've spent some time rewriting my first chapter of Ghost in the Park. I've … Continue reading
Wyrd House update and writing news
So far, March has brought sun, warmth, rain, cold, and snow to my neck of the woods. Oh, the joy of living in the Ohio River Valley! Crappy weather has me indoors, dealing with some mundane life issues and doing … Continue reading
Would you read this book?
I have a favor to ask. I've been working on the description for Ghost in the Park that goes into the query letters I have been sending to agents. Thanks to the good people over at Absolute Write's Water Cooler, … Continue reading
Writing News
I just wanted to post a little update as to how things in my writing world are going. If you are waiting on another chapter of Wyrd House, I plan to post another one by the end of this week. … Continue reading
Posted in Ghost in the Park news, Uncategorized, writing, Wyrd House Tagged Ghost in the Park, writing, wyrd house 2 Comments
Writing news- Winter and Ghost in the Park
Winter, my post-apocalyptic short story has been accepted by Das Krakenhaus Publishing, and will appear in their book The End of the World as We Know It in the spring. Yay for me! I'm very excited that it's been accepted, … Continue reading
Posted in Ghost in the Park news, writing Tagged gatlinburg, Ghost in the Park, Query letters, winter, writing Leave a comment
Wyrd House update, and writing news
The fifth chapter of Wyrd House, my paranormal romance novel, is completed and posted. You can find it here: Wyrd House- Chapter Five I have been a busy little writer. My post apocalyptic short story, Winter, has been tweaked and … Continue reading
Posted in Ghost in the Park news, romance novel, Short Stories, writing, Wyrd House Tagged Ghost in the Park, romance, romance novels, winter, wyrd house Leave a comment


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Learn another language the way you learnt your own. You learnt your own language naturally and enjoyably: now you can learn Portuguese in the same way. You'll stick with it because you'll love it.
Join the millions of people worldwide who have learnt a new language with the Michel Thomas Method.
©2008 Thomas Keymaster Languages LLC;; (P)2008 Hodder and Stoughton
"A genuinely new way of learning a language." (Daily Mail)
"Michel Thomas is a precious find indeed." (The Guardian)
"Real fun."
The lesson was interesting and engaging. I was taught so much in such a short space of time.
"Would not Play"
It would have been better if it could be played or downloaded
What was most disappointing about Virginia Catmur's story? It would not play or download.
Who might you have cast as narrator instead of Virginia Catmur and Paulo Santos ?
People with louder voices
If you could play editor, what scene or scenes would you have cut from Michel Thomas Method?
There was none to cut.
"Suck it and see"
The Michel Thomas system for learning languages is so quick, you'll know within the length of this sample whether or not the method suits you. Audible offer these snapshots for free, so you can try out not only languages you currently need or want to learn but some you might always have fancied learning. The method quickly has you speaking accurately in your chosen language with no writing, exercises or book-learning. It relies on repetition and springboarding off what you already know, using your own language as a foundation. Try it for yourself.
"Empowering Method for Learning a Language"
This free sample is well worth your time. I enjoyed learning general themes of the Portuguese language in a refreshing and pleasant format. The audio format is excellent. I was very tempted to use my Audible credit for a purchase before I realized it was for one lesson lasting about an hour. This method will cost over $100 in credits to listen to several lessons. That is a steep purchase price. I searched in vain for these recordings at a local library. I am guessing, that this price is beyond their budgets, too.
The lesson was fun. I like the emphasis on language as it is spoken in Portugal since that is where I will be traveling to.
This is the first one.
Language learning need not be tedious nor difficult.
Thank you for providing the free sample.
Report Inappropriate Content
If you find this review inappropriate and think it should be removed from our site, let us know. This report will be reviewed by Audible and we will take appropriate action.


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Worthy Christian Devotions » Urgency
Be God's Messenger!
Jonah preached his 8 word sermon and the Ninevites were struck with the fear of the Lord and moved to complete repentance.
The king stood up (a sign of his serious intent), removed his royal robes (a sign of humility)...read more...continue reading this Christian devotional


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BMFcast331 – Evil Dead CopBy
Maniac Cop (1988) is a BMF alumni-packed murderfest, where an evil Robert Z'Dar is on a murderous rampage and only Tom Atkins, Laurene Landon and Bruce Campbell can stop him! Sort of. William Smith is also along to act menacing.
Second half we talk about a plethora of movies including Harlo's thoughts on The Brothers Grimsby, Zootopia, and the documentary Nintendo Quest, Maki discusses Trumbo and gives his thoughts on a solo viewing of Samurai Cop 2, and Chuck watches 2015's Fantastic Four and White Line Fever. You can probably figure out which one he liked better. Enjoy!
Podcast: Play in new window | Download
Subscribe: Apple Podcasts | Android | RSS
Maniac Cop was fun and the podcast is hilarious. The rant about today's ironic, self-aware low budget movies confirms my wariness of Samurai Cop 2 and the like. Keep up the great work!


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Temp Sending Unit; 93-98 Jeep Grand Cherokee ZJ
Temperature Sending Unit (With Temp Gauge), 1993-1998 Grand Cherokee (5.2L), 1996-Grand Cherokee (4.0L), 1998 Grand Cherokee (5.9L)
Pricing: $23.99
Direct OE replacement Jeep parts and accessories built to the original specifications by Omix-ADA. Limited five year Manufacturer's warranty.
- 17217.02
- 17217.02
- 804314058760
- Omix-ADA
- New
Shipping Information
- Item Requires Shipping
- 0.1 lbs.
- W2.0000" x H2.0000" x L4.0000"
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Listing information
- Beds: 2
- Stories: 3
- Foreclosure: No
- Lot Area: 0.32
- County: Warren
- Body of Water: Hudson River
- Style: Contemporary
- Baths: 2 (Full: 2 3/4: 0 1/2: 0 Other: 0)
- Parking Type Description: Attached 1 car
- Total Square Footage: 1,440 ($138.89 per sq. foot)
- Lot Size: 100x128
- High School: Hadley/Luzerne
- Year Built: 1988
- Status: Active
- New Construction: No
- Above Ground Sq. Feet: 1,440
- Estimated Annual Taxes: $3,522
- Elementary School: Hadley/Luzerne
Appliances Included Dishwasher, Refrigerator, Stove - Gas
Attic No Attic
Basement Garage, No Basement
Cooling Type Window Units
Documents Plan/Plat, Plot
Exterior Feature Deck Pressure Treated, Insulated Glass
Exterior Finish Clapboard, Wood
Flooring Laminate, Linoleum, Wall/Wall Carpeting
Garage Attached 1 Car, Garage Door Opener
Heat Distribution Baseboard, Warm Air
Heat System Fuel Electric, Propane
Interior Features Blinds, Ceiling Cathedral, Dry Wall, Dryer Connection, Fireplace - Propane, Internet - Cable, Washer Connection
Kitchen Features Eat-In, Semi-Modern
Land Features Somewhat Hilly
Other Amps - 200
Pool Hot Tub - Outdoor
Possession At Closing
Roof Asphalt
Sewer Septic Tank/Leach Field
Style Contemporary
To Be Built No
Water Well Drilled
Water Heater Propane
Water Type Direct Water Front
Waterfront Dock w/Sundeck, Lake/River Across Road, Riverfront, Water Front Deck
Zoning Residential
Listing Courtesy of Davies-Davies & Associates Real Estate, Llc


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pretty good...
today went pretty good, i havent been nausious except in the early morning..then i had to go to the dentist...which i DONT LIKE lol . ive never been good with the pain in my mouth and my husband said that i needed to go...especially now being pregnant. So i got there and i need a root canal and 2 teeth extracted:( i hate that kinda pain but im actually glad i can finally get it taken care of... who is with me on not liking the dentist????lol
Bummer about having so much done :( At least you will get it all settled! Personally I don't mind the dentist. I had braces forever and teeth pulled and filled quite a bit, so I think I'm just desensitized. It helped that they knocked me out for the wisdom teeth!
Aw.. I don't like the dentist either.. ick! Make sure to check about medications when getting those procedures done so early on in your pregnancy.. some meds aren't very good for the baby when you aren't very far along!
i was feeling good yesterday and ok i will definetly check into them. he told me i can only take tylenol for the pain and he also said its best to get the procedures done while im pregnant so insurance will cover more
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About Tiffany Velez
Birth: September 28
On Moms.com since: Sep 5, 2014
I am a stay at home mommy of a crazy 5 year old, stepmommy to a genius 9 year old lol, married to my high school sweetheart, mommy to an angel whom i never met, and now pregnant again! love baking and making ppl beautiful!
All Recent Tiffany's Posts


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Event Photos - The Sound of Music Arts and Culture Salon
The Paper Mill Playhouse Arts and Culture Salon series is an opportunity to learn how artists create the magic seen on the Paper Mill stage. During The Sound of Music, several actors gave donors a personal look back on their acting careers and audition process, including the open auditions for the Von Trapp children. Jonathan Charette (Friedrich standby), Donna English (Elsa), Ben Davis (Captain Von Trapp), Anthony Federov (Rolf) all stopped by to tell their stories.
For more information on this and other donor events, please contact Leigh Schaedel, Special Events and Donor Relations Manager, at 973-315-1663 or email@example.com.
Donor Events
There are no upcoming events currently scheduled.~ View full calendar ~


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Shred School is a cost-effective, two-day training program that covers a range of records and information management and secure destruction topics. To learn more about the program or related initiatives, please fill out the form below. A representative will be in touch with you shortly.
About NAID
NAID® is the non-profit, international watchdog trade association for the secure information destruction industry (shredding and sanitization, including paper, film, hard drives, etc.). NAID advocates for a standard of best practices across governments and by service providers as well as suppliers of products, equipment and services to destruction companies. NAID members assist all types of organizations in fulfilling their legal obligation to prevent unauthorized access to discarded personal and competitive information. NAID's mission is to promote the proper destruction of discarded information and to encourage the outsourcing of destruction needs to qualified contractors, including those that are NAID certified. Shred School is a registered trademark of NAID.
Learn more about NAID and all its programs at www.NAIDonline.org >>


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Films In Da Housewritten by yokekei on 2013-08-14
My films in my fridge till 13 July 2013. I wish no more film discontinued news but film relaunch news. Hope that film price stop increasing. I want to collect and try more films in future.16 20
Cameras in da Housewritten by yokekei on 2013-07-18
My cameras collection till 6th July 2013. I wish to collect more vintage cameras in the future :D I love Analog Camera ^_^12 31
Happy 29th Birthday LOMO LC-Awritten by yokekei on 2013-06-20
Happy Birthday to you~ Happy Birthday to you~ Happy Birthday to LC-A~ Happy Birthday to you~~~ ^_^8
Adorable Baby Fisheye 110 Bauhauswritten by yokekei on 2013-06-07
Wow~ Baby Fisheye 110 Bauhaus is so lovely and cute. I love it ^_^4 20
What I learnt and tried on 1st March 2013written by yokekei on 2013-03-03
Thanks for who always share their tips on Lomography. I had learnt something easy and fun through "Fuji Instax In Freezer" article which published on tipster section :)3 8