Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His8) was purified, and its kinetic constants were determined: the Vmax was 51.7 ± 7.6 μmol min−1 mg−1, and the Km values for propionyl-PO42− and acetyl-PO42− were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.
A number of bacterial genera including Salmonella, Klebsiella, Shigella, Yersinia, Listeria, Lactobacillus, and Lactococcus include members that grow on 1,2-propanediol (1,2-PD) in a coenzyme B12-dependent fashion. 1,2-PD is a major product of the fermentation of rhamnose and fucose, which are common sugars in plant cell walls, bacterial exopolysaccharides, and the glycoconjugates of intestinal epithelial cells. Accordingly, the ability to degrade 1,2-PD is thought to provide a selective advantage in anaerobic environments such as the large intestines of higher animals, sediments, and the depths of soils. Recent studies with Salmonella enterica have shown that 1,2-PD degradation is one of the most complex metabolic processes known (6, 14, 15, 18, 19, 31). The degradation of this small molecule requires catabolic enzymes, a system for recycling inactive cobalamins to coenzyme B12, and an unusual polyhedral body-microcompartment that is composed of metabolic enzymes encased within a multiprotein shell.
A pathway for 1,2-PD degradation by S. enterica has been proposed based on enzymatic studies of crude cell extracts and genetic analyses (25, 37). In the first step of the pathway, 1,2-PD is converted to propionaldehyde via coenzyme B12-dependent diol dehydratase (1). Propionaldehyde is then converted to 1-propanol and propionic acid by a reaction series thought to involve 1-propanol dehydrogenase, coenzyme A (CoA)-dependent propionaldehyde dehydrogenase, phosphotransacylase (PTAC), and propionate kinase (25, 37). This pathway generates one ATP, an electron sink, and a three-carbon intermediate (propionyl-CoA), which feeds into central metabolism via the methylcitrate pathway (16).
The genes specifically required for 1,2-PD utilization (pdu) by S. enterica form a single contiguous cluster, the pdu locus. DNA sequence analyses indicate that this locus includes 23 genes (6, 7, 10). Based on experimental and/or bioinformatic analyses, six pdu genes are thought to encode enzymes needed for the 1,2-PD degradative pathway (6), two are involved in transport and regulation (5, 10), two are probably involved in diol dehydratase reactivation (6), two are used for the conversion of vitamin B12 to coenzyme B12 (18, 31), four are of unknown function, and seven share similarity to genes involved in the formation of carboxysomes, a polyhedral body found in certain cyanobacteria and chemoautotrophs (6, 10). Although the methylcitrate pathway is required for the growth of S. enterica on 1,2-PD as a sole carbon source, the genes for this pathway map outside the pdu locus (16).
It was recently shown that S. enterica forms polyhedral bodies (PhBs) during growth on 1,2-PD (6, 15, 36). These bodies are extremely large macromolecular complexes that are 100 to 150 nm in cross section and consist of metabolic enzymes encased within a protein shell. Purification of these structures showed that they are composed of at least 14 different polypeptides (PduABB′CDEGHJKOPTU) (14). This includes four enzymes and as many as seven shell proteins, all of which are encoded by the pdu locus. The first two steps of 1,2-PD degradation are thought to occur in the lumen of the PhBs, and the remaining steps are thought to occur in the cytoplasm of the cell (Fig. 1) (14). The function of PhBs is uncertain. Previous studies suggested that they act as microcompartments that sequester propionaldehyde (an intermediate of 1,2-PD degradation) in order to minimize toxicity and/or prevent carbon loss (9, 14, 15, 28). Interestingly, recent genomic analyses tentatively indicated that there are seven functionally distinct polyhedral bodies distributed among over 40 genera of bacteria (4). Thus, polyhedral bodies may be a general mechanism of metabolic organization in the Bacteria.
Model for 1,2-propanediol degradation by S. enterica. Coenzyme B12 (AdoCbl), coenzyme B12-dependent diol dehydratase (PduCDE), propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), propionate kinase (PduW), and 1-propanol dehydrogenase (PduQ) are depicted. The first two steps of 1,2-PD degradation (conversion of 1,2-PD to propionyl-CoA) are proposed to occur within a polyhedral body-microcompartment. The dashed line indicates the shell of the polyhedral body, which is composed of up to seven different polypeptides. ATP is generated during the conversion of propionyl-PO42− to propionate. Given a suitable terminal electron acceptor such as O2, propionyl-CoA is degraded via the methylcitrate pathway, where biosynthetic precursors and additional energy are produced. Two additional enzymes associated with the polyhedral bodies (but not shown in the figure) are a putative diol dehydratase reactivase (PduGH) and an ATP:cob(I)alamin adenosyltransferase (PduO) involved in B12 recycling.
As described above, previous enzymatic analysis of crude cell extracts indicated that a PTAC is used for 1,2-PD degradation (25, 37). However, the enzyme involved was not identified. Here, we show that the pduL gene encodes a conserved, evolutionarily distinct PTAC that functions in 1,2-PD degradation by S. enterica.
Chemicals and reagents.Antibiotics, vitamin B12, and acetyl-PO42− were obtained from Sigma Chemical Company (St. Louis, MO). Propionyl-PO42− was synthesized as described previously (27). Isopropyl-β-d-thiogalactopyranoside (IPTG) was obtained from Diagnostic Chemicals Limited (Charlotteville, PEI, Canada). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA). Coomassie brilliant blue R-250, EDTA, ethidium bromide, 2-mercaptoethanol, and sodium dodecyl sulfate (SDS) were obtained from Bio-Rad (Hercules, CA). Other chemicals were obtained from Fisher Scientific (Pittsburgh, PA).
Bacterial strains, media, and growth conditions.The bacterial strains used in this study are listed in Table 1. The rich medium used was Luria-Bertani/Lennox (LB) medium (Difco, Detroit, MI) (23). The minimal medium used was no-carbon-E (NCE) medium containing supplements indicated in the text and figure legends (3, 38). MacConkey-1,2-PD indicator medium contained MacConkey agar base (Difco), 1% 1,2-PD, and 200 ng ml−1 of vitamin B12.
General molecular methods.Agarose gel electrophoresis was performed as described previously (30). Plasmid DNA was purified by the alkaline lysis procedure (30) or by using QIAGEN (Chatsworth, CA) products according to the manufacturer's instructions. Following restriction digestion or PCR amplification, DNA was purified using Promega Wizard PCR Preps (Madison, WI) or QIAGEN gel extraction kits. Restriction digests were carried out using standard protocols (30). For ligation of DNA fragments, T4 DNA ligase was used according to the manufacturer's directions. Electroporation was carried out as previously described (6).
Protein methods.Polyacrylamide gel electrophoresis (PAGE) was performed using Bio-Rad Redigels and Bio-Rad Mini-Protean II electrophoresis cells according to the manufacturer's instructions. Following gel electrophoresis, Coomassie brilliant blue R-250 was used to stain proteins. The protein concentration of solutions was determined using Bio-Rad protein assay reagent (Bio-Rad).
P22 transduction.Transductional crosses were performed using P22 HT105/1 int-210 (12), a mutant phage that has high transducing ability (32), as described previously. Transductants were tested for phage contamination and sensitivity by streaking on green plates against P22 H5.
Construction of plasmids for production of PduL and PduL-His8.PCR was used to amplify the pduL coding sequence from template pMGS2 (15). The primers used for amplification were 5′-GCCGCCAGATCTATGGATAAAGAGCTTCTGCAATCA-3′ and 5′-GCCGCCAAGCTTATTATCGCGGGCCTACCAGCCG-3′. These PCR primers introduced BglII and HindIII restriction sites that were used for cloning into vector pLAC22 (39). Following ligation, clones were introduced into Escherichia coli DH5α by electroporation, and transformants were selected by plating onto LB agar supplemented with 100 μg ml−1 ampicillin (Amp) (18). Pure cultures were prepared from selected transformants. The presence of insert DNA was verified by restriction analysis or PCR, and the DNA sequence of selected pduL clones was determined. Clones with the expected DNA sequence were used for further study.
A similar procedure was used to clone PduL fused to eight C-terminal histidine residues (PduL-His8) with the following differences. The primers used for PCR amplification were 5′-GCCGCCAGATCTATGGATAAAGAGCTTCTGCAATCA-3′ and 5′-GCCGCCAAGCTTATTAATGATGATGATGATGATGATGATGTCGCGGGCCTACCAGCCG-3′. The PCR product was ligated into T7 expression vector pTA925 (18). Transformation was done by electroporation, and LB plates supplemented with 25 μg ml−1 kanamycin (Kan) were used to select for transformants.
Growth of the PduL-His8 production strain.A T7 expression plasmid (pTA925) was used for the production of PduL-His8. The host used for protein expression was E. coli BL21(DE3)RIL (Stratagene). This strain expresses T7 RNA polymerase as well as rare tRNAs for arginine, isoleucine, and leucine. Expression and control strains (BE554 and BE119) were grown in 400 ml LB broth containing 25 μg/ml Kan and 10 μg/ml chloramphenicol. Cultures were incubated at 15°C with shaking at 275 rpm in a 1-liter baffled Erlenmeyer flask. Cells were grown to an optical density at 600 nm of 0.6 to 0.8, and protein expression was induced by the addition of 1 mM IPTG. Cells were incubated for an additional 16 h and harvested by centrifugation at 8,000 × g for 10 min and at 4°C using a Beckman JA-10 rotor and an Avanti J-25 centrifuge.
Purification of PduL-His8.The PduL-His8 production strain (BE554) was grown as described above. One gram of cells (wet weight) was suspended in 3 ml of buffer containing 50 mM Tris-HCl (pH 7.2), 200 mM (NH4)2SO4, and 0.4 mM AEBSF [4-(2-aminoethyl)benzenesulfonylfluoride·HCl]. Cells were broken using a French pressure cell at 20,000 lb/in2, and the resulting cell extract was centrifuged at 20,000 × g using a Beckman JA-17 rotor. The supernatant fraction was filtered through a 0.45-μm syringe filter. A chromatography column containing 1 ml of Ni-nitrilotriacetic acid resin (QIAGEN) was equilibrated with 5 ml of buffer A [50 mM Tris-HCl (pH 7.7), 25 mM KCl, 300 mM NaCl, 200 mM (NH4)2SO4, 10 mM imidazole, and 5 mM 2-mercaptoethanol]. Filtered cell extract (2.5 ml, about 50 mg protein) was applied to the column. The column was washed with 15 ml of buffer A supplemented with 10% glycerol and 80 mM imidazole. The column was eluted with 10 ml of buffer A (in two of 5-ml steps) supplemented with 10% glycerol and 400 mM imidazole.
Preparation of cell extracts of S. enterica.Cells were grown under conditions that induce the pdu operon (5). Cell paste was suspended in 25 mM Tris-HCl (pH 7.2) containing 0.4 mM protease inhibitor AEBSF (3 ml of buffer per gram cells [wet weight]). Cells were broken using a French pressure cell (Thermo Electron Corp., Waltham, MA) at 20,000 lb/in2.
PTAC assays.PTAC assays measured the conversion of acyl-PO42− plus coenzyme A (HS-CoA) to acyl-CoA and were performed as described previously (21). Standard assay mixtures contained 50 mM Tris-HCl (pH 7.2), 20 mM KCl, 0.2 mM HS-CoA, and 1 mM acyl-PO42− in a total volume of 1 ml. For kinetic studies, the concentrations of certain assay components varied as indicated in the text. Activity was determined by monitoring the absorbance at 232 nm over time and by using an E232 of 5.5 mM−1 cm−1 for calculations.
Construction of a nonpolar PduL deletion.Bases 22 to 612 of the pduL coding sequence were deleted via a PCR-based method (24). The deletion was designed to leave all predicted translational start and stop signals of pdu genes intact. The following primers were used for PCR amplification of the flanking regions of the pduL gene: 5′-GCTCTAGAGCCGAAATCAGCCTAATCGATGGCG-3′ (primer 1), 5′-CGTTCATCGCGGGCCTACCAGCCGATCCATTACGCTTCACCTCGC-3′ (primer 2), 5′-CGGCTGGTAGGCCCGCGATGAACG-3′ (primer 3), and 5′-CGAGCTCGCCAGATGCATGATTTACTC-3′ (primer 4). Primers 1 and 2 were used to amplify a 498-bp region upstream of the pduL gene, and primers 3 and 4 were used to amplify 527 bases downstream of the pduL gene. The upstream and downstream amplification products were purified and then fused by a PCR that included 1 ng/μl of each product and primers 1 and 4. The fused product was digested with XbaI and SacI (these sites were designed into primers 1 and 4, respectively) and ligated into suicide vector pCVD442 that had been similarly digested. The ligation mixture was used to transform E. coli S17.1 by electroporation, and transformants were selected on LB medium supplemented with Amp (100 μg/ml). Six transformants were screened by restriction analysis, and all transformants released an insert of the expected size (1,058 bp). One of these transformants was used to introduce the pduL deletion into the S. enterica chromosome using the procedure described previously by Miller and Mekalanos (24), with the following modifications. For the conjugation step, strain BE47 was used as the recipient, and exconjugants were selected by plating onto LB agar supplemented with Amp (100 μg/ml) and chloramphenicol (20 μg/ml). Deletion of the pduL coding sequence was verified by PCR using chromosomal DNA as a template. Finally, the thr-480 dCAM insertion used for selection of exconjugants was "crossed off" by P22 transduction using a phage lysate prepared with the wild-type strain and by selecting for prototrophy on NCE glucose minimal medium.
Aerobic growth curves.Growth media are described in the figure legends. For strains carrying pLAC22, media were also supplemented with 100 μg/ml Amp and 0.2 mM IPTG. To prepare the inoculum, LB cultures (2 ml) were incubated overnight at 37°C, and cells were then collected by centrifugation and resuspended in growth curve medium. Media were inoculated to a density of 0.15 absorbance units, and growth was monitored by measuring the optical density at 600 nm using a BioTek Synergy microplate reader with 48-well flat-bottom plates (Falcon), 0.5 ml of growth medium per well at 37°C, and shaking set at level 4. A relatively low volume of growth medium (0.5 ml) in a 48-well microplate was necessary for adequate aeration, and controls showed that growth under these conditions was similar to growth in shake flasks (data not shown).
Anaerobic growth curves.A procedure similar to that for aerobic growth curves was used to measure the fermentation of 1,2-PD but with the following differences. Cultures were inoculated to an initial density of 0.1 absorbance units. Microplates were sealed with polyolefin membranes (Fisher Scientific) inside an anaerobic growth chamber (Coy Laboratory Products, Grass Lake, MI). To monitor whether anaerobic conditions were maintained within the microplate, NCE glycerol minimal medium was added to several wells and inoculated with wild-type S. enterica. S. enterica cannot ferment glycerol; therefore, growth on glycerol would indicate the presence of oxygen. For the experiments described in this study, no growth in wells containing glycerol minimal medium was observed, indicating that the headspace contained minimal amounts of oxygen (data not shown).
DNA sequencing and analysis.DNA sequencing was carried out at the Iowa State University (ISU) DNA Facility using automated sequencing equipment from Applied Biosystems Inc. The template for DNA sequencing was plasmid DNA purified using QIAGEN 100 tips or QIAGEN Mini-Prep kits. Blast software was used for sequence similarity searching (2).
High-pressure liquid chromatography (HPLC).A Microsorb C18 column (150 by 4.6 mm) was used with a Varian ProStar system that included a model 230 solvent delivery module, a model 430 autosampler, and a model 325 UV-Vis detector (Varian, Palo Alto, CA). Buffers A and B contained 10 mM NH4 formate (pH 4.6) and 10% or 90% methanol, respectively. The flow rate was 1 ml min−1, and the buffer composition varied as follows (minute:percent buffer): 0:0, 5:0, 17:100, 22:100, 23:0, and 28:0.
HPLC electrospray ionization mass spectrometry (HPLC-ESI-MS).An Agilent (Palo Alto, CA) ion trap model 1100 mass spectrometer was operated in the positive mode. The ion source parameters were optimized for the formation of [M + H]+ ions with a source temperature of 310°C, a capillary voltage of 3.2 kV, and a cone voltage of 25 V. Nitrogen was used as the nebulizing gas and as the drying gas at flow rates of 15 and 400 liters h−1, respectively.
The pta gene is nonessential for 1,2-PD degradation.Prior biochemical studies indicated that PTAC was needed for 1,2-PD degradation by S. enterica (25, 37). In the proposed biochemical pathway, this enzyme catalyzed the conversion of propionyl-CoA to propionyl-phosphate (transfer of a three-carbon acyl group) (Fig. 1). An enzyme with homology to known PTAC enzymes is not encoded by the pdu locus (6). However, S. enterica produces a phosphotransacetylase (Pta) encoded by the pta gene, which maps outside the pdu operon at centisome 50 (20). Pta is used for growth on acetate and inositol and plays a key role in the interconversion of acetyl-CoA and acetyl-PO42− (transfer of a two-carbon acyl group) (20). In vitro, Pta not only mediates the interconversion of acetyl-CoA and acetyl phosphate, it also catalyzes the interconversion of propionyl-CoA and propionyl-PO42− (20, 35). This raised the question of whether pta plays a role in 1,2-PD degradation. Two independent pta mutants (BE281 and BE527) were examined for growth on 1,2-PD. Aerobically, both grew slightly faster than wild-type S. enterica. The doubling times for the wild type were typically 7.5 to 8.5 h, and those for the pta mutants were 6 to 7 h. Both pta mutants were reconstructed via P22 transduction to ensure that they were isogenic with the wild type and that both still exhibited a small increase in the growth rate on 1,2-PD (not shown). These results confirmed previous studies that showed that pta is not essential for aerobic growth of S. enterica on 1,2-PD minimal medium (26).
Similarly, pta was unnecessary for the fermentation of 1,2-PD. Strains with pta mutations and wild-type S. enterica both had doubling times of 5 to 6 h during 1,2-PD fermentation. Phenotypic test showed that the pta mutants used in the above-mentioned studies were impaired for growth on acetate or inositol minimal medium and lacked detectable pta activity in cell extracts, as is expected for pta mutants (20). In addition, the growth defects on acetate and inositol were corrected by a pta minimal clone, and the chromosomal location of both pta insertions was verified by PCR (data not shown). Hence, we conclude that the pta gene is not essential for B12-dependent 1,2-PD degradation under either aerobic or anaerobic growth conditions.
Strains with pduL mutations produce less propionic acid on MacConkey-1,2-propanediol indicator medium.The finding that the pta gene was unnecessary for 1,2-PD degradation suggested that S. enterica expresses an additional PTAC that is sufficient to mediate this process. As part of ongoing studies of 1,2-PD degradation by S. enterica, we constructed a series of precise pdu deletion mutations using PCR-based methods (11, 24). Tests with MacConkey-1,2-PD medium (17) indicated that three independent pduL deletion mutants each produced less propionic acid from 1,2-PD than did wild-type S. enterica. The enzymes that convert 1,2-PD to propionic acid were proposed to include coenzyme B12-dependent diol dehydratase, propionaldehyde dehydrogenase, propionate kinase, and PTAC (Fig. 1) (25, 37). Of these, only the gene for PTAC was not identified (7, 19, 26). Hence, these results tentatively suggested that the pduL gene encodes a PTAC enzyme.
Enzyme assays indicate that pduL encodes a PTAC enzyme.Wild-type S. enterica as well as pduL and pta null mutants were grown under conditions that induce the pdu operon (5). Cell extracts were prepared and PTAC activity was measured using an assay that monitors the conversion of acyl-phosphate plus HS-CoA to acyl-CoA and inorganic phosphate (the reverse reaction with respect to 1,2-PD degradation). Both propionyl-PO42− and acetyl-PO42− were used as substrates. Cell extract from the wild-type strain had 8.9 μmol min−1 mg−1 PTAC activity with propionyl-PO42− (Table 2). Extracts from pta or pduL mutants had partial activity (6.0 and 2.6 μmol min−1 mg−1, respectively) (Table 2). PTAC activity was undetectable in cell extracts from the double mutant (pta pduL). The simplest interpretation of these results is that pduL and pta each encode PTAC enzymes, which was previously shown for pta (20). Furthermore, the pta pduL double mutant lacked detectable PTAC activity, demonstrating that the activity in Pta− PduL+ cell extracts required PduL.
Enzyme assays also showed that the PTAC in Pta− PduL+ cell extracts was 15-fold more active with propionyl-PO42− than with acetyl-PO42− (6 μmol min−1 mg−1 compared to 0.4 μmol min−1 mg−1) (Table 2). This is consistent with a role for PduL in 1,2-PD degradation. On the other hand, the PTAC activity in Pta+ PduL− cell extracts was 1.6-fold more active with acetyl-PO42−, consistent with the role of Pta in acetyl group metabolism (20).
For the above-described enzyme assays, controls showed that detectable PTAC activity required acyl-phosphate and HS-CoA, and in all cases, PTAC activity was linear with the enzyme concentration (data not shown).
PduL mutants are impaired for aerobic growth on 1,2-PD.Compared to wild-type S. enterica, a pduL mutant (BE188) was impaired for aerobic growth on 1,2-PD (Fig. 2A). The doubling time for S. enterica was 7.9 ± 0.5 h, and that of a pduL mutant was 12.6 ± 0.6 h. Further impairment of growth was not seen in the pduL pta double mutant even though this mutant lacked measurable PTAC activity (data not shown). These findings are consistent with the idea that PduL is a PTAC involved in 1,2-PD degradation. Aerobically, growth on 1,2-PD is expected to proceed in the absence of PTAC, since propionyl-CoA (which is formed prior to the PTAC reaction) can be metabolized via the methylcitrate pathway (Fig. 1) (16). The observed growth impairment of the pduL mutant was likely due to reduced ATP synthesis resulting from a block in the oxidative branch of the 1,2-PD degradative pathway, although other explanations cannot be ruled out.
Growth of a pduL null mutant on 1,2-PD. (A) Aerobic growth of wild-type S. enterica and a pduL mutant (BE188) on 1,2-PD minimal medium. (B) Fermentation of 1,2-PD. BE188 carries a precise deletion of the pduL gene made by a PCR-based method. The observed growth defects of BE188 were corrected by a pduL minimal clone. Each growth curve was performed three times in quadruplicate. Representative curves are shown. Log-phase doubling times with standard deviations are given in the text. The aerobic growth medium was NCE supplemented with 52 mM 1,2-PD, 200 ng ml−1 vitamin B12, and 0.3 mM valine, isoleucine, leucine, and threonine. The fermentation medium was NCE supplemented with 52 mM 1,2-PD, 200 ng ml−1 vitamin B12, and 0.2% yeast extract. The inoculation and incubation procedures are described in Materials and Methods. OD600, optical density at 600 nm.
pduL mutants are unable to obtain energy from 1,2-PD under fermentative conditions.In the absence of an exogenous electron acceptor, the methylcitrate pathway is inoperative, and S. enterica is unable to grow on 1,2-PD as a sole carbon source (29). Under these conditions, S. enterica converts 1,2-PD to 1-propanol and propionate (Fig. 1). This stimulates growth on minimal medium supplemented with a small amount of yeast extract by providing a source of ATP (29). The production of ATP via the conversion of 1,2-PD to propionate is expected to require PTAC (Fig. 1). Therefore, we tested the effect of a pduL mutation on this process. The doubling times of wild-type S. enterica in the presence and absence of 1,2-PD under fermentative conditions were 5.5 ± 0.35 h and 10.7 ± 0.29 h, respectively (Fig. 2B); the fermentation of 1,2-PD stimulated the growth rate about twofold, which is consistent with previous studies (29). The doubling times of the pduL mutant (BE188) under fermentative conditions in the presence and absence of 1,2-PD were 13.1 ± 1.0 and 13.3 ± 0.95 h, respectively. This indicated that energy production via 1,2-PD fermentation was completely eliminated by the pduL mutation. This is consistent with a role for PduL in 1,2-PD degradation.
The pduL mutant used in the above-described studies contained a wild-type pta gene, indicating that Pta did not substitute for PduL under the conditions used to measure 1,2-PD fermentation. Furthermore, a pta null mutation (alone or in combination with a pduL null mutation) did not affect the fermentation of 1,2-PD (data not shown). These results indicate that the Pta enzyme is insufficient to support the fermentation of 1,2-PD and that PduL is required.
The observed phenotypes of a pduL mutant are complemented by a pduL minimal clone.Growth tests showed that the aerobic and anaerobic growth phenotypes described above for a pduL mutant (BE188) were fully corrected by the expression of a pduL minimal clone using a tightly regulated expression vector, pLAC22. Under aerobic conditions, the doubling times for BE287 (wild-type S. enterica/pLAC22-no insert) and BE286 (wild-type S. enterica/pLAC22-pduL) were 5.4 ± 0.12 h and 6.0 ± 0.32 h, respectively. The doubling times for strains for BE285 (pduL/pLAC22-no insert) and BE284 (pduL/pLAC22-pduL) were 8.2 ± 0.71 and 5.5 ± 0.25 h, respectively. Under anaerobic conditions, the doubling times for S. enterica/pLAC22-no insert, S. enterica/pLAC22-pduL, pduL/pLAC22-no insert, and pduL/pLAC22-pduL were 5.0 ± 0.22, 5.2 ± 0.37, 11.1 ± 0.19, and 5.3 ± 0.40 h, respectively. Thus, under aerobic and anaerobic conditions, a pduL minimal clone complemented the growth defect of the pduL mutation. On the other hand, vectors without an insert did not correct the observed growth defects of the pduL mutant. These results show that the observed phenotypes of the pduL mutant (BE188) resulted from the deletion of the pduL gene but not from polarity or a mutation acquired during strain construction.
PduL substitutes for Pta in vivo during acetate utilization.Growth tests were performed to determine whether PduL could substitute for Pta in vivo. Previous studies showed that pta mutants grow very slowly on minimal medium with >30 mM acetate (20). Here, we show that ectopic expression of PduL corrects this defect (Fig. 3). The doubling times of the Pta+ strain and the pta mutant on acetate minimal medium were 4.4 ± 0.41 and 28.6 ± 1.4 h, respectively. The doubling time of a pta mutant producing PduL from expression vector pLAC22 was 3.7 ± 0.1 h, which was slightly faster than the control (4.4 ± 0.41 h). Hence, ectopic expression of pduL fully corrected the growth defect of the pta mutant on acetate minimal medium. Since Pta is a well-studied PTAC enzyme, this finding indicates that PduL also has PTAC activity.
Ectopic expression of a pduL minimal clone corrects the growth defect of a pta mutant on acetate minimal medium. Data for S. enterica harboring expression vector pLAC22 without an insert (control), a pta mutant carrying pLAC22 without an insert (Pta−/no insert), and a pta mutant harboring a pduL minimal clone in pLAC22 (Pta−/pduL) are shown. The relevant strains are BE287, BE529, and BE530 (Table 1). Controls showed that pLAC22 with or without the pduL insert did not observably affect the growth of the wild-type strain on acetate minimal medium (data not shown). In addition, the growth defect of the pta mutant was fully corrected by a pta minimal clone, showing that the effects of this mutation were not due to polarity (data not shown). The growth medium was NCE supplemented with 0.2% Na acetate and 0.2 mM IPTG. Each growth study was performed twice in quadruplicate. Representative curves are shown. Log-phase doubling times with standard deviations are given in the text. OD600, optical density at 600 nm.
Propionyl-CoA is a product of the PduL reaction.The finding that HS-CoA was required for PduL activity (see above) indicated that an acyl-CoA was formed in assay mixtures. Moreover, the PTAC assay used to measure PduL activity monitored the absorbance at 232 nm, which monitors the formation of thioester bonds (13). To confirm that acyl-CoA was formed in PduL enzyme assays, HPLC and HPLC-mass spectrometry were performed. Assay mixtures contained cell extract from BE527 (Pta− PduL+) or BE291 (Pta− PduL−), HS-CoA, propionyl-PO42−, and standard components; therefore, propionyl-CoA was the expected acyl-CoA product. Reverse-phase HPLC with UV detection at 260 nm was used to identify CoA compounds present in reaction mixtures. A single major compound with a retention time of 10.9 min was detected when Pta− PduL+ cell extracts were used. This compound coeluted with authentic propionyl-CoA following coinjection and produced a mass spectrum characteristic of propionyl-CoA via HPLC-ESI-MS (Fig. 4). The major peaks for authentic propionyl-CoA and the PduL reaction product that eluted at 10.9 min were at m/z 824.1 or 824.2, respectively. These peaks correspond to [M + H]+ for propionyl-CoA. In contrast, propionyl-CoA was undetectable by HPLC in assay mixtures containing cell extract from BE291 (Pta− PduL−), and the major HPLC peak observed corresponded to HS-CoA (retention time, 4.5 min). Our interpretation of the above-described results is that propionyl-CoA is a product of the PduL reaction.
Propionyl-CoA is produced by the PduL reaction. (A) HPLC-ESI-MS of propionyl-CoA standard. (B) HPLC-ESI-MS of a PduL assay after 10 min of incubation at 30°C. The assay mixture initially contained 10 μg cell extract from BE527 (Pta− PduL+), 0.4 mM HS-CoA, 1 mM propionyl-PO42−, and buffer.
Production of PduL-His8 protein.E. coli strain BE554 was constructed to produce high levels of recombinant PduL fused to eight C-terminal histidine residues (PduL-His8). Protein expression by BE554 and a control strain (BE119) was examined by SDS-PAGE (not shown) and enzyme assays. Relatively high amounts of a protein with a molecular mass of nearly 27 kDa were present in the inclusion body fraction from the pduL expression strain, which is near the predicted molecular mass for PduL-His8 (24 kDa). However, a modest amount of soluble protein near this mass was produced.
To better assess the production of PduL-His8, cell extracts were tested for PTAC activity. Soluble extracts from the PduL production strain typically contained about 50 μmol min−1 mg−1 activity, whereas the control strain produced only 2 μmol min−1 mg−1 PTAC activity. Hence, a reasonable amount of PduL-His8 appeared to be soluble and active under the production conditions used. On the other hand, although the inclusion body fraction from the expression strain contained high amounts of PduL-His8 protein, the PTAC activity of this fraction was typically about twofold lower than that of the soluble fraction, indicating that most of the PduL in this fraction was inactive. No PTAC activity was detected in the inclusion body fraction from the control strain. The PTAC assay used measured the conversion of propionyl-PO42− and HS-CoA to propionyl-CoA and HPO42−. No activity could be detected when enzyme, HS-CoA, or propionyl-PO42− was omitted from the assay mixture. In each case, PTAC activity was linear with regard to the enzyme concentration (data not shown).
Purification and kinetic characterization of PduL-His8.PduL-His8 was purified from cell extracts of strain BE554 by nickel affinity chromatography (Fig. 5). Purified PduL-His8 appeared to be homogenous following SDS-PAGE and Coomassie blue staining (Fig. 5). Kinetic analysis showed that purified PduL-His8 was 3.8-fold more active with propionyl-PO42− than with acetyl-PO42− (Vmax of 51.7 and 13.5 μmol min−1 mg−1, respectively) (Table 3). It also had a lower Km for propionyl-PO42− than for acetyl-PO42− (0.61 ± 0.06 versus 0.97 ± 0.26 mM). No activity was detected when PduL-His8, HS-CoA, or propionyl-PO42− (or acetyl-PO42−) was omitted from the assay mixture. In each case, the PduL-His8 activity was linear with the enzyme concentration (data not shown).
SDS-PAGE analysis of PduL-His8 purification. Lane 1, molecular mass markers; lane 2, soluble crude extracts from the PduL-His8 production strain (BE554) (10 μg protein); lane 3, fraction obtained from the first elution step using buffer with 400 mM imidazole (2 μg protein); lane 4, fraction obtained from the second elution step using buffer with 400 mM imidazole (2 μg protein). A 12% acrylamide gel was stained with Coomassie brilliant blue R-250.
For PduL-His8, crude cell extracts typically had PTAC activity of about 50 μmol min−1 mg−1. Nickel affinity chromatography yielded purified enzyme with specific activities of about 45 to 55 μmol min−1 mg−1. Visual inspection of SDS-PAGE gels indicated that PduL-His8 comprised about 2 to 5% of the total cell protein (Fig. 5); hence, these results suggest that PduL-His8 lost activity during the course of purification. Indeed, PduL-His8 was unstable in the purified form and lost about 50% of its activity 48 h after purification. The reported kinetic constants are based on assays done as soon as possible after purification. As the activity of PduL-His8 decreased, its Km for HS-CoA and propionyl-PO42− remained unchanged within experimental error. Its activity with propionyl-PO42− and acetyl-PO42− decreased in direct proportion. We infer that the Km values reported here are representative but that the Vmax values are probably underestimated.
Here, we presented genetic and biochemical evidence that the pduL gene encodes a PTAC involved in 1,2-PD degradation. Growth tests showed that a pduL mutant was impaired for 1,2-PD degradation under aerobic and anaerobic conditions, indicating a role for pduL in 1,2-PD degradation. Growth studies also showed that ectopic expression of PduL corrected the growth defect of a pta mutant on acetate minimal medium, indicating that PduL has phosphotransacetylase activity in vivo. Enzyme assays of crude cell extracts demonstrated that pduL was necessary for the production of detectable PTAC activity in a genetic background that contained a pta mutation. HPLC-ESI-MS showed that propionyl-CoA was produced from propionyl-PO42− and HS-CoA in PduL enzyme assays. PduL-His8 was purified and found to have a specific activity of 57.4 μmol min−1 mg−1 with propionyl-PO42− as a substrate, indicating the PduL is sufficient for PTAC activity. Thus, genetic and biochemical studies indicate that PduL is a PTAC involved in 1,2-PD degradation.
BLAST and PSI-BLAST analyses (five iterations) showed that PduL lacks significant similarity to known PTAC enzymes. Thus, PduL is evolutionarily distinct. It either evolved independently of known PTAC enzymes or became highly divergent from them over time. Sequence analyses also showed that PduL homologues (expect <7 × 10−30) are found in 49/337 (14.5%) complete microbial genomes present in the GenBank database. No homologues of PduL were found among the Archaea or Eukarya. Hence, PduL is apparently a conserved protein specific to the Bacteria. In addition, BLAST analyses identified four PduL homologues that are fused to sequences with homology to acetate kinases. These may be dual-function enzymes that convert acyl-CoA to its corresponding organic acid with the synthesis of ATP.
Including PduL, there are three known classes of phosphotransacylases. The S. enterica PduL, Pta, and EutD enzymes are representatives of these classes. PduL is comprised of 210 amino acids, while Pta and EutD are 714 and 338 amino acids in length, respectively (8). EutD and the C-terminal region of Pta are homologous. PduL lacks significant similarity to EutD and Pta. The N-terminal region of Pta contains BioD-like and "DRTGG" domains. These two domains are absent from EutD and PduL. The physiological role of these domains is uncertain. In enteric bacteria, acetyl group metabolism is linked to two-component signal transduction systems and DNA damage and repair in a complex manner (22, 34, 40). The N-terminal domains of Pta may be needed to accommodate expanded physiological roles.
Kinetic studies with purified PduL-His8 were performed (Table 3). The Vmax of PduL with propionyl-PO42− as a substrate was 51.7 ± 7.6 μmol min−1 mg−1. Previously purified Pta enzymes were reported to have specific activities between 32 and 9006 μmol min−1 mg−1 when acyl-PO42− was used as a substrate (33). The activity of PduL-His8 is at the low end of this range but may be underestimated due to its instability (see Results).
The Km values of PduL-His8 for HS-CoA and propionyl-PO42− were determined to be 0.61 ± 0.06 and 0.032 ± 0.06 mM, respectively. Reported Km values of Pta homologues for HS-CoA range from 0.03 to 1.7 mM, and those for acetyl-PO42− range from 0.024 to 4.7 mM (33). Thus, Km values of PduL for HS-CoA and propionyl-PO42− appear to be within a physiologically meaningful range. Kinetic studies found that the kcat/Km ratio of Pdu-His8 was about sixfold higher for propionyl-PO42− than for acetyl-PO42−. The selectivity of PduL for propionyl-PO42− is consistent with a role in 1,2-PD degradation. Furthermore, its relatively low activity with acetyl-PO42− may be important to minimize the perturbation of acetyl group metabolism (22, 40).
Two findings in this report were surprising to us: (i) the observation that pduL mutants were significantly impaired for growth on 1,2-PD minimal medium (Fig. 2) and (ii) the finding that a pduL pta double mutant was not further impaired for growth on 1,2-PD. Because Pta is known to be active with propionyl-PO42−, we expected that Pta would partly substitute for PduL during growth of S. enterica on 1,2-PD. However, in the studies reported here, it did not. A simple explanation is that PduL is more efficient at propionyl group metabolism in vivo. Two explanations that we think are more interesting are the differential regulation of pta and pduL as well as the possibility that PduL is specifically adapted to function in concert with the polyhedral body-microcompartment involved in 1,2-PD degradation.
We thank the ISU DNA Sequencing and Synthesis Facility for assistance with DNA analyses. We thank Ann Perera and the ISU Metabolomics Laboratory for help with mass spectrometry.
We thank the ISU Office of Biotechnology for financial support.
↵▿ Published ahead of print on 8 December 2006.
Abeles, R. H., and H. A. Lee. 1961. Intramolecular oxidation-reduction requiring a cobamide coenzyme. J. Biol. Chem. 236:2347-2350.
Berkowitz, D., J. M. Hushon, H. J. Whitfield, Jr., J. Roth, and B. N. Ames. 1968. Procedure for identifying nonsense mutations. J. Bacteriol. 96:215-220.
Bobik, T. A., M. Ailion, and J. R. Roth. 1992. A single regulatory gene integrates control of vitamin B12 synthesis and propanediol degradation. J. Bacteriol. 174:2253-2266.
Bobik, T. A., Y. Xu, R. M. Jeter, K. E. Otto, and J. R. Roth. 1997. Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J. Bacteriol. 179:6633-6639.
Chen, P., D. I. Andersson, and J. R. Roth. 1994. The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176:5474-5482.
Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics: a manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Dawson, R. M. C., D. C. Elliott, W. H. Elliott, and K. M. Jones (ed.). 1969. Data for biochemical research, 2nd ed. Oxford University Press, Oxford, United Kingdom.
Havemann, G. D., and T. A. Bobik. 2003. Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 185:5086-5095.
Havemann, G. D., E. M. Sampson, and T. A. Bobik. 2002. PduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 184:1253-1261.
Horswill, A., and J. Escalante-Semerena. 1997. Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the σ54 family of activators, and the prpBCDE genes constitute an operon. J. Bacteriol. 179:928-940.
Jeter, R. M. 1990. Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium. J. Gen. Microbiol. 136:887-986.
Leal, N. A., G. D. Havemann, and T. A. Bobik. 2003. PduP is a coenzyme A-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. Arch. Microbiol. 180:353-361.
LeVine, S. M., F. Ardeshir, and G. F. Ames. 1980. Isolation and characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 143:1081-1085.
Lundie, L. L., Jr., and J. G. Ferry. 1989. Activation of acetate by Methanosarcina thermophila. J. Biol. Chem. 264:18392-18396.
McCleary, W. R., J. B. Stock, and A. J. Ninfa. 1993. Is acetyl phosphate a global signal in Escherichia coli? J. Bacteriol. 175:2793-2798.
Obradors, N., J. Badia, L. Baldoma, and J. Aguilar. 1988. Anaerobic metabolism of the l-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium. J. Bacteriol. 170:2159-2162.
Palacios, S., V. J. Starai, and J. C. Escalante-Semerena. 2003. Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol. J. Bacteriol. 185:2802-2810.
Paoli, P., P. Cirri, L. Camici, G. Manao, G. Cappugi, G. Moneti, G. Pieraccini, G. Camici, and G. Ramponi. 1997. Common-type acylphosphatase: steady-state kinetics and leaving-group dependence. Biochem. J. 327:177-184.
Price-Carter, M., J. Tingey, T. A. Bobik, and J. R. Roth. 2001. The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar Typhimurium on ethanolamine or 1,2-propanediol. J. Bacteriol. 183:2463-2475.
Sampson, E. M., C. L. Johnson, and T. A. Bobik. 2005. Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase. Microbiology 151:1169-1177.
Schmieger, H. 1971. A method for detection of phage mutants with altered transducing ability. Mol. Gen. Genet. 110:378-381.
Schomburg, I., A. Chang, C. Ebeling, M. Gremse, C. Heldt, G. Huhn, and D. Schomburg. 2004. BRENDA, the enzyme database: updates and major new developments. Nucleic Acids Res. 32:D431-433.
Shi, I. Y., J. Stansbury, and A. Kuzminov. 2005. A defect in the acetyl coenzyme A↔acetate pathway poisons recombinational repair-deficient mutants of Escherichia coli. J. Bacteriol. 187:1266-1275.
Shimizu, M., T. Suzuki, K. Y. Kameda, and Y. Abiko. 1969. Phosphotransacetylase of Escherichia coli B, purification and properties. Biochim. Biophys. Acta 191:550-558.
Shively, J. M., C. E. Bradburne, H. C. Aldrich, T. A. Bobik, J. L. Mehlman, S. Jin, and S. H. Baker. 1998. Sequence homologs of the carboxysomal polypeptide CsoS1 of the thiobacilli are present in cyanobacteria and enteric bacteria that form carboxysomes-polyhedral bodies. Can. J. Bot. 76:906-916.
Toraya, T., S. Honda, and S. Fukui. 1979. Fermentation of 1,2-propanediol and 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B12-dependent diol dehydratase. J. Bacteriol. 139:39-47.
Warren, J. W., J. R. Walker, J. R. Roth, and E. Altman. 2000. Construction and characterization of a highly regulable expression vector, pLAC11, and its multipurpose derivatives, pLAC22 and pLAC33. Plasmid 44:138-151.
Wolfe, A. J., D. E. Chang, J. D. Walker, J. E. Seitz-Partridge, M. D. Vidaurri, C. F. Lange, B. M. Pruss, M. C. Henk, J. C. Larkin, and T. Conway. 2003. Evidence that acetyl phosphate functions as a global signal during biofilm development. Mol. Microbiol. 48:977-988.


text:
OnePlus to Launch OnePlus Watch Along with OnePlus 9 Series
By Shekhar Vaidya
PC Hardware Expert
After a ton of leaks and rumors, OnePlus is finally ready to reveal the new first-gen OnePlus Watch along with the OnePlus series on 23rd March 2021. OnePlus has been working for this for more than 2 years. The smartphone was supposed to be announced with the OnePlus 8 series last year, but OnePlus decided to delay the announcement.
A recent tweet by OnePlus, confirms the announcement of the OnePlus Watch on 23rd March along with the OnePlus 9 series. This year OnePlus is working hard for new gadgets and improved gadgets from last year. For example, OnePlus is taking the camera perspective of the new OnePlus 9 series very seriously, last year, CEO Pete promised that the next-gen OnePlus smartphone will be a camera smartphone, and from recent events, the OnePlus seems to be obeying the CEO's word.
Read: OnePlus Partners with Hasselblad for The OnePlus 9 Series, Launch Date, and More
You asked for it. You're getting it.
— OnePlus (@oneplus) March 12, 2021
For a couple of years, we have been seeing OnePlus tirelessly increasing their product like the OnePlus TV, Mid-Range OnePlus Nord, OnePlus Fitness Band, etc. And now with the smartwatch, OnePlus is trying to cover the wearable lineup as well.
OnePlus Watch Teaser
In a post on its official forums, OnePlus said its "fresh addition to the OnePlus family is healthy, punctual and even takes care of you while you sleep." These are other confirmatory phrases that the OnePlus Watch is for real this time.
Back in January, leakster Ishan Agarwal revealed a few watches renders that were dug from the OnePlus Health app, which means that could the OnePlus Watch. But other than that we don't have much info about the upcoming OnePlus Watch.
OnePlus also announced a contest on its forum and 9 winners will be awarded the new upcoming OnePlus Watch. Entries will be accepted until March 23, 9:59 AM EDT.
Unfortunately, that's pretty much everything we so far know about the OnePlus Watch design and features. So, let's wait for the official announcement on 23rd March for more details.
List of All iPhones in Order (2007-Present)
Fix: Apple Watch Stuck on Apple Logo
Best Note Taking Apps to Use Right Now
Best Smartwatch To Buy Right Now

redpajama_set_name: RedPajamaCommonCrawl


Desperate for answers, the children are forced to steal Hal's keys to access the library, where they find the film. It contains a debriefing of Jacques by a V. . . volunteer, with Jacques informing the interviewer that someone has survived a fire, which may or may not be the one that killed the Baudelaire's parents. However, before they can watch any further, they are interrupted by Esme; as they try to escape, the library is ruined. Violet is captured by Olaf while Klaus and Sunny hide in a chute, in possession of the film.
He is really didn't expect this armor beast so strong. He had been in the elven forests by visiting time actually seen armor beast. Just when he know somebody else and he is not a class, so far far saw, run. And he got from dragon scales, power increased greatly, after a bit crazy, began to don't mind the beast's eye on rails. Spirit all want to direct to the sword in the mouth to steel beast, see it tongue is also so hard. But the thought of the somebody else's throat inside that fire, he immediately dropped the idea. Is the flame of steel beast directly spray as saying that he has a dragon scales protection will not be burned into coke, at most is also a eight mature.
Clearly those in the professions should be careful to distance themselves from the lay sector but not primarily on ethical grounds. The 'clear blue water' that separates the two should reflect the commitment of the former to mainstream knowledge and disciplines and the commitment of the latter to alternative ideas about the human mind and body just as we see with orthodox and alternative medical practitioners. SOME FURTHER PROFESSIONAL AND ETHICAL GUIDELINES In Chapter 1, we mentioned that there are various national societies throughout the world that reserve their membership for doctors, psychologists, dentists and other mainstream health care professionals. We do not necessarily advocate that readers join one of these; this will depend on the extent to which they use hypnosis in clinical practice. Many practitioners limit their use of hypnosis to relaxation procedures and will find it unnecessary to belong to a hypnosis organisation. However, if you are using hypnosis for a wider range of applications, then it may be helpful to register with a national society. The societies that we are referring to are those that are affiliated to the International Society of Hypnosis (ISH).
The cousin's grandson Reid and granddaughter Chana they brought from shopping plastic dinosaur Citipati mongoliensisr. Is for a sore throat should serve polpril and ethambutol for thirteen months old child. Buy now program in which, in promotions search spare accessories for set care of the navel of a newborn baby. You have a large dose of humor in manga Handy Manny or Teenage ninja turtles. Most tested rheumatologist, street dobra Lomza see you later. Where inLa Tuque acquire electronic weight rcs 7010. Pay attention to: manhattan toy my snuggly blank ellie the elephant is unique gift.
In a written submission, Hollywood's MPAA shared a few notable ideas. The group calls for more cooperation from Internet services, including hosting providers, who should filter infringing content and block referral traffic from pirate sites, among other things. In addition, hosting providers should not challenge suspension court orders, when copyright holders go up against pirate sites. Going a step further, hosts should keep an eye on high traffic volumes which may be infringing, and ban referral traffic from pirate sites outright. We went on opening night and the theater was packed. I remember thinking that the way the movie opened with the old film footage of a younger Mr Incredible, Elastigirl and Frozone being interviewed was such a different way to open an animated film, and it just got better from there. The whole thing was so stylized and just.
I had completed my Masters, majoring in Physics from IISERK, last May and have moved to Singapore to join the Centre for Quantum Technologies ( CQT ) at NUS for a PhD. My current work is on ultracold molecules, where we are aiming to probe quantum many-body physics in an ultracold Fermi-Fermi mixture of 40 K and 6 Li. After all, why would you want to work if you have a "successful" online business. Yahoo, MSN and Altavista's popularity dwindled in front of the steep rise of Google and now most savvy web users do not even use those "outdated" search engines anymore. According to Wikipedia: Social media is media designed to be disseminated through social interaction, created using highly accessible and scalable publishing techniques. In Economics, everything is based on the "other hand". Ask people for feedback on SEO and you will get two answers: 1.
But the King preferred to maintain a distance from these jihadi elements'. He told these tribals that while he fully shared their sentiments, the time was not ripe for an open confrontation with the British. To win over the tribal chieftains, he distributed a huge largesse amongst them. The British government then roped in some pliable members of the Muslim ulema and managed to persuade them to issue a fatwa which forbade the tribal chiefs to participate in a jihad against the British without obtaining the blessings of their King Amir Habibullah. It was also a striking pointer to the fact that the British in particular and the West as a whole has never hesitated in exploiting religious sentiments whenever they found it expedient. 106? Colonialism and the Call to Jihad in British India King Habibullah was enticed and lured by expensive gifts and above all he was promised that his son, the prince, would be anointed his successor bypassing the claims of his brother.


Greetings! I need help with illustration design for my website. The image I like should be the background for the whole website. Im thinking some kind of sci-fi style of it. The image should blend in smoothly with my logo.
Here are some keywords I got.
The image could be of any style, drawn or footage but of course needs to me custommade and unique.
In a way I want the image to "stick out" and I welcome everyone who are able to think a little outside the box so to speak to create something more original.
If you are interested please share a link to portfolio or other examples of previous work.
My hope is to find an artist that can be my go-to artist/designer whenever I need artwork/design to be done. So a succesful job can of course generate more income for you later on.


Conifer Student in Top 10 in Snowboard Design Contest - Vote to help her win!
We have two labs, located in A211 and A007.
Each lab has 32 computers, a teacher station, printer, and a projector and screen. The computers have Word, Excel, and PowerPoint.
Chromebooks - Feature full internet access, fast startup, easy Google access, and a Chrome browser. No Word/Excel/Powerpoint or printing capability. The Flip Chromebooks have a flippable screen.
Laptops - Feature full internet access, Chrome browser, Word/Excel/Powerpoint and printing capability. They are slow to start up and there is only 1 cart of 15.
iPads - Feature full internet access and many pre-loaded, district approved apps. No printing capability.
If you have any problems or questions about this process, please stop by the Library and we'll give you a hand.


text:
FILM PRODUCTION ANNOUNCEMENT
Christmas Story LLC is preparing to film the made-for-television movie tentatively titled "Shooting Christmas" and we are looking for North Georgians to join our Cast and Crew! The three-week production will begin filming September 26th and will be shooting in and around the Dahlonega area.
"Shooting Christmas" is a heartwarming film that tells the story of a Hollywood movie production that takes over a small Midwestern town and disrupts its peaceful holiday traditions until the town's Mayor and Movie Star fall in love.
Below is a list of positions we're looking to fill. If interested, please email your resume and headshot (if applicable) to
ChristmasStoryLLC@gmail.com
NO LATER THAN SUNDAY, SEPTEMBER 18TH.
IMPORTANT: In the subject line, please list the position you are interested in. Thank you!
TALENT:
Extras: We're seeking male and female background extras, all ages and ethnicities. If you do not have a resume, please send the following details:
Wardrobe Sizes
• Script Supervisor (Previous Experience Required)
• Production Assistants
• Production Secretaries
• Office Production Assistants
• Teachers/Welfare Workers (must be state certified)
• Graphic Designer
• Professional Christmas Light Hangers (Set Dressers)
• Costumer
• Hair Assistant
• Print Shop
The 2016 Great American Main Street Award winners have succeeded in making their towns an exciting place to live, work, play and visit through implementing historic preservation-based methodology for downtown revitalization.
I had such a great experience filming in Dahlonega. The city and county representatives are extremely helpful and eager to support film productions in any way possible. It's pretty remarkable to come to a small, quaint mountain town like this to find such a professional, production ready atmosphere. Not only are you dealing with great people, but you're also in one of the prettiest areas in Georgia! I hope to be back soon!
Walter Thompson, Locations Manager
Downtown Dahlonega is a sparkling example of how careful planning, small business support and historic preservation can combine to create a thriving downtown. Its downtown district, art galleries, restored theater and courthouse stand out in North Georgia, making Dahlonega a viable place to do business.
Patrice Frey, National Main Street Center
© 2016 by North Georgia Film, Inc.
Production Office for Dahlonega & the Georgia Mountain Region


Beautiful contemporary wedding photography by Elli Dean. Oakham-based photographer covering Rutland, Leicestershire, Lincolnshire and beyond.
Your wedding will be one of the most important and happiest days in your life. It is your special day and I aim to capture it in the most honest and sensitive style. Each photo I take tells part of your day's story and brings it back to life. I document the day as it flows, with a little help and direction only when essential. I shoot reportage to capture the vibe, atmosphere, laughter and emotions but will get involved when shooting groups and your portraits as a married couple (always with a smile!). I pay attention to the smallest and biggest details of your day, including the ones you never noticed! When you look back at the photos I want you to be reminded of your excitement while getting ready, your partner's expression when they first saw you walking down the aisle, the kiss, your guests and the atmosphere throughout the day. I want you to remember your venue, the cake, the flowers and all the small and big details that made your day so special to you. I offer four packages to choose from, but if your requirements are different, or you would like any of them tailored to your own needs, please call or email me to discuss. Prices from £500-£1,700. I designed my packages to be straight-forward and transparent, there are no hidden costs or unexpected charges beyond what you agreed to pay. With all my packages you will receive ALL the photos from the day as fully-edited JPGs on a USB (ie. no copyright watermarks on them), so you are not restricted to choose only a limited number or worry about additional costs. The digital files are yours to keep for good.
Thank you for looking at my profile and website. If you like my style and think I could be the right photographer for your big day, please get in touch.
I really have too many I absolutely loved and it won't be fait to mention just one!
Enjoy the planning process but don't let it take over your life and stress you out.
A Super Heroes themed wedding where the bride and her friends created all the table & cake decorations, button holes and the flowers out of pages from comics magazines. The groom was a serious super hero fun. It was absolutely stunning!
Wellies and umbrellas. These are some of the best photos!
I studied design with fine art in London and started a career as a commercial photographer in London. When I moved to Rutland 8 years ago I started to do more and more weddings and I now shoot both commercial and weddings.


text:
Lions Tigers And Bears Mourns Loss Of Tiger
September 29, 2015 By sdcnews
Phevos the Tiger
ALPINE–Phevos, a 17 year-old male tiger who was rescued from a failing zoo in Greece last year has died. Bobbi Brink, founder/director of San Diego's non-profit accredited sanctuary Lions Tigers and Bears, announced the passing last week. "It is with great sadness, that I must share the news about the passing of Phevos. He fought a long and hard battle in this lifetime, and had been through so much. I am honored to have been able to provide him with compassionate care for the time that he was here with us." states Brink. "Phevos' health had been on a slow decline over the last two months. We have been keeping a very watchful eye over him, but he had progressively shown signs of pain and became increasingly withdrawn from his normal interests and activities," said Dr. Jane Meier, the sanctuary's lead veterinary. "His appetite had steadily decreased, making our abilities to provide pain medication difficult, since medication was administered through his daily diet. Before his state deteriorated further, the decision was made with a heavy heart and much contemplation, to humanely euthanize our dear friend, Phevos." Sanctuary officials describe the feline as "a tiger that truly defied all odds thrown his way." Born in captivity, Phevos travelled around with an Italian touring circus group until he was seized by Greek authorities in 2002, along with his mate Athena, and five other tigers. The pair were sent to the Trikala Zoo, outside of Athens, Greece where they lived together until March 2014. Due to the surge of the economic crisis in Greece, the conditions of the zoo deteriorated over time. Neither tiger received necessary care, and signs of abuse were apparent. Athena passed away due to medical neglect in March 2014, after a gaping wound in her paw caused by a Christmas ornament, went untreated for over a year. David Barnes, a UK citizen who initially helped place the two tigers at the Trikala Zoo , and who first brought the plight of Phevos to LTB's attention, helped to arrange the monumental rescue that would provide this suffering tiger a second chance. Eight months after Athena's death, Phevos was rescued from the Trikala Zoo and brought to LTB, where he would live out the remainder of his life. Phevos' rescue made international headlines, as he was "the tiger that travelled halfway across the world for a better life." LTB keepers and staff made every effort to provide the 300 pound tiger with specialized care and a better life at the sanctuary. He was described as a gentle giant, with a calm and reserved demeanor. Phevos enjoyed sunning himself in the cool grass, lounging in the shallows of his pool, and taking naps in his beds of hay. He grew fond of the sanctuary's two year old male tiger, Maverick, as the pair enjoyed spending time together through their habitat fences. "Phevos' passing is heartbreaking to our keepers, staff, volunteers and members, all of whom played a special role in helping provide this beautiful animal with a better life." A memorial will be held at the sanctuary on October 3 after the morning member visit. Members, family and volunteers are invited to pay tribute to Phevos through a celebration of life.
Filed Under: Animal/Wildlife News Tagged With: Animal News


text:
Cuban Fournier Wins Bronze Medal at 2nd World Rowing Cup
Poznan, Poland, Jun 23 (Prensa Latina) Angel Fournier, from Cuba, on Sunday won the bronze medal in the single weight open modality at the 2nd World Rowing Cup, which ended today in the Polish city of Poznan. The 31-year-old Cuban won the bronze medal after clocking 7:24.790 minutes, behind Sverri Nielsen, from the Netherlands, who clocked 7:21.960 minutes, and Pilip Pavukou, from Belarus, who stopped the clock at 7:24.100 minutes.
According to the website of the event, the world multi-medal rower paddled the first 1,000 meters in the fifth position on the water track of Lake Malta, while already in the third segment, at 1,500 meters route, he speeded up until reaching the third place, where the competition ended.
The 2nd edition of the World Rowing Cup began last Friday and ended on Sunday after the completion of 18 finals.
jg/omr/mem/rps
Cuba, Sports, Europe, Portada Deportes, Nota Informativa, Interior Deportes,


text:
With blood on my hands, I held her.
With death in my soul, I took from her.
With the devil in my heart, I coveted her.
There're many definitions of Hell.
My list was exhaustive, my definitions tragic.
Tonight I was adding something new to the very top.
Girl number six thirty-two.
She arrived on my birthday, the same day, every year, I play Russian Roullete and pull the trigger.
She was my omen.
My end game.
She made it personal.
And for the first time in my life, I gave in.
A virgin mob boss with no soul.
My name is Andrei Petrov.
They call me the devil.
All I want is for the pain to end.
All she wants is for me to share it.
I am the last remaining heir to a dynasty that should burn in Hell.
And my last wish remains for it to die with me.
Review: 4.75 of 5 Stars
I have to confess, when I opened this book I wondered how I was going to feel about it. I absolutely adore the Eagle Elite series and the characters. It is by far the best mafia series out there. But the last couple of books, while great, did not hit me with as much emotion and impact as some of the earlier ones. But then I opened Debase. And damn, Andrei Petrov.
Andrei is probably the darkest character that Rachel has written about in the mafia. Don't get me wrong, they are all ruthless and have their own issues, but they are still fun loving and funny. I know it sounds weird but it works. But Andrei was different. He had some serious issues and I was heartbroken for him and the more I learned the more I wanted to help him.
Girl Six Thirty Two is one of the best female characters as well. She has her own set of things she has gone through and being born in a family is a big part of it. But I just love her strength and tenacity. I loved watching the story of these two unfold. Even though this book is intense, it is amazing and you won't be disappointed that you read it. Well done, Rachel!
From USA Today bestselling author Jillian Dodd comes the next book in a sizzling series filled with action and adventure. Fans of The Selection and The Hunger Games will discover a heart-pounding thrill ride of espionage and suspense set in glittering high society.
A single sentence is muttered from the lips of an assassin dangling from a four-story building, "It starts in Montrovia."
That sentence led Black X to send a spy to protect Prince Lorenzo.
And that sentence haunts Huntley as she realizes her time is running out.
When athletes from around the world descend on Montrovia for the Olympics, another group is coming together, its years of planning a new world order finally coming to fruition.
A disease is released.
People start dying.
And, with rumors of an impending coup, the country falls into chaos.
Alliances are betrayed.
Lives are lost.
Loves are challenged.
And hearts are turned.
Can Huntley stop what has started, or will Montrovia and the world fall?
I just LOVE everything about the Spy Girl series. I love the main character Huntley, I love the mysterious plot line and every ounce of suspense that comes with constantly trying to figure out the the end game. It's utterly addicting. The Phoenix dives right into the continuation of Huntley's story and honestly it never slows down. Major plot lines are revealed, character identities are answered and the mystery still continues. It's like solving a puzzle and this book gives you almost all the pieces but in the end it's really just enough to keep you coming back for more.
Huntley's strength and passion continue to amaze me. She has grown so much as a person, not only does she follow her instincts but she hardly every jumps to the wrong conclusion. She is very careful to examine things from all angles before making a decision, but what if even after doing all that she still doesn't see the end game? What if she isn't able to save Montrovia? Part of me wonders if at the end of this mission the real lesson was learning one person alone cannot be responsible for Montrovia's safety but instead it comes from trusting the right people, depending on the people you love and working together? It's just one theory running through my mind as to what we will find in book #7.
Jillian Dodd throws us readers some serious curve balls with The Phoenix. The rumors are all true - yes this book ends on a cliffhanger, yes characters die and yes you will be nervous about book #7 because I have real fears things are going to get even worse for Huntley before this mission is over. This series is awesome, full of interesting characters you will find yourself wanting to live vicariously through and mystery in spades! Thank you Jillian for this guilty pleasure, I for one love it!!
Labels: Book Review, Jillian Dodd, Spy Girl, Suspense
What else can a virgin do when she's ditched at the altar? Seattle heiress Mackenzie Dupont is treating herself to a single-girl honeymoon in Mexico and a desire to relinquish her innocence to a gorgeous one-night stand. Fake names. True pleasure. But when she wakes up alone, Mackenzie realizes just how much anger is left in her broken heart.
Suffering a tragic personal loss, pro soccer player Slade Rodriguez has his reasons for vanishing without a goodbye. Right or wrong, he's blaming the beautiful and infuriating stranger he never wants to see again. They're both in for a shock when Mackenzie shows up as his new personal assistant. And they both have a lot to learn about each other. Because they share more than they could possibly know, including a common enemy who's playing his own games. And he's not afraid to get dirty.
Now there's only one way Mackenzie and Slade can win: to trust in each other and to stop hiding from the lies they've told, the secrets they've kept, the mistakes they've made, and the attraction that still burns between them.
I have to admit I loved this one! It was a classic Rachel Van Dyken book. The perfect amount of angst, humor, chemistry and drama all rolled into one HEA. Rachel is one of my favorite go to authors, mainly because she seems to nail it every single time.
Mackenzie is left at the alter on her wedding day - sure she's upset but not in the way you'd expect. Fast forward several months and in the hopes of self re-discovery she's off on her honeymoon alone. Here's where Slade Rodriguez comes into the picture and I have to say this meet cute was absolute perfection!
These two connect on a deeper level than either would expect, but to say it's a short lived romance would be putting it mildly. Here's where the story gets juicy! Yes, I could give you a play by play but I'm not because this story is one that deserves to be read and experiences first hand!
I loved watching Mackenzie work through her struggles and watching her grow. From beginning to end it was a beautiful transformation of a character who's been incredibly lost become a strong independent women, who yes would love the support of the man she's in love with, but who finally not only knows what she wants but also has the courage to fight for herself.
Slade is broody, exhausted with the circus that's his life and so angry he honestly cannot stand himself. But underneath all this is this amazing book boyfriend who's passionate and caring - and let's not forget his looks, basically he's the total package.
They are both lost in their own circumstances but together they make one heck of a couple. Risky Play is going to be one of those RVD book's you're absolutely going to want to read again and again. Such a fun escape - thanks Rachel - this is why we all love you so much!!!
Labels: Book Reviews, Rachel Van Dyken, Review


text:
The 3 positive developments revealed at the latest Covid press conference
14% of the total population has now received a Covid jab, Arlene Foster said
Shaun KeenanDerry reporter
More than a quarter-of-a-million people in Northern Ireland have received their first dose of the coronavirus vaccine, it emerged on Thursday.
Speaking earlier, First Minister Arlene Foster said lockdown measures were beginning to make a difference in efforts to suppress the virus.
The DUP leader addressed the media at the Executive's daily Covid-19 press briefing from Ranfurly House in Dungannon.
It comes as the Department of Health reported a further 10 coronavirus related deaths.
According to the latest update, four of the fatalities are said to have occurred within the current reporting period, and six outside of it.
The death toll in Northern Ireland now stands at 1,899, according to the DoH.
Coronavirus Northern Ireland: When will lockdown end and life return to normal
Northern Ireland students to receive £500 coronavirus payment
However, speaking yesterday Health Minister Robin Swann said the pandemic was "far from over".
In a stark address to the public, Minister Swann repeated the message that has echoed since March.
"We should never underestimate Covid-19 and the damage it can do," Minister Swann added.
Lockdown measures making "major difference", says Arlene Foster
As Northern Ireland entered week seven under lockdown regulations, Arlene Foster said the strict measures implemented in December were beginning to have a "major difference" on infection rates.
Mrs Foster said, however, now was not the time to "ease up" but to "push on" against the virus.
Lockdown restrictions, due to Covid-19, continue across Belfast and Northern Ireland. (Image: Jonathan Porter/PressEye)
The First Minister said that hospitals and intensive care units across the country were still continuing to face huge pressures.
She said: "In real terms, this means that many thousands of people have been protected from this deadly virus, some of whom would have not survived contact."
The "R" number continues to stay below 1
Meanwhile, the Stormont minister said the public had managed to "maintain the rate of infection to well below one."
The R - or reproduction - number is now sitting between 0.75 and 0.85.
Mrs Foster added: "Last month, I made an appeal to everyone in Northern Ireland to join us in keeping the fight against Covid and push down the curve of infection and take those difficult daily decisions to do the right thing, to stay at home and everybody has.
"To all of you who have taken action, who changed how you managed your everyday life, I say thank you because three weeks on it has made a major difference."
More than 250,000 in NI have received Covid jab
One in seven people in Northern Ireland has received their first coronavirus vaccine, Arlene Foster said.
The First Minister said 263,735 people have received their first jab, with 25,539 people receiving both.
Stock image (Image: GettyImages)
It was also revealed that 90% of care homes residents have had their second dose.
When it comes to those 80 or over, Arlene Foster revealed that 90% had received their first jab.
"I commend all those involved in the vaccine programme for their efforts. It is something that everyone in Northern Ireland should be proud of."
For the latest breaking news straight to your inbox, sign up for our newsletter here.


text:
Gerald Croft
The meaning of the word catalyst in a dictionary is "substance that speeds up a chemical reaction without itself changing" (Collins gem English dictionary, p 80). The inspector is a catalyst, as he is asking the questions which cause the character he is asking to answer and tell the story of their involvement with Eva Smith and the listening characters to react upon the response, without actually being involved himself. He speeds up the process by having all the characters present when one is being questioned, I can tell this as when he is talking to Mr Birling, Gerald asks "Wouldn't you rather I was out of this?" (Gerald, act one, p 13), the inspector says, "I'd prefer if you stay." (Inspector Goole, act one, p 13). I think this is because he wants all the characters to know the whole story of Eva Smiths life from when she was fired by Mr Birling, this way they can all fill in the part where they are involved in order when the inspector asks them to.
I think the inspectors general way of questioning is very clever as he either shows them a photograph or gets them to confirm they knew her by other means such as a changed name is Gerald's case. Once he has the confirmation he gives them a small amount of information to trigger the characters memory, he then begins to ask his questions which are very short and to the point. After he has asked the question the characters seem to tell him everything they know or knew about her including their input on her life. So really he asks the question then lets the character dig themselves a grave as it were.
This is the basic outline of the inquest in to each character, but of course there are slight variations between each character and small differences. Which is why I am going to examine the way the inspector interacted with each of the characters in turn. Starting with Mr Birling as he has the longest history with Eva Smith and ending with Mrs Birling as she has the shortest history with Eva Smith as she only met her two weeks ago.
I will show two things that the inspector did, one is how Inspector Goole drew the truth out of them and two is how he made each of the characters face up to their responsibility concerning the suicide of Eva Smith.Mr Birling: Mr Birling is the first person the inspector speaks to when he enters the dining room, this is because he is the head of the household and it was customary for people to speak to him first as he was considered the most important over his family and Gerald. Inspector Goole draws the truth out of Mr Birling by telling Gerald, Eric and Mr Birling the details of Eva Smith's suicide, including all the gory details.
This doesn't have much of an effect on Mr Birling and he seems to show impatience more than anything, "(rather impatiently) Yes, yes. Horrid business. But I don't understand why you should come here Inspector" (Mr Birling, act one, p 12).The inspector informs them that he has "been round to the room she had, and she'd left a letter there and a sort of diary…
her real name – was Eva Smith." (Inspector Goole, act one, p 12). The inspector only asks him three questions regarding Eva Smith, the first one is "Do you remember her, Mr Birling?" (Inspector Goole, act one, p 12), Mr Birling doesn't remember her much, inspector Goole reminds him that she was once employed at one of his factories; this still doesn't trigger Mr Birling's memory so Inspector Goole shows him a photograph. He then says, "I think you remember Eva Smith now, don't you, Mr Birling?" (Inspector Goole, act one, p 13), this is his second question.Mr Birling then tells them a small bit of information, which the inspector already seems to know as he confirm the date that Mr Birling says he discharged her on.
There is a gap here in the questioning when Eric interrupts and Gerald asks if he should leave, the inspector says no. This seems to trigger an emotion in Mr Birling and he gets protective of himself proclaiming "Look – there's nothing mysterious – or scandalous – about this business – or at least not as far as I'm concerned." (Mr Birling, act one, p 11).
There is a small part when Eric begins to repeat what Mr Birling had told them earlier about the "a man has to mind his own business and look after his own" (Mr Birling, act one, p 10) speech. Mr Birling doesn't seem to want to talk about this in front of the inspector so he tells the rest of his story without any interruptions. That is how Inspector Goole drew the truth out of Mr Birling.Sheila;Sheila enters the room just as Mr Birling is asking what happened to her after he had sacked her, right on cue in my opinion as this is where Sheila also comes into Eva Smiths life. I think Priestley set it out like this to make things nice and simple for the audience to understand. Sheila seems to genuinely care about the girl, so when she recognises Eva Smith from the photograph the inspector shows her and realises she has destroyed her life after her father has already done so she is so grief stricken she runs out of the room crying. This makes Mr Birling very angry, understandably, as she had been so happy just hours ago.
He had tried to get her out of the room as he believed she had nothing to do with the investigation.When she eventually comes back with tear stains on her face she has calmed down, she first asked if what she did made much of a difference to Eva Smith's life, she is told that "you're partly to blame. Just as your father is." (Inspector Goole, act one, p 23). Its Eric who actually asks what she had done, and Sheila begins to tell them that she had "gone to the manager at Milwards and told him that if they didn't get rid of that girl, I'd never go near the place again" (Sheila, act one, p 23). The inspector then asks her questions, partly for their knowledge and partly to make her feel guiltier. Then he asks her "What happened?" (Inspector Goole, act one, p 23), she then tells the whole story and how she only did it because she was jealous.
By asking short questions like these inspector Goole was able to extract all the information he wanted from her.Once he knows the story concerning Sheila he makes her feel horrid by rubbing in the fact that it was so mean, by saying to her, "you might be said to have been jealous of her" and had "used the power you had, as the daughter of a good customer and also of a man well known in the town, to punish the girl just because she made you feel like that"(Inspector Goole, act one, p 24) and when Sheila says "Yes…if I could help her now, I would" (Sheila, act one, p 24) he makes her feel even worse by harshly saying, "Yes, but you can't, It's too late. She's dead."(Inspector Goole, act one, p 24).
By doing this he made her face up to the responsibility she has towards the suicide of Eva Smith.Gerald; After the inspector had questioned Sheila and had all of the information he wanted from her he begins to tell them what happened to Eva Smith after Sheila had got her fired from her job at Milwards, "She changed her name to Daisy Renton" (Inspector Goole, act one, p 25) then Gerald says (startled) "What?"(Gerald, act one, p 25). This gives him away as knowing of her immediately and the inspector of course notices this and after he gets back from briefly going to find Mr Birling he begins the questioning. "Well?" (Inspector Goole, act one, p 26).
Here act two begins. Gerald wants to get Sheila out of the room so he can't hear his secret, the inspector says she can but she wants to hear the rest of the questioning. Mrs Birling comes in before he has answered so there is a gap in which Sheila and Mrs Birling argue about Eric's drinking.After inspector Goole has explained what everyone knows so far to Mr and Mrs Birling he asks Gerald "when did you first get to know her?" (Inspector Goole, act two, p 33). Gerald first acts dumb pretending he doesn't know what the inspector is on about, which makes the inspector 's job of getting the truth out of them a bit harder, though when Inspector Goole says that he already knew and Sheila gave him away he decides its best to tell the truth, "All right, if you must have it." (Gerald, act two, p 34). Sheila is more of a hindrance then a help to the inspector as she wants to hear all about it and Gerald is rather put of with her around making comments like "Well, we didn't think you meant Buckingham Palace." (Sheila, act two, p 34).
He tells his story, with the inspector turning on anyone who got in the way, for example with Mr Birling, when he says (angrily) "I really must protest -", (Mr Birling, act two, p 37) "Why should you do any protesting? It was you who turned the girl out in the first place." (Inspector Goole, act two, p 37). I don't think that the inspector needed to make Gerald face up to any responsibility as he was probably the one bright spark in her life since when she was fired, he took pity on her and liked her. The only thing he needs to feel guilty about in my eyes is the fact that he cheated on Sheila.
I think that this play is more about the Birling family characters facing up to responsibility more than anyone else, and that they are the main ones who need to feel guilty, not Gerald Croft.
Gerald and the Inspector
The Birling family
Business control
Gerald using short, sharp, snappy questions
Drinking problem
Elizabethan society