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Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate ("phenylphosphate carboxylase"). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcusstrains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.
Phenolic compounds are important plant constituents. For example, lignin represents about 20 to 30% of the biomass of higher plants and consists of phenylpropane units with various hydroxyl or methoxyl groups. Phenol is formed from lignin and several other natural or synthetic substrates during microbial degradation. The best-known phenol-generating enzyme is tyrosine phenol lyase, whose activity is used in bacterial taxonomic studies. Because of its toxic effects on cells, phenol was used as an antiseptic in clinical applications for a long time. In the chemical industry, phenol is a basic substance for the production of phenolic compounds from coal. Because of its broad application in industry, phenolic compounds are among the most prominent man-made groundwater contaminants derived from coal treatment processes, such as gasifying coal.
The aerobic metabolism of phenol has been studied extensively; in all known aerobic pathways, mono-oxygenases initiate the degradation of phenol by hydroxylation to catechol (for a recent review on phenolortho hydroxylases, see reference 60). Among ortho-hydroxylating phenol hydroxylases, one-component flavoproteins or multicomponent enzyme systems are recognized. The following ring cleavage is a key reaction in microbial degradation of aromatic compounds. Catechol is oxygenolytically cleaved by dioxygenases by either ortho or metacleavage.
Anaerobic growth on phenol has been observed for various bacteria (4, 24, 37, 55, 59, 62, 63, 64, 68). In all cases studied, phenol appeared to be carboxylated to 4-hydroxybenzoate and growth on phenol was dependent on the presence of CO2(62). Consortia of fermenting bacteria convert phenol to benzoate (24, 68) and decarboxylate 4-hydroxybenzoate to phenol (24, 68). They also catalyze an isotope exchange between D2O and the proton at C-4 of the aromatic ring of phenol (23).
Phenol carboxylation to 4-hydroxybenzoate is a paradigm for a new type of biological carboxylation reaction. The process has been studied in the denitrifying bacterium Thauera aromatica (3,32-34, 63) (Fig. 1). The gram-negative, rod-shaped bacterium belongs to the β-group of the proteobacteria and was enriched and isolated under denitrifying conditions with phenol as substrate (1, 62; for similar strains, see references 61 and64). It has a broad substrate spectrum and is able to grow anaerobically on many aromatic compounds. Cells grown with phenol were simultaneously adapted to growth with 4-hydroxybenzoate, whereas 4-hydroxybenzoate-grown cells did not metabolize phenol. Induction of the capacity to metabolize phenol required several hours.
Phenol carboxylation proceeds in two steps and seems to involve formation of phenylphosphate as the first intermediate (equation 1) (32, 33). An enzyme activity catalyzing an isotope exchange between [14C]phenol and the phenyl moiety of phenylphosphate was identified in extracts of phenol-grown cells (equation 3) and was lacking in 4-hydroxybenzoate-grown cells. [32P]Phosphate did not exchange with phenylphosphate. This suggests a phosphorylating enzyme, E1, with a ping-pong mechanism which becomes phosphorylated in an essentially irreversible step (equations 2 and 3). The phosphorylated enzyme E1 is postulated to transform phenol to phenylphosphate in a reversible reaction (equation 3). The whole reaction is understood as the sum of equations 2 and 3. Unfortunately, the phosphoryl donor X∼P is unknown so far. The enzyme E1was termed "phenylphosphate synthase." Equation 1 Equation 2 Equation 3Phenylphosphate is postulated to be the substrate of a second enzyme, E2 (32). It requires Mn2+ and catalyzes the carboxylation of phenylphosphate to 4-hydroxybenzoate (equation 4). An enzyme activity catalyzing an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (equation 6) was present in phenol-grown cells; [14C]phenol did not exchange with 4-hydroxybenzoate. It was assumed that the carboxylation reaction (equation 4) and the CO2 isotope-exchange reaction (equation 6) are catalyzed by the same enzyme, reaction 6 being a partial reaction of reaction 4. The CO2 isotope-exchange activity again suggests a ping-pong mechanism for enzyme E2(equations 5 and 6), involving an enzyme E2-phenolate intermediate which is formed in a (presumably) exergonic reaction (equation 5) followed by a reversible carboxylation (equation 6). Equation 4 Equation 5 Equation 6The actual substrate is CO2 rather than bicarbonate (as is the case in biotin-dependent carboxylases), and the carboxylating enzyme was not inhibited by avidin, a potent inhibitor of biotin-dependent carboxylases. Both results suggest that biotin is not involved in carboxylation. The enzyme E2 was termed "phenylphosphate carboxylase." Synthesis of both the phosphorylating and the carboxylating enzymes is strictly regulated. All activities were only present in cells grown anoxically on phenol and were lacking, e.g., in 4-hydroxybenzoate-grown cells (29, 34). The phenol-carboxylating enzyme in T. aromatica does not belong to any group of the studied carboxylases, as it seems to proceed via a phosphorylated free intermediate, it is extremely oxygen sensitive and sensitive to radical-trapping agents (S. Breinig, M. Hügler, and G. Fuchs, unpublished results), and it is not dependent on biotin or thiamine diphosphate. It differs from most known carboxylases by using carbon dioxide as substrate and by the requirement of a metal as cocatalyst.
Further metabolism of 4-hydroxybenzoate proceeds in two steps to the intermediate benzoyl coenzyme A (CoA) (Fig. 1). A specific CoA ligase forms 4-hydroxybenzoyl-CoA (8), which becomes reductively dehydroxylated to benzoyl-CoA by a molybdo-flavo-iron-sulfur protein, 4-hydroxybenzoyl-CoA reductase (11, 14). A 2 [4Fe-4S] ferredoxin serves as electron donor for this reaction (14). The ligase and reductase are both induced in phenol- and in 4-hydroxybenzoate-grown cells, but not in benzoate-grown cells (29). Benzoyl-CoA is a common intermediate in the metabolism of many aromatic compounds. It is reductively dearomatized (9,10) and further metabolized to three acetyl-CoA molecules and one CO2 molecule (note that one CO2 was originally introduced into phenol) (reviewed in reference 28). The anaerobic metabolism of phenol is shown in Fig. 1.
Nothing was known so far about the molecular background of anaerobic phenol metabolism. Based on the N-terminal and internal peptide sequence information of five phenol-induced proteins, it was our aim to find the genes coding for phenol-induced proteins. Analysis of the DNA sequences should provide new insights into the function of the phenol-induced proteins and into the regulation of anaerobic phenol metabolism.
Materials.Chemicals were reagent grade and purchased from Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Germany), Serva (Heidelberg, Germany), or Sigma-Aldrich (Deisenhofen, Germany). Biochemicals were from Boehringer (Mannheim, Germany). Scintillation cocktail and acrylamide stock solution were purchased from Roth (Karlsruhe, Germany). [14C]Na2CO3(2 GBq mmol−1) was purchased from American Radiolabeled Chemicals Inc./Biotrend Chemikalien GmbH (Cologne, Germany). Nitrogen, helium, and a N2-H2 gas mixture (95% nitrogen, 5% hydrogen) were purchased from Linde (Höllriegelskreuth, Germany).
Bacterial strains, growth conditions, cell harvesting, and storage.Strains of T. aromatica, K172 (1,62), S100 (62), SP (54), LG356 (unpublished results), and T1 (61), and Azoarcus evansii KB740 (12) were cultured anaerobically at 30°C in a mineral salt medium with 0.5 mM phenol, 10 mM bicarbonate, and 2 mM nitrate or with 5 mM 4-hydroxybenzoate and 15 mM NaNO3 as described elsewhere (11, 62). Growth was measured by following the optical density at 578 nm (OD578). An OD578 of 1.0 corresponded to a cell concentration of approximately 0.3 g of dry cell mass per liter. The harvested cells were immediately frozen and stored in liquid nitrogen. Growth conditions of Escherichia coli strains (XL1-blue MRF′ [Δ(mcrA)183 Δ(mcr-CB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI q ZΔM15 Tn10(Tetr)]]; K38 [hfrC phoA4 pit-10 tonA22 ompF627 relA1 spoT λ−]; P2392 [E14− (McrA−) hsdR514 supE44 supF58 lacYl or Δ(lacIZY)6 galK2 galT22 metB1 trpR55 (P2 lysogen)] and XLOLR [Δ(mcrA) 183 Δ(mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI q ZΔM15 Tn10(Tetr)] Su− (nonsuppressing) λr(lambda resistant)]) were as previously described (50).
Preparation of cell extract.All steps were performed under strictly anaerobic conditions. Frozen cells (20 g) were suspended in 20 ml of 20 mM imidazole-HCl (pH 6.5), 10% glycerol, 0.5 mM dithionite, and traces of DNase I, disrupted by a passage through a French press (137 MPa), and ultracentrifuged (1.5 h at 100,000 × g and 4°C). The supernatant containing the soluble protein fraction (55 mg of protein/ml) was used immediately or stored at −20°C for later experiments. The soluble protein fraction (cell extract) contained all 4-hydroxybenzoate: 14CO2 exchange and phenylphosphate carboxylase activity.
Enzymatic tests.All tests were conducted at 30°C under strictly anaerobic conditions. The assays used 1 ml of assay mixture in a stoppered vial with a gas phase of approximately 250 μl. The assays for 14CO2:4-hydroxybenzoate isotope exchange and carboxylation of phenylphosphate were performed as described elsewhere (32, 43). The amount of radioactivity in acid-stable products (4-hydroxybenzoate) in a sample of 250 μl was analyzed by liquid scintillation counting. The amount of labeled 4-hydroxybenzoate formed was calculated from the amount of fixed radioactivity, taking into account the known specific radioactivity of bicarbonate in the assay (80 Bq/nmol).
Partial purification of three dominant phenol-induced proteins, F1, F2, and F3.All chromatographic steps were performed under anaerobic conditions in a glove box under an N2-H2 atmosphere (95%/5% [vol/vol]) at 4°C. All buffers were filtered and degassed and then supplemented with dithionite to a final concentration of 0.5 mM. Generally used buffers were as follows. Buffer A contained 20 mM imidazole-HCl (pH 6.8), 20 mM KCl, 0.5 mM MnCl2, and 10% glycerol. Buffer B contained buffer A plus 500 mM KCl. The purification was started with extract (approximately 20 ml) from 20 g of frozen cells grown on phenol and nitrate.
Ion-exchange chromatography 1.Cell extract (20 ml) was applied on a DEAE-Sepharose fast-flow column (35 ml; Pharmacia), which was run at a flow rate of 0.5 ml/min. The column was washed with 50 ml of buffer A, followed by a flat linear gradient from 20 mM KCl (100% buffer A) to 200 mM KCl (60% buffer A, 40% buffer B) in 100 ml and a steeper gradient from 200 mM KCl (60% buffer A, 40% buffer B) to 500 mM KCl (100% buffer B) in another 100 ml. Fractions of 2.5 ml were collected, and 6-μl samples of every second fraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Compared with extract of 4-hydroxybenzoate-grown cells, three dominant phenol-induced proteins, F1 (60 kDa), F2 (58 kDa), and F3 (67 kDa), were separated. F1 eluted between 60 and 100 mM KCl, F2 eluted between 140 and 190 mM KCl, and F3 eluted between 250 and 350 mM KCl. Fractions containing F1, F2, and F3 were pooled and tested for phenylphosphate carboxylase and CO2 isotope exchange activities.
Ion-exchange chromatography 2.Two milliliters of the pooled fractions of F1, F2, and F3 after DEAE-Sepharose fast-flow chromatography was applied to a high-performance liquid chromatography (HPLC) system equipped with a MonoQ HR5/5 column (1 ml; Pharmacia), which was run at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected in each run, and aliquots of 10 μl were analyzed by SDS-PAGE. For F1 purification, the column was equilibrated with buffer A containing 60 mM KCl and eluted with this buffer until the UV absorption baseline at 280 nm was stable. Samples of every fraction were tested for both activities and analyzed by SDS-PAGE. F1 eluted after 5 to 10 column volumes. For F2 purification, the column was equilibrated with buffer A containing 150 mM KCl. The column was washed with this buffer until the UV absorption baseline at 280 nm was stable. F2 eluted after 10 to 14 column volumes. For F3 purification, the column was equilibrated with buffer A containing 260 mM KCl and washed until the baseline at 280 nm was stable. Then, a linear gradient of 260 to 400 mM KCl in buffer A (8 column volumes) was applied. F3 eluted early in the gradient between 260 and 330 mM KCl.
Affinity chromatography on blue Sepharose.Ten milliliters of the F2 pool after chromatography on DEAE was loaded on a blue Sepharose column (48 ml; Pharmacia), equilibrated with 20 mM Tris-HCl, pH 7.5, at a flow rate of 2 ml/min. Because of the instability of the color matrix in the presence of reducing agents, no dithionite was added. The column was washed with 1 volume of the same buffer, and then F2 was eluted with 10 mM phenylphosphate in 20 mM Tris-HCl, pH 7.5, in 3 volumes. Fractions of 5 ml were collected, and 100-μl samples of each fraction were analyzed by SDS-PAGE (10% acrylamide).
SDS-PAGE.Polyacrylamide gels (normally 11.5% acrylamide) were prepared as previously described (35). Staining of the proteins was done according to a previously described method (67).
Two-dimensional gel electrophoresis.The first dimension (isoelectric focusing) was done with cell extract (120 μg of protein) according to Görg et al. (25) by using the Immobiline Dry Strip system (linear pH gradient 3 to 10; Pharmacia) according to the manufacturer's protocol. The second dimension (SDS-PAGE) was performed as described above. Comparison of extracts of cells grown on phenol and 4-hydroxybenzoate allowed the identification of phenol-induced proteins.
Protein transfer onto polyvinylidene difluoride membranes.Proteins of cell extract and of fractions after chromatography were electrophoretically separated on SDS-polyacrylamide gels and were then transferred to an Immobilon-Psq transfer membrane (Millipore, Bedford, Mass.) for N-terminal sequencing of the phenol-induced proteins by using the Nova Blot System (Multiphor II; Pharmacia LKB, Freiburg, Germany) according to the manufacturer's protocol.
Amino acid sequencing and tryptic digest.Protein spots from cell extract of phenol-grown cells obtained after two-dimensional electrophoresis and fractions containing the phenol-induced proteins F1 to F3 after chromatography on MonoQ were transferred to a polyvinylidene difluoride membrane. Induced proteins were excised and sequenced using an Applied Biosystems 473A sequencer or an automated gas phase sequencer. The sequencing was performed according to the Edman method. The separated amino acid derivatives were analyzed by C8 reversed-phase HPLC. For peptide sequencing, the pooled fractions containing F2 after chromatography on blue Sepharose were digested with modified trypsin (Promega, Mannheim, Germany) as follows: 500 μg of protein in 200 μl of 20 mM Tris-HCl, pH 7.5, was adjusted to pH 8 with 3 μl of triethylamine, and 10 μg of trypsin in 10 μl of H2O (Promega sequencing grade modified) was added. The digest was carried out at 37°C for 4 h. The reaction was stopped by heating for 5 min to 100°C. After centrifugation, volumes of 5, 70, and 100 μl were sequentially applied to the HPLC. The peptides generated were separated on a reversed-phase C18 Superpac-Sephasil HPLC column (Amersham Pharmacia Biotech, Uppsala, Sweden).
Cloning, DNA sequencing, and nucleic acid manipulations.Plasmids used for transformation of E. coli strains were pBK-CMV phagemid [fl(−)ori lacZ CMV SV40 poly(A) colE1 ori NeorKanr], pBluescript II KS/SK(±) [fl(±)ori lacZ colE1 oriAmpr], and pGP1-2 [Kanr T7 Gen1]. Oligonucleotides used for amplification of the probes for screening via PCR were as follows (>, forward; <, reverse; all in 5′ to 3′ direction): fubrei p374 (>GCGGCGCTTTCGGCACA), fubrei 375 (<GCGACTTCGCGCATCTTGTT), F2H (>ATGGAYCTSCGSTACTTCATC), F2t43R (<CATSAGGAAYTCSGCCTGCTG), λ15H (>TCGCCGGCGACGACGCCG), and λ15R (<CCGCGCGCTGCGCCGCCG). PCR products used as probes were labeled with [α-32P]dCTP (Amersham). Standard protocols were used for DNA cloning, transformation, amplification, and purification (50). Recombinant plasmids were generated in pBluescript vectors and were maintained in E. coli XL1-blue.T. aromatica DNA has a high GC content (67%). This made sequencing difficult because of the compression phenomenon. Three methods were applied. First, sequencing of plasmid DNA by the Sanger method was performed with 35S-dATP. Second, sequencing of a PCR product with internal Cy5-dATP labeling, 0.5 μg of template/kbp, was performed according to the manufacturer's protocol and analyzed with an ALF express (Pharmacia). Third, cycle sequencing of PCR DNA according to the manufacturer's protocol (Pharmacia) was performed. Sequences were analyzed with an ALF express (Pharmacia). Part of the sequencing was performed with an ALF Automated Sequencer (Pharmacia).
Cloning strategy.Based on the N-terminal amino acid sequences of F2 and of two internal fragments, degenerate oligonucleotides were designed according to the codon usage of T. aromatica. A 500-bp PCR product (F2 forward and F2T6 reverse primers) of the N-terminal sequence of F2 was labeled with [32P]dCTP and used as a probe for screening the λ EMBL3 gene library. One phage hybridized with the probe. From the positive λ clone, two adjacent BamHI fragments of 2.7 and 4.0 kbp and an EcoRI fragment of 5.25 kbp were subcloned in pBluescript and sequenced (for restriction sites, see Fig. 5). The boundary between the BamHI fragments was verified by amplification and sequencing of an overlapping DNA fragment. A double-stranded contiguous sequence of ca. 9 kbp was obtained from the insert of this λ clone. To obtain sequence information of the 5′ and 3′ flanking regions of the first λ clone, gene libraries of T. aromatica were rescreened. A 500-bp probe of the 5′ boundary of the known sequence was amplified and used to screen the λ EMBL3 library again. Two positive clones were obtained whose inserts were subcloned in pBluescript. Plasmids containing parts of the known sequence were identified by PCR assays and used for DNA sequencing. A total of 6.2 kbp of DNA was sequenced from two overlapping subclones containing either a 3.7-kbp PstI fragment or a 9-kbpBamHI fragment. A 600-bp probe of the 3′ boundary of the known sequence was amplified and used to screen a λ ZAP library. One positive phage was detected and converted to a phagemid as recommended by the supplier (Stratagene). It contained an insert of 8 kbp which overlapped with the known sequence and was used to sequence a further 400 bp of the 3′ flanking region.
Southern hybridization.DNA fragments resolved by agarose gel electrophoresis were partially hydrolyzed by gently rinsing in 0.25 M HCl for about 20 min, followed by denaturing in 0.5 M NaOH–1.5 M NaCl for another 20 min. Finally, the gel was neutralized by excessively rinsing it in 1 M Tris-HCl–3 M NaCl, pH 7, three times for 10 min each. Then, the DNA was vacuum blotted onto a nylon/NC membrane with the help of a vacuum gel dryer system (Slab Gel Dryer 2003; Pharmacia LKB). After the blot procedure, the slots of the gel were marked on the membrane and the membrane was allowed to dry at room temperature. Fixation of the DNA on the membrane was achieved by incubating for 2 h at 80°C. In addition, chromosomal DNA was also blotted by capillary blotting, which guaranteed a better transfer of the large fragments (50).
Screening of gene banks of T. aromatica.A λEMBL3 gene bank (Stratagene) and a λZAPExpress gene bank (Stratagene) were prepared and screened according to the manufacturer's protocol.
PCR.PCR conditions were as follows: 100 ng of target DNA, 200 nM concentrations of each primer, 200 μM (each) dATP, dCTP, dTTP, and dGTP, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 1 U of Taq DNA polymerase (Amersham Pharmacia Biotech). The assay was overlaid with 75 μl of paraffin oil. PCR parameters were as follows: 30 s at 95°C, 1 min at 40°C, 2.5 min at 72°C, 30 cycles. For amplification of fragments larger than 3 kbp,Taq DNA polymerase from Qiagen (Hilden, Germany) was used under the following conditions (Taq PCR kit from Qiagen): 100 ng of target DNA, a 200 nM concentration of each primer, 200 μM (each) dATP, dCTP, dTTP, dGTP, (NH4)2SO4, and KCl, 4.5 mM MgCl2, 10 mM Tris-HCl (pH 8.7), 1× Q solution, and 1 U ofTaq DNA polymerase (Qiagen). PCR parameters were as follows: 30 s at 95°C, 1 min at 45°C, 2.5 min at 72°C, 30 cycles.
Expression of the T. aromatica genes.Heterologous expression of the genes was performed as previously described (13) for E. coli K38 containing the pGP1-2 plasmid, which contains the gene for the heat-inducible T7 RNA polymerase. Strain K38 was transformed according to a previously described method (18) with the pBluescript vector containing different parts of the phenol-induced gene cluster of T. aromatica under the control of a T7 promoter.
Computer analysis.The nucleotide and amino acid sequences were analyzed using the PC/gene software package (Genofit) and the Open Reading Frame Finder (ORF Finder;http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Similar sequences were identified using the BLAST search using the TBLASTN algorithm provided by the National Center for Biotechnology Information.
Nucleotide sequence accession number.All sequence data have been deposited in the EMBL nucleotide sequence database under accession no. AJ272115.
Detection of phenol-induced proteins.Growth on phenol and nitrate obviously requires induction of proteins that are required for anaerobic phenol metabolism. This follows from the observation that 4-hydroxybenzoate-grown cells were not able to metabolize phenol immediately, but only after a prolonged adaptation period of several hours. Furthermore, phenol-grown cells contained several protein bands in SDS-PAGE that were missing in 4-hydroxybenzoate-grown cells (not shown). Therefore, the soluble fraction (supernatant obtained after centrifugation at 100,000 × g) and the membrane fraction (pellet obtained after centrifugation at 100,000 × g, not shown) of extracts of cells that were grown anaerobically on phenol and 4-hydroxybenzoate, respectively, were compared in more detail by two-dimensional gel electrophoresis (Fig.2).
The soluble fraction contained up to 11 phenol-induced proteins. Three of them, called F1, F2, and F3, showing apparent molecular masses of about 60, 58, and 67 kDa, respectively, were strongly visible and were stained with similar intensity on SDS-polyacrylamide gels. The pI values were estimated to be 6.5, 7.0, and 5.5, respectively. Two further induced proteins, called F4 and F5, with apparent molecular masses of about 20 kDa and pI values of approximately 6.5 and 8.0, respectively, could be identified. Another two protein spots (no. 10 and 11 in Fig. 2) were only weakly stained and, because of their size (apparently about 14 kDa), were hardly visible. Their pI values were estimated to be 5.5 and 9.0. Up to four additional phenol-induced proteins were not clearly resolved from neighbor proteins; their apparent molecular masses were in the range of 30 to 55 kDa, and their pI values were between 5.8 and 7.0. The membrane fraction did not contain proteins that were strongly phenol induced.
Partial purification of phenol-induced proteins.The high content of phenol-induced proteins encouraged us to purify phenylphosphate carboxylase and CO2 isotope exchange activity, despite its oxygen sensitivity. A chromatographic separation of cell extract on a DEAE-Sepharose fast-flow column is shown in Fig.3A. Both activities coeluted in two fractions, and both contained phenol-induced proteins F1 and F2, although at different ratios. Both activities seemed to require the presence of some amount of the protein F1. Neither activity was measured in a third fraction containing the protein F3. The subfractions of the first two fractions were pooled and tested for both activities. In the first fraction, 35% of the residual activity of the CO2 isotope exchange reaction and 15% of the phenylphosphate carboxylase activity could be detected, and in the second fraction, 20 and 10% of the activities, respectively, were detected. No combination of the three fractions restored full activity.
The two pooled DEAE fractions containing mainly F1 and F2 were chromatographed isocratically on MonoQ HR5/5, and the pooled DEAE fraction containing mainly F3 was chromatographed on MonoQ HR5/5 under refined conditions by using a linear KCl gradient. This resulted in further purification of the three proteins (Fig. 3B), but activity was completely lost. When the three DEAE fractions were applied to blue Sepharose at pH 7.0, F1 to F3 bound to the column and were eluted with 1 M KCl. At pH 7.5, F2 was specifically eluted with 10 mM phenylphosphate (Fig. 3C).
N-terminal sequencing of five phenol-induced proteins (F1 to F5), peptide sequencing of F2, and synthesis of degenerate primers.Three phenol-induced proteins, F1 to F3, were enriched by chromatographic techniques in the enzymatically inactive form, and two other phenol-induced proteins, F4 and F5, were obtained from two-dimensional gels. The material was sufficient for N-terminal sequencing and peptide sequencing of F2. Up to 40 amino acids were determined, with only a few ambiguous amino acid residues. Comparison of the experimentally determined sequences with the sequences deduced from the genes (see below) showed very good correspondence, with only very few mismatches, and most uncertain amino acids were confirmed to be correct (Fig. 4). The results of the N-terminal sequencing were used to design degenerate primers, taking into account the known codon usage of T. aromatica. Amino acid sequences that were used as templates are underlined in Fig. 4.
Screening of a λ EMBL3 and a λ ZAP express gene library.Based on the N-terminal amino acid sequences of F2 and of an internal fragment of F2, degenerate oligonucleotides were designed. The primers used for the generation of labeled probes to screen the libraries are listed in Materials and Methods. A labeled 500-bp PCR product corresponding to the N-terminal sequence of F2 (F2H/F2T43R) was used as a probe for screening a λ EMBL3 gene library. One phage hybridized with the probe. The insert DNA was prepared and subcloned in pBluescript vectors. About 9 kbp of sequence information of both DNA strands was obtained.
To get more sequence information 5′ of the first segment, a new probe was amplified by PCR (fubrei p374 and fubrei 375) and used for gene bank screening. Two positive phages whose DNA was subcloned in pBluescript vectors were obtained. The sequences overlapped with the 5′ sequences of the clone obtained by the first screen.
To obtain more sequence information 3′ of the sequences of the first screen, a λZAPExpress gene library was screened with a 600-bp probe derived from the 3′ end of the sequences (λ15H/λ15R). One positive clone was detected, and the phagemid DNA was prepared. The size of the DNA insert was estimated to be at least 8 kbp by agarose gel electrophoresis. The 5′ sequences of the phagemid insert overlapped with the 3′ sequences of the first screen.
DNA sequencing and identification of the genes coding for putative proteins involved in anaerobic phenol metabolism.Screening of the gene libraries and sequencing of the subcloned DNA resulted in sequence information for 14,272 bp. Eleven open reading frames (ORFs) were detected (Fig. 5). Table1 summarizes the location and orientation of ORFs 1 to 11 and, the number of amino acids encoded by each ORF, as well as the percent similarity and percent identity of the amino acid sequences of ORFs 1 to 11 with known sequences in the databases.
Ten ORFs are transcribed in the same direction. The first six ORFs (orf1 to orf6) are separated by less than 65 bp and amount to 7,210 bp. This cluster of putative genes is followed by a 658-bp noncoding region whose transcript contains two putative hairpin mRNA secondary structures. Another cluster of four putative genes with fewer than 40 bp of intergenic regions amounts to 2,702 bp. Downstream of orf10, 840 bp which did not code for proteins were sequenced. Upstream of orf1 and transcribed in the opposite direction, another putative gene was found, orf11, which was separated by 428 bp from orf1.
Comparison of deduced amino acid sequences of ORFs 1 to 11 with the experimentally determined N-terminal and internal amino acid sequences of phenol-induced proteins revealed that the following genes code for known proteins: orf1 for F3, orf4 for F2,orf5 for F4, orf6 for F1, and orf8 for F5 (Fig. 4). The predicted molecular masses agree reasonably well with the experimentally determined masses (Table 1). The similarities of the gene products possibly involved in anaerobic phenol metabolism with other proteins in the databases, comparison of deduced and experimentally determined sequences, and some extrapolated parameters of the proteins compared to those of the phenol-induced protein spots are summarized in Table 1.
Protein F3 (encoded by orf1) and the orf2 gene product show similarity to parts of phosphoenolpyruvate synthase (PEP synthase) of E. coli. The reaction catalyzed by PEP synthase is shown in Fig. 5. The C-terminal half of F3 is similar to the central part of PEP synthases and contains a highly conserved histidine (amino acid 569) which is known to become phosphorylated in the PEP synthase reaction (histidine 421 in the central part of PEP synthase of E. coli). The orf2 gene product shows similarity to the N-terminal domain of PEP synthase. In E. coli, this domain is probably involved in ATP binding. We propose that ORFs 1 and 2 code for two proteins belonging to the phenylphosphate synthase system, whereby phenol ultimately becomes phosphorylated. This proposal seems plausible, considering the structural similarity between the enol form of pyruvate and phenol, respectively.
ORFs 4, 6, and 7 code for F2, F1, and a not-yet-identified protein, respectively. Sequence alignment shows that each of these gene products is overall similar to the ubiD gene product of E. coli, 3-octaprenyl-4-hydroxybenzoate carboxy lyase. This enzyme is involved in the biosynthesis of ubiquinone; the reaction catalyzed is shown in Fig. 4. orf8 codes for F5 and shows high similarity to ubiX, a gene which codes for an isoenzyme of 3-octaprenyl-4-hydroxybenzoate carboxy lyase (UbiD). UbiX and F5 were also similar to phenylacrylic acid decarboxylase fromSaccharomyces cerevisiae. An alignment of a conserved amino acid sequence motif showed that all three proteins have a common E-X-P motif. We propose that the proteins encoded by orf4 andorf6 to orf8 are involved in the carboxylation of phenylphosphate or similar phenolic compounds; this proposal is based on the similarity of the known reactions of the similar proteins, decarboxylation of 3-octaprenyl-4-hydroxybenzoate or phenylacrylic acid, to the phenylphosphate carboxylase reaction.
The function of four gene products is enigmatic. Orf3 showed similarity to a putative inosine-5′-monophosphate dehydrogenase ofMethanobacterium thermautotrophicum. Orf5 (F4) and Orf9 showed similarities to hypothetical and conserved proteins whose functions are unknown. Orf10 has high similarity to the putative Mutator T protein (MutT) of Methanococcus jannaschii.
orf11 codes for a protein that shows strong similarity to the DmpR gene regulator of Pseudomonas putida, which regulates expression of genes involved in aerobic metabolism of phenolic compounds (41, 57, 58). Therefore, we assume that Orf11 is the regulator of anaerobic phenol metabolism.
Expression of F1 to F5 proteins in E. coli.Five of the 11 ORFs were identified as genes coding for F1 to F5. However, there was no proof that the other putative genes were expressed as well. Therefore, several DNA fragments were ligated into pBluescript KS under the control of the heat-inducible T7 polymerase promoter and transformed into E. coli K38, and expression was induced. The following DNA fragments were used (compare with Fig. 5): 3.7-kbpPstI fragment containing orf1 (which codes for the F3 protein) and orf2; a 2.7-kbp BamHI fragment containing orf3 and orf4 (which codes for F2); a 4.0-kbp BamHI fragment containing orf5(which codes for F4), orf6 (which codes for F1), andorf7; and a 5.25-kbp EcoRI fragment containing the 3′ end of orf6, the intergenic region betweenorf6 and orf7, and the complete sequences oforf7, orf8 (coding for F5), orf9, andorf10. The proteins were separated by SDS-PAGE and localized by autoradiography (Fig. 6).
orf1 and orf2 were obviously transcribed by the T7 polymerase. On the SDS-polyacrylamide gel, the resulting proteins F3 and Orf2 migrated at apparent molecular masses of 67 and 41 kDa, respectively, which agreed reasonably well with the predicted molecular masses. orf3 and orf4 were also both transcribed by the T7 polymerase. F2 (encoded by orf4) was strongly induced and had an apparent molecular mass of approximately 58 kDa. This was slightly larger than expected according to the nucleotide sequence (52 kDa), but agreed with the apparent molecular mass of F2 prepared from T. aromatica. In contrast, expression of orf3 was not strongly induced and ORF3 was hardly visible on the autoradiogram. However, the apparent molecular mass of 24 kDa agreed with the predicted molecular mass. The experiment with the 4.0-kbp BamHI fragment containingorf5 to orf7 showed the expression of only two proteins. Their apparent sizes of approximately 19 and 60 kDa corresponded to those of the phenol-induced proteins F4 and F1, respectively. As in cell extract, F1 ran with a molecular mass larger than that expected from the nucleotide sequence (54 kDa). No expression was observed for orf7, which would have resulted in a 39-kDa protein. The putative secondary mRNA structures between orf6and orf7 may have prevented translation of orf7.
Using the 5.25-kbp EcoRI fragment, none of the ORFs fromorf7 to orf10 were expressed. This may be due to the fact that the secondary structures downstream of orf6may have prevented transcription of orf7 toorf10. Despite several attempts, we did not succeed in subcloning the DNA sequence of orf7 to orf10without the putative secondary structures and thus could not express these genes. However, expression of orf8 is clearly induced in T. aromatica in the presence of phenol, as F5 is one of the phenol-induced proteins. This suggests that the second gene cluster consisting of orf7 to orf10 indeed codes for structural phenol-induced proteins.
Genomic Southern hybridization of chromosomal DNA ofT. aromatica and other bacterial strains.Genetic work with T. aromatica is troublesome because of poor growth on agar plates. To obtain data on a possible correlation of the phenol-induced genes and their function in anaerobic phenol metabolism, a 32P-labeled 750-bp PCR product (F2H/F2t43R) that corresponded to the 5′ part of the gene coding for F2 (orf4) was used as a probe for Southern hybridization. The probe should only hybridize with the DNA of those strains that were closely related to T. aromatica and able to metabolize phenol. The 16S rRNA of Thauera sp. strain S100 is 100% identical and that of strain SP is more than 98.9% identical to that of T. aromatica type strain K172 (E. Stackebrandt and G. Fuchs, unpublished results). The phenol-induced genes should not be present in strains that could not have been shown to grow on phenol, like Thauera sp. strain LG356 (which has 100% 16S rRNA sequence identity with strain SP) and Azoarcus evansii type strain KB740.
The result of the genomic Southern experiment was surprising (Fig. 7). DNA of all organisms hybridized with the labeled probe. Moreover, the sizes of the hybridizing fragments seemed to be very similar in most of the organisms. In all organisms, the probe hybridized with the undigested DNA and/or fragments larger than 15 kbp. The experimentally determined sizes of hybridizing fragments of the digested chromosomal DNA of T. aromatica were consistent with the expected sizes. Only after EcoRI digestion did the probe hybridize with two fragments instead of one, as expected. The strongly hybridizing fragment of about 5 kbp corresponded to the expected one, and the 7-kbp fragment hybridized only weakly, which may be due to unspecific binding of the probe. T. aromatica type strain K172 and strain S100 showed the same sizes and patterns of hybridizing fragments; strain SP differed slightly in its hybridization pattern. Hybridization of strain SP DNA was expected, because this organism is also able to grow on phenol. Slight differences in the hybridization patterns may be explained with slight differences in the genome sequences of the three organisms.
Unexpectedly, chromosomal DNA of strain LG356 also hybridized. Moreover, the hybridization pattern of strains LG356 and SP were quite different, although these organisms have 100% identical 16S rRNA sequences. Even A. evansii DNA hybridized; however, the hybridization pattern was totally different from those of theThauera strains, which may be due to major differences in the genome sequences of Thauera andAzoarcus species. The results of the genomic Southern experiment showed that there is no simple correlation between the presence of the F2 gene and reported growth on phenol.
Genes and gene organization.Eleven ORFs were cloned and sequenced, and up to 11 phenol-induced proteins were identified by two-dimensional gel electrophoresis. Table 1 summarizes the deduced sizes and isoelectric points of these gene products. Five of the genes coded for the identified phenol-induced proteins F1 to F5. Comparison of the experimentally determined and deduced values showed that the molecular masses agreed reasonably well with the prediction.
However, the deduced pI values (as determined by diverse computer programs) did not fit exactly to those estimated experimentally. In general, the deduced pI values were 0.5 to 1.0 U lower than the experimentally determined values. This discrepancy may be due to inaccurate determination of the pI values, which were extrapolated assuming an immobilized linear pH gradient of 3 to 10. Six phenol-induced protein spots could not be directly attributed to the corresponding genes by purification or extraction mainly due to the poor separation of the proteins. Although the estimated molecular masses and pI values fit reasonably well with those predicted from ORFs 2, 3, 7, and 9 to 11, the identification is very preliminary and may be incorrect in some cases. For example, protein spot no. 7 may essentially fit to the gene product of orf11, which encodes the putative regulator protein of phenol metabolism, but it is unlikely that a regulator protein is induced in such amounts.
Ten of the 11 genes are transcribed in the same direction and are organized in two gene clusters. To underline the correlation between the genes and the induced proteins, the genes were cloned under the control of the T7 polymerase promoter and were expressed in E. coli. All genes of the first gene cluster (orf1 toorf6) were transcribed and expressed. Probably due to the possible RNA hairpin secondary structures between the two clusters, we did not observe expression of the second cluster. However, the fact that the induced protein F5 (which is encoded by orf8 of the second gene cluster) is expressed in the presence of phenol in T. aromatica leaves no doubt that the second gene cluster is expressed as well.
Possible functional assignment of the genes.The two essential steps in phenol metabolism are the phosphorylation of phenol to phenylphosphate and the subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate. Two gene products showed similarity to PEP synthase and four gene products showed similarity to 3-octaprenyl-4-hydroxybenzoate carboxy lyase, respectively, both ofE. coli. Because of the similar chemistry involved in the respective reactions, we suggest that these genes are coding for phenol-phosphorylating and phenylphosphate-carboxylating enzymes, respectively.
Orf1 and Orf2, homologues of PEP synthase.Two ORFs,orf1 (coding for F3) and orf2, code for proteins of 70.2 and 40.4 kDa with similarity to the central and the N-terminal parts, respectively, of PEP synthase. Thus, the gene products appear to belong to the family of the structurally and functionally related PEP-utilizing enzymes, which comprises three groups of proteins (19, 20): (i) PEP synthase (or pyruvate:water dikinase [EC 22.214.171.124]), (ii) pyruvate:orthophosphate dikinase (EC18.104.22.168), and (iii) PEP-protein phosphotransferase (EC 184.108.40.206), which is enzyme I of the PEP-dependent phosphotransferase system. Two sequence signatures are shared by all these proteins and by no other proteins in the current protein databases. The first sequence signature (PROSITE, accession no. PS00370) contains an active-site histidine residue that mediates phosphoryl transfer. The second sequence signature (PROSITE, accession no. PS00742) is a conserved region, the exact role of which is unknown. The functional similarity of the enzymes lies in the transfer of a phosphoryl group via a phospho-histidine intermediate and in the utilization of PEP or pyruvate as a substrate; the corresponding active sites are located on independently folded domains. The E. coli PEP synthase (175 kDa) is a dissociable dimer of identical subunits (87.4 kDa), and histidine residue 442 has been identified as the phosphorylation site (6, 42). In the thermophilic archaebacteriumStaphylothermus marinus, PEP synthase is a homomultimeric enzyme complex whose subunits have a similar structure as the subunits of the homodimeric E. coli enzyme (20).
T. aromatica Orf1 (protein F3) shows 54% similarity to the central part of PEP synthase of E. coli and carries the conserved sequence motif of PEP-utilizing enzymes (PROSITE, accession no. PS00370) that contains the active-site histidine residue. The PEP synthase of B. subtilis contains the (putative) active-site histidine at the C-terminal part of the protein, as does F3. The N-terminal part of F3 shows no similarity to any region of the known PEP synthase sequences or to other sequences in the databases. Orf2 showed 60% similarity to the N-terminal part of PEP synthase ofE. coli. We believe that the gene products oforf1 and orf2 together form an active enzyme complex responsible for phosphorylation of phenol. The phosphoryl donor for this reaction remains unknown, although a number of potential cosubstrates have been tested (33).
Orf4, -6, -7, and -8, homologues of 3-octaprenyl-4-hydroxybenzoate carboxy lyase.The gene products of orf4 (F2),orf6 (F1), and orf7 showed 47 to 63.8% similarity to UbiD (55.2 kDa) (Table 1 and Fig.8), and the product of orf8(F5) showed 86.8% similarity to UbiX (20.7 kDa). UbiD catalyzes the third reaction in ubiquinone biosynthesis, namely, the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to 3-octaprenylphenol (Fig. 5). The gene ubiX encodes an isoenzyme of UbiD that catalyzes the same reaction. So far, only a few biochemical data are available on both enzymes. The holoenzyme UbiD of E. colihas a molecular mass of 340 kDa, and its activity is dependent on the presence of Mn2+ and an unidentified, heat-stable factor with a mass of <10 kDa (36). Moreover, the activity was increased by adding membrane preparations or phospholipids, indicating that the enzyme normally functions in association with the membrane.
We assume that some or all of those four genes code for enzymes involved in carboxylation of phenylphosphate to 4-hydroxybenzoate because decarboxylation of 3-octaprenyl-4-hydroxybenzoate to 3-octaprenyl-phenol can be compared to the reverse reaction of phenylphosphate carboxylase. Interestingly, the four genes of T. aromatica are distributed on two different clusters of genes. F1 and F2 are encoded in one cluster together with the genes possibly encoding the phenol-phosphorylating enzymes (F3 and Orf2), whereas Orf7 and F5 are encoded in a second gene cluster where no phosphorylating enzymes seem to be encoded (Fig. 5). Both clusters are expressed in the presence of the substrate phenol, as gene products of both were identified as strongly phenol-induced proteins.
Like the E. coli enzyme UbiD, the phenylphosphate carboxylase and isotope exchange activities were both dependent on Mn2+. Phenylphosphate carboxylase of T. aromatica could be purified by gel filtration chromatography with little loss of activity. This indicated that, unlike 3-octaprenyl-4-hydroxybenzoate decarboxylase, no low-molecular-weight factor is required for enzyme activity.
There are recent reports that enzymes involved in anaerobic degradation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) (17), 3,4-dihydroxybenzoic acid (28a) and 4-hydroxybenzoic acid (30) show similarity to the UbiD/UbiX system of E. coli, and this similarity is extended to the phenol system inT. aromatica (Fig. 8). The vdcBCD gene cluster inStreptomyces sp. strain D7 codes for proteins specifically involved in vanillic acid decarboxylation to 2-methoxyphenol (guaiacol). VdcC is similar to UbiD in E. coli and to Orf4, -6, and -7 in T. aromatica. VdcB is similar to UbiX and phenylacrylic acid decarboxylase of S. cerevisiae and to Orf8 of T. aromatica. VdcD shows similarity to an unknown protein of Sphingomonas and YclD (function not known) ofBacillus subtilis; in T. aromatica, no gene product similar to VdcD was found. Wiegel and coworkers identified the gene coding for the (reversible) 4-hydroxybenzoate decarboxylase (54.4 kDa) in Clostridium hydroxybenzoicum (30). This enzyme was similar to the three UbiD-like gene products ofT. aromatica and to UbiD from E. coli (53.4 kDa) (17). Two other ORFs, orf2 andorf3, downstream of the gene for the 4-hydroxybenzoate decarboxylase were similar to the vdcDB genes ofStreptomyces sp.; however, orf3 seems not to be complete. A sequence motif with highly conserved amino acids was also found in three decarboxylases from Lactobacillus plantarum (15), B. subtilis (16), and Bacillus pumilus (66). In all listed protein sequences, an E-X-P motif is conserved and may therefore play a role in substrate binding or catalysis of (hydroxy-) arylic acid decarboxylation or carboxylation of the corresponding phenolic compounds.
Obviously, in the Clostridium, Streptomyces, andThauera species, the genes coding for UbiD-like decarboxylases are arranged in one or two clusters together withubiX homologues. In E. coli, however,ubiD and ubiX are widely separated on the chromosome. There remain open questions about T. aromatica: why there are four genes with similarity to (reversible) decarboxylases organized in two clusters and why both clusters are transcribed in the presence of phenol. Perhaps phenol acts as a gratuitous inducer of the second gene cluster that may play a role in the metabolism of other phenolic compounds (for a classical example of gratuitous induction, see reference 5).
Orf10, homologue of a mutator T protein.The gene product oforf10 showed approximately 60% similarity to the mutator protein MutT of M. jannaschii (Table 1 and Fig.9). Mutator proteins are enzymes involved in DNA metabolism which are widespread in prokaryotes and eukaryotes. The 15 members of the MutT family of proteins are believed to act in nucleoside phosphate metabolic pathways. 8-Oxo-7,8-dGTP (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism and causes AT-to-GC transversion mutations when it is incorporated into DNA (7, 22,53). The MutT protein of E. coli and related mammalian enzymes specifically hydrolyze 8-oxo-dGTP to 8-oxo-dGMP and PPi, thereby preventing the occurrence of transversion mutation (38, 43). Members of this class of enzymes require two divalent cations per active site for activity, one coordinated by the enzyme and the other by the enzyme-bound nucleoside triphosphate (39). The substrate of proteins of the MutT family varies from 8-oxo-dGTP and diphosphoinositol polyphosphate (49) to adenosine(5′)triphospho(5′)adenosine, ADP-ribose, and NADH (43). One can only speculate which role Orf10 plays in the anaerobic metabolism of phenol in T. aromatica, e.g., in phenol activation.
Orf11, a putative regulator protein.One ORF located 5′ of the two gene clusters and transcribed in the opposite direction codes for a protein with 72% similarity to the regulator protein DmpR of P. putida CF600, which regulates aerobic (dimethyl)phenol metabolism (Fig. 10). DmpR is a member of the NtrC family of transcriptional activators and activates transcription of the dmp operon, which contains all 15 structural genes needed for the degradation of (dimethyl)phenol, in response to aromatic effector compounds (58). These effectors are phenol, o-cresol, m-cresol,p-cresol, and 3,4-dimethylphenol, and also ethylphenol, catechol, and 3- and 4-methylcatechol. MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250, also belongs to the NtrC family (51). Like all regulators of the NtrC family, Orf11 contains several conserved protein domains (40). The highly conserved central (C) domain probably binds and hydrolyzes ATP and promotes open-complex formation with RNA polymerase. The C-terminal (D) domain is expected to bind to the corresponding target DNA sequence ("enhancer"). The highly variable sensory domain is joined to the C domain by short interdomain linker B. NtrC-like regulators sense their respective signals by direct binding of the effector to the A domain (56). Effector binding to the A domain then leads to release of the C domain (21, 56), thus permitting the binding of ATP followed by an ATP-driven cycle of multimerization and demultimerization of the regulator at the enhancer sites (45). This supports open-complex formation of ς54-containing RNA polymerase with the promoter (65). We propose that Orf11 controls the expression of the first or both gene clusters by a similar mechanism and acts as a transcriptional activator, when phenol or a similar compound plus ATP is present.
In P. putida strains P35X and CF600, the difference in the spectrum of aromatic growth substrates seems to be due to the different specificity of effector binding to the regulator protein rather than due to the different substrate specificity of the catabolic enzymes (58). However, this hypothesis has yet to be proven. All DmpR-like effector proteins (>20 known) mediate their action through a single binding site within the A domain (44). Depending on the effector, this results in differential effects on ATPase activity of the C domain and its productive coupling to the transcriptional apparatus.
Promoter region.Transcriptional activators of the NtrC-like family usually regulate transcription from ς54-dependent promoters, as was shown in P. putida for the positive regulator DmpR (57). The structural dmpgenes of the aerobic phenol degradation pathway are clustered in a single operon that lies downstream of a nif/ntr-like promoter sequence. Upon inspection of the first gene cluster inT. aromatica, a similar nif/ntr-like promoter sequence was indeed found 5′ of orf1 (Fig.11). Primer extension analysis has to be performed to show whether transcription really starts at the expected site. So far, nothing is known about promoter sequences inT. aromatica, so the −24 and −12 consensus sequences are speculative. Upstream of the second gene cluster, nonif/ntr-like promoter sequences were found. All ς54-dependent promoters analyzed so far are regulated by positive transcriptional activators that usually bind to specific DNA sequences ("enhancer sequences," mostly inverted repeats) located relatively far upstream of the promoter (100 to 200 bp) (31).
Regulation of transcription.We suggest that phenol acts as an effector of a transcriptional activator protein, as there is anntr-like regulator gene present upstream of the two gene clusters and a nif/ntr-like promoter sequence upstream of the first cluster of genes. In addition to the regulation by phenol, there may be additional catabolite repression, but no experimental data are available. As T. aromatica could not be shown to metabolize phenol in the presence of oxygen, it has to be shown whether O2 represses the induction of transcription of the genes involved in phenol metabolism.
Presence of genes possibly involved in anaerobic phenol metabolism in different strains of T. aromatica and variousAzoarcus species.So far, it has not been possible to unequivocally correlate genes coding for phenol-induced proteins and their function. Southern hybridization of a labeled probe oforf4 (coding for F2) with chromosomal DNA of different strains seemed to indicate that all testedThauera and Azoarcus strains have a gene similar to orf4, whether or not they have been shown to metabolize phenol. One plausible assumption is that all strains are able to utilize phenol, but so far the appropriate growth conditions have not been applied. Alternatively, rearrangement and deletion may be the reason why some Thauera and Azoarcus strains are able to metabolize phenol and some are not.
Related carboxylases acting on aromatic compounds.Carboxylation of the aromatic ring is widespread among anaerobic microorganisms, but the respective enzymes have not been studied yet. The initial reaction in anaerobic o-cresol (2-methylphenol) metabolism in denitrifying Azoarcus sp. strain U 120 (formerly a Paracoccus strain) (48) is carboxylation in para-position to 3-methyl-4-hydroxybenzoate. Sulfate-reducing and methanogenic bacteria have been reported to carboxylate m-cresol (3-methylphenol) in para-position to the phenolic hydroxy group to 2-methyl-4-hydroxybenzoate (46, 47). ADesulfococcus sp. carboxylated hydroquinone (1,4-dihydroxybenzene) to gentisate (2,5-dihydroxybenzoate) (27). Some sulfate-reducing bacteria carboxylate catechol (1,2-dihydroxybenzene) to protocatechuate (3,4-dihydroxybenzoate), whereas phenol is not metabolized (26). Aniline is carboxylated to 4-aminobenzoate in Desulfobacterium anilini (52). An aerobicortho-carboxylation of aniline to 2-aminobenzoic acid was reported for Rhodococcus erythropolis(2). In sulfate-reducing consortia, the initial reaction in the anaerobic metabolism of naphthalene is carboxylation specifically at C2 to form 2-naphthalene carboxylic acid; phenanthrene carboxylation leads to phenanthrene carboxylic acid, and the carboxylation site is unknown (69). Growth on all of these substrates is likely to be CO2 dependent.
This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and cooperation with E. I. DuPont de Nemours.
We thank Hans Heider for helpful suggestions and critical reading of the manuscript. Thanks are also due to Hermann Schägger, Frankfurt, Germany, for amino acid sequencing of some of the proteins, to Juliane Alt-Mörbe, DNA-Analytik Freiburg, for DNA sequencing, to Christa Ebenau-Jehle and Victor Gadon for help in growing cells, and to Sima Sariaslani, Heinz Hefter, and coworkers from DuPont for their support.
- Received 28 March 2000.
- Accepted 31 July 2000.
- Copyright © 2000 American Society for Microbiology


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This is a better day. Yesterday turned into a kind of disaster as last night I heard from our main speaker for the coming weekend, Dr. Stephan Patterson, that he felt a $750 honorarium was too little for the weekend. He is coming in later Friday evening after the event is finished on that day and leaving during dinner on Saturday afternoon. I don't consider that a weekend. It's just one full day. Plus we are paying for his flight, his meals and his motel. I could only apologize since the man who obtained him had not told him what we were giving him for the honorarium. That man was in charge of obtaining a speaker. I thought he would talk to him about the entire deal. Evidently not. When I discussed it with the speaker, I learned he was expecting a lot more. There's nothing I could do about it. We were stretching ourselves as it was. We were told he was coming as a favor to Don, who knew him as a friend. I don't need friends like that personally.
Anyhow, what's done is done.
Today I learned that the church that is hosting our Independence, Kansas, Baccalaureate service has their all purpose and basement rooms all torn up building a day care center and they will not be finished for a reception after the service on May 13th. They want us to forego the reception. I hate that but I don't have an option. I won't worry about that until after this weekend. One worry at a time is enough.
I went to Independence this morning and got my hair done and had breakfast with Juanita. Then I went to Wal Mart and bought a print cartridge for my printer and came back home.
We will pick up a bucket of chicken tonight and go out to John and Leslie's new house to see how it looks with all the furniture in place.
Wednesday, April 15, 2009
Refreshing Wednesday
Posted by Margie's Musings at 11:16 AM
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You Are In Safe Hands
Check out our Website: UAC Local Movers serving Davie FL
At UAC Apartment Movers, they know that each change of address is unlike.
Their website UACMovingCompanyFL.com is contributed to present our users with access to top quality moving specialist. Nationwide Movers have earned a reputation for over 15 years through our pleased customers. Moving Company FL focuses in local and long distance relocating of house, office and piano relocations. Storage is also available for one piece or your complete house. Our workforce includes certified as well as skilled labor that is coached to handle all the goods, commercial or personal, with greatest care. Whether you need a local moving company or just full service and outstanding service. They make moving easy and stress free at a inexpensive rate.
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For Hassle Free Move, And Complete Peace of Mind, Look No Further, Let The Professionals Do The Job, Call (954) 780-3048 ? Now, We Will Take All The Stress Out Of Moving, The Job Is Not Finished Until You Are Happy


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The Fashion Industries Favorite Business Network ...
Is your wedding knocking the door? Are you worried about the bridal dress that you will wear on your special day? Are you too much tensed about the fittings and the hem design? Are you still thinking what type of accessories you will pair with your dress? Have you thought about the shoes and clutch? Have you decided the color and fabric? Okay, it is not the end. There are millions of questions th…
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To make the filing process quicker and easier, I recommend you gather your information before you start. This will make filing the Form 2290 online even simpler than it already is.
Tag: suspended
2290 Instructions Simplified
The IRS hasn't been known for making anything simple. Like, at all. Ever. In all their existence. When it comes to the Form 2290 instructions, it's okay that the IRS is not simple because you have me to make it simple for you.
My goal of this article is to take IRS jibberish that seems to be written in this fashion-
IRS: The motor vehicle parked adjacent to the standing infrastructure on the northeast corner of the 1600 block is painted by an irrelevant painter *insert irrelevant name here* to whom chose the shade of red to coat the exterior portion of the motor vehicle to give it a distinct, vibrant shade according to the 11th jurisdiction of the United States.


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Quick and Best Tour around Urumqi Turpan
Travel to
Urumqi Turpan
- Information
- Tour Plan
- Location
- Gallery
- Reviews
- Similar Tours
What's included
- Accommodation-4 star Hotel
- Entrance tickets to monuments and tour spots
- Professional English speaking tour guide
- Transportation with air-conditioned car/bus
- Drinks & Meal on Tour
- Hotel in Urumqi
- Services not specifically stated in the itinerary
- Tips to guide and driver
- Day1 Urumqi Tour to Heavenly lake
- Day2 Urumqi to Turpan,190km
- Day3 Turpan to Urumqi
Heavenly lake
Heavenly lake is about 110 km away from Urumqi, located on the top of Tianshan mountain. The surface of the lake is 1910 meters above sea level, just one kilometer higher than Urumqi. It takes about 2-3 hours to reach to the lake since we have to take the shuttle bus for the last 30 km of the trip. You can have a hiking around the lake for hours, blue sky, natural forests and grassland view will relax your mind.
Sultan minaret, Karez underground irrigation system, Jiaohe ruined city
In the morning, we drive to Turpan city, it will take us about 3.5 hours by car, but you will enjoy the landscapes and biggest wind farm in China on the way, you can see semi desert, lakes, grassland, grassless mountain, sand and stone desert.
Turpan is about 1 km lower than Urumqi by altitude, when you arrive to Turpan, you will have a lunch at local restaurant, enjoy from the Uighur cuisine. then will visit Sultan minaret (the only highest minaret in Xinjiang, was build 18th century), which very close to the Turpan city. Then you will go to the Karez irrigation (an underground water cannel, was build 2000 years ago) and Jiaohe Ancient city, Jiao was added to UNESCO 2014, is a best preserved soil structured city ruins in China. After dinner, you can walk around the food streets of Turpan city. You will find that it is very different from Urumqi and relaxing.
Flaming mountains, Bezeklik Thousand Buddha Caves and Tuyuk village
Today, we will have a unique Turpan tour, after breakfast, we will drive to east and see the marvelous landscape Flaming mountains, then go to the Bezeklik Thousand Buddha Caves, which was Buddhist temple with ancient paintings, after that, we drive east again to visit Tuyuk village and have a village local lunch. The environment is not luxury but the food is quite tasty and experience with locals are good.
After visiting there, you will drive back to Urumqi. It is about 250 kilometers from the village. Finish the tour


text:
Item Information
Title: Yasuo Kuniyoshi
Date: ca. 1945
Physical Details: 1 photographic print : b&w ; 36 x 28 cm.
Description: Headshot portrait of Yasuo Kuniyoshi.
Creator: Valente, Alfredo, 1899-1973, photographer
Forms part of: Downtown Gallery records, 1824-1974, bulk 1926-1969
Rights Statement: Current copyright status is undetermined
Citation: Yasuo Kuniyoshi, ca. 1945 / Alfredo Valente, photographer. Downtown Gallery records, 1824-1974, bulk 1926-1969. Archives of American Art, Smithsonian Institution.
Digital ID: 3984


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It's raining light! To be precise, a beautiful, delicate shower of light that hangs from the ceiling like a soft cloud.
This is the I.Rain, a wonderfully inspiring light designed by Thierry Gaugain. The I.Rain takes the form of dozens of suspended, pendant lights illuminated with organic light-emitting diodes. The flat OLED screens give the impression of rain drops flattening out as they hit an invisible surface. This is one rain shower we are happy to stay under all day long.


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Don't Waste Your Christmas
We love this video and the idea from Advent Conspiracy. If you're looking for a creative way to invest eternally this Christmas, consider giving gifts from our SOS Christmas Catalog. Whether you give to SOS or any other wonderful cause, this is a great time of year to show that you value Jesus more than anything because of the great ways that He has shown His love to us!


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by Alex Gwyther
22nd & 23rd July
John Thaw Studio
96% of all male rape cases go unreported.
This is Jack's explosive story as he steps into the absurd world of modern masculinity, masks his trauma and reinvents himself as a 'real man'. How far will Jack go to fit in, whilst hoping his past never catches up with him?
Written by and starring Alex Gwyther (Most Promising New Playwright shortlist - Off West End Awards), Ripped exposes this national crisis and the pressures put on young men to live up to outdated ideals. Directed by Max Lindsay.
Ripped will preview at The Actors Centre prior to premiering at the Edinburgh Festival Fringe with Underbelly this summer.
"Gwyther is a terrific storyteller" - WhatsOnStage
"A wonderful actor and writer" - Times Square Chronicles


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Self-help books are my guilty pleasure
You have probably spotted people like me, on my own on the subway. At first glance, I don't look like some desperate self-diagnosed loser, but every few minutes when I come across a particularly strong passage, I will face-and-neck-blush with shame. So I hunch over my e-reader a little more; I'm reading something totally cringe-worthy, and I am desperate for you not to look over my shoulder.
I have a long suffering, not ironic love for self-help. From pamphlets by swamis to sassy relationship books called things like It's Called A Breakup Because It's Broken, when a crisis strikes, my instinct is to curl up with a book. Because if you're sniveling in bed waiting for your ex to call, you need a total stranger to quote cute aphorisms at you—like "even with all the mayonnaise in the world, you can't make chicken salad out of chicken s–t."
Some of them have been helpful. When reading The Ethical Slut the other day, I balked at the idea of attending a sex party. But the authors wrote brilliantly about the crazy emotions that accompany adventurous love lives. "Jealousy is the mask worn by the your most difficult inner conflict" practically jumped off the page at me, and was not a lesson that I had to wife-swap to understand.
Other self-help books I read have been a bit more stupid. When iconic magazine editor Helen Gurley Brown died, I mourned her by diving into her retro handbook, Sex and the Single Girl. None of the advice made any sense, but damn, if that tatty old hardcover wasn't worth its weight in shipping fees. Apparently, "one of the paramount reasons for staying attractive is to have somebody to go to bed with," and "money, if it does not bring you happiness, will at least help you be miserable in comfort."
But hey. It made me laugh.
The worst a self-help book can do is remind you that you are already an expert in a few matters of the heart. I willingly admit that the only book I've ever trashed was the The Seven Levels of Intimacy. Being told "we can never get enough of what we don't really need" sounded less like a Pinterest quote and more like a reminder to stop buying vanilla lattes.
OK: I admit these books are not right for everyone. Most people (including me, sometimes) suspect self-help is written for the weak, the desperate, and the gullible. But much as I'm embarrassed by my self-help habit, I still love getting to the final page of a book and realizing I'm no longer intimidated by my problems.
Who knows—maybe the next one will help me get over my genre shame.
Ellie Broughton is a 28-year-old journalist and editor from London, UK. She loves writing about books and also likes making readers laugh. She tweets at @___ellie.
(Image via Stephen Kelleher.)


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OCEAN CITY, MD 21842MLS Number: MDWO102044
Beds: 3
Baths: 2 Full / 1 Half
View the Virtual Tour
Beds: 3
Baths: 2 Full / 1 Half
View the Virtual Tour
Request Information
DescriptionWhen only the best will do! Aronimink #301 is arguably one of the nicest waterfront condos in all of Ocean City that conveys with two slips, one being a 50~ deep-water slip and the second a 25~ slip with lift. Its top floor, end unit location offers the best placement in the building with southern exposure and great views of the open bay, harbor, and marina. The condo presents a unique and well-thought-out floor plan with high-end appointments and features throughout. The large living room boasts cathedral ceilings, gas fireplace and lots of windows for great light and views. The gourmet kitchen offers custom cabinets, granite tops and stainless-steel appliances. A welcoming dining area adjoins the kitchen and living room to round out the living space. The split bedroom configuration has a large, waterfront master suite with master bathroom and two additional, generously sized guest bedrooms with their own full bathroom and a half bathroom. Interior appointments include Brazilian hardwood and tile floors, GE profile appliances, tray ceilings, crown moldings, granite tops, beautiful tile work and much more. For outdoor living there are two decks with electric awnings that are perfect for relaxing after a long day on the water or at the beach. Aronimink is a small six-unit building that offers a secure lobby, elevator and privacy at the end of 13th St. This is a special property for the discerning buyer that wants the ultimate in waterfront living.
AmenitiesElevator,Extra Storage,Other
General DetailsBedrooms: 3
Bathrooms, Full: 2
Bathrooms, Half: 1
Lot Area: 0.00
County: WORCESTER
Year Built: 2007
Sub-Division: NONE AVAILABLE
Area: 1872
Security: Main Entrance Lock
Sewer: Public Sewer
Community InformationZoning: CM
Exterior FeaturesDock: 2
Foundation: Pilings
Garage(s): N
Pool: No Pool
Roof: Metal
Style: Coastal
Exterior Features: Outside Shower,Secure Storage,Other
Waterfront: Y
Waterview: Y
Interior FeaturesAppliances: Built-In Microwave,Cooktop - Down Draft,Dishwasher,Disposal,Dryer,Oven - Wall,Refrigerator,Stainless Steel Appliances,Washer,Water Heater
Basement: N
Flooring: Ceramic Tile,Hardwood
Heating: Heat Pump(s)
Cooling: Y
Fireplace: N
Furnished: No
Water: Public
Interior Features: Ceiling Fan(s),Crown Moldings,Dining Area,Entry Level Bedroom,Kitchen - Gourmet,Master Bath(s),Walk-in Closet(s),Other
School InformationSchool District: WORCESTER COUNTY PUBLIC SCHOOLS
Fees & TaxesCity Taxes: $2,771
Condo Fee: $7,500
County Taxes: $4,970


text:
Martyn The Hat
Martyn is a Presenter at Hot Radio.
Can you help update this entry?
Do you know Martyn's Twitter name? Please add it to our directory.
This is a user-edited website.
Thank you to Glen M for their edits on this page. Edit this page yourself. See the changelog.


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Newest front in measles fight: camp
Several counties require campers to be vaccinated
The battle to contain the worst U.S. measles outbreak in 27 years has a new front: summer camp.
Vaccinations have been made mandatory this summer for campers and staff in several counties north of New York City that annually fill up with kids from the Orthodox Jewish communities that have been hit hardest by measles.
Ulster County took the extra step of mandating the measles vaccine or proof of immunity at all day camps and overnight camps, becoming the latest county in the area to issue immunization requirements. Rockland County announced a similar order this month, following mandates from Sullivan and Orange counties.
"We have to make sure our t's are crossed and our i's are dotted in making sure all these vaccination records are in and have been fine-combed through to make sure everything is in compliance," said Rabbi Hanoch Hecht, of Ulster County's Camp Emunah, which hosts many girls from a Chabad community in Brooklyn's Crown Heights.
The state of New York requires summer camps to keep immunization records for all campers, but doesn't bar children from attending if they haven't gotten a measles shot.
Children are required to get the measles vaccine to attend schools in New York, however, and Gov. Andrew Cuomo signed legislation Thursday eliminating an exemption for kids whose parents object to vaccinations on religious grounds.
The CDC recommends everyone over a year old should get the vaccine, except for people who had the disease as children. Those who have had measles are immune.
The vaccine, which became available in the 1960s, is considered safe and highly effective. Hecht and others stressed that vaccinations are widely accepted by most members of the Orthodox community, echoing rabbis in Brooklyn and Rockland County who say it is a relatively small group of parents influenced by anti-vaccination propaganda — not religious teachings — who have resisted inoculations.


text:
Kings Tutors
Contact details
- Location
- 19 Horder Road, London SW6 5ED
- Telephone
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- firstname.lastname@example.com
- View website
Kings Tutors Limited is based in London and provides private academic tuition to children and young adults across the United Kingdom. Our aim is to help our tutees reach their full potential academically. This is achieved by tailoring our high level of tuition to suit every individual's needs and create an effective, as well as motivating, learning environment. All of our tutors are all highly qualified specialists in their particular subjects with a wealth of teaching experience. Our principal objective is to maintain the highest standard of tuition at all times.
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text:
Coach and weightlifter.
When UW Oshkosh track and field assistant coach Mary Theisen-Lappen isn't coaching her Titan athletes, she is putting in the hours training herself as a professional weightlifter.
The dedication she shows has helped empower and inspire her athletes.
Theisen-Lappen next competes Oct. 26 in Niles, Illinois at the American Weightlifting Championships and on Dec. 8 in Salt Lake, Utah at the American Open Finals.


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Welcome Home! best describes this charming house located in the heart of Ridglea Hills on a nicely landscaped, oak treed corner lot one block up from Luther lake. This home offers an inviting front porch with great living spaces as well as a very private side gated driveway entrance with plenty of on site parking.
Listing courtesy of Cooper Collins Realty.


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My business-to-business credentials date back to 1991, spanning two continents and brands such as ABB, Agfa, Digital, Dow Chemical, Ericsson, IBM and Sony. Typically I have been both a copywriter and a member of the account team, working with product managers and marketing executives to define goals and set strategies.
Being a former editor makes it easier to practice and promote consistent branding. I maintained four separate legacy brands in my work at Deluxe Corporation, and in my current job I have developed brand guideline documents for numerous banks, credit unions and regional utilities.
Telling stories is easy, but integrating complex ideas and selling points is hard. I strive for profiles, articles and case studies that are both engaging and smart.
I've written more than 6,000 email campaigns, a "Best of NH" website, social media, videos, ad campaigns, direct mail, trade mags … you get the picture.
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Gold (Informational)
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Do you know who is a bankruptcy attorney is? He is basically a lawyer who deals with the working of bankruptcy. Sometimes, the situation gets happened when there is no way to come out from their financial crises, and then people chose the option of getting insolvent. If this is going to happen with you within sometime, then don't forget to hire the bankruptcy lawyer san diego. The attorneys are aware of all the legal and another working of insolvency.
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Now when it comes to hiring the bankruptcy lawyer, San Diego then look at these signs for meeting with the right person.


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Brotherhood of Saturn
View Single Post
Senior Member
Join Date: Apr 2008
Location: Inactive
Posts: 36,483
Likes: 237 (190 Posts)
Capricorn (♑) is the 10th
astrological sign in the zodiac, originating from the constellation of Capricornus..Exaltation Mars-Fall Jupiter.. It spans the 270–300th degree of the zodiac, corresponding to celestial longitude. Capricorn is ruled by the planet Saturn..The Encyclopedia Britannica states that the figure of Capricorn derives from the half-goat, half-fish representation of the Sumerian god Enki (Ea)..The planet Mercury, associated with Babylonian Nabu (the son of Marduk) was in Sumerian times, identified with Enki..The exact meaning of his name is uncertain: the common translation is "Lord of the Earth": the Sumerian en is translated as a title equivalent to "lord"; it was originally a title given to the High Priest; ki means "earth"; but there are theories that ki in this name has another origin, possibly kig of unknown meaning, or kur meaning "mound"..
The sign that is positioned directly opposite a planet's sign of exaltation is considered to be its fall..In astrology, exaltation is one of the 5 essential dignities of a planet..Essential dignity, in astrology, refers to the relative strength or weakness of a planet or point's zodiac position by sign and degree, or its essence—what the pre-eminent 17th-century astrologer William Lilly called "the strength, fortitude or debility of the Planets [or] significators".. In other words, essential dignity seeks to view the strengths of a planet or point as though it were isolated from other factors in the sky of the natal chart..In astrology, a natal chart is a stylized map of the universe with the "native" (the individual or subject to be studied) at the center.. It is calculated for the exact time and location of the native's birth for the purposes of gaining insight into the native's personality and potential..For example, the essential dignity of Mars, if located at 27 degrees of Capricorn would take into account the fact that Mars is exalted in Capricorn, and also that it is the bounds ruler of the 27th degree of Capricorn and also the face ruler of the 27th degree of Capricorn.. This is a
Considerably dignified Mars...
Messier 10 or M10
(designated NGC 6254) is a globular cluster of stars in the equatorial constellation of Ophiuchus.. Its name is from the Greek Ὀφιοῦχος "serpent-bearer", and it is commonly represented as a man grasping the snake that is represented by the constellation Serpens.. It is unique among the modern constellations in being split into two non-contiguous parts, Serpens Caput (Serpent's Head) to the west and Serpens Cauda (Serpent's Tail) to the east. Between these two halves lies the constellation of Ophiuchus, the "Serpent-Bearer". In figurative representations, the body of the serpent is represented as passing behind Ophiuchus between μ Ser in Serpens Caput and ν Ser in Serpens Cauda..The brightest star in Serpens is Unukalhai or Cor Serpentis "Serpent's Heart", with an apparent magnitude of 2.63. Part of the Milky Way passes through Serpens Cauda, which is therefore rich in deep-sky objects, such as the
Eagle Nebula
(IC 4703) and its associated star cluster Messier 16..There were two "serpent" constellations in Babylonian astronomy, known as Mušḫuššu and Bašmu. It appears that Mušḫuššu was depicted as a hybrid of a dragon, a lion and a bird, and loosely corresponded to Hydra..There are two daytime meteor showers that radiate from Serpens, the Omega Serpentids and the Sigma Serpentids.. Both showers peak between December 18 and December 25..In Pythagoreanism, the number 10 played an important role and was symbolized by the tetractys..As a mystical symbol, it was very important to the secret worship of the Pythagoreans.."Bless us, divine number, thou who generated gods and men! O holy, holy Tetractys, thou that containest the root and source of the eternally flowing creation! For the divine number begins with the profound, pure unity until it comes to the holy four; then it begets the mother of all, the all-comprising, all-bounding, the first-born, the never-swerving, the never-tiring holy 10,
the keyholder of all"...
There was an almost continual outpouring of the Holy Spirit through my mind..There was also the Poem "A Birthday", written on
August 10th for Leila Waddell,
who was then twenty-six.. She had gone to England to fulfil an engagement as leader of the Ladies' Orchestra in The Waltz Dream...
Last edited by lightgiver; 22-08-2014 at
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text:
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text:
Getting Pregnant Category Sitemap
Getting Pregnant 1
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Health Forums
Getting Pregnant
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text:
From Name:
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VerveLogic is a web and mobile app development company in Dubai, UAE focusing on iPhone, iPad & Android applications. We
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837 Superior Blvd, Wyandotte
Charm and Character abound in this beautiful 3 bedroom colonial! This home offers a spacious Living Room and Formal Dining Room. Refinished beautiful hardwood floors throughout. The original wood doors and trim have been wonderfully taken care of and preserved. Home also offers newer Furnace, AC, Stove, Fridge, Washer, Dryer, Hot water heater and brand new Garage door! Private back yard that backs up to a field with large 2 car detached garage. A wonderful area of Wyandotte that is close to downtown, schools, shopping, dining and endless parks! Don't hesitate, come see for yourself!