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Rhodobacter capsulatus nifA1 Promoter: High-GC -10 Regions in High-GC Bacteria and the Basis for Their Transcription. Cynthia L. Richard, 2004.It was previously shown that the Rhodobacter capsulatus NtrC enhancer-binding protein activates the R . capsulatus housekeeping RNA polymerase but not the Escherichia coli RNA polymerase at the nifA1 promoter . We have tested the hypothesis that this activity is due to the high G+C content of the -10 sequence . A comparative analysis of R . capsulatus and other  -proteobacterial
promoters with known transcription start sites suggests that
the G+C content of the -10 region is higher than that for E . coli .
Both in vivo and in vitro results obtained with nifA1
promoters with -10 and/or -35 variations are reported here . A major
conclusion of this study is that
-proteobacteria
have evolved a promiscuous sigma factor and core RNA polymerase that
can transcribe promoters with high-GC -10 regions in addition
to the classic E . coli Pribnow box . To facilitate studies of
R . capsulatus transcription, we cloned and overexpressed all
of the RNA polymerase subunits in E . coli, and these were reconstituted
in vitro to form an active, recombinant R . capsulatus RNA
polymerase with properties mimicking those of the natural polymerase .
Thus, no additional factors from R . capsulatus are necessary
for the recognition of high-GC promoters or for activation by R .
capsulatus NtrC . The addition of R . capsulatus
 70
to the E . coli core RNA polymerase or the use of -10 promoter
mutants did not facilitate R . capsulatus NtrC activation of
the nifA1 promoter by the E . coli RNA polymerase . Thus,
an additional barrier to activation by R . capsulatus NtrC
exists, probably a lack of the proper R . capsulatus NtrC-E .
coli RNA polymerase (protein-protein) interaction(s) .
Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme. Lilian Schoefer, 2004.Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus . The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity . The insert of a clone conferring phloretin hydrolase activity was sequenced . Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure . The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens . The phloretin hydrolase was heterologously expressed in Escherichia coli and purified . The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer . The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid . The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively . The Km for phloretin was 13 ± 3 µM and the kcat was 10 ± 2 s–1 . The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one . The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively . Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively . Glycerol-3-Phosphate Acquisition in Spirochetes: Distribution and Biological Activity of Glycerophosphodiester Phosphodiesterase (GlpQ) among Borrelia Species. Tom G. Schwan, 2003.Relapsing-fever spirochetes achieve high cell densities (>108/ml) in their host's blood, while Lyme disease spirochetes do not (<105/ml) . This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood . Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively . In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm . Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes . Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group . A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B . hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids . Sequence analysis of B . hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B . burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P . Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol . The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes .
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