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Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction. Keietsu Abe, 2002.Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO2 . This trait is encoded on a 25-kb plasmid, pD1 . We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-ß-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences . The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter  aspD
aspT . The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-ß-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis . Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions . The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp . Expression of the asp operon in E . coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine . Histidine-tagged AspD (AspDHis) was also expressed in E . coli and purified from cell extracts . The purified AspDHis clearly exhibited activity of L-aspartate-ß-decarboxylase . Recombinant AspT was solubilized from E . coli membranes and reconstituted in proteoliposomes . The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine . Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high .
Expression of the Azotobacter vinelandii Poly-ß-Hydroxybutyrate Biosynthetic phbBAC Operon Is Driven by Two Overlapping Promoters and Is Dependent on the Transcriptional Activator PhbR. Martín Peralta-Gil, 2002.The Azotobacter vinelandii phbBAC genes encode the enzymes for poly-ß-hydroxybutyrate (PHB) synthesis . The phbR gene, which is located upstream of and in the opposite direction of phbBAC, encodes PhbR, a transcriptional activator which is a member of the AraC family of activators . Here we report that a mutation in phbR reduced PHB accumulation and transcription of a phbB-lacZ fusion . We also report that phbB is transcribed from two overlapping promoters, pB1 and pB2 . The region corresponding to the -35 region of pB1 overlaps the pB2 -10 region . In the phbR mutant, expression of phbB from the pB1 promoter is significantly reduced, whereas expression from the pB2 promoter is slightly increased . Two phbR promoters, pR1 and pR2, were also identified . Transcription from pR2 was shown to be dependent on  S . Six conserved 18-bp sites, designated R1 to R6, are present within the phbR-phbB intergenic region and are proposed to be putative binding targets for PhbR . R1 overlaps the -35 region of the pB1 promoter . A model for the regulation of phbB transcription by PhbR is proposed .
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