REGULATION OF AROMATIC AMINO ACID BIOSYNTHESIS IN ESCHERICHIA COLI
Abstract
The Escherichia coli aroF gene encodes the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (TYR), one of the three isozymes that control carbon flow through the shikimate pathway. Carbon flow into the shikimate pathway is controlled by feedback inhibition and repression of DAHP synthase. Transcription of aroF is repressed by the tyrR gene product complexed to tyrosine. The configuration around the anomeric carbon of DAHP, the product of DAHP synthase (TYR), has been determined as alpha. The nucleotide sequences for the regulatory and coding regions of the aroF gene have been determined. Constitutive aroF mutants with lesions linked to aroF were isolated. The nucleotide sequences for the aroF regulatory regions from six such mutants have been determined. The mutations lie in two 18 bp imperfect palindromes called aroFo1 and aroFo2 which are located upstream of the aroF transcription start by 61 and 113 bp, respectively. Nuclease S1 mapping has identified the 5' end of the aroF transcript 51 bp upstream of the aroF translation start. Aromatic amino acid biosynthesis has been studied in vivo by whole cell ('13)C-nuclear magnetic resonance spectroscopy. In wild type E. coli, free aromatic amino acids are not detectable by this method. E. coli strains harboring a feedback-resistant allele of aroF (HE202) or cells which carry a multiple copy plasmid (HE102/pKB45) that encodes a wild type aroF allele synthesize comparable amounts of aromatic amino acids. Since strain He102/pKB45 synthesizes 50 times as much DAHP synthase as HE202, these in vivo measurements with a truly noninvasive probe suggest feedback inhibition as the quantitatively major mechanism controlling carbon flow in the shikimate pathway. A consequence of deregulating aromatic amino acid biosynthesis is the biosynthesis of trehalose. The amount of trehalose synthesized correlates directly with the amount of deregulated DAHP synthase. Catabolism of trehalose by E. coli is shown to be independent of aromatic amino acid biosynthesis yet dependent on cAMP.
Degree
Ph.D.
Subject Area
Microbiology
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