Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N-acetylgalactosamine metabolism in Escherichia coli K-12

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Summary

The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N-acetylgalactosamine and galactosamine in Escherichia coli. In some strains of E. coli, particularly the common K-12 strain, a portion of this cluster is missing because of a site-specific recombination event that occurred between sites in agaW and agaA. Strains that have undergone this recombination event have lost the ability to utilize either N-acetylgalactosamine or galactosamine as sole sources of carbon. Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators. Promoters upstream of agaR, agaZ and agaS were characterized. All three promoters had elevated activity in the presence of N-acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA-binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting. In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP-CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA-binding or response to inducer. These residues of AgaR were targeted for site-directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non-inducible. The apparent ability to produce negatively dominant and non-inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function.

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