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Mol Microbiol. 2004 Feb;51(3):813-26.

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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  • 1Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.

Abstract

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.

PMID:
14731281
[PubMed - indexed for MEDLINE]
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