Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in XXXX metabolism in @entity2624 .

@entity268 @entity11485 @entity526 @entity535 @entity505 @entity2624 @entity10811
The agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of @entity10811 and @entity11485 in @entity526 . In some strains of @entity526 , particularly the common @entity2624 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 @entity10811 or @entity11485 as sole sources of @entity505 . 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 @entity10811 or @entity11485 , 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 @entity268 -CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved @entity535 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.
