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Media type:
E-Article
Title:
Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC β‐lactamase
Contributor:
Bartowsky, E.;
Normark, S.
imprint:
Wiley, 1991
Published in:Molecular Microbiology
Language:
English
DOI:
10.1111/j.1365-2958.1991.tb01920.x
ISSN:
0950-382X;
1365-2958
Origination:
Footnote:
Description:
<jats:title>Summary</jats:title><jats:p>AmpR, the transcriptional regulator for the <jats:italic>Citrobacter freundii</jats:italic> ampC β‐lactamase gene, was purified. The purified AmpR had DNA‐binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate polyacrylamide gel electrophoresis as previously described, and N‐terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA‐binding and β‐lactamase induction and which map in the amino‐ and carboxyl‐terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix‐turn‐helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C‐terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the <jats:italic>ampR</jats:italic> gene. This G102E mutant leads to constitutive β‐lactamase expression in the absence of both β‐lactam inducer and <jats:italic>ampG</jats:italic>, a gene essential for induction in wild‐type enterobacteria. Another mutant protein, D135Y, showed wild‐type properties In an <jats:italic>ampG</jats:italic>* and an <jats:italic>ampG::kan</jats:italic> background, but could, unlike wild‐type AmpR, activate the <jats:italic>ampC</jats:italic> gene in an <jats:italic>ampG1</jats:italic> mutant background. It is thought that <jats:italic>ampG1</jats:italic> is a missense mutant. These two types of <jats:italic>ampR</jats:italic> mutants suggest that activation of <jats:italic>ampC</jats:italic> transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.</jats:p>