AmpR is a trans-acting transcriptional regulator of ampC β-lactamase. As it binds to the intercistronic region of ampC-ampR genes, elucidation of its binding nature to the region will give aids to solve the transcription activation process. For this study, Citrobacter freundii AmpR was highly purified through affinity chromatography, quantified at every purification step, subjected to binding, cross-linking and kinetic studies. AmpR was purified up to 99% from DNA(1.5 kilobase-pair BamHI fragment of pNU305)-sepharose chromatography and was identified on SDS-PAGE gel. The first 15 N-terminal amino acids were in agreement with that predicted from the nucleotide sequence. Interestingly, DNA binding ability of AmpR was not dependant on purification degree. Cross-linking of purified AmpR with 0.005% glutaraldehyde shows dimer and tetramer bands on SDS-PAGE gel. On the binding studies with 236 base-pair Clal-Sacl fragment of pNU305, which carries binding sites, sigmoidal binding curve was observed suggesting that AmpR binds the DNA cooperatively. 3 protein-DNA ladders at high AmpR concentration may suggest that there would be 3 binding sites. The apparent equilibrium binding constant was calculated as 150nM. On the plot of Hill equation, the Hill coefficient and the apparent dissociation constant were calculated as 1.95 and $5 \times10^{-14}M^2$, respectively. The association and dissociation rate constants were $6.4 \times10^{10}/M^2·sec$ and $9.0 \times10^{-3}/sec$, respectively, from the binding kinetic studies. These results mean that AmpR binds very tightly to the intercistronic region of ampR - ampC genes at least at two sites, cooperatively, as dimeric or tetrameric form, with very high DNA sequence specificity. Additional protein was found to bind tightly to this region. The protein is presumed to be one of regulatory factor that can change the conformation of AmpR-DNA complex, and is needed for the transcription of ampC gene.
It is reported that the transcription activation of ampC gene follows to the binding of unidentified cytoplasmic effector moecule to AmpR protein.
Some chemicals such as glycine, D-alanine, D-methionine, D-tryptophan, and DAP(meso-diaminopimelic acid) was reported as inducer of β-lactamase. However, all of these chemicals didn't induce the enzyme significantly. They didn't increase or decrease the DNA binding of AmpR, and neither they activate the transcription by AmpR. Therefore, none of them seems to be an actual cytoplasmic inducer.
Low pH of medium, $Ca^2+$, O-phospho-D-tyrosine, and D-galactosamine significantly increased the rate of β-lactamase induction from E.Coli SNO3(pNU305). $Ca^2+$ and O-phospho-D-tyrosine abolished the upper two protein-DNA ladders in the gel retardation assay. D-galactosamine increased the retardation very significantly. It was also observed that AmpR didn't bind to the DNA at low pH. However the 3 chemicals and low pH didn't increase the amount of transcript in in vitro transcription.
Aspirin is of special interest because it decrease the induction rate by cefoxitin as much as 50-60%, and reduced the DNA binding of AmpR significantly. Consequently, the physiological role of aspirin is presumed to be as a negative effector because of more convincing observation that it decreased the amount of ampC transcript. It seems that AmpR binds to the intercistronic region of ampC - ampR genes at its maximum at 3 sites as dimer or tetramer cooperatively with very high sepecificity. Low pH give effects on the induction of the β-lactamase. $Ca^2+$, O-phospho-D-tyrosine, and D-galactosmine are suggested to be novel putative inducers of β-lactamase. ProteinX is also another candidate for the regulator protein of β-lactamase induction system.
