Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD

Inhibition of spermidine uptake in Escherichia coil, which occurs in the pr esence of accumulated polyamines, has been studied using the spermidine upt ake operon consisting of the potA, -B, -C, and -D genes. Transcription of t he potABCD operon was inhibited by PotD, a spermidine-binding protein usual ly found in the periplasm, and the inhibitory effect of PotD was increased by spermidine, Transcription was not affected by bovine serum albumin, PotA , or PotF, suggesting that the effects of PotD are specific to the PotD pro tein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:Pot D. It was found that PotD bound to regions -258 to -209 nucleotides upstrea m and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermi dine. Overexpression of PotD in Escherichia coil DH5 alpha inhibited the up take of spermidine, the synthesis of Pot-ABCD mRNA, and expression of a lac Z reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as Pot D precursor in spheroplasts. Our results indicate that PotD precursor can a lso inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using m utated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the sperm idine transport (potABCD) operon is inhibited in vivo by PotD precursor rat her than PotD through its binding to two regions close to the transcription al initiation site of the operon.