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

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually 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 protein. 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:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream 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 spermidine. Overexpression of PotD in Escherichia coli DH5 alpha inhibited the uptake of spermidine, the synthesis of Pot-ABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.