The speA gene of Escherichia coli encodes biosynthetic arginine decarboxylase (ADC), the first of two enzymes in a putrescine biosynthetic pathway. The activity of ADC is negatively regulated by mechanisms requiring cyclic AMP (cAMP) and cAMP receptor protein (CRP) or putrescine. A 2.1-kb BamHI fragment containing the speA-metK intergenic region, speA promoter, and 1,389 bp of the 5' end of the speA coding sequence was used to construct transcriptional and translational speA-lacZ fusion plasmids. A single copy of either type of speA-lacZ fusion was transferred into the chromosomes of Escherichia coli KC14-1, CB806, and MC4100, using bacteriophage lambda. The speA gene in lysogenized strains remained intact and served as a control. Addition of 5 mM cAMP to lysogenic strains resulted in 10 to 37% inhibition of ADC activity, depending on the strain used. In contrast, the addition of 5 or 10 mM cAMP to these strains did not inhibit the activity of j8-galactosidase (i.e., ADC::4-galactosidase). Addition of 10 mM putrescine to lysogenized strains resulted in 24 to 31% repression of ADC activity and 41 to 47% repression of j8-galactosidase activity. E. coli strains grown in 5 mM cAMP and 10 mM putrescine produced 46 to 61% less ADC activity and 41 to 52% less j-galactosidase activity. cAMP (0.1 to 10 mM) did not inhibit ADC activity assayed in vitro. The effects of cAMP and putrescine on ADC activity were additive, indicating the use of independent regulatory mechanisms. These results show that cAMP acts indirectly to inhibit ADC activity and that putrescine causes repression of speA transcription.Putrescine (diaminobutane) is an aliphatic cation that is required for optimum growth of all cells. Putrescine and other polyamines are involved in many biological processes, but their physiological role(s) remains unclear. Most eucaryotes possess a single putrescine biosynthetic pathway (pathway I), in which ornithine decarboxylase (ODC), encoded by speC in Escherichia coli, converts ornithine to putrescine. Bacteria and plants also have a second putrescine biosynthetic pathway (pathway II). In E. coli, arginine is converted to agmatine by biosynthetic arginine decarboxylase (ADC), encoded by the speA gene. Agmatine is hydrolyzed to putrescine and urea by agmatine ureohydrolase (AUH), encoded by the speB gene (25). The presence of two parallel putrescine biosynthetic pathways is necessary in E. coli because the bacterium lacks arginase and therefore cannot convert arginine to ornithine. In the presence of exogenous arginine, as in the intestine, putrescine synthesis from pathway I would also decrease as the intracellular pool of ornithine becomes limiting as a result of arginine feedback repressing arginine biosynthesis (9, 14). Thus, pathway II ensures that putrescine is produced as nutritional conditions change (26).Native ADC is a 280,000-Da tetramer that requires magnesium and pyridoxal phosphate for activity (24, 43). Monomers of ADC are synthesized as 74,000-Da precursor polypeptides that are posttranslationally proces...