The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH). AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E. coli. Sequencing of a 2.97-kilobase-pair fragment of the E. coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as weUl as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3. ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis. Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4. The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity. The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.In Escherichia coli putrescine is synthesized either by decarboxylation of ornithine or by decarboxylation of arginine to agmatine followed by hydrolysis of agmatine to putrescine and urea (14). The last two reactions are catalyzed by the enzymes arginine decarboxylase and agmatine ureohydrolase (AUH), respectively. The AUH protein has previously been purified from an E. coli isolate that was transformed with the plasmid pKA5; the enzymatic properties of AUH have been characterized previously (9). The subunit size of AUH, as deduced from its mobility on a sodium dodecyl sulfate-polyacrylamide gel, is 38 kilodaltons. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cyclic AMP receptor protein, cyclic AMP represses the expression of the speB gene, while agmatine induces it. These two modulators appear to act independently from each other (10); the mechanism of this differential regulation is unknown. The speB gene coding for AUH is located at approximately 63.5 min on the E. coli chromosome. Although a large chromosomal fragment corresponding to this region, including the speB gene, is present in the pKA5 plasmid, the exact location of speB was not previously established. Here we report the nucleotide sequence of the speB structural gene and identify the promoter responsible for transcription of one of two mRNAs encoding AUH. We also report the mapping of the mRNA resulting from transcription that initiated at this promoter. MATERIALS AND METHODSBacterial hosts, media, and growth conditions. E. coli CB806(A1acZ galK phoA8 rpsL thi recA56) (11) was used for all experiments involving the promoter vector pCB267 and its derivatives. E. coli DHSa [F- The bacteria wer...

The speB gene of Escherichia coli encodes agmatine ureohydrolase (AUH), a putrescine biosynthetic enzyme. The speB gene is transcribed either from its own promoter or as a polycistronic message AMP (cAMP) repressed AUH activity of chromosomally encoded AUH but had no effect on plasmid-borne speB nor 4b(speB-phoA). It is concluded that ORF1 encodes a protein which is a posttranscriptional regulator of speB, agmatine induces speB independent of speA, and cAMP regulates speB indirectly.In Escherichia coli putrescine can be synthesized from arginine by decarboxylation of arginine to agmatine, catalyzed by arginine decarboxylase; agmatine is hydrolyzed to putrescine, catalyzed by agmatine ureohydrolase (AUH) (13). Arginine decarboxylase and AUH are encoded by the speA and speB genes, respectively, located at approximately minute 63.5 on the E. coli chromosome (1). We have sequenced the speB gene and characterized its pattern of transcription (12). The speB gene is encoded by one open reading frame (ORF); however, it can be transcribed as either a monocistronic or a polycistronic message. The speB promoter, initiating the monocistronic transcript, is unusual in that it does not require any sequence specificity upstream from the -10 Pribnow box for its activity. The polycistronic message is initiated from the promoter of speA residing immediately upstream from speB (12).The source of the speB gene was plasmid pKA5, which contains a 7.5-kb EcoRI fragment of E. coli chromosome subcloned into pBR322 (2). It had been previously established that this EcoRI fragment contains the speB, speA, and metK genes, encoding AUH, arginine decarboxylase, and methionine adenosyltransferase, respectively. In the initial search for the speB gene on pKA5, a 3.0-kb fragment subcloned into pBR322 resulted in overproduction of AUH activity (12). A small deletion at one end of this fragment caused a significant decrease in AUH activity. After establishing the location of the speB gene within the 3.0-kb fragment, it became apparent that the deletion did not map within the speB gene. Rather, the deletion included the 5' region of ORF1, the first of the two ORFs (ORF1 and ORF2) * Corresponding author. located on the strand complimentary to speB. These observations prompted us to investigate the nature of the relationship between ORF1, ORF2, and expression of speB. The data reported here indicate that ORF1 encodes a protein which stimulates expression of AUH at a posttranscriptional level.Agmatine is a substrate of the reaction catalyzed by AUH. A number of E. coli strains either bearing a chromosomal copy of speB or transformed with pKA5 show increased AUH activity following agmatine supplementation (9). Our results show that agmatine is an inducer of the speB, but not of the speA, promoter.The involvement of cyclic AMP (cAMP) in regulation of AUH in E. coli has been the subject of controversy. Halpern and Metzer (4) reported that cAMP supplementation of strain CS101B of E. coli K-12 induced the expression of AUH activity. In contrast, Satis...