The regulation of the putrescine biosynthetic gene, speB, encoding agmatine ureohydrolase in Escherichia coli

Satishchandran, C. (1985) The regulation of the putrescine biosynthetic gene, speB, encoding agmatine ureohydrolase in Escherichia coli. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Agmatine ureohydrolase (EC.3.5.3.11) was purified 3,300 fold from an E.coli strain by ammonium sulphate precipitation, heat treatment, ion-exchange column chromatography, gel permeation column chromatography and chromatofocusing. The active enzyme is probably a dimer of identical subunits with a molecular weight of 38,000, and a pI of 8.3. The Km for agmatine was 1.3 mM, L-arginine was a competitive inhibiter and ornithine inhibited in a mixed manner. In crude extracts the enzyme was associated with another factor, probably a protein, which reduced the pI to 5.5 and decreased the activity of the enzyme. This factor also blocked reaction with antibodies raised against the purified enzyme in rabbits. In a strain known to express both biosynthetic and biodegradative ornithine decarboxylase and arginine decarboxylase, there was evidence for only the biosynthetic form of agmatine ureohydrolase. -- Agmatine ureohydrolase was negatively regulated by cAMP and the cAMP receptor protein, CRP. The specific activity of agmatine ureohydrolase was determined in crude extracts prepared from wild type strains of E.coli, from strains carrying a mutation in the structural gene for adenylcyclase (EC.4.6.1.1) (cya) or from strains carrying mutations in both cya and cAMP receptor protein gene (crp). Cyclic AMP when added to a glucose based medium repressed the specific activity of agmatine ureohydrolase, in contrast, cAMP induced the specific activity of β -galactosidase in both the wild type and the cya mutant, but not in the crp mutant. Addition of 1 mM agmatine to a glucose based medium induced an the specific activity of agmatine ureohydrolase in wild type, ∆cya or ∆cya, ∆crp strains. Chloramphenicol (150 μg/ml) abolished the inducibility of agmatine ureohydrolase by agmatine. In mutants blocked in the steps leading to the biosynthesis of polyamines, the addition of putrescine repressed the specific activities of arginine decarboxylase and ornithine decarboxylase, but did not affect agmatine ureohydrolase activity. The negative regulation of speA, speB and speC (encode arginine decarboxylase, agmatine ureohydrolase and ornithine decarboxylase respectively) by cAMP was shown not to be mediated by the repressive effect of cAMP on glutamine synthetase (EC.6.3.1.2), as cAMP repressed the expression of all three genes in a strain deleted for glnA (encodes glutamine synthetase). -- The speB gene was cloned into the plasmid vector pBR322 at the BamHI site, and was localised by exonuclease digestion of the plasmid pKA5. The extent of digestion of the plasmid was determined and was related to the ability of these religated plasmids to restore agmatine ureohydrolase activity to an speBsup- strain of E.coli. Using a cell-free transcription and translation system the direction of transcription and the approximate location of the promoter was determined. The direction of transcription was also determined by cloning fragments in both directions into the promoterless genes of lacZ (encodes β -galactosidase) and galK {encodes galactokinase). The ability of these hybrid genes to confer enzymatic activities (β -galactosidase or galactokinase) to strains deleted in their respective genes confirmed the location of the speB promoter. -- cAMP:CRP was shown to interact with the promoters of speA and speB using a cell-free transcription and translation system in which cloned copies of the genes served as templates. This was also shown in vivo, by demonstrating that cAMP, inhibited the expression of β -galactosidase and galactokinase in plasmids carrying fusions of the speA and speB promoters to the structural genes of these enzymes. In addition, cAMP supplementation of minicells carrying the plasmid pKA5 (carries speB) caused repression of agmatine ureohydrolase synthesis. Cyclic AMP decreased and agmatine increased the steady state mRNA concentrations of speB, while those of lacZ were increased by cAMP in the ∆cya strain. In contrast, cAMP did not affect the steady state concentrations of lacZ or speB mRNAs in the ∆crp strain.

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