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A strain of Escherichia coli was constructed in which all of the genes involved in polyamine biosynthesis--speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase)--had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.

Wild-type Escherichia coli cells grow normally in 95% O(2)/5% CO(2). In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O(2)/5% CO(2). Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of H(2)O(2) that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen.

Null mutants of Escherichia coli were constructed that cannot synthesize spermidine, because of deletions in the gene encoding S-adenosylmethionine decarboxylase. These mutants are still able to grow at near normal rates in purified media deficient in polyamines. These results in E. coli differ from recent findings that null mutants of Saccharomyces cerevisiae and of Neurospora crassa have an absolute growth requirement for spermidine.

We have shown that toxicity of paraquat for Escherichia coli is increased over 10-fold in strains defective in the biosynthesis of spermidine compared to isogenic strains containing spermidine. The increased sensitivity of these spermidine-deficient mutants to paraquat is eliminated by growth in medium containing spermidine or by endogenous supplementation of spermidine by the use of a speE+D+ plasmid. No paraquat toxicity is seen in the absence of oxygen, even in amine-deficient strains, indicating that superoxide is the agent responsible for the increased toxicity. However, the specific mechanisms responsible for the increased paraquat toxicity in the spermidine-deficient mutants remain to be determined. The marked sensitivity to paraquat of E. coli deficient in spermidine is of particular interest, since such mutants have no other phenotypic properties that can be easily assayed. This increased sensitivity has been used as the basis of a convenient method for scoring for mutants in polyamine biosynthesis and for the detection of plasmids containing the biosynthetic genes.

Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site.

We have obtained Escherichia coli mutants lacking spermidine synthase (putrescine aminopropyltransferase) and have found that the mutated gene (speE) is located immediately upstream from the gene coding for S-adenosylmethionine decarboxylase (speD); these genes are located at 2.7 minutes on the E. coli chromosome. Both genes are present in a 1795-base-pair fragment of E. coli DNA that was cloned into pBR322. Deletion of 105 bases upstream of speE caused a coordinate loss of both activities, indicating that speE and speD constitute a single operon. speE and speD have also been cloned separately in a high-expression vector; strains carrying these plasmids overproduce the respective enzymes.

A covalently linked pyruvoyl group is essential for the enzymatic activity of S-adenosylmethionine decarboxylase from Escherichia coli. A rapid purification method based on affinity chromatography is described for the isolation of this enzyme from an E. coli K12 strain which contains a plasmid containing the structural gene for S-adenosylmethionine decarboxylase, and which overproduces this enzyme. The purified enzyme contains one pyruvate moiety on each of six subunits. The enzyme is inactivated by incubation with carbonyl group reagents such as NaBH4 and phenylhydrazine; after inactivation, 1 mol of lactate or 1 mol of phenylhydrazone is found/mol of enzyme subunit. The enzyme is also inactivated by NaCNBH3 but only in the presence of either substrate or product and the divalent metal ion activator Mg2+; inactivation is accompanied by incorporation of 1 mol of the product, decarboxylated adenosylmethionine, per mol of enzyme subunit, suggesting that the pyruvoyl group participates in catalysis by formation of a Schiff base with the substrate. Equilibrium dialysis studies indicated a single substrate (or product) binding site/enzyme subunit.

A procedure is presented for the rapid screening of bacterial colonies to detect mutants unable to produce 14CO2 from a labeled precursor. The method is especially useful for mass screening for mutants that cannot be easily detected by their phenotypic characteristics.

Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli. We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes. We have now studied the upstream promoter and the downstream terminator regions of this operon more completely. We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene. Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t. An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t. By comparing our DNA fragments with those prepared from this region of the E. coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E. coli chromosome. We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E. coli chromosomal map.

Mutants of Escherichia coli deficient in adenosylmethionine decarboxylase, an enzyme in the biosynthetic pathway for spermidine, were isolated after mutagenesis of E. coli K 12 with N-methyl-N-nitro-N-nitrosoguanidine or with the bacteriophage Mu. The mutated gene, designated speD, is at 2.7 min on the E. coli chromosome map. In several of the mutants resulting from Mu insertion both adenosylmethionine decarboxylase activity and spermidine were undetectable. The absence of spermidine from speD strains proves the essential role of adenosylmethionine decarboxylase in the biosynthetic pathway for spermidine. Despite the complete absence of spermidine, these mutants grew at 75% of the wild type rate.

The presence of certain rpsL (strA) mutations in a strain of Escherichia coli that cannot synthesize putrescine or spermidine because of deletions in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase, converts a partial requirement for polyamines for growth into an absolute requirement.

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

An Escherichia coli metK mutant, designated metK110, was isolated among spontaneous ethionine-resistant organisms selected at 42 degrees C. The S-adenosylmethionine synthetase activity of this mutant was present at lower levels than in the corresponding wild-type strain and was more labile than the wild-type enzyme when heated or dialyzed. A mixture of mutant and wild-type enzyme preparations had an activity equal to the sum of the component activities. These facts strongly suggest that the mutated gene in this strain is the structural gene for this enzyme. Genetic mapping experiments placed the metK110 mutation near or at the site of other known metK mutants (i.e., 63 min), confirming its designation as a metK mutant. A revised gene order has been established for this region, i.e., metC glc speC metK speB serA.

Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.

A method is described for markedly enriching a bacterial population for cells containing any given Mu insertion mutation. The method involves the transfer of a small piece of deoxyribonucleic acid from a Mu-infected Hfr donor donor strain to a suitable F- strain and a subsequent selection of those recombinant organisms that have received a Mu prophage from the donor. The method is particularly usefule for isolating mutants whose selection requires "brute-force" assay, since only a few hundred colonies have to be screened.