Deletion mutations in the speED operon: spermidine is not essential for the growth of Escherichia coli.

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.

Spermidine biosynthesis in Saccharomyces cerevisae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase).

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant.

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK).

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 enzymatic method permitting early determination of histocompatibility in mixed lymphocyte culture.

The standard mixed lymphocyte culture assay, which measures the incorporation of [3H]thymidine into DNA, usually requires 5 days. We describe a more rapid assay based on changes in the activity of ornithine decarboxylase. An increase in the activity of ornithine decarboxylase was observed in mixed lymphocyte cultures from genetically defined, major histocompatibility complex (MHC)-nonidentical miniature swine as early as 18 hr after plating. No increase was found in mixed cultures from inbred MHC-identical animals. Similar results were obtained with the enzyme S-adenosylmethionine decarboxylase with the increase in activity starting at about 32 hr. There was a good correlation between the ornithine decarboxylase values at 18 hr and the results of the [3H]thymidine incorporation assay on day 5. Preliminary experiments with human lymphocytes revealed similar results.

Mutants of Saccharomyces cerevisiae deficient in polyamine biosynthesis: studies on the regulation of ornithine decarboxylase.

We have isolated the following mutants in the polyamine biosynthetic pathway in yeast: (i) spe10 mutants, which have no ornithine decarboxylase activity and therefore cannot make putrescine; (ii) spe2 mutants, which have no adenosylmethionine decarboxylase and therefore cannot make spermidine or spermine; (iii) spe3 mutants, which have no putrescine aminopropyltransferase and therefore cannot make spermidine and spermine, and (iv) spe4 and spe40 mutants (suppressors of spe10 mutations), which have no spermidine aminopropyltransferase and therefore cannot make spermine. These mutants show that (i) yeast has an absolute requirement for these amines for growth (ii) putrescine in the absence of spermidine and spermine supports growth at one-sixth the wild type rate; (iii) intracellular spermine controls the ornithine decarboxylase activity and thus mutants that cannot make spermine are derepressed for ornithine decarboxylase; (iv) Saccharomyces cerevisiae can make putrescine only by one pathway, i.e., ornithine decarboxylase; (v) spermidine and spermine are synthesized by different aminopropyltransferases in yeast; and (vi) spermidine and/or spermine are absolutely required for both sporulation and maintenance of the double-stranded RNA "killer" plasmid. We have purified ornithine decarboxylase to homogeneity and shown that loss of ornithine decarboxylase activity resulting from growth with added spermidine and spermine is the result of post-translational modification.