For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. In previous reports we have described the use of microarray and proteomic techniques together with our polyamine-requiring mutants of Saccharomyces cerevisiae to find which gene and proteins are upregulated or downregulated by spermidine supplementation to polyamine -deficient cultures. We have also reported the construction of a strain of Escherichia coli that contained deletions in all of the genes involved in polyamine biosynthesis;namely, 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). Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free media;albeit at a slightly (ca 40-50%) reduced growth rate. Recently, we are also carrying out microarray studies with our Escherichia coli mutants that lack all of the enzymes involved in polyamine-biosynthesis. For this purpose we have developed the use of a chemostat and also we have found it is important to use a mixture of air and nitrogen to avoid cell death due to oxidative stress during polyamine deprivation. As opposed to a number of studies reported from other laboratories, we feel that it is very important not to have the results complicated by changes in the growth rates after spermidine addition. Lysine and arginine auxotrophs have also being constructed in the polyamine mutant background to permit the use of the SILAC technique for proteomic studies to compare the protein arrays between polyamine plus and deprived culture. In a preliminary study, polyamine mutants also show higher frequency of fluorouracil resistant mutants in the absence of added amines as compared to polyamine supplemented culture.
