A genetic and physiological study of enzymes involved in the catabolism of nucleosides in Escherichia coli.
thesisposted on 19.11.2015, 08:53 by Shamim Iqbal. Ahmad
In Escherichia coli thymidine phosphorylase, purine nucleoside phosphorylase, deoxyribomutase and deoxyriboaldolase are involved in the catabolism of nucleosides and deoxynucleosides and the sugar moiety is used as a carbon source. All four enzymes are induced by deoxynucleosides and the actual inducer appears to be dRib-5-P. Purine nucleoside phosphorylase and deoxyribomutase are also induced by purine ribonucleosides. Overwhelming evidence is available which favours the catabolic role of these enzymes. Four genes viz. tpp, dra, drm and pup specifying thymidine phosphorylase, deoxyriboaldolase, deoxyribomutase and purine nucleoside phosphorylase respectively were mapped by transduction with phage P1. All pairs showed greater than 90% co-transduction. The gene order was found to be dra-tpp-drm-pup and the gene cluster lies between the hsp and serB loci on the chromosome map of E.coli. A regulatory mutant which leads to constitutive synthesis of at least three of the four enzymes (and perhaps the fourth, deoxyribomutase) was analysed. The regulatory gene is designated nucR and is located near galE. The amount of thymine required for growth (colony formation) of thy- strains is affected by the nucR mutation. The amount required by the thy- drm- strain is reduced about four fold if it carries the constitutivity mutation. The amount required by a thy- drm+ strain is increased at least two fold. These differences in nutritional requirement provide a method for selecting constitutive from non-constitutive strains and vice versa. By this method a thymidine phosphorylase constitutive mutant was selected. The data obtained from the analysis of this second regulatory mutant indicate that it is similar to an Oc type mutation of the lac operon. The mutation of the putative operator, which is located to the left of the dra, renders only thymidine phosphorylase and deoxyriboaldolase constitutive. Purine nucleoside phosphorylase is not affected by this mutation and therefore it is believed that the four genes constitute at least two operons, one operon containing dra and tpp and the other pup and drm. A double mutant of E.coli was produced which lacks cytosine deaminase activity and requires pyrimidine for growth. This mutant is not able to grow on cytosine as sole pyrimidine source. This observation indicates that the only pathway involved in the incorporation of exogenous cytosine is via its first step conversion to uracil. A gene, udp, specifying uridine phosphorylase and a gene, cod, specifying cytosine deaminase were located at 74-75 and around 87 minutes respectively on the E.coli linkage map. Fluorouracil was used to isolate various mutants lacking nucleoside and deoxynucleoside phosphorylase activities. Using these mutants, it was shown that there are three routes via which FU can be converted to compounds which inhibit cell growth.