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Bacillus subtilis grown at temperatures below 37°contains two thymidylate synthetases, TSaseA and TSaseB. Their presence is dependent on functional thyA and thyB genes, respectively. When cells are grown at 460 they only contain TSaseA activity. This allows an easy positive selection for thyA and thyA,thyB mutants. The two TSases have been physically separated, and they show similar overall requirements for activity. However, they differ significantly in both their kinetic and their physicochemical properties. Forsogsanlaeg, Ris0, Denmark. RESULTS Selection of thy Mutants in B. subtilis. Thymine-requiritng mutants of enteric bacteria are easily obtained by selecting for mutants resistant to the folate analogue trimethopritn in the presence of thymine at 37°. Resistant mutants, of wvhich the majority are Thy-, appear spontaneously with a frequency of about 10-6 (10, 11). When B. subtilis cells are spread on plates containing trimethoprim (10 Mg/ml) and thymine (50 pg/ml) and incubated at 37c, resistant mutants appear with a frequency of less than 10-to. However, if the selection is made at 460, trimethoprim-resistant mutants appear with a frequency of about 10-6. The majority of these mutants are Thy-at 46 but Thy+ and trimethoprim-sensitive at 37' (tsThv-). One of these mutants, ED43. derived from QB16. was kept for further studies. When ED43 is used in a second selection for trimethoprim-resistance, this time at 37D, mutants appear with a frequency of about 10-6. These mutants have an absolute requirement for thymine at all temperatures. One of these mtutants, ED53, wvas kept for further studies.Genotypes of the tsThy-and Thy-Mutants. By using phage PBS 1 grown on QB798 (citfthy + ,ilvA + ) as a donor and ED43 as a recipients we found that the tsThv+ phenotype cotransduces 25-505So with the citB rtarker bhst it gives no cotransduction with ilvA. This stsggests that the tsThy-phenotype is due to a mutation in thyA. In a second transduction, we transduced the Thy-mutant ED5:3 with the same phage as above, selecting for Ilv+ at 37'. Of the 88 transdllctants tested. 88 had become Thy+ at 37' but still Thy-at 46'-i.e., we had restored the tsThy-phenotype (thyA genotype) of the parent Abbreviation: TSasc. thymidylate synthetase. * Ots leave from the

The phosphoribosyltransferases catalyze the formation of nucleotides by reaction with 5-phosphoribosyl-1-diphosphate (PRPP) and a nitrogenous base, releasing pyrophosphate as the second reaction product. The enzymes participate in the biosynthesis of the amino acids histidine and tryptophan, pyridine coenzymes, and all nucleotides by de novo synthesis and salvage pathways [1]. They are generally unregulated enzymes obeying hyperbolic saturation kinetics for the substrates, but there are exceptions. The glutamine PRPP amidotransferase, which catalyses the first step specific for de novo purine nucleotide synthesis, is feedback inhibited by AMP and GMP, and uracil phosphoribosyltransferase (UPRTase; a salvage enzyme that makes UMP from uracil and PRPP) was shown to be activated by GTP in several organisms e.g. The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60°C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number k cat and raised the K m values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two-and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (K D equal to 2 and 0.5 lm, respectively). The binding of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.Abbreviations IPTG, isopropyl thio-b-D-galactoside; GdHCl, guanidinium chloride; PP i , inorganic diphosphate or pyrophosphate; PRPP, 5-phosphoribosyl-1-adiphosphate; UPRTase, uracil phosphoribosyltransferase or UMP synthase (EC 2.4.2.9).

The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyre gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300‐base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the ‘minicell’ technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC‐encoded and pyrD‐encoded polypeptides are 38000–40000 and 36000–38000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The ‘minicell’ experiments positively identified a protein chain of Mr 23000–24000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13000–14000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45000 ± 4000, this latter polypeptide (Mr 13000–14000) is hardly a component of the enzyme. Pyr‐lac + operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter‐clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

A mutant of Salmonella typhimurium with a defect in the regulation of pyr-gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. The mutant possesses 4-fold elevated pools of the pyrnmidine nucleoside triphosphates, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40-fold and 7-fold higher in the mutant than in the parent strain when grown in minimal media. Fur#iennore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5' -monophosphate decarboxylase are O3-fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the (3 and ,B' subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control ofpyr-gene expression in S. typhimurium.