Comparative effects of 5-fluorouracil on strains of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Comparative effects of 5-fluorouracil on strains of Bacillus megaterium. J. Bacteriol. 87:1011-1018. 1964.-Growth of Bacillus megaterium strain KM is severely inhibited by 5-fluorouracil (FU). Both thymidine and uridine are required to overcome this inhibition. The addition of uridine alone to a FU-inhibited culture permits good ribonucleic acid (RNA) and protein synthesis for the first 2 hr, but rather poor deoxyribonucleic acid synthesis. Uridine enhances the bactericidal effect of FU, promoting a decrease in the viable count of from 4 to 5 decades in 5 hr. Death begins after a 1-hr lag and is accompanied by hydrolysis of RNA and cell lysis, commencing during the 2- to 5-hr interval. The combination of FU and uridine is not bactericidal, when a methionine auxotroph is deprived of its required amino acid. Substrains of KM, partially resistant to FU, were isolated. Strain T(2) requires only thymidine to overcome the inhibitory effects of FU, whereas strain FU/2 requires only uridine. With a uridine auxotroph of strain KM, FU partially replaces uridine by permitting a small, but reproducible, increase in the amount of protein.     

Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1118-1122. 1964.-When strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, is allowed to undergo thymineless death on a minimal medium, the survivors are greatly enriched in polyauxotrophic mutants. Cells were irradiated with ultraviolet light, grown in the presence of thymidine and a complete amino acid mixture, and then starved for thymidine in the absence of amino acids. Doubly auxotrophic mutants (thymine(-) amino acid(-)) may account for more than 90% of the survivors. The most reproducible results were obtained when sucrose (0.4 m) was added to both growth and starvation media. Although the percentage of mutants among the survivors increases with the time of thymine starvation, the absolute number of double auxotrophs per milliliter decreases. It is probable that the extent of cross-feeding determines both the mutant yield and the mutants types. Substrains of KM:T(-) having additional requirements for each of the following amino acids have been isolated: histidine, threonine, tyrosine, tryptophan, arginine, isoleucine, methionine, serine, and cysteine.     

Thymineless death in Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Thymineless death in Bacillus megaterium. J. Bacteriol. 87:1079-1086. 1964.-Strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, rapidly loses the ability to multiply when incubated in the absence of thymine, on an otherwise sufficient medium. At 37 C, there is a lag of approximately 60 min, prior to the onset of exponential death (decrease of 1 decade per 50 min). The extent of the decrease in viable count varies from 4 to 5 decades after 5 hr of starvation. The cells die more slowly at 30 C (decrease of 1 decade per 120 min) after a lag of approximately 90 min. Thymine starvation permits substantial net ribonucleic acid (RNA) and protein synthesis, but only slight deoxyribonucleic acid synthesis. In contrast with the changes occurring at 30 C, thymineless death at 37 C is eventually accompanied by a rapid hydrolysis of RNA and by cell lysis. Chloramphenicol inhibits thymineless death at 37 C. Strain T(-)R(1), a derivative of strain KM:T(-), undergoes a very low rate of thymineless death at 37 C (decrease of 1 decade per 240 min). Neither hydrolysis of RNA nor cell lysis occurs during 8 hr of thymine starvation. Strain KM:T(-)H(-) (doubly auxotrophic for thymidine and histidine) requires histidine for maximal thymineless death at 37 C. Preincubation of this strain on the basal medium supplemented with thymidine alone enables the population to become increasingly immune to subsequent thymineless death.     

Isolation and characterization of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase.

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.     

Factors affecting transformation of Bacillus licheniformis

Thorne, Curtis B. (Fort Detrick, Frederick, Md.), and Harold B. Stull. Factors affecting transformation of Bacillus licheniformis. J. Bacteriol. 91:1012-1020. 1966.-Transformation systems involving two types of transformable mutants of Bacillus licheniformis 9945A were compared. Each system required its specific growth medium, but a single transformation medium could be used for both. Cells from a culture of optimal age were not competent, at least to any great extent, but they developed competence during incubation in a transformation medium. With each system, 3 to 5% of the recipient cells were transformed upon exposure to wild-type deoxyribonucleic acid (DNA) for 2 to 3 hr. When competent cells were exposed to DNA for 30 min, 1 to 2% of them were transformed. The data are interpreted to mean that cells were heterogeneous with respect to development of competence, and when properly grown cells were incubated in transformation medium some of them gained competence, whereas others lost it. If DNA was present during the entire period, the cells were transformed as they became competent and the transformants accumulated. However, during any short period of exposure to DNA, only those cells that were competent at the time were potential transformants. The high frequencies of transformation obtained in these studies made it feasible to prepare marked strains by transforming markers into recipient cells. These experiments demonstrated that the characteristics of the two transformation systems could not be attributed to specific nutritional markers. Presumably, each of the two series of highly transformable auxotrophic mutants also carried at least one other mutation that resulted in development of competence under the specific conditions.     

Properties of 5-fluorouracil-containing ribonucleic acid and ribosomes from Bacillus subtilis

Growth of a strain of Bacillus subtilis that requires uracil, thymine, adenine, and tryptophan in the presence of 5-fluorouracil (FU) results in the synthesis of ribonucleic acid (RNA) and ribosomes in which 55 to 65% of the RNA uracil has been replaced by the fluorine derivative. Examination of analogue-containing ribosomes by sucrose density gradient centrifugation and thermal denaturation studies suggests that, as far as the size, shape, and packing structure are concerned, extensive FU substitution has little or no effect. FU appears to replace uracil in RNA without selectivity for one RNA class over another, as determined by methylated albumin-kieselguhr column chromatography and sucrose density gradient centrifugation. The total amino acid content of the cells is markedly affected by growth in the presence of FU. The possibility of an FU effect on genetic translation is discussed.     

Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil

Reich, Melvin (The George Washington University School of Medicine, Washington, D.C.), and H. George Mandel. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar concentrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.     

Effect of Ethyl Picolinate on Germination, Growth and Sporulation of Bacillus spp.

Ethyl picolinate inhibited outgrowth and sporulation of Bacillus cereus T and laboratory isolates of B. megaterium AH2 and AV1 and B. brevis AG4. The outgrowth-inhibition was relieved by supplementing the medium before inoculation with any of the following: aspartate, asparagine, leucine, lysine, phenylalanine, tyrosine and vitamin-free casamino acids. The uptake of radioactive precursors in the synthesis of proteins, ribonucleic acid and deoxyribonucleic acid was prevented when ethyl picolinate was added before inoculation. Cultures incubated for a short period before the addition of ethyl picolinate assimilated radioactive precursors, outgrew and divided after a lag. None of the cultures containing ethyl picolinate sporulated. Growth was not inhibited when a portion of vegetatively growing culture was transferred to medium containing ethyl picolinate.     

A method for the enrichment of auxotrophic mutants of Serratia marcescens with the antibiotic azthreonam

A new method for the enrichment of cultures of Serratia marcescens for auxotrophic mutants has been developed. The method is based on the formation of filaments by growing cells in minimal medium M70 containing azthreonam. Auxotrophic mutants unable to grow in M70 do not form filaments. Mutants are collected from the culture by filtration.     

Occurrence in Bacillus megaterium of uridine 5''-diphospho-N-acetylgalactosamine and uridine 5''-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer.

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.     

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.     

Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization

Summary Strains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.Abbreviations and Symbols FU 5-fluorouracil - FUR 5-fluorouridine - FUdR 5-fluoro-2'deoxyuridine - FCR 5-fluorocytidine - FCdR 5-fluorodeoxycytidine - THUR 3, 4, 5, 6-tetrahydrouridine - UMP uridine monophosphate - CMP cytidine monophosphate - dUMP deoxyuridine monophosphate. Genes coding for: cytidine deaminase - edd uridine phosphorylase - udp thymidine phosphorylase - tpp purmnucleoside phosphorylase - pup uridine kinase (=cytidine kinase) - udk UMP-pyrophosphorylase - upp. CytR regulatory gene for cdd, udp, dra, tpp, drm and pup Enzymes EC 2.4.2.1 Purine nucleoside phosphorylase or purine nucleoside: orthophosphate (deoxy)-ribosyltransferase - EC 2.4.2.4 thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - EC 2.4.2.3 uridine phosphorylase or uridine: orthophosphate ribosyltransferase - EC 3.5.4.5 cytidine deaminase or (deoxy)cytidine aminohydrolase - EC 4.1.2.4 deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - EC 2.4.2.9 UMP-pyrophosphorylase or UMP: pyrophosphate phosphoribosyltransferase - EC 2.7.1.48 uridine kinase or ATP: uridine 5-phosphotransferase     

UV-mutagenesis in Bacillus subtilis. VII. Induction of auxotrophic mutants]

An experimental testing of material from thin-layered, transparent in passing light, colonies which appear with some frequency after plating Bacillus subtilis cells on agar medium with limited enrichment, has shown that such colonies are formed by auxotrophic mutants. The growth requirements for many of them has been identified. The most of mutants can be reversed to original phenotype by UV-irradiation. The frequency of auxotrophs increases after UV-irradiation of suspension of original cells. The sensitivity of auxotrophic mutants to inactivating action of UV-light is near to that of original cells, hence the increase of the frequency of mutants with dose is a result of induction, but not of selection of preexisting spontaneous auxotrophic mutants. The frequency of induced auxotrophs, in contrast to that of suppressor revertants, badly give way to declining in the time of temporary inhibition of postradiation growth. In the case of Bac, subtilis, the system of induced auxotrophic mutants on the medium with limited enrichment is rather comfortable in use and can be recommended for studying UV-induced mutagenesis in structural genes as well as for testing mutagenic activities.     

Reduction of the antineoplastic fluoropoyrimidine, 5-fluorouracil,to 5,6-dihydro-5-fluorouracil inEscherichia coli

A two-step culture ofE. coli, in presence of 5-fluorouracil (FU), allowed to obtain 5,6-dihydro-5-fluorouracil in the external medium. The first step led to large quantities of cells in which FU catabolic pathway was induced (complex culture medium, presence of thymidine, and growth up to the stationary phase); the second step was carried out in conditions in which FU catabolic pathway was favoured (minimal medium).     

Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium

Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium. J. Bacteriol. 87:1044-1050. 1964.-Penicillin and cycloserine do not inhibit the growth of protoplasts of Bacillus megaterium, indicating that inhibition of cell-wall synthesis is the only significant process by which they inhibit growth of bacteria. In contrast, bacitracin and vancomycin inhibit growth of protoplasts and bacteria at similar concentrations, indicating that they have important sites of action other than their known inhibition of cell-wall synthesis. At concentrations which inhibit mucopeptide synthesis, penicillin, bacitracin, and vancomycin each cause an increased rate of efflux of K ions from growing bacteria. This effect of penicillin is prevented by chloramphenicol or hypertonic sucrose, whereas the effects of bacitracin and vancomycin are unchanged under these conditions. It is concluded that bacitracin and vancomycin have direct effects on the cytoplasmic membrane, and it is proposed that their inhibition of cell-wall synthesis could be a consequence of these effects. Bacitracin and vancomycin do not compete with penicillin for binding to cells of B. megaterium, a further indication that they have a different primary site of action.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

Nutritional requirements ofCytophaga johnsonae and some of its auxotrophic mutants

A defined medium for growing cells ofCytophaga johnsonae was developed for use in studies of the genetics of gliding motility of this organism. The defined medium, containing only mineral salts and any of six sugars, supports good growth of actively motile cells of several strains ofC. johnsonae. Characteristics of growth in the defined medium and ability of cells to use alternative sources of carbon and nitrogen are reported. Certain compounds, when added to solid media, inhibit colony spread ofC. johnsonae without affecting motility of individual cells. Amino acid requirements were determined for 41 of 43 auxotrophic mutants isolated following penicillin selection of unmutagenized cells growing in the minimal medium.     

Selective Protection by Uridine of Growth Inhibition by 5-Fluorouracil (5FU) Mediated by 5FU Incorporation into RNA,But Not the Thymidylate Synthase Mediated Growth Inhibition by 5FU-Leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.     

Netropsin stimulates the formation of an extracellular proteinase and suppresses protein turnover in sporulating Bacillus megaterium

Abstract Netropsin stimulated the rate of synthesis of an extracellular metalloproteinase in Bacillus megaterium incubated in a sporulation medium. The antibiotic delayed but did not suppress the decrease in the ability to synthesize the proteinase occurring at later sporulation stages. Netropsin also stimulated the synthesis of the proteinase when added to a growing culture; it inhibited the increase of protein turnover which was switched on between the 2nd and 3rd hour in the sporulating population. No refractile spores were developed during 6 h at 35°C in the antibiotic-treated culture. In the control 60% of sporulating cells were observed under similar conditions.     

Selective protection by uridine of growth inhibition by 5-fluorouracil (5FU) mediated by 5FU incorporation into RNA, but not the thymidylate synthase mediated growth inhibition by 5FU-leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.