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Volume 132,
Issue 5,
1986
Volume 132, Issue 5, 1986
- Biochemistry
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- Biochemistry
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Control of the cAMP Pathway by the Cell Cycle Start Function, CDC25, in Saccharomyces cerevisiae
M. L. Tripp and R. PiñtonWe investigated the relationship in Saccharomyces cerevisiae between the cell cycle start function, CDC25, and two mutants defining components of the cAMP pathway. The thermolabile adenylate cyclase mutant cyr1-2(ts) is phenotypically similar to the temperature-sensitive mutant cdc25(ts) in that both mutants, when shifted to the restrictive temperature, arrest in G1 of the cell cycle and permit the initiation of meiosis and sporulation. The mutant bcy1 [a lesion resulting in a low level of regulatory (R) subunit and a high level of active, catalytic (C) subunit of the CAMP-dependent protein kinase] suppresses the temperature-sensitive phenotype of cyr1-2(ts) and confers an asporogenous phenotype. We found that cdc25(ts) complemented cyr1-2(ts), and, unlike cyr1-2(ts), was not suppressible by bcy1, demonstrating that cyr1 and CDC25 must encode different functions. Also our results indicate that CDC25 does not encode the R subunit of the CAMP-dependent protein kinase. In addition, although the cdc25(ts) bcy1 double mutant was temperature sensitive like cdc25(ts), we found that the cdc25(ts) bcy1 homozygous diploid was asporogenous like bcy1/bcy1. The inability of the cdc25(ts)bcy1 double mutant to sporulate demonstrated that CDC25 does not encode the C subunit of the cAMP kinase, and indicated that the CDC25 function modulates the cAMP pathway to control meiosis and sporulation. Further, the temperature-sensitive phenotype of the double mutant, and hence the inability of bcy1 to suppress cdc25(ts), suggested that a second CDC25 cell cycle function exists which is independent of the cAMP pathway. Taken together, our genetic data indicated that the CDC25 protein has a dual regulatory role: (1) to control the decision between proliferation and differentiation by controlling the cAMP pathway; and (2) to control the cell cycle positively by a mechanism independent of the cAMP pathway. cdc25(ts) and cyr1-2(ts) conferred resistance to ammonium and methylamine. This suggests that CDC25 and the cAMP pathway work in concert as an ammonium signal-response system. We conclude that CDC25 controls meiosis and sporulation in response to nutritional stimuli by modulating the cAMP pathway.
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- Physiology And Growth
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Selectivity to K+ and Na+ of Protoplast Fractions Isolated from Different Regions of Aspergillus nidulans Hyphae
More LessThe selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1·5 mm-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.
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A New Method for Inducing Synchronous Growth of Dictyostelium discoideum Cells Using Temperature Shifts
More LessA method employing temperature shifts was used to obtain extremely synchronous growth of amoebae of Dictyostelium discoideum Ax-2 in axenic medium. When exponentially growing cells at 22·0 °C were shifted to 11·5 °C and shaken for 20 h, they exhibited good synchrony upon further culture at 22·0 °C. A relatively narrow range of low temperature (about 11·5 °C) and of treatment time (20 · 0°5 h) was necessary for inducing the best synchrony. This temperature-shift method is simple and practical and should be useful for detailed cellular and molecular investigations of growth regulation and of the relationship between the cell cycle and the differentiation of developing cells.
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Patterns of Individual Cell Growth in Marine Centric Diatoms
More LessRates of cell volume increase in individuals of five genera of centric marine diatoms were measured using time-lapse video microscopy. In continuous light, size increased continuously in Thalassiosira weissflogii and Lauderia borealis, while steps were observed during the growth of Stephanopyxis turris, Biddulphia aurita and Coscinodiscus sp. In the latter three species, the duration of the periods of no growth were well correlated with the generation time for individual cells. When the species exhibiting continuous growth in constant light were grown in a light/dark cycle of 12:12 h, the rate of size increase during the dark period was on average slower than during the light. Behaviour of individual cells was highly variable, however, and in L. borealis appeared to be related to the previous light history of each cell. The results suggest a regulatory coupling between the cell cycle and the time evolution of cell volume.
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Orthanilic Acid and Analogues as Carbon Sources for Bacteria: Growth Physiology and Enzymic Desulphonation
More LessCarbon-limited aerobic batch enrichment cultures were grown and 17 bacteria able to degrade orthanilic acid (2-aminobenzenesulphonic acid), sulphanilic acid, sulphonamide, 4-sulphobenzoic acid, and benzene-, toluene- and phenolsulphonic acids were isolated. The organisms could each use one to three of the substances. Strain O-1, a Pseudomonas sp., which utilized three of these compounds, was studied in detail. A complete mass balance was obtained for the growth of the organism in medium containing, for example, orthanilic acid, and a specific growth rate of 0·1 h-1 was observed. Cell extracts desulphonated six aromatic sulphonates. The enzyme(s) was soluble and was not synthesized in succinate-grown cells. Enzyme activity [about 40 μkat (kg protein)-1] was dependent on the presence of catalytic amounts of NAD(P)H.
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Effects of Ethanol and Other 1-Alkanols on Cell Autolysis and Macromolecular Synthesis by Bacillus subtilis
More LessThe addition of 1-alkanols to exponentially growing cultures of Bacillus subtilis led to an immediate reduction in growth rate and a rapid reduction in the rate of autolysis of cells in suspension. 1-Alkanols had no effect on autolysis when added to the non-growing suspensions. The effects were proportional to the concentrations added to cultures and to the lengths of the carbon chains up to octanol. The growth rate was immediately restored to that of the control when 0·7 m-ethanol was removed from cultures but the rate of cell autolysis only fully recovered after two to three generations. Ethanol (0·7 m) protected the organism against lysis and the bactericidal effects of β-lactam antibiotics; leakage of the intracellular pool was also greatly reduced. Despite some properties in common with the phenotype of lyt mutants or of cultures treated with cerulenin, the individual bacteria separated well, and were of normal shape and highly motile. Turnover of the walls was only slightly affected. Protein synthesis was not specifically inhibited but export of proteins was increased and included the appearance of novel proteins. Peptidoglycan synthesis was less affected than growth; after 3 h about twice as much peptidoglycan was present per unit of dry weight compared with control cultures. It seems unlikely that ethanol inhibited synthesis of autolytic enzymes although less activity could usually be extracted from the cells by 5 m-LiCl, since treatment with very low concentrations of neutral detergents reactivated cell autolysis. The walls of ethanol-grown cells were fully susceptible to autolytic enzymes.
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Environmental Regulation of the Methanol Oxidase System of Methylophilus methylotrophus
More LessMethylophilus methylotrophus was grown in continuous culture at different dilution rates under either methanol or oxygen limitation. Methanol dehydrogenase and cytochrome oxidase aa 3 were maximally repressed in oxygen-limited cultures and also in methanol-limited cultures at dilution rates close to mUmax, whereas cytochrome oxidase co was maximally repressed in methanol-limited cultures and also in oxygen-limited cultures at dilution rates close to μmax. The observed changes in the activity and concentration of methanol dehydrogenase were accompanied by approximately parallel changes in the whole cell respiration rate with ethanol, formaldehyde or acetaldehyde, but not with methanol. These and other results suggest that whole cell methanol oxidase activity reflects respiration from both methanol and its oxidation product, formaldehyde, and is regulated via repression–derepression of methanol dehydrogenase and the cytochrome oxidases. The methanol oxidase system is thus precisely tuned to the concentration of methanol or oxygen in the culture such that the latter is able to maintain a rate of respiration that satisfies the energy demands of the imposed growth rate.
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The Rate of Killing of Escherichia coli byβ-Lactam Antibiotics Is Strictly Proportional to the Rate of Bacterial Growth
E. Tuomanen, R. Cozens, W. Tosch, O. Zak and A. TomaszNongrowing bacteria evade the bactericidal activity of βbT-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two βbT-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0·7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to βbT-lactam antibiotics as the generation time was extended.
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Pathways and Regulation of Ammonium Assimilation in Streptomyces clavuligerus
More LessGlutamine synthetase, glutamate synthase and alanine dehydrogenase activities were detected in crude extracts of Streptomyces clavuligerus. Glutamate dehydrogenase and alanine: 2-oxoglutarate aminotransferase could not be found. Glutamate synthase levels were independent of the nitrogen source in the culture medium. Glutamine synthetase activity was repressed in the presence of ammonium, and a rapid inactivation of the enzyme was seen after ammonium shock. Alanine dehydrogenase was induced by alanine or ammonium concentrations higher than 20 mm. Glutamate and, in lower amounts, glutamine, alanine, ?gM-aminobutyrate and aspartate were the main constituents of the intracellular pool of free amino acids. Only the pool concentrations of glutamine and alanine were markedly influenced by the nitrogen source used for growth. Mutants devoid of glutamine synthetase, glutamate synthase, or alanine dehydrogenase were isolated. The pattern of utilization of nitrogen sources by the three classes of mutants indicated that the glutamine synthetase–glutamate synthase pathway is the only means of assimilation of ammonium in S. clavuligerus. Alanine dehydrogenase is not directly involved in that process, but a possible related function is discussed.
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Comparison of Extracellular Protein Profiles of Seven Serotypes of Mutans Streptococci Grown under Controlled Conditions
More LessExtracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass ≥ 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS–PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185–200 kDa), P2 (130–155 kDa) and P3 (60–95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.
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Mutants of Streptococcus pneumoniae That Contain a Temperature-sensitive Autolysin
More LessTwo mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-l-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 °;C or at 30 °C, although only the cell extracts obtained at 30 °;C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 °;C, mutant cultures grown at 30 °;C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 °;C.
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