- Volume 132, Issue 5, 1986
Volume 132, Issue 5, 1986
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Identification and Partial Purification of a Dipeptidyl Aminopeptidase from Streptococcus faecalis
More LessA dipeptidyl aminopeptidase was identified in Streptococcus faecalis JH2SS and was partially purified (approximately 245-fold) by HPLC. Gel filtration chromatography indicated an M r of 140 000. The partially purified enzyme exhibited a requirement for Co2+. The pH optimum for the hydrolysis of l-Val-l-Ala-p-nitroanilide was approximately 9·5. The apparent K m for this substrate was 0·22 mm. The enzyme preferentially hydrolysed X-Ala-Y substrates, but also utilized X-Pro-Y substrates, and therefore is most closely related to the mammalian dipeptidyl aminopeptidase II (EC 3.4.14.-). The enzyme was inhibited by p-chloromercuribenzoate, but not by iodoacetate, N-ethylmaleimide or the serine protease inhibitor phenylmethylsulphonyl fluoride.
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The Oxidation of Glucose by Acinetobacter calcoaceticus: Interaction of the Quinoprotein Glucose Dehydrogenase with the Electron Transport Chain
More LessThe coupling of the quinoprotein glucose dehydrogenase to the electron transport chain has been investigated in Acinetobacter calcoaceticus. No evidence was obtained to support a previous suggestion that the soluble form of the dehydrogenase and the soluble cytochrome b associated with it are involved in the oxidation of glucose. Analysis of cytochrome content, and of reduction of cytochromes in membranes by substrates, and of sensitivity to cyanide indicated that glucose, succinate and NADH are all oxidized by way of the same b-type cytochrome(s) and cytochrome oxidases (cytochrome o and cytochrome d). Mixed inhibition studies [with KCN and hydroxyquinoline N-oxide (HQNO)] showed that the b-type cytochrome(s) formed a binary complex with the o-type oxidase and that there was thus no communication between the electron transport chains at the cytochrome level. Measurements of the reduction of ubiquinone-9 by glucose and NADH, and inhibitor studies using HQNO, indicated that the ubiquinone mediates electron transport from both the glucose and NADH dehydrogenases. In some conditions the quinone pool facilitated communication between the ‘glucose oxidase’ and ‘NADH oxidase’ electron transport chains, but in normal conditions these chains were kinetically distinct.
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- Biochemistry
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Deficiency of Uncoupler-stimulated Adenosine Triphosphatase Activity in Yeast Mitochondria
More LessOligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi–ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.
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Control of the Flux in the Arginine Pathway of Neurospora crassa: Effects of Co-ordinate Changes of Enzyme Concentration
More LessThe flux to arginine was determined in growing mycelium of Neurospora crassa carrying the aga mutation, by measuring the exponential growth rate and the arginine content of the free pool and that in protein. Derepression of the enzymes in the pathway by a factor of about three resulted in a 14% increase in the flux. Using a mutant (cpc-1), which affected the cross-pathway control, the pathway enzymes were found to have about threefold lower activities. This resulted in a 16% decrease in the measured flux. The effect of arginine acting as a feedback inhibitor of acetylglutamate kinase was estimated in an arg-12 mutant by varying the steady-state arginine concentration using histidine as a competitive inhibitor of the citrulline uptake. It is concluded that the feedback loop is mainly responsible for the small response of the flux to the large coordinate changes in the pathway enzymes. The results are discussed in terms of control analysis of metabolic systems.
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Purification and Characterization of the Outer Membrane Associated Alkaline Phosphatase of Lysobacter enzymogenes
More LessThe alkaline phosphatase (EC 3.1.3.1) associated with the outer membrane of Lysobacter enzymogenes was solubilized from a crude membrane preparation with detergent and purified 219-fold using ion-exchange chromatography and gel filtration. The yield of the purified enzyme was 21% and the specific activity was 438 units mg-1. The enzyme was most active at pH 8·5, readily hydrolysed 5'-, 2'- and 3'-ribose and deoxyribose nucleotides, glucose 6-phosphate, glycerophosphates and p-nitrophenylphosphate and was strongly inhibited by EDTA and 8-hydroxyquinoline. The EDTA-inhibited enzyme could be reactivated to some extent by CoCl2 and more effectively by ZnCl2. The phosphatase was slightly activated by p-nitrophenylphosphate and from kinetic studies using this substrate two K m values, 0·56 x 10-4 m and 3·4 x 10-4 m, were estimated. The enzyme retained activity in the presence of SDS, unless it was heated, and had an apparent M r of 69 000, as determined by SDS-PAGE. Gel filtration data suggested that the native enzyme might consist of at least two subunits. The properties of the enzyme were consistent with the view that it is held in the outer membrane by hydrophobic interactions
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- Development And Structure
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Glycogen and Trehalose Accumulation during Colony Development in Streptomyces antibioticus
More LessStreptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.
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- Ecology
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Water Potential, Growth and Cellulolysis of Fungi Involved in Decomposition of Cereal Residues
N. Magan and J. M. LynchThe effect of water potential and temperature on growth and cellulolysis of 10 soil fungi which colonize cereal crop residues was determined in vitro. On 2% (w/v) milled straw agar all species grew in the range -0·7 to -7·0 MPa at both 10 and 20 ·C. Growth rates differed depending on the solute type used to control water potential. In general, but with the exception of the Penicillium spp., growth decreased with decreasing water potential. Hyphal growth on sterile straw internode segments was much less than that on 2% straw agar. Fusarium culmorum and Trichoderma harzianum colonized straw pieces best at high potential (-0·7 MPa) while only F. culmorum and the Penicillium spp. grew at low potential (-7·0 MPa). Trichoderma spp., Gliocladium spp. and Chaetomium globosum cleared cellulose agar most rapidly, depth of clearing decreasing with water potential in the range -0·7 to -2·8 MPa at 20 ·C. The relationship between dry weight loss of inoculated cellulose filter paper and water potential was more variable.
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The Effect of Lowering the pH on the Composition and Metabolism of a Community of Nine Oral Bacteria Grown in a Chemostat
More LessNine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0·05 h-1 (mean generation time 13·9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7·0, pH control was discontinued. The pH then decreased until it stabilized at pH 4·1 after 9 d (16 generations), while the E h rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7·0, ‘Streptococcus mitior’, Veillonella alcalescens and S. sanguis were most numerous while at pH 4·1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4·6. The survival at pH 4·1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7·0, the community growing at pH 4·1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.
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- Biochemistry
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- Biochemistry
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Characterization of Aspergillus nidulans Mutants in Carbon Metabolism Isolated after d-Galacturonate Enrichment
More LessA selective method for the isolation of Aspergillus nidulans mutants defective in the pyruvate dehydrogenase complex was devised. The essential steps in the procedure were a mutagenic treatment of conidia with X-rays to about 50% survival, followed by filtration enrichment in minimal medium with d-galacturonate as sole carbon source, and rescue on complete medium with acetate. The mutants thus isolated were phenotypically characterized on the basis of growth tests, and different genotypes were assigned on the basis of complementation tests. The majority of the mutants that were unable to utilize galacturonate were defective in one of the components of the pyruvate dehydrogenase complex. In addition, mutants defective in pyruvate carboxylase, mutants defective in glycerol catabolism and some novel mutants which were only unable to use d-galacturonate as carbon source were found. At least two genes were shown to be involved in d-galacturonate metabolism.
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- Genetics And Molecular Biology
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Conjugational Fertility and Location of Chloramphenicol Biosynthesis Genes on the Chromosomal Linkage Map of Streptomyces venezuelae
More LessIn Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physicallyundetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by ‘lethal zygosis’ elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1–2 o'clock region of the S. coelicolor A3(2) map.
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pIN32: a Cointegrate Plasmid with IncHI2 and IncFII Components
An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 °C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 °C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184·3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.
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Genetic, Functional and Sequence Analysis of the xylR and xylS Regulatory Genes of the TOL Plasmid pWW0
More LessMutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of ~ 37 kDa.
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Functions Encoded by the Yeast Plasmid pSB3 Isolated from Zygosaccharomyces rouxii IFO 1730 (Formerly Saccharomyces bisporus var. mellis)
More LessFunctions encoded by the yeast plasmid pSB3 were analysed in Saccharomyces cerevisiae and Zygosaccharomyces rouxii. The autonomously replicating sequence (ARS) and partitioning mechanisms of pSB3 worked as satisfactorily in Z. rouxii ME3 as in the native host Z. rouxii IFO 1730 (formerly Saccharomyces bisporus var. mellis). The ARS in Z. rouxii ME3 was located within a 168 bp BanII–HindIII fragment spanning part of the inverted repeat (IR) and a unique region contiguous to it. The FLP enzyme (responsible for the intramolecular recombination at IRs) of pSB3 was functional in Z. rouxii ME3. In spite of the similarity of the putative recognition site for the FLP enzyme in pSB3 and pSR1, the FLP enzyme of pSB3 did not recognize the recombination site of pSR1, and the FLP enzyme of pSR1 did not use the recombination site of pSB3. Three transcripts of 3·4, 1·8 and 1·1 kb from pSB3 in Z. rouxii ME3 were identified by Northern blotting; they encompassed the A, B and C genes, respectively. pSB3 contained a region from which no poly(A)-containing RNA was transcribed.
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Cloning and Expression of Clostridium acetobutylicum Endoglucanase, Cellobiase and Amino Acid Biosynthesis Genes in Escherichia coli
More LessClostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4·9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli. Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel. The endoglucanase activities observed in cell extracts of E. coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C. acetobutylicum P270. Complementation of E. coli arg and his mutations by cloned C. acetobutylicum DNA was also observed.
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Estimation of the M r of Isocitrate Lyase Messenger RNA from Chlorella fusca
More LessThe mRNA for the adaptive enzyme isocitrate lyase (ICL) from Chlorella fusca has been identified by fractionation of total poly(A)-containing RNA and in vitro translation followed by immune precipitation. The M r of ICL mRNA was approximately 8·0 x 105, which is in good agreement with a previous estimate obtained by in vivo double-labelling.
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- Pathogenicity And Medical Microbiology
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Enhancement of Streptococcus faecalis Infection and Complement Depletion in Yeast-treated Mice
More LessEnhancement of Streptococcus faecalis infection and lowering of the complement level have been demonstrated in mice injected with a heat-treated suspension of baker's yeast (Saccharomyces cerevisiae). The leucocyte response to the infection was not affected. The yeast preparation showed, in vitro, an intense anti-complementary activity on mouse serum and interfered with the microbial killing function of the mouse peritoneal macrophages. No significant stimulation of the growth of S. faecalis in vitro in the presence of the yeast was observed. The enhancement of the infection in mice treated with the yeast seems to be mediated, mainly, by complement depletion.
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Effects of in vitro Growth Phase on the Pathogenesis of Salmonella typhimurium in Mice
More LessThe growth phase of a bacterial (Salmonella typhimurium) culture was shown to have pronounced effects on the pathogenic properties of the harvested bacteria. Salmonellae obtained from a culture in primary (exponential) growth phase (PP) were more readily cleared from the blood and more readily killed by phagocytes than were salmonellae obtained from a more slowly growing secondary growth phase (SP) culture. PP salmonellae were observed to cause death of mice sooner than SP salmonellae. This appeared to be because the more rapid growth of PP, as compared to SP, salmonellae continued in the liver and spleen for several hours following intravenous injection, and more than compensated for their high in vivo death rate. As a result, within 4 h there were approximately 10-fold more live salmonellae in the spleens and livers of mice that had received PP, as compared to SP, salmonellae. This 10-fold difference was maintained until the death of the mice, indicating that after the first 4 h post-inoculation, the net in vivo growth of the salmonellae was the same regardless of their growth phase in the inoculating culture. This transition between PP and SP salmonellae occurred long before a dense stationary phase culture was obtained. Salmonellae grown in minimal media exhibited the biological properties of SP salmonellae and never entered as rapid a growth phase as did salmonellae in complete media.
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Iron Regulated Outer Membrane Proteins of Escherichia coli: Variations in Expression Due to the Chelator Used to Restrict the Availability of Iron
More LessIron restriction was induced in Escherichia coli O111, E. coli O164 and E. coli C by growing the organisms in trypticase soy broth containing ovotransferrin, desferal, EDDA (ethylenediamine-dihydroxyphenylacetic acid) or α,α'-dipyridyl. There were marked qualitative and quantitative differences in the iron regulated outer membrane proteins expressed in the presence of the various iron chelators. Differences in the kinetics of growth were also noted. E. coli C was devoid of a ferric enterobactin iron uptake system.
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Lipopolysaccharide Alteration is Associated with Induced Resistance of Neisseria gonorrhoeae to Killing by Human Serum
More LessOn SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 °C with low M r fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher M r component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher M r component and a weaker reaction with the component of low M r. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high M r LPS moiety.
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A Proposed Model to Explain Persistent Infection of Host Cells with Coxiella burnetii
More LessL929 mouse fibroblast cells and J774 macrophage-like cells are both susceptible to persistent infection with the Q fever agent Coxiella burnetii. Previously this laboratory has shown that persistently infected cell populations multiply with unaltered generation times or cell cycle progression. It has also been reported by others and us that highly infected cells typically exhibit one large parasite-containing vacuole. We now report that lightly and heavily infected cells are capable of division and in the process segregate the parasite-containing vacuole into one of the emerging daughter cells; the companion daughter cell emerges parasite-free. This asymmetric division of infected cells, revealed via photomicrography of stained cells, accounts for the appearance of uninfected cells within persistently infected host cell populations that were previously 100% infected. Some of the persistently infected L929 populations were maintained in culture for over two years without the addition of normal cells.
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- 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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