Effect of 2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone onMycobacterium smegmatis

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells ofMycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associate d with this cycle (glutamate, asparagine). DDA inhibits the incorporation of both¹⁴C-leucine and¹⁴C-adenine into appropriate macromolecules ofM. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity ofU-¹⁴C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth ofM. smegmatis probably through an interference with the cell energy-carbon metabolism.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

The pathway ofD-cycloserine biosynthesis inStreptomyces garyphalus

Incubation experiments using washed cells and toluene treated cells ofStreptomyces garyphalus showed that O-acetyl-L-serine and hydroxyurea are intermediates in the biosynthesis ofD-cycloserine. The formation of [¹⁴C]O-ureidoserine from O-acetyl-L-serine and hydroxyurea was demonstrated by incubating an enzyme solution with¹⁴C-labelled substrates. Desalted cell-free extract catalyzed the conversion of O-ureido-D-serine toD-cycloserine in a reaction requiring ATP and Mg²⁺. The results suggested the following pathway forD-cycloserine biosynthesis.     

Evidence for the role of intracellular glutamine level in the regulation of glutamine uptake in the cyanobacteriumAnabaena 7120

The role of intracellular glutamine concentration in the regulation of¹⁴C-glutamine uptake was studied in a diazotrophic cyanobacteriumAnabaena 7120. The uptake pattern was found to be biphasic, consisting of a rapid first phase lasting up to 60 s followed by a slower second phase. Azaserine, which could not inhibit in vitro and in vivo glutamine synthetase (GS) activity effectively, inhibited the¹⁴C-glutamine uptake. Glutamine uptake was also not significantly affected when glutamate, methylglutamate, aspartate, arginine, lysine, hydroxylysine, ornithine, and GS inhibitor,L-methionine-DL-sulfoximine (MSX) were simultaneously available during uptake assay, suggesting that glutamine uptake takes place via a general amino acid permease which does not, however, transport basic and acidic amino acids. The azaserine-treated cells had increased and decreased levels of glutamine and glutamate, respectively, suggesting that the increased intracellular glutamine level is responsible for the inhibition of¹⁴C-glutamine uptake and provides evidence here for the role of an intracellular glutamine pool in the regulation of¹⁴C-glutamine uptake inAnabaena 7120.     

Biosynthesis of nicotianamine in the suspension-cultured cells of tobacco (Nicotiana megalosiphon)

Summary Seven kinds of suspension cell cultures from five species ofNicotiana were screened for the occurrence of nicotianamine. Nicotianamine was detected in the cultured cells ofN. megalosiphon andN. plumbaginifolia.l-[l-¹⁴C]Methionine, which is the precursor of the mugineic-acid-family phytosiderophores and nicotianamine in barley plants, was incorporated into nicotianamine by the cultured cells ofN. megalosiphon both in vivo and in vitro. The advantage of the cultured tobacco cells for the study of the biosynthesis of nicotianamine and the mugineic-acid-family phytosiderophores is discussed.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Increased root exudation of14C-compounds by sorghum seedlings inoculated with nitrogen-fixing bacteria

Summary Organic components leaked fromSorghum bicolor seedlings (‘root exudates’) were examined by recovering¹⁴C labelled compounds from root solutions of seedlings inoculated withAzospirillum brasilense, Azotobacter vinelandii orKlebsiella pneumoniae nif-. Up to 3.5% of the total¹⁴C recovered from shoots, roots, and nutrient solutions was found in the root solutions. Inoculation with Azospirillum and Azotobacter increased the amounts of¹⁴C and decreased the amounts of carbohydrates in the root solutions. When sucrose was added as a carbon source for the bacteria, the increase of¹⁴C in the solutions did not occur. Quantities of¹⁴C found in the root solutions were proportional to amounts of mineral nitrogen supplied to the plants. Bacterial growth also was proportional to nitrogen levels. When sorghum plants were grown in soil and labelled with¹⁴CO₂, about 15% of the total¹⁴C recovered within 48 hours exposure was found in soil leachates.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Inhibition of cell division inMyxococcus xanthus by mecillinam

Increase in cell numbers is inhibited by adding mecillinam to vegetative cultures ofMyxococcus xanthus. However, both cell length and volume continue to increase in the presence of the antibiotic. Different size classes of cells increase in proportion to their initial size. Incorporation ofmeso-diamino[¹⁴C]pimelic acid into peptidoglycan and [³H]uridine into RNA is not immediately affected by mecillinam. It is suggested that mecillinam inhibits the formation of new sites of wall synthesis, and these are necessary for cell division to occur.     

Na+ requirement for glutamate-dependent sugar transport byFusobacterium nucleatum ATCC 10953

Resting cells ofFusobacterium nucleatum ATCC 10953, when provided with glutamic acid (Na⁺ salt) as fermentable energy source, rapidly accumulated [¹⁴C]glucose, from the medium. Sugar accumulation was not observed when Na⁺ glutamate was replaced by ammonium glutamate. However, addition of Na⁺ (chloride) to the latter system elicited uptake of [¹⁴C]glucose by the organism. Of other monovalent cations tested, only Li⁺ was found to be slightly stimulatory, but K⁺, Rb⁺, and Cs⁺ ions were ineffective. For determination of the role(s) of Na⁺ in sugar accumulation, the transport of [¹⁴C]glucose and [¹⁴C]glutamic acid by the cells was studied independently, with lysine as an alternate (and Na⁺-independent) energy source. In the presence of lysine, cells ofF. nucleatum 10953 accumulated [¹⁴C]glucose from a Na⁺-free medium, but, in contrast, uptake and fermentation of [¹⁴C]glutamic acid was Na⁺-dependent. The glucose transport system is Na⁺-independent. However, our data indicate dual role(s) for Na⁺ in the transport and intracellular metabolism of glutamic acid. The Na⁺-dependent glutamate fermentation pathway provides the necessary energy for active transport of glucose by the resting cell.     

A rapid and simple procedure for the preparation of the two bacterial cell wall peptidoglycan nucleotide precursors labeled in their amino sugars

The two bacterial cell wall peptidoglycan precursors UDP-MurNAc-l-Ala-d-iso Glu-l-Lys-d-Ala-d-Ala and UDP-GlcNAc labeled in their amino sugars with either tritium or carbon-14 accumulated in cells ofMicrococcus luteus that were incubated for short periods of time in a minimal medium to which [¹⁴C]glucose or [³H]glucose together with Vancomycin were added. The radioactive nucleotides were extracted from the cells with cold trichloroacetic acid, and their purification was achieved by paper electrophoresis followed by paper chromatography.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

In vivo and in vitro studies of nitrate reductase regulation in Asperillus nidulans.

Summary Induced wildtype cells ofA. nidulans rapidly lost NADPH — linked nitrate reductase activity when subjected to carbon and or nitrogen starvation. A constitutive mutant at the regulatory gene for nitrate reductase,nirA ^c1, rapidly lost nitrate reductase activity upon carbon starvation. This loss of activity is thought to be due to a decrease in the NADPH concentration in the cells. Cell free extracts from wild-type cells grown in the presence of nitrate, rapidly lost their nitrate reductase activity when incubated at 25° C. NADPH prevented this loss of activity. Wildtype cells grown in the presence of nitrate and urea have a higher initial NADPH: NADP⁺ ratio and cell free extracts from such cells lost their nitrate reductase activity slower than extracts of cells grown with nitrate alone.The Pentose Phosphate Pathway mutant,pppB-1, had a lower NADPH concentration compared with the wildtype grown under the same conditions and cell free extracts lost their nitrate reductase activity more rapidly than the wildtype. Cell free extracts ofnirA ^c-1 and a non-inducible mutant for nitrate reductase,nirA ^--14, upon incubation lost little of their nitrate reductase activity.     

Utilization of glucose and amino acids byBacteroides intermedius andBacteroides gingivalis

Growth ofBacteroides intermedius was promoted only moderately by glucose, and the incorporation of¹⁴C-glucose into cells was limited. WithBacteroides gingivalis growth promotion was negligible and glucose incorporation even more restricted. Both species grew prolifically on protein hydrolysates containing peptides, but grew poorly on acid-hydrolyzed casein even when supplemented with amino acids. These results are discussed in relation to the ecological distribution of these species compared to saccharolytic bacteroides.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Application of percoll density gradients in studies of the adhesion ofStreptococcus pyogenes to human epithelial cells

A new assay was used to study the adhesion ofStreptococcus pyogenes strains to epithelial cells. [³H]thymidine-labeled bacteria were incubated with standardized preparations of epithelial cells collected from oral-pharyngeal surfaces of human volunteers. The mixtures were then centrifuged in 50% Percoll to form a density gradient. Epithelial cells with attached bacteria formed a band near the top of the tube, whereas unattached bacteria were located near the bottom. The epithelial cells were collected on membrane filters, and the number of adherent bacteria was then determined by scintillation counting.The abilities of M-protein-positive (M⁺) and M-protein-negative (M^–) strains ofS. pyogenes to attach to human pharyngeal, buccal, and tongue epithelial cells were compared. The results obtained confirmed the significant difference previously shown to exist between the attachment of M⁺ and M^– strains to human epithelial cells. M⁺ strains ofS. pyogenes exhibited a much greater ability to bind to pharyngeal epithelial cells than did M^– variants. Also, M⁺ strains were bound in higher numbers to pharyngeal epithelial cells than to buccal or tongue epithelial cells. The adhesion ofS. pyogenes strains to epithelial cells was time dependent, and a significant increase in the adhesion of M⁺ strains occurred after 3–4 h of exposure of the bacteria to epithelial cells.The adsorption ofS. pyogenes strains to epithelial cells was described by a Langmuir isotherm. With this model, the number of binding sites and the affinities of the streptococci for epithelial cells were estimated. Significantly higher numbers of binding sites were calculated to be present on pharyngeal epithelial cells for M⁺ strains ofS. pyogenes than on buccal cells. However, the affinity of the organisms was similar for both types of cells.Adsorption of M⁺ strains to human pharyngeal epithelial cells was inhibited by certain galactosides and fucose, but not by glucose or xylose. This suggests that saccharide moities play a role in the binding of M⁺ strains ofS. pyogenes to human pharyngeal epithelial cells.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Hg2+ and Cd2+ induced inhibition of light induced proton efflux in the cyanobacteriumAnabaena flos-aquae

Light induced proton efflux in intact cells ofAnabaena flos-aquae is inhibited by the heavy metals Hg²⁺ and Cd²⁺. Furthermore, Hg²⁺ and Cd²⁺ reduced the¹⁴CO₂ fixation, oxygen evolution and carbonic anhydrase activity responsible for H⁺ efflux.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Action Spectrum of Photosynthesis for Skeletonema costatum Obtained with Carbon-14

The quantized action spectrum of photosynthesis for Skeletonema costatum was obtained from values of photosynthetic ¹⁴CO₂ uptake at various wavelengths of light isolated “with a diffraction grating monochromator. The quantized action spectrum of photosynthesis exhibited maxima at wavelengths similar to maxima in the absorption spectrum, in vivo, of a suspension of S. costatum cells. While the ¹⁴CO₂ technique will provide an accurate action spectrum of photosynthesis for diatoms, a large number of samples is required in order to minimize sampling error.