Chloramphenicol inhibition of Pythium ultimum and Rhodotorula glutinis

Summary The growth of Rhodotorula glutinis is inhibited by both D-threo chloramphenicol and an L-threo isomer of chloramphenicol (lacking the dichloroacetyl group), causing an increase in the mean generation time, in a variety of media, approximately proportional to the concentration of antibiotic. The antibiotic is not removed from the growth medium in any quantity during this inhibition of growth. The oxygen uptakes of normal and chloramphenicol-grown cells of R. glutinis are similar when expressed on a dry weight basis. The oxygen uptake of normal and L-threo isomer-grown cells is strongly inhibited by antimycin A, whereas D-threo chloramphenicol-grown cells are unaffected. There was no evidence to suggest that any uncoupling of phosphorylation occurred with either isomer. Pythium ultimum mycelium also showed similar oxygen uptakes per unit dry weight whether grown in the presence or absence of D-threo chloramphenicol. The D-threo chloramphenicol-grown mycelium was also insensitive to antimycin A in contrast to the normal mycelium which was strongly inhibited. P. ultimum grows slowly in the presence of 100 g/ml D-threo chloramphenicol in a glucose salts medium, but is completely inhibited by a similar concentration in a glycerol salts medium. The L-threo isomer does not inhibit the growth of P. ultimum.The mitochondria of Rhodotorula glutinis show a progressive disorganization when grown in the presence of increasing concentrations of D-threo chloramphenicol up to 1000 g/ml. There is an associated over synthesis of cell wall material in the higher concentrations of the antibiotic. The L-threo isomer produces no obvious fine structural abnormalities even at concentrations of 1000 g/ml.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition

The possible role of L-ascorbate (AsA) as a biochemical signal during the interactions between photosynthesis and respiration was examined in leaf discs of Arabidopsis thaliana. AsA content was either decreased as in AsA-deficient vtc1 mutants or increased by treatment with L-galactono-1, 4-lactone (L-GalL, a precursor of AsA; EC 1.3.2.3). In mutants, photosynthesis was extremely sensitive to both antimycin A (inhibitor of the cytochrome c oxidase pathway [COX pathway]) and salicylhydroxamic acid (SHAM, inhibitor of the alternative pathway [AOX pathway]), particularly at high light conditions. Mitochondrial inhibitors lowered the ratio of reduced AsA to total AsA, at high light, indicating oxidative stress in leaf discs. Elevation of AsA by L-GalL decreased the sensitivity of photosynthesis at high light to antimycin A or SHAM, sustained photosynthesis at supraoptimal light and relieved the extent of photoinhibition. High ratios of reduced AsA to total AsA in L-GalL-treated leaf discs suggests that L-GalL lowers oxidative stress. The protection by L-GalL of photosynthesis against the mitochondrial inhibitors and photoinhibition was quite pronounced in vtc1 mutants. Our results suggest that the levels and redox state of AsA modify the pattern of modulation of photosynthesis by mitochondrial metabolism. The extent of the AOX pathway as a percentage of the total respiration in Arabidopsis mesophyll protoplasts was much higher in vtc1 than in wild type. We suggest that the role of AsA becomes pronounced at high light and/or when the AOX pathway is inhibited. While acknowledging the importance of the COX pathway, we hypothesize that AsA and the AOX pathway may complement each other to protect photosynthesis against photoinhibition.     

Studies on the respiratory system of Aspergillus oryzae I. Development of respiratory activity during germination in the presence and absence of antimycin A

Active growth of Aspergillus oryzae was observed when conidiawere inoculated into a medium containing antimycin A. Immediatelyafter adding antimycin A, to young mycelia germinated in itsabsence, growth stopped, but began again after several hours.This restored growth was antimycin A-insensitive. Percentagegermination was the same in the presence and absence of thisdrug. It seems that drug-resistant germination and growth donot result from selection of resistant cells but result frominduction of antimycin A-insensitive mitochondria in the wholepopulation. Endogenous respiration of cells germinated in theabsence of antimycin A was inhibited by this drug, whereas thatof cells grown in the presence of antimycin A was completelyinsensitive. Antimycin A-sensitivity of cellular respirationseems to determine the effect of this drug on mycelial growth.Mitochondria were isolated from mycelia grown in the presenceand absence of this drug. The difference in antimycin A-sensitivityin endogenous respiration was attributed to a difference inproperties of the mitochondrial respiratory systems. ¹Present address: Department of Chemistry, Institute of MedicalScience, University of Tokyo, Tokyo, Japan (Received December 21, 1969; )     

The effect of respiratory inhibitors on the fermentative ability of Pichia stipitis,Pachysolen tannophilus and Saccharomyces cerevisiae under various conditions of aerobiosis

Summary The growth and ethanol production by the d-xylose-fermenting yeasts Pichia stipitis and Pachysolen tannophilus under various conditions of aerobiosis responded similarly to the addition of the respiratory inhibitors potassium cyanide (KCN), antimycin A (AA), sodium azide and rotenone. However, the d-glucose-fermenting yeast Saccharomyces cerevisiae differed markedly from these yeasts in response to the inhibitors. In general the growth of the d-xylose-fermenting yeasts was inhibited by the respiratory inhibitors while ethanol production was either stimulated (especially when oxygen was available) or unaffected or inhibited by rotenone or AA or KCN and sodium azide, respectively. However, by exception KCN and AA stimulated ethanol production under aerobic conditions by Pichia stipitis and Pachysolen tannophilus respectively. Stimulatory or inhibitory effects by respiratory inhibitors were less marked in S. cerevisiae. These data suggest that unimpaired mitochondrial function is necessary for growth on d-xylose and optimal d-xylose fermentation. A requirement for membrane generated energy during d-xylose utilisation is indicated by 2,4-dinitrophenol inhibition of growth and fermentation.     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.     

Stimulation of amino acid transport inSaccharomyces cerevisiae by metabolic inhibitors

Inhibitors of energy metabolism (3-ohlorophenylhydrazonomalononitrile, antimycin A, iodoacetamide, dicyclohexylcarbodiimide) but not of transport (uranyl ions) stimulate at low concentrations the uptake ofl-leucine,l-glutamic acid,l-argimne and, to a lesser degree, of 2-aminoisobutyric acid inSaccharomyces cerevisiae. The effect is apparent only after augmenting the energy reserves of cells by preincubation withd-glueose or, more strikingly, with ethanol. It is absent in a mutant (op₁) lacking the translocation system for ADP-ATP in mitochondria. The presence of two different energy reserves for amino acid transport is indicated (one in energy-poor, the other in energy-rich cells). The stimulating effect appears to be caused by a retarded degradation of the transport proteins as occurs at a lowered level of mitochondria-produced ATP.     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

Utilization of lactose in non-respiring cells of the yeast Debaryomyces polymorphus

The facultative anaerobic yeast Debaryomyces polymorphus ferments glucose and galactose but does not utilize the disaccharide lactose under anaerobic conditions. The activity of the intracellulary located -galactosidase was not affected by anaerobiosis. Hence, the transport of lactose appears to be limiting for lactose utilization. The uptake of lactose (and of its metabolizable analogue, 4-nitrophenol--d-galactoptranoside) was mediated by an inducible transport system and it was strictly dependent on metabolic energy. Anaerobic conditions inhibited the transport of lactose completely as did the uncoupler carbonylcyanide-m-chlorophenyl-hydrazone, the electron transport chain inhibitors rotenone, antimycin A, potassium cyanide and the ATPase inhibitor diethylstilbestrol under aerobic conditions. Transport inhibited by antimycin A was resumed by adding ascorbate+tetramethyl-p-phenylenediamine. Glucose was taken up by a constitutive transport system, even in anaerobic cells it was still about five times faster than the uptake of lactose in respiring cells. Thus, monosaccharides can energize their uptake by glycolysis and represent, in contrast to lactose, fermentable, substrates in D. polymorphus.Abbreviations 4NPßgal 4-nitrophenol--d-galactopyranoside - TMPD tetramethyl-p-phenylenediamine Dedicated to Professor Augustin, Betz at the occassion of his 65th birthday.     

Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes

Mitochondria are the main source of reactive oxygen species (ROS). The aim of this work was to verify the ROS generation in situ in HeLa cells exposed to prooxidants and antioxidants (menadione, tert-butyl hydroperoxide, antimycin A, vitamin E, N-acetyl-l-cysteine, and butylated hydroxytoluene) using the ROS-sensitive probes 6-carboxy-2,7-dichlorodihydrofluorescein diacetate di-acetomethyl ester (DCDHF) and dihydrofluorescein diacetate (DHF). Mitochondria were counterstained with the potential-sensitive probe tetramethylrhodamine methyl ester perchlorate (TMRM). Both DCDHF and DHF were able to detect the presence of ROS in mitochondria, though with distinct morphological features. DCDHF fluorescence was invariably blurred, smudged, and spread over the cytoplasm surrounding the major mitochondrial clusters. On the contrary, DHF fluorescence was sharp and delineated thin filaments which corresponded in all details to TMRM-stained mitochondria. These data suggest that DCDHF does not reach the mitochondrial matrix but is oxidized by ROS released by mitochondria in the cytosol. On the other hand, DHF enters mitochondria and reacts with ROS released in the matrix. Cytosolic (DCDHF+) ROS but not matrix (DHF+) ROS, were significantly decreased by vitamin E. N-acetyl-l-cysteine was effective in reducing DCDHF and DHF photooxidation in the medium, but was unable to reduce intracellular ROS. ROS generation was accompanied by partial mitochondrial depolarization.     

l-lactate or pyruvate stimulated growth of novikoff rat hepatoma cells

Summary Novikoff rat hepatoma cells (subline N1S1-67) grew when 30mm l-lactate or pyruvate was substituted ford-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lackingl-lactate, pyruvate ord-glucose, cell growth of 0.42 generation was obtained. Growth withl-lactate was dependent on thel-lactate concentration up to 30mm at which the greatest increase in cell number occurred. Significant growth did not occur whend-lactate, glycerol, acetate, α-ketoglutarate, succinate or malate, each at 30mm, was substituted ford-glucose. Growth in the medium containingl-lactate was not due to the utilization ofd-glucose or some other substrate carried into the culture with the inoculum. Medium contamination byd-glucose was insufficient to explain the growth obtained in the medium containingl-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration ofl-lactate in the medium remained unchanged. The increase in cell number cannot be explained byl-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoproteins in the medium.l-Lactate does not serve as a significant carbon or energy source in the growth of these cells. This investigation was supported by grants from the National Institute of Allergy and Infectious Disease, the National Science Foundation, and the United States Public Health Service.     

Regulation of biosynthesis of monensins on chemically defined medium

Addition ofL-valine andDL-isoleucine to the cultivation medium ofStreptomyces cinnamonensis was found to affect the ratio of synthesized monensins A and B. In the presence ofL-valine monensin A is synthesized predominantly, whereas in the presence ofDL-isoleucine the production of monensin B increases.     

Structure and Function of Mitochondria in Crithidia fasciculata*

SYNOPSIS. In correlating mitochondrial structure with composition and function of the electron-transport system in Crithidia fasciculata, failure to find cytochrome oxidase in isolated mitochondria coincided with the presence of longitudinally-oriented lamellar cristae in the mitochondria in intact cells. Cytochromes b and c were detected spectrophotometrically. Respiration of intact cells and mitochondria, measured polarographically, was sensitive to 10⁻⁴ M antimycin A and 5 × 10⁻⁴ M KCN. The difficulty in detecting cytochrome oxidase and the biogenesis of mitochondria are briefly discussed.     

Influence of 2,4-D,TIBA and 3,5-D on the growth response of cultured maize embryos

Induction of embryogenic calli from immature zygotic embryos of maize requires the presence of 2,4-D or similar auxin-like growth regulators in the culture medium. Pulse-chase experiments with 2,4-D, using various concentrations of 2,4-D in the induction medium were tested in relation to induction of callus in the embryogenic inbred line A188 and the non-embryogenic inbred A632. Interactions of 2,4-D, 3,5-D and the auxin transport inhibitor TIBA were also studied. Pulse-chase experiments showed that exposure to 2,4-D influenced the culture response from 0.5 h onwards. After a pulse of 0.5 h, shoot and root elongation of the embryo was stimulated. A pulse of 16 h or longer induced outgrowths and callus formation at the basal side of the scutellum. Pulses of 7 days and longer resulted in the induction of friable embryogenic Type II callus in A188. Embryos were cultured at 2,4-D concentrations ranging from 0.002 to 2000 mg l⁻¹ and optimal concentration for the induction of embryogenic callus in A188 was 2 mg l⁻¹. At lower concentrations there was a transition between callus formation and germination; at increasing concentrations, callus induction was reduced and finally growth responses became blocked. When TIBA was added to medium without 2,4-D, root elongation decreased in a dose-dependent way suggesting the need of polar transport of endogenous auxins for root elongation. When added to medium with 2,4-D, TIBA caused suppression of callus formation, again pointing to the necessity of polar transport of 2,4-D. In combination with 2,4-D, cultures with 3,5-D resembled cultures at lower 2,4-D concentrations, pointing to a competitive interaction between 3,5-D and 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interspecific hybrid between Allium cepa and Allium sativum

Summary Interspecific hybrids between Allium cepa and Allium sativum were obtained using the fertile clone A. sativum as the male parent. The nascent embryos which formed shortly in interspecific hybridization between A. cepa and A. sativum were rescued by ovule culture at an early stage. The zygotes or proembryos developed in Murashige and Skoog medium containing 5.7×10^-8 M indole-3-butyric acid (IBA). Once developed, the embryos were taken out of the ovule and cultured on embryo culture medium where they regenerated into whole plants. The hybridity of the plants obtained was examined by morphological observation, chromosome analysis, and ribosomal RNA gene analysis. The analyses proved that the plants were mature sexual hybrids between A. cepa and A. sativum. Each hybrid plant had keeled but fistulose leaves and formed a bulb resembling that of A. cepa. The hybrids produced not only S-propenyl-l-cysteine sulfoxide, which is the major flavor precursor in A. cepa, but also S-allyl-l-cysteine sulfoxide (alliin), which is characteristic of A. sativum.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Uptake of phenol by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

Phenol was absorbed unspecifically by active and by inactivated cells of a strictly anaerobic, phenol-degrading consortium to reach about twice the concentration of the medium. The absorption was temperature-dependent. A Q₁₀ of 1.7 was determined, indicating that accumulation was due to diffusion or facilitated diffusion and not to an active transport process. At increasing phenol concentration in the medium, concentrated cell suspensions adsorpted phenol proportionally until saturation was reached at about 25 nmol phenol/mg cell dry weight. At a phenol concentration in the medium of 2 mm, the washed cell pellet contained 3.5 mm phenol. Under conditions that allowed phenol metabolism (presence of CO₂), [¹⁴C]4-hydroxybenzoyl-coenzyme A and [¹⁴C]4-hydroxybenzoate were found as early intermediates of [U-¹⁴C]phenol degradation for the first time. [¹⁴C]Benzoate was excreted stoichiometrically if phenol degradation to acetate was prevented by H₂. Absolutely no ¹⁴C-label was found in the phenylphosphate peak after HPLC separation, which excluded phosphorylation of phenol during uptake or during degradation in the cells. Correspondence to: J. Winter