Anaerobically-grown Cells Research Articles

Anaerobic Growth and Maintenance of Mammalian Cell Lines.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2-3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Similarities between Exogenously- and Endogenously-Induced Envelope Stress: The Effects of a New Antibacterial Molecule, TPI1609-10

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.

Differential effects of hydrogen peroxide and ascorbic acid on the aerobic thermosensitivity of yeast cells grown under aerobic and anoxic conditions

We have previously demonstrated that in aerobically-grown cells of the yeast Saccharomyces cerevisiae, hydrogen peroxide (H(2)O(2)) increases and ascorbic acid decreases cellular thermosensitivity, as determined by the inducibility of a heat shock (HS)-reporter gene. In this work, we reveal that the aerobic thermosensitivity of anaerobically-grown yeast cells also increases in the presence of H(2)O(2), albeit differentially between cells with two different lipid profiles. In comparison to aerobically-grown fermenting cells treated with the same H(2)O(2) concentration, both these types of anaerobically-grown cells were found to be considerably less sensitive to aerobic heat shock and considerably more thermotolerant. Paradoxically, and in contrast to ascorbate-pretreated aerobically-grown yeast cells, when anaerobically-grown cells were heat-shocked aerobically in the presence of the same ascorbic acid concentration, they exhibited increased thermosensitivity and decreased intrinsic thermotolerance with respect to their untreated counterparts. These findings are discussed with respect to what is currently known about the redox and physiological status of yeast cells grown aerobically and cells reoxygenated following anoxic growth.

Proteome analysis of aerobically and anaerobically grown Saccharomyces cerevisiae cells

The yeast Saccharomyces cerevisiae is able to grow under aerobic as well as anaerobic conditions. We and others previously found that transcription levels of approximately 500 genes differed more than two-fold when cells from anaerobic and aerobic conditions were compared. Here, we addressed the effect of anaerobic growth at the post-transcriptional level by comparing the proteomes of cells isolated from steady-state glucose-limited anaerobic and aerobic cultures. Following two-dimensional gel electrophoresis and mass spectrometry we identified 110 protein spots, corresponding to 75 unique proteins, of which the levels differed more than two-fold between aerobically and anaerobically-grown cells. For 21 of the 110 spots, the intensities decreased more than two-fold whereas the corresponding mRNA levels increased or did not change significantly under anaerobic conditions. The intensities of the other 89 spots changed in the same direction as the mRNA levels of the corresponding genes, although to different extents. For some genes of glycolysis a small increase in mRNA levels, 1.5-2 fold, corresponded to a 5-10 fold increase in protein levels. Extrapolation of our results suggests that transcriptional regulation is the major but not exclusive mechanism for adaptation of S. cerevisiae to anaerobic growth conditions.

The effect of oxygen on the growth and physiology of Porphyromonas gingivalis.

Oxygen constitutes a constant challenge for the survival of strict anaerobes in the oral environment. The aim of this study was to investigate the effect of oxygen on the physiology and growth of Porphyromonas gingivalis in a continuous culture system when grown under conditions of hemin limitation and excess. Results showed that, when grown in the presence of hemin at 0.5 mg/l, P. gingivalis could tolerate low levels of oxygen, being able to reach steady-state when 6% oxygen was present in the incoming gas mixture. When the hemin concentration was increased to 5 mg/l, the culture tolerated 10% oxygen. Anaerobically-grown cells were coccoid in shape, whereas those grown in the presence of oxygen were bacillary. Acetate was the predominant end-product in cultures grown in the presence of oxygen or in cultures hemin-limited. Despite some changes in the activity of Arg- and Lys-gingipain, most of the proteolytic activity was retained in the presence of oxygen. Activity of each of the three anti-oxidant enzymes tested (NADH oxidase, NADH peroxidase and SOD) was detected under all conditions and usually increased under oxygenated environments. Higher activities were also seen in the hemin-limited cultures. These results show some of the changes that occur in the physiology of P. gingivalis as a result of oxidative stress and confirm that hemin has a protective effect on the growth of the microorganism in the presence of oxygen.

Non-respiratory oxygen consumption pathways in anaerobically-grown Saccharomyces cerevisiae: evidence and partial characterization.

Despite the absence of an alternative mitochondrial ubiquinol oxidase, Saccharomyces cerevisiae consumes oxygen in an antimycin A- and cyanide-resistant manner. Cyanide-resistant respiration is typically used when the classical respiratory chain is impaired or absent (i.e in anaerobically-grown cells shifted to normoxia or in respiratory-deficient cells). We characterized the non-respiratory oxygen consumption pathways operating during anoxic-normoxic transitions in glucose-repressed resting cells. High-resolution oxygraphy confirmed that the cellular non-respiratory oxygen consumption pathway is sensitive to high concentrations of cyanide, azide, SHAM and TTFA, and revealed several new characteristics. First, the use of sterol biosynthesis inhibitors showed that this pathway makes a considerable contribution (about 25%) to both endogenous and glucose-dependent oxygen consumption. Anaerobically-grown glucose-repressed cells exhibited high apparent oxygen affinities (K(m) for oxygen = 0.5-1 micro M), even in mutants deficient in respiration or sterol synthesis. Exogeneously added glucose and endogenous stored carbohydrates were the only substrates that were efficient for cellular oxygen consumption (apparent K(m) for exogenous glucose = 2-3 mM). On the other hand, fluorimetric measurements of the cellular NAD(P)H pool showed that the cellular oxygen consumption (sterol biosynthesis and unknown pathways) was dependent more on the intracellular level of NADPH than of NADH. High oxygen affinity NADPH-dependent oxygen consumption systems were thought to be mainly localized in microsomal membranes, and several data indicated a significant contribution made by uncoupled p450 systems, together with still uncharacterized systems. Such activities are associated in vitro with a massive production of O(2) (.-) and, to a lower extent, H(2)O(2) and a likely concomitant production of H(2)O.

Synthesis ofRhodobacter sphaeroidescytochromec2inEscherichia coli

The cytochrome c2 structural gene, cycA, from Rhodobacter sphaeroides was expressed in Escherichia coli. CycA-specific mRNA was detected in E. coli both under aerobic and anaerobic conditions with trimethylamine-N-oxide as electron acceptor. However mature holocytochrome c2 was only detected in anaerobically-grown cells. The mature form of cytochrome c2 (Mr= 12,500) was secreted into the periplasm of E. coli suggesting that the signal polypeptide was processed. The cytochrome c2 synthesized in E. coli exhibited absorbance maxima in the reduced from at 550 nm (α-band) and 521 nm (β-band) and contained covalently attached haem c. The results indicate that a foreign c-type cytochrome can be secreted and assembled in E. coli under anaerobic conditions.

Synthesis of Rhodobacter sphaeroides cytochrome c2 in Escherichia coli

The cytochrome c2 structural gene, cycA, from Rhodobacter sphaeroides was expressed in Escherichia coli. CycA-specific mRNA was detected in E. coli both under aerobic and anaerobic conditions with trimethylamine-N-oxide as electron acceptor. However mature holocytochrome c2 was only detected in anaerobically-grown cells. The mature form of cytochrome c2 (Mr = 12,500) was secreted into the periplasm of E. coli suggesting that the signal polypeptide was processed. The cytochrome c2 synthesized in E. coli exhibited absorbance maxima in the reduced form at 550 nm (alpha-band) and 521 nm (beta-band) and contained covalently attached haem c. The results indicate that a foreign c-type cytochrome can be secreted and assembled in E. coli under anaerobic conditions.

The effect of oxygen on fructose-2,6-bisphosphate concentration and fructose-6-phosphate, 2-kinase activity in yeast cells

Fructose-2,6-bisphosphate concentration and fructose-6-phosphate,2-kinase activity were measured in yeast cells grown aerobically or anaerobically using glucose as a carbon source. A new improved analytical method using HPLC was employed to measure fructose-2,6-P 2 concentration. Anaerobically-grown yeast cells contain approximately 4-fold higher levels of fructose-2,6-P 2 as compared to aerobically-grown cells in the growth phase of culture. Similarly, fructose-6-P,2-kinase activity is approximately 7-fold higher in the anaerobically-grown cells. These results suggest that the presence of oxygen in the growth medium decreases the content of fructose-2,6-P 2 through inactivation of fructose-6-P,2-kinase.

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30 degrees C (generation time 1.1 h) or 20 degrees C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.

Anaerobic transport in Escherichia coli membrane vesicles.

Anaerobic beta-galactoside transport in whole cells and membrane vesicles from E. coli ML 308-225 is coupled to the oxidation of alpha-glycerol-P or D-lactate with fumarate as an electron acceptor. Alternatively, anaerobic beta-galactoside transport may be coupled to the oxidation of formate utilizing nitrate as electron acceptor. Both anaerobic electron-transfer systems are induced by growth of the organisms under appropriate conditions. Components of both systems are loosely bound to the membrane, necessitating the use of a modified procedure for vesicle preparation in order to demonstrate anaerobic transport in vitro. Addition of ATP or an ATP-generating system to vesicles prepared from anaerobically-grown cells or inclusion of ATP or the ATP-generating system during preparation of vesicles does not stimulate transport. The results support the conclusion that active transport under anaerobic conditions is coupled primarily to electron flow.

Oxidative phosphorylation in yeast VIII. Osmotic and permeability properties of mitochondria isolated from wild-type yeast and from a respiration-deficient mutant

1. Close similarities between yeast and mammalian mitochondria were found with respect to (a) osmotic response in impermeable solutes, sorbitol and KCl, (b) substrate translocation, (c) properties of the adenine nucleotide translocation system. A separate transport system for succinate, distinct from the dicarboxylate translocator, may be present in yeast mitochondria. 2. Substrate translocation was found to be preserved in pro-mitochondria from anaerobically-grown cells and in mitochondria from a respiration-deficient mutant. Adenine nucleotide translocation was demonstrated not to be affected by the cytoplasmic mutation. Along with previous data of other investigators, these results allow a general conclusion that neither the presence of a functional respiratory chain nor mitochondrial protein synthesis are prerequisite for the proper assemblage of the translocation systems in the mitochondrial membrane and for determining its permeability characteristics.

Ultraviolet irradiation studies on the cytoplasmic determinant of the yeast mitochondrion

Ultraviolet induction of the cytoplasmic petite mutation in yeast was studied under a variety of growth conditions both before and after irradiation. The induction curves were found to be single-event for anaerobically-grown cells, and multiple-event for aerobic cells. By using a split dose technique, both photo-reactivation and dark-repair were investigated. No significant difference in repair ability was found between aerobic and anaerobic cells. A target theory interpretation of the induction curves suggests that mitochondrial DNA, assumed to be the genetic determinant, exists as a single heritable unit in anaerobic cells, and as multiple units in aerobic cells; moreover, the number of genetically effective copies in the aerobic cells appears to be far smaller than the number of mitochondria. These results are shown to be in accord with an independent estimate of the number of cytoplasmic determinants derived from recent genetic studies on mitochondrial recombination in aerobic and anaerobic yeast cells.

Role of oxygen and heme in heme synthesis and the development of hemoprotein activity in an anaerobically-grown staphylococcus

Abstract 1. 1. Cell suspensions of anaerobically-grown Staphylococcus epidermidis exhibit very low catalase and respiratory activity and low content of protoheme when compared to cells grown aerobically. Protoheme was determined by extraction with acid-acetone and conversion to the pyridine hemochromogen. Large amounts of coproporphyrin are formed during growth under anaerobic conditions. If added hemin is present in the medium during anaerobic growth, the resulting cells exhibit a marked increase in catalase and respiratory activity. 2. 2. When resting suspensions of cells grown anaerobically without hemin supplementation are aerated at 37°, they exhibit a rapid, 30-fold increase in catalase activity, a 4-fold increase in respiratory activity, and, in the presence of δ-amino-levulinic acid, a 5-fold increase in protoheme content. These effects of aeration do not require addition of amino acids and are not appreciably inhibited by chloramphenicol, which causes a marked inhibition of protein synthesis as indicated by [14C]leucine incorporation. 3. 3. When hemin is added under anaerobic conditions to resting suspensions of anaerobically-grown cells a marked rise in catalase and respiratory activity also occurs. This effect of hemin is not blocked by chloramphenicol. 4. 4. These results suggest that when heme synthesis is impaired by growth in the absence of oxygen, this organism forms all the enzymes required for heme synthesis, and some of the apoenzymes of the functional hemoproteins (minus the heme prosthetic group). Oxygen may be required as substrate for a terminal step in heme synthesis in this organism.

On the relationship between respiratory activity and lipid composition of the yeast cell

1. 1. Phospholipids, total fatty acids, sterols and hydrocarbons were determined in respiratory-competent and respiratory-deficient cells of Saccharomyces cerevisiae grown aerobically and anaerobically. 2. 2. In a respiratory-competent strain, the previously-reported reduction in the content of phospholipids, fatty acids, and sterols of cells grown anaerobically on glucose in medium supplemented with ergosterol and Tween 80, relative to aerobically-grown cells, was confirmed. Anaerobically-grown cells from medium lacking ergosterol contained more phospholipids and fatty acids, and from medium lacking both ergosterol and Tween 80 contained more phospholipids, than cells from the supplemented medium. 3. 3. Changes in the lipid fractions during respiratory adaptation of anaerobically-grown respiratory-competent yeast were followed. The level of phospholipids rose during the first 2 h of adaptation, the level of other fractions rising during several hours approximately parallel with the development of respiratory activity. 4. 4. When anaerobically-grown respiratory-competent yeast was aerated in the presence of acriflavin or chloramphenicol, no respiratory adaptation took place. However, the content of phospholipids and of total fatty acids increased in a manner similar to that in cells during respiratory adaptation, whereas the content of sterols increased less. The cells aerated in the presence of cycloheximide had no respiratory ability, the content of phospholipids was similar to that in the cells aerated with acriflavin or chloramphenicol, the content of fatty acids was intermediate between aerobically- and anaerobically-grown cells, and the content of sterols was markedly reduced and approximated that of non-aerated anaerobically-grown cells. 5. 5. The respiratory-deficient mutant, derived from the competent strain, showed the same pattern of lipid composition as the original strain. The amount of lipids was reduced in anaerobically-grown cells. The lipid composition of aerobically-grown mutant cells was the same as that of aerobically-grown wild yeast, except for the phospholipid content which was lower in the mutant.

Subcellular particles carrying mitochondrial enzymes in anaerobically-grown cells of Saccharomyces cerevisiae

Subcellular particles carrying mitochondrial enzymes in anaerobically-grown cells of Saccharomyces cerevisiae

Subcellular particles carrying mitochondrial enzymes in anaerobically-grown cells of Saccharomyces cerevisiae

Subcellular particles carrying mitochondrial enzymes in anaerobically-grown cells of Saccharomyces cerevisiae

Effect of cysteine on respiration and catalyse synthesis by Saccharomyces cerevisiae.

Catalase synthesis can be induced in anaerobically grown cells of Saccharomyces cerevisiae by aeration (Chantrenne, 1954). In a study of the effect of various nitrogenous substances on the ability of anaerobically grown yeast cells to synthesize catalase, it was found that cysteine inhibited the catalase induction severely (Bhuvaneswaran, Rege & Sreenivasan, 1961). In the present paper, the effect of cysteine on catalase synthesis and on respiration is examined in more detail.